技术领域technical field
本发明属于生物技术领域，具体涉及高凝血状态的生物标志物及其应用。The invention belongs to the field of biotechnology, and in particular relates to a biomarker of a hypercoagulation state and an application thereof.
背景技术Background technique
机体受到生理或病理因素刺激时，会产生一系列的变化，其中可监测的变化被称为生物标志物。生物标志物的最根本特征是变化。当机体受到刺激后，体内原本的平衡状态被打破，细胞、组织和器官会产生一些异常的代谢产物，并将其排出体外，以恢复原有的平衡，维持内环境稳态。与血液相比，尿液由于不受机体稳态机制的调节，可以积累大量变化，能够更好地反应机体的变化，是一种良好的生物标志物的来源。同时，尿液具有大量、无创、可连续收集等优势，对尿液中各类代谢产物的研究越来越受到人们的重视。When the body is stimulated by physiological or pathological factors, it will produce a series of changes, and the changes that can be monitored are called biomarkers. The most fundamental characteristic of a biomarker is change. When the body is stimulated, the original balance in the body is broken, cells, tissues and organs will produce some abnormal metabolites and excrete them from the body to restore the original balance and maintain the homeostasis of the internal environment. Compared with blood, urine can accumulate a large number of changes because it is not regulated by the body's homeostasis mechanism, and can better reflect changes in the body, and is a good source of biomarkers. At the same time, urine has the advantages of large volume, non-invasiveness, and continuous collection, and the research on various metabolites in urine has attracted more and more attention.
虽然尿液在生物标志物研究方面具有很多优势，但由于蛋白质组具有明显的个体差异，且多种生理性因素，如年龄、性别、运动、饮食等，均会使尿液蛋白质组产生变化，这就使得直接研究人类的尿液蛋白质组学比较困难。而采用动物模型进行尿蛋白质组研究，筛选可能的候选疾病生物标志物，就可通过严格控制实验条件，将个体差异和环境等因素对尿蛋白质组的影响降到最低，从而在较短的时间内获得比较可信的实验结果，是目前较为流行的一种寻找尿液生物标志物的方法。Although urine has many advantages in biomarker research, due to the obvious individual differences in the proteome, and various physiological factors, such as age, sex, exercise, diet, etc., will cause changes in the urine proteome. This makes it difficult to directly study human urine proteomics. However, using animal models to conduct urinary proteome research and screen possible candidate disease biomarkers can strictly control the experimental conditions to minimize the impact of individual differences and environmental factors on the urinary proteome, so that in a short period of time It is currently a popular method to find urine biomarkers to obtain more credible experimental results.
血液高凝状态(hypercoagulable states)是指血液处于非常容易凝结的状态，通常被认为是血栓前状态，最终会导致动静脉血栓的形成。血栓性疾病常常是心血管疾病的严重并发症。高凝状态的相关生物标志物研究，目前可见报道主要是一些血液生物标志物的相关报道，而未见相关尿液生物标志物研究报道。同时，临床上对于高凝状态的诊断方法，主要集中于血液检测方面，如凝血因子检测、D-二聚体检测、凝血时间检测等。但Hypercoagulable states refer to the state in which the blood is very easy to coagulate, which is generally considered to be a prethrombotic state, which will eventually lead to the formation of arterial and venous thrombosis. Thrombotic disease is often a serious complication of cardiovascular disease. The studies on biomarkers related to the hypercoagulable state are currently reported mainly on some blood biomarkers, but there are no related studies on urine biomarkers. At the same time, the clinical diagnosis methods for hypercoagulable state mainly focus on blood testing, such as blood coagulation factor detection, D-dimer detection, coagulation time detection and so on. but
发明内容Contents of the invention
本发明使用三种不同的促凝血药物(酚磺乙胺、氨甲环酸和氨基己酸)，分别对大鼠的凝血状态进行干预，成功得到大鼠急性高凝血状态模型。然后采用完全一致的分析策略，分析了不同促凝血药物干预组的大鼠尿液蛋白质组的变化，首次系统研究了高凝血状态下，尿液蛋白质组的变化特征，并寻找到高凝血状态相关尿液生物标志物。The present invention uses three different coagulation-promoting drugs (ethamine, tranexamic acid and aminocaproic acid) to respectively intervene on the coagulation state of rats, and successfully obtains a model of acute hypercoagulation state of rats. Then, using a completely consistent analysis strategy, the changes in the urine proteome of rats in different procoagulant drug intervention groups were analyzed. For the first time, the changes in the urine proteome in the hypercoagulable state were systematically studied, and the correlation between the hypercoagulable state was found. Urine biomarkers.
本发明首先提供了检测X含量的系统的新用途。The present invention first provides a new use of the system for detecting X content.
本发明提供了检测X含量的系统在制备诊断或辅助诊断高凝血状态的产品中的应用；The invention provides the application of the system for detecting X content in the preparation of products for diagnosing or assisting in diagnosing hypercoagulation state;
本发明还提供了检测X含量的系统在制备诊断或辅助诊断血栓性疾病和/或心血管疾病的产品中的应用；The present invention also provides the application of the system for detecting X content in the preparation of products for diagnosis or auxiliary diagnosis of thrombotic diseases and/or cardiovascular diseases;
本发明还提供了检测X含量的系统在诊断或辅助诊断高凝血状态中的应用；The present invention also provides the application of the system for detecting X content in diagnosis or auxiliary diagnosis of hypercoagulation state;
本发明还提供了检测X含量的系统在诊断或辅助诊断血栓性疾病和/或心血管疾病中的应用；The present invention also provides the application of the system for detecting X content in the diagnosis or auxiliary diagnosis of thrombotic diseases and/or cardiovascular diseases;
所述X为如下X1)或X2)或X3)：Said X is as follows X1) or X2) or X3):
X1)为高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3、核糖核酸酶4、氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基；X1) is Golgi protein I, carbonic anhydrase I, glypican-3, ribonuclease 4, chloride intracellular channel protein 1, biglycan, Rab GDP dissociation inhibitor, phosphoric acid Glycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triad nucleotide binding protein 1, myosin light chain 6, L-xylulose reductase and hemoglobin β subunit;
X2)为胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基、血管紧张素转换酶2同系物、超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)；X2) Cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 subunit, actin-like protein, guanine nucleotide-binding protein G(k)α subunit, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunit, angiotensin converting enzyme 2 Homolog, superoxide dismutase [Cu-Zn], amyloid 2, calbindin, fumaryl acetoacetase, myosin light chain 6, Frizzled 2, apolipoprotein M, myoglobin, liver ligand B1, selenium-binding protein 1, and calmodulin (CaM);
X3)为细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白、硫氧还蛋白(Trx)、肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4。X3) is cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin, thioredoxin (Trx), peptidyl prolyl cis-trans isomerase A, ABHD14B protein, Frizzled protein 4 , α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transferase P, alkaline phosphatase, 14-3-3 protein ζ/δ, Quinolone Oxidoreductase, Rho GDP-Dissociation Inhibitor 1, Peroxide Reductase-6, Inositol Polyphosphate Phosphatase 1, Glucose-6-Phosphate Isomerase, E3 Ubiquitin-Protein Ligase UBR4, Prostaglandins F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triad nucleotide binding protein 1, linker adhesion molecule C, stanniocalcin-1, glutamate-cysteine ligase catalysis Subunit, N(G), N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetylgalactosaminidase, fumaryl acetoacetase, 1,2-dihydroxy- 3-keto-5-methylthiopentene dioxygenase, selenium binding protein 1, and chondroitin sulfate proteoglycan 4.
上述应用中，所述检测X含量的系统可为利用现有技术中的方法检测所述X含量所需的试剂和/或仪器，如利用质谱或其相关技术检测所述X含量所需的试剂和/或仪器，或利用免疫杂交技术检测所述X含量所需的试剂和/或仪器，或利用ELISA技术检测所述X含量所需的试剂和/或仪器，或利用蛋白芯片或试纸检测所述X含量所需的试剂和/或仪器；所述X含量为尿液中X含量。In the above application, the system for detecting the content of X can be the reagents and/or instruments required to detect the content of X by methods in the prior art, such as the reagents required for detecting the content of X by using mass spectrometry or related technologies and/or instruments, or the reagents and/or instruments required for detecting the X content by using immunoblotting technology, or the reagents and/or instruments required for detecting the X content by using ELISA technology, or using protein chips or test papers to detect the X content Reagents and/or instruments required for the above X content; the X content is the X content in urine.
本发明发现促凝血药物(酚磺乙胺、氨甲环酸和氨基己酸)干预后，大鼠凝血功能发生改变，导致尿液蛋白质组发生变化，说明尿液可以反映血液的变化。尿液作为积累体内代谢物的最终场所，不受稳态机制调控，可以反映大量的变化，大量的变化意味着大量的潜在生物标志物。The present invention finds that after the intervention of procoagulant drugs (ethanolsulfate, tranexamic acid and aminocaproic acid), the coagulation function of rats changes, resulting in changes in urine proteome, indicating that urine can reflect changes in blood. Urine, as the final site for the accumulation of metabolites in the body, is not regulated by homeostatic mechanisms and can reflect a large number of changes, which means a large number of potential biomarkers.
本发明所涉及的酚磺乙胺，中文别名：止血敏、止血定、羟苯磺乙胺、氢醌磺乙胺，化学名称为2,5-二羟基苯磺酸二乙胺盐，其分子式：C10H17NO5S，分子量为263.31，分子结构示意图如图1所示。本发明使用酚磺乙胺对大鼠的凝血状态进行干预，成功得到大鼠急性高凝血状态模型，基于酚磺乙胺构建的大鼠高凝血状态动物模型的蛋白质组学分析，得到20个显著差异蛋白，其中，有13个为人类同源蛋白，即上述X1)中所述的13个蛋白，并将其作为酚磺乙胺诱导所致高凝血状态的生物标志物。酚磺乙胺诱导所致高凝血状态指的是由于初级凝血功能增强导致的血液高凝状态，即通过促进血管收缩，促进血小板的聚集、粘附和活性因子的释放，所导致的高凝状态，也可称为血栓前状态。The ethylamine phensulfonate involved in the present invention has Chinese aliases: Zhixuemin, Zhixueding, oxybenzenesulfonylethylamine, hydroquinonesulfethylamine, and its chemical name is 2,5-dihydroxybenzenesulfonic acid diethylamine salt. Its molecular formula : C10 H17 NO5 S, the molecular weight is 263.31, and the schematic diagram of molecular structure is shown in Fig. 1 . The present invention intervenes the coagulation state of rats by using acesulfame, and successfully obtains the acute hypercoagulation state model of rats. Based on the proteomic analysis of the rat hypercoagulation state animal model constructed by acesulfame, 20 significant Among them, 13 are human homologous proteins, namely the 13 proteins described in X1) above, and they are used as biomarkers of the hypercoagulation state induced by etamsulfame. The hypercoagulable state induced by ethylamine refers to the hypercoagulable state caused by the enhancement of the primary coagulation function, that is, the hypercoagulable state caused by promoting the aggregation, adhesion and release of active factors of platelets by promoting vasoconstriction , also known as the prethrombotic state.
本发明所涉及的氨甲环酸，化学名称为4-(氨甲基)环己甲酸，又名为凝血酸、传明酸、止血环酸，分子式为C8H15NO2，分子量为157.21，其分子结构示意图如图1所示；氨甲环酸能与纤溶酶和纤溶酶原上的纤维蛋白亲和部位的赖氨酸结合部位(LBS)强烈吸附，阻抑了纤溶酶、纤溶酶原与纤维蛋白结合，从而强烈地抑制了由纤溶酶所致纤维蛋白分解，从而达到血液高凝态。本发明使用氨甲环酸对大鼠的凝血状态进行干预，成功得到大鼠急性高凝血状态模型，基于氨甲环酸构建的大鼠高凝血状态动物模型的蛋白质组学分析，得到28个显著差异蛋白，其中，有18个为人类同源蛋白，即上述X2)中所述的18个蛋白，并将其作为氨甲环酸诱导所致高凝血状态的生物标志物。氨甲环酸诱导所致高凝血状态指的是通过抑制纤溶酶活性所导致的高凝状态。The tranexamic acid involved in the present invention has a chemical name of 4-(aminomethyl)cyclohexanecarboxylic acid, also known as tranexamic acid, tranexamic acid, and tranexamic acid, with a molecular formula ofC8H15NO2 and a molecular weight of157.21 . The schematic diagram of its molecular structure is shown in Figure 1; tranexamic acid can strongly adsorb with the lysine binding site (LBS) of the fibrin affinity site on plasmin and plasminogen, inhibiting plasmin, Plasminogen combines with fibrin, thereby strongly inhibiting the decomposition of fibrin caused by plasmin, thereby achieving a hypercoagulable state of blood. The present invention uses tranexamic acid to intervene on the coagulation state of rats, and successfully obtains the rat acute hypercoagulation state model. Based on the proteomic analysis of the rat hypercoagulation state animal model constructed by tranexamic acid, 28 significant Among them, 18 are human homologous proteins, that is, the 18 proteins described in X2 above, and they are used as biomarkers of hypercoagulation state induced by tranexamic acid. The hypercoagulable state induced by tranexamic acid refers to the hypercoagulable state caused by inhibiting the activity of plasmin.
本发明所涉及的氨基己酸，中文别名：6-氨基己酸，氨己酸、ε-氨基已酸、抗血纤溶酸，分子式为C6H13NO2，分子量为131.17，其分子结构示意图如图1所示；氨基己酸可抑制纤溶酶原激活，从而使纤维蛋白溶酶原不能激活为纤维蛋白溶酶，从而抑制纤维蛋白的溶解，产生高凝血状态。本发明使用氨基己酸对大鼠的凝血状态进行干预，成功得到大鼠急性高凝血状态模型，基于氨基己酸构建的大鼠高凝血状态动物模型的蛋白质组学分析，得到65个显著差异蛋白，其中，有34个为人类同源蛋白，即上述X3)中所述的34个蛋白，并将其作为氨基己酸诱导所致高凝血状态的生物标志物。氨基己酸诱导所致高凝血状态指的是通过抑制纤维蛋白溶酶原的激活因子导致的高凝血状态。Aminocaproic acid involved in the present invention, Chinese aliases: 6-aminocaproic acid, aminocaproic acid, ε-aminocaproic acid, anti-blood fibrinolytic acid, molecular formula is C6 H13 NO2 , molecular weight is 131.17, its molecular structure The schematic diagram is shown in Figure 1; aminocaproic acid can inhibit the activation of plasminogen, so that plasminogen cannot be activated into plasmin, thereby inhibiting the dissolution of fibrin and producing a hypercoagulable state. The present invention uses aminocaproic acid to intervene in the coagulation state of rats, and successfully obtains the acute hypercoagulation state model of rats. Based on the proteomic analysis of the rat hypercoagulation state animal model constructed with aminocaproic acid, 65 significantly different proteins are obtained. , 34 of which are human homologous proteins, namely the 34 proteins described in X3 above, and they are used as biomarkers of the hypercoagulation state induced by aminocaproic acid. The hypercoagulable state induced by aminocaproic acid refers to the hypercoagulable state caused by inhibiting the activator of plasminogen.
在实际应用中可以根据上述获得的高凝血状态的生物标志物按照如下(1)-(3)中任一种方法来判断受试者是否处于高凝血状态：In practical application, it can be judged whether the subject is in a hypercoagulable state according to any of the following methods (1)-(3) according to the biomarkers of the hypercoagulable state obtained above:
(1)若受试者尿液的高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3和核糖核酸酶4的含量均高于健康人至少一倍，且氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于酚磺乙胺诱导所致高凝血状态。(1) If the content of Golgi protein I, carbonic anhydrase I, glypican-3 and ribonuclease 4 in the urine of the subject is at least twice that of healthy people, and the chloride ion intracellular Channel protein 1, biglycan, Rab GDP dissociation inhibitor, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triad nucleotide binding protein 1, myosin light chain 6. If the content of L-xylulose reductase and hemoglobin β subunit is at least one time lower than that of healthy people, the subject is in or is expected to be in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to phenol Ethylamine-induced hypercoagulable state.
若受试者尿液的高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3和核糖核酸酶4的含量未均高于健康人至少一倍，或氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于酚磺乙胺诱导所致高凝血状态。If the content of Golgi protein I, carbonic anhydrase I, glypican-3 and ribonuclease 4 in the urine of the subject is not at least double that of healthy people, or the chloride ion intracellular channel protein 1. Biglycan, Rab GDP dissociation inhibitor, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triplet nucleotide binding protein 1, myosin light chain 6, The contents of L-xylulose reductase and hemoglobin β subunit are not at least one times lower than that of healthy people, then the subject is not in or candidate is not in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to phenol Hypercoagulable state induced by sulphonethylamine.
(2)若受试者尿液的胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基和血管紧张素转换酶2同系物的含量均高于健康人至少一倍，且超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于氨甲环酸诱导所致高凝血状态。(2) If the test subject's urine contains cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, and guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 Subunit, actin-like protein, guanine nucleotide-binding protein G(k)α subunit, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunit and angiotensin-converting enzyme 2 homologues are at least twice as high as those in healthy people, and superoxide dismutase [Cu-Zn], amyloid 2, calcium binding protein, fumaryl acetoacetase, myosin The content of light chain 6, Frizzled protein 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1 and calmodulin (CaM) is at least one time lower than that of healthy people, then the subject is in or The candidate is in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by tranexamic acid.
若受试者尿液的胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基和血管紧张素转换酶2同系物的含量未均高于健康人至少一倍，或超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于氨甲环酸诱导所致高凝血状态。If the subject's urine contains cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 subunit, Actin-like proteins, guanine nucleotide-binding protein G(k)α subunits, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunits, and angiotensin The content of homologues of protein converting enzyme 2 was at least double that of healthy people, or superoxide dismutase [Cu-Zn], amyloid 2, calcium binding protein, fumaryl acetoacetase, myosin light chain 6. If the content of Frizzled protein 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1 and calmodulin (CaM) is not at least one time lower than that of healthy people, the subject is not in or The candidate is not in a hypercoagulable state; and the mechanism of this hypercoagulable state is related to the hypercoagulable state induced by tranexamic acid.
(3)若受试者尿液的细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白和硫氧还蛋白(Trx)的含量均高于健康人至少一倍，且肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于氨基己酸诱导所致高凝血状态。(3) If the content of cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin and thioredoxin (Trx) in the subject's urine is at least twice that of healthy people, and Peptidylprolyl cis-trans isomerase A, ABHD14B protein, Frizzled 4, α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transfer Enzyme P, alkaline phosphatase, 14-3-3 protein ζ/δ, quinolone oxidoreductase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, polyphosphate inositol phosphatase 1, glucose- 6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triad nucleotide binding protein 1, linker adhesion molecule C , stanniocalcin-1, glutamate-cysteine ligase catalytic subunit, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetyl The contents of galactosaminidase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase, selenium binding protein 1 and chondroitin sulfate proteoglycan 4 were all lower than At least twice that of healthy people, the subject is or is expected to be in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by aminocaproic acid.
若受试者尿液的细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白和硫氧还蛋白(Trx)的含量未均高于健康人至少一倍，或肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于氨基己酸诱导所致高凝血状态。If the content of cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin and thioredoxin (Trx) in the urine of the subject is not at least double that of healthy people, or the peptide group Prolyl cis-trans isomerase A, ABHD14B protein, Frizzled 4, α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transferase P , alkaline phosphatase, 14-3-3 protein ζ/δ, quinolone oxidoreductase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, polyphosphoinositide phosphatase 1, glucose-6- Phosphate isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triplet nucleotide binding protein 1, junctional adhesion molecule C, statin Calcein-1, glutamate-cysteine ligase catalytic subunit, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetylgalactose The contents of aminase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase, selenium-binding protein 1 and chondroitin sulfate proteoglycan 4 were not lower than those in healthy The subject is not or is not in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by aminocaproic acid.
本发明的另一个目的是提供以上述X作为高凝血状态标志物的新用途。Another object of the present invention is to provide a new use of the above-mentioned X as a marker of hypercoagulation state.
本发明提供了以上述X作为高凝血状态标志物在制备诊断或辅助诊断高凝血状态的产品中的应用；The present invention provides the application of the above-mentioned X as a hypercoagulation state marker in the preparation of products for diagnosis or auxiliary diagnosis of hypercoagulation state;
本发明还提供了以上述X作为高凝血状态标志物在制备诊断或辅助诊断血栓性疾病和/或心血管疾病的产品中的应用。The present invention also provides the use of the above-mentioned X as a hypercoagulation state marker in the preparation of products for diagnosing or assisting the diagnosis of thrombotic diseases and/or cardiovascular diseases.
本发明还提供了以上述X作为高凝血状态标志物在诊断或辅助诊断高凝血状态中的应用；The present invention also provides the use of the above-mentioned X as a hypercoagulable state marker in the diagnosis or auxiliary diagnosis of hypercoagulable state;
本发明还提供了以上述X作为高凝血状态标志物在诊断或辅助诊断血栓性疾病和/或心血管疾病中的应用。The present invention also provides the use of the above-mentioned X as a hypercoagulation state marker in the diagnosis or auxiliary diagnosis of thrombotic diseases and/or cardiovascular diseases.
本发明还有一个目的是提供诊断或辅助诊断高凝血状态的产品。Another object of the present invention is to provide products for diagnosing or assisting in the diagnosis of hypercoagulable states.
本发明提供的诊断或辅助诊断高凝血状态的产品为上述检测X含量的系统。The product for diagnosing or assisting the diagnosis of hypercoagulation state provided by the present invention is the above-mentioned system for detecting X content.
所述产品包括记载有如下内容的诊断卡：The product described includes a diagnostic card that states:
(1)若受试者尿液的高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3和核糖核酸酶4的含量均高于健康人至少一倍，且氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于酚磺乙胺诱导所致高凝血状态。(1) If the content of Golgi protein I, carbonic anhydrase I, glypican-3 and ribonuclease 4 in the urine of the subject is at least twice that of healthy people, and the chloride ion intracellular Channel protein 1, biglycan, Rab GDP dissociation inhibitor, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triad nucleotide binding protein 1, myosin light chain 6. If the content of L-xylulose reductase and hemoglobin β subunit is at least one time lower than that of healthy people, the subject is in or is expected to be in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to phenol Ethylamine-induced hypercoagulable state.
若受试者尿液的高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3和核糖核酸酶4的含量未均高于健康人至少一倍，或氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于酚磺乙胺诱导所致高凝血状态。If the content of Golgi protein I, carbonic anhydrase I, glypican-3 and ribonuclease 4 in the urine of the subject is not at least double that of healthy people, or the chloride ion intracellular channel protein 1. Biglycan, Rab GDP dissociation inhibitor, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triplet nucleotide binding protein 1, myosin light chain 6, The contents of L-xylulose reductase and hemoglobin β subunit are not at least one times lower than that of healthy people, then the subject is not in or candidate is not in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to phenol Hypercoagulable state induced by sulphonethylamine.
(2)若受试者尿液的胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基和血管紧张素转换酶2同系物的含量均高于健康人至少一倍，且超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于氨甲环酸诱导所致高凝血状态。(2) If the test subject's urine contains cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, and guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 Subunit, actin-like protein, guanine nucleotide-binding protein G(k)α subunit, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunit and angiotensin-converting enzyme 2 homologues are at least twice as high as those in healthy people, and superoxide dismutase [Cu-Zn], amyloid 2, calcium binding protein, fumaryl acetoacetase, myosin The content of light chain 6, Frizzled protein 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1 and calmodulin (CaM) is at least one time lower than that of healthy people, then the subject is in or The candidate is in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by tranexamic acid.
若受试者尿液的胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基和血管紧张素转换酶2同系物的含量未均高于健康人至少一倍，或超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于氨甲环酸诱导所致高凝血状态。If the subject's urine contains cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 subunit, Actin-like proteins, guanine nucleotide-binding protein G(k)α subunits, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunits, and angiotensin The content of homologues of protein converting enzyme 2 was at least double that of healthy people, or superoxide dismutase [Cu-Zn], amyloid 2, calcium binding protein, fumaryl acetoacetase, myosin light chain 6. If the content of Frizzled protein 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1 and calmodulin (CaM) is not at least one time lower than that of healthy people, the subject is not in or The candidate is not in a hypercoagulable state; and the mechanism of this hypercoagulable state is related to the hypercoagulable state induced by tranexamic acid.
(3)若受试者尿液的细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白和硫氧还蛋白(Trx)的含量均高于健康人至少一倍，且肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于氨基己酸诱导所致高凝血状态。(3) If the content of cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin and thioredoxin (Trx) in the subject's urine is at least twice that of healthy people, and Peptidylprolyl cis-trans isomerase A, ABHD14B protein, Frizzled 4, α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transfer Enzyme P, alkaline phosphatase, 14-3-3 protein ζ/δ, quinolone oxidoreductase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, polyphosphate inositol phosphatase 1, glucose- 6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triad nucleotide binding protein 1, linker adhesion molecule C , stanniocalcin-1, glutamate-cysteine ligase catalytic subunit, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetyl The contents of galactosaminidase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase, selenium binding protein 1 and chondroitin sulfate proteoglycan 4 were all lower than At least twice that of healthy people, the subject is or is expected to be in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by aminocaproic acid.
若受试者尿液的细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白和硫氧还蛋白(Trx)的含量未均高于健康人至少一倍，或肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于氨基己酸诱导所致高凝血状态。If the content of cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin and thioredoxin (Trx) in the urine of the subject is not at least double that of healthy people, or the peptide group Prolyl cis-trans isomerase A, ABHD14B protein, Frizzled 4, α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transferase P , alkaline phosphatase, 14-3-3 protein ζ/δ, quinolone oxidoreductase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, polyphosphoinositide phosphatase 1, glucose-6- Phosphate isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triplet nucleotide binding protein 1, junctional adhesion molecule C, statin Calcein-1, glutamate-cysteine ligase catalytic subunit, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetylgalactose The contents of aminase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase, selenium-binding protein 1 and chondroitin sulfate proteoglycan 4 were not lower than those in healthy The subject is not or is not in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by aminocaproic acid.
本发明产品具体可为试剂盒。The product of the present invention can specifically be a kit.
上述产品在制备诊断或辅助诊断血栓性疾病和/或心血管疾病的产品中的应用也属于本发明的保护范围。The application of the above products in the preparation of products for diagnosis or auxiliary diagnosis of thrombotic diseases and/or cardiovascular diseases also falls within the protection scope of the present invention.
上述产品在制备检测或评估受试者高凝血状态的产品中的应用也属于本发明的保护范围。The application of the above-mentioned products in the preparation of products for detecting or evaluating the hypercoagulation state of subjects also belongs to the protection scope of the present invention.
上述产品在诊断或辅助诊断血栓性疾病和/或心血管疾病中的应用也属于本发明的保护范围。The application of the above products in the diagnosis or auxiliary diagnosis of thrombotic diseases and/or cardiovascular diseases also belongs to the scope of protection of the present invention.
上述产品在检测或评估受试者高凝血状态的产品中的应用也属于本发明的保护范围。The application of the above products in products for detecting or evaluating the hypercoagulation state of subjects also falls within the protection scope of the present invention.
本发明的最后一个目的是提供一种检测或评估受试者高凝血状态的方法。A final object of the present invention is to provide a method for detecting or assessing the hypercoagulable state of a subject.
本发明提供的检测或评估受试者高凝血状态的方法是检测受试者中上述X含量。The method for detecting or evaluating the hypercoagulation state of the subject provided by the present invention is to detect the above-mentioned X content in the subject.
上述方法中，所述检测受试者中上述X含量为检测受试者尿液中上述X含量。In the above method, the detection of the content of X in the subject is the detection of the content of X in the urine of the subject.
上述方法中，评估受试者高凝血状态的方法参照上述产品中的诊断卡所记载的内容。In the above method, the method for evaluating the hypercoagulation state of the subject refers to the content recorded in the diagnosis card in the above product.
上述方法中，所述受试者是哺乳动物；哺乳动物是人或非人哺乳动物。In the above method, the subject is a mammal; the mammal is a human or a non-human mammal.
本发明的试验结果表明：三种不同促凝药物分别干预后，共得到105个显著变化的差异蛋白，这105个差异蛋白中有65个能够在人类中找到对等蛋白，即人类同源蛋白，在这65个人类同源蛋白中，有35个是人类核心尿蛋白质组成员，说明血液高凝状态下，人类核心尿蛋白质组可能会发生变化。本发明还发现差异蛋白的人类同源蛋白分布于各个不同组织中，但多集中于泌尿系统、消化系统和呼吸系统，说明凝血功能的改变，可能会影响这些系统的功能，也说明高凝血状态的机体干预范围是比较广泛的，机体高凝血状态作为一种显著的病理生理变化，可对机体多条代谢途径产生影响。本发明初步揭示了高凝血状态的机体干预范围和变化因子，而这些既可以提示相关生理病理机制、信号途径的变化特征，同时，也可以作为重要的生物标志物，应用于高凝血状态相关病理变化的检测和/或诊断，或者为此方面提供辅助信息。The test results of the present invention show that after the intervention of three different procoagulant drugs, a total of 105 significantly changed differential proteins were obtained, and 65 of the 105 differential proteins could find equivalent proteins in humans, that is, human homologous proteins , among the 65 human homologous proteins, 35 are members of the human core urine proteome, indicating that the human core urine proteome may change in the state of blood hypercoagulation. The present invention also found that the human homologous proteins of differential proteins are distributed in different tissues, but most of them are concentrated in the urinary system, digestive system and respiratory system, indicating that changes in blood coagulation function may affect the functions of these systems, and also indicate a hypercoagulable state The range of body intervention is relatively wide. As a significant pathophysiological change, the hypercoagulation state of the body can affect multiple metabolic pathways of the body. The present invention preliminarily reveals the scope of body intervention and change factors of hypercoagulation state, and these can not only prompt the change characteristics of related physiological and pathological mechanisms and signal pathways, but also can be used as important biomarkers in the pathology related to hypercoagulation state detection and/or diagnosis of changes, or to provide ancillary information in this regard.
附图说明Description of drawings
图1为酚磺乙胺、氨甲环酸和氨基己酸的结构示意图。A为酚磺乙胺的结构示意图；B为氨甲环酸的结构示意图；C为氨基己酸的结构示意图。Fig. 1 is the schematic structural diagram of phensulfame, tranexamic acid and aminocaproic acid. A is the structural schematic diagram of phenetamine; B is the structural schematic diagram of tranexamic acid; C is the structural schematic diagram of aminocaproic acid.
图2为酚磺乙胺、氨甲环酸和氨基己酸致大鼠高凝血状态动物模型的建立方法的示意图。A为酚磺乙胺致大鼠高凝血状态动物模型的建立方法的示意图；B为氨甲环酸致大鼠高凝血状态动物模型的建立方法的示意图；C为氨基己酸致大鼠高凝血状态动物模型的建立方法的示意图。Fig. 2 is a schematic diagram of a method for establishing an animal model of rat hypercoagulant state induced by etamide, tranexamic acid and aminocaproic acid. A is a schematic diagram of the establishment method of rat hypercoagulant state animal model induced by phensulfame; B is a schematic diagram of the establishment method of tranexamic acid-induced rat hypercoagulable state animal model; C is aminocaproic acid-induced rat hypercoagulant state Schematic diagram of the establishment method of the state animal model.
图3为酚磺乙胺、氨甲环酸和氨基己酸对大鼠APTT和CT的影响。A为酚磺乙胺对大鼠APTT和CT的影响；B为氨甲环酸对大鼠APTT和CT的影响；C为氨基己酸对大鼠APTT和CT的影响。Fig. 3 is the effect of phensulfame, tranexamic acid and aminocaproic acid on APTT and CT of rats. A is the effect of fensulfame on APTT and CT of rats; B is the effect of tranexamic acid on APTT and CT of rats; C is the effect of aminocaproic acid on APTT and CT of rats.
图4为对照组(生理盐水组)和实验组的尿液蛋白质SDS-PAGE分析。A为生理盐水组和酚磺乙胺组的尿液蛋白质SDS-PAGE分析；B为生理盐水组和氨甲环酸组的尿液蛋白质SDS-PAGE分析；C为生理盐水组和氨基己酸组的尿液蛋白质SDS-PAGE分析。Fig. 4 is the SDS-PAGE analysis of urine protein in the control group (normal saline group) and the experimental group. A is the urine protein SDS-PAGE analysis of normal saline group and phenetamine group; B is the urine protein SDS-PAGE analysis of normal saline group and tranexamic acid group; C is the normal saline group and aminocaproic acid group Urine protein SDS-PAGE analysis.
图5为高凝血状态尿液差异蛋白的组织表达分析。A为酚磺乙胺高凝血状态尿液差异蛋白的组织表达分析；B为氨甲环酸高凝血状态尿液差异蛋白的组织表达分析；C为氨基己酸高凝血状态尿液差异蛋白的组织表达分析。Figure 5 is the tissue expression analysis of urine differential proteins in hypercoagulable state. A is the tissue expression analysis of differential protein in urine of hypercoagulable state of etamsulfame; B is the tissue expression analysis of differential protein in urine of hypercoagulable state of tranexamic acid; C is the tissue of differential protein in urine of aminocaproic acid hypercoagulable state expression analysis.
具体实施方式detailed description
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。The quantitative tests in the following examples were all set up to repeat the experiments three times, and the results were averaged.
下述实施例中的SPF级雄性Wistar大鼠，180-200g，购自北京维通利华实验动物技术有限公司，标准环境下适应性饲养一周后开始实验。The male Wistar rats of SPF grade in the following examples, 180-200 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and the experiments were started after one week of adaptive feeding under standard conditions.
下述实施例中采用活化部分凝血活酶时间(Activated Partial ThromboplastinTime，APTT)和凝血时间(Clotting Time，CT)评估大鼠的凝血状态，以确定大鼠高凝血模型是否成功建立，具体测定方法如下：In the following examples, activated partial thromboplastin time (Activated Partial ThromboplastinTime, APTT) and coagulation time (Clotting Time, CT) were used to evaluate the coagulation status of rats, so as to determine whether the hypercoagulation model of rats was successfully established, and the specific measurement methods were as follows :
1、活化部分凝血活酶时间的测定1. Determination of activated partial thromboplastin time
大鼠内眦静脉采血后，按照血液:柠檬酸钠抗凝剂＝9:1(体积比)的比例加入柠檬酸钠抗凝剂，上下轻柔颠倒混匀5～6次，离心：4℃，2500g，15min，离心所得上清液即为血浆。取100μL血浆用于部分凝血活酶时间的测定。After blood collection from the inner canthus vein of rats, sodium citrate anticoagulant was added according to the ratio of blood: sodium citrate anticoagulant = 9:1 (volume ratio), mixed up and down gently for 5-6 times, centrifuged at 4°C, 2500g, 15min, the supernatant obtained by centrifugation is plasma. Take 100 μL of plasma for the determination of partial thromboplastin time.
测定方法遵循试剂盒的产品说明书：37℃温浴100μL鞣花酸试剂(不超过15min)，然后加入100μL待测血浆，37℃温浴5min(不少于5min)，之后加入37℃预热的0.5mol/LCaCl2溶液100μL，混匀并开始计时。当出现絮状沉淀时立即停止计时，即为活化部分凝血活酶时间。The determination method follows the product manual of the kit: 100 μL of ellagic acid reagent was warmed at 37°C (not more than 15 minutes), then 100 μL of plasma to be tested was added, warmed at 37°C for 5 minutes (not less than 5 minutes), and then 0.5 mol of 37°C preheated was added /LCaCl2 solution 100 μL, mix well and start timing. Stop timing immediately when flocculent precipitation appears, which is the activated partial thromboplastin time.
2、凝血时间测定2. Determination of coagulation time
采用毛细玻璃管法测定凝血时间。大鼠内眦静脉采血后，立即将25μL未加抗凝剂的静脉血吸入毛细玻璃管中，开始计时。计时过程中，与水平面呈±60°角度来回颠倒毛细玻璃管，直到玻璃管内的血液不再来回移动，停止计时，即为凝血时间。Coagulation time was determined by the capillary glass tube method. Immediately after blood collection from the inner canthus vein of rats, 25 μL of venous blood without anticoagulant was aspirated into a capillary glass tube, and the timing was started. During the timing process, turn the capillary glass tube back and forth at an angle of ±60° from the horizontal plane until the blood in the glass tube no longer moves back and forth, and stop timing, which is the coagulation time.
下述实施例中构建大鼠高凝血状态动物模型所用的试剂如表1所示，仪器及耗材如表1所示。Table 1 shows the reagents used to construct the animal model of rat hypercoagulation state in the following examples, and the instruments and consumables are shown in Table 1.
表1、构建大鼠高凝血状态动物模型所用的试剂Table 1. Reagents used in the establishment of animal models of rat hypercoagulation state
表2、构建大鼠高凝血状态动物模型所用的仪器及耗材Table 2. Instruments and consumables used in the establishment of rat hypercoagulation state animal models
实施例1、大鼠急性高凝血状态模型的建立Embodiment 1, establishment of rat acute hypercoagulation state model
一、基于酚磺乙胺构建大鼠高凝血状态动物模型1. Constructing an animal model of hypercoagulable state in rats based on phensulfame
1、实验方法1. Experimental method
将20只雄性wistar大鼠随机分成两组，每组10只，称重。其中一组大鼠腹腔注射酚磺乙胺注射液(250mg/kg体重)，记为实验组；另一组大鼠腹腔注射相应体积生理盐水注射液，记为对照组。第一次注射时间记为0h，每隔3h腹腔注射一次，共注射3次(即在0h、3h和6h各一次)。在第一次注射后9h(即第三次注射后3h)，立即内眦静脉采血，并检测大鼠的凝血功能，确定高凝血状态大鼠动物模型形成规格(流程图如图2所示)。Twenty male wistar rats were randomly divided into two groups, 10 in each group, and weighed. One group of rats was intraperitoneally injected with phensulfame injection (250 mg/kg body weight), and was recorded as the experimental group; the other group of rats was injected with a corresponding volume of normal saline injection, and was recorded as the control group. The time of the first injection was recorded as 0h, and intraperitoneal injection was performed every 3h, for a total of 3 injections (that is, once at 0h, 3h and 6h). 9 hours after the first injection (that is, 3 hours after the third injection), blood was collected from the inner canthus vein immediately, and the coagulation function of the rat was detected to determine the specifications for the formation of the hypercoagulable state rat animal model (the flow chart is shown in Figure 2) .
通过APTT和CT两个重要指标来评估大鼠凝血功能的改变。APTT，即活化部分凝血活酶时间，是通过对内源性凝血的全部条件进行体外模拟，测定血浆凝固的时间，从而反映出内源凝血因子是否正常行使生物学功能。CT，即凝血时间，是指血液离开血管，在体外发生凝固所需的时间，用于检测内源性凝血途径中，各种凝血因子的数量和功能正常与否，同时还可检测是否有多余的抗凝物质。The changes of coagulation function in rats were evaluated by two important indicators, APTT and CT. APTT, that is, activated partial thromboplastin time, is to measure the time of plasma coagulation by simulating all the conditions of endogenous coagulation in vitro, so as to reflect whether the endogenous coagulation factors normally perform biological functions. CT, that is, coagulation time, refers to the time required for blood to leave the blood vessel and coagulate in vitro. It is used to detect whether the quantity and function of various coagulation factors in the intrinsic coagulation pathway are normal or not, and it can also detect whether there are redundant anticoagulant substances.
2、实验结果2. Experimental results
通过上述实验步骤，给予促凝药物酚磺乙胺之后，如图3所示的大鼠的活化部分凝血活酶时间(APTT)和凝血时间(CT)检测结果显示，wistar大鼠的APTT和CT均显著缩短(p<0.01)，表明大鼠的正常凝血功能被扰乱，处于高凝血状态，建模成功。Through the above experimental steps, after the procoagulant drug phenetamine was administered, the activated partial thromboplastin time (APTT) and coagulation time (CT) detection results of the rats shown in Figure 3 showed that the APTT and CT of wistar rats All of them were significantly shortened (p<0.01), indicating that the normal coagulation function of rats was disrupted, and the rats were in a state of hypercoagulation, and the modeling was successful.
二、基于氨甲环酸构建大鼠高凝血状态动物模型2. Constructing an animal model of hypercoagulation state in rats based on tranexamic acid
1、实验步骤1. Experimental steps
雄性wistar大鼠20只，随机等分成两组，每组10只，称重。实验组大鼠灌胃氨甲环酸(315mg/kg)，对照组灌胃相应体积生理盐水。6h一次，共2次。将第一次灌胃时间记为0h，第二次灌胃时间则为6h。第一次灌胃后12h，立即内眦静脉采血，并检测大鼠的凝血功能，确定高凝血状态大鼠动物模型形成规格(流程图如图2所示)。Twenty male wistar rats were randomly divided into two groups, 10 in each group, and weighed. Rats in the experimental group were given tranexamic acid (315 mg/kg) orally, and rats in the control group were given a corresponding volume of normal saline. Once every 6 hours, a total of 2 times. Record the time of the first gavage as 0h, and the time of the second gavage as 6h. 12 hours after the first gavage, blood was collected from the inner canthus vein immediately, and the coagulation function of the rat was tested to determine the specifications for the formation of a rat animal model in a hypercoagulable state (flow chart shown in Figure 2).
2、实验结果2. Experimental results
通过上述实验步骤，经氨甲环酸作用之后，如图3所示的大鼠的活化部分凝血活酶时间(APTT)和凝血时间(CT)检测结果显示，Wistar大鼠的APTT和CT均显著缩短(p<0.01)，表明灌胃氨甲环酸使大鼠凝血功能改变，血液处于高凝血状态，高凝态模型建模成功。Through the above experimental procedures, after the action of tranexamic acid, the activated partial thromboplastin time (APTT) and coagulation time (CT) detection results of the rats shown in Figure 3 showed that the APTT and CT of Wistar rats were significantly shortened (p<0.01), indicating that intragastric administration of tranexamic acid changed the coagulation function of rats, and the blood was in a hypercoagulable state, and the hypercoagulable state model was successfully established.
三、基于氨基己酸构建大鼠高凝血状态动物模型3. Establishment of animal model of hypercoagulation state in rats based on aminocaproic acid
1、实验方法1. Experimental method
雄性wistar大鼠20只，随机等分成两组，每组10只，称重。实验组大鼠灌胃氨基己酸(4.2g/kg)，对照组灌胃相应体积生理盐水。6h一次，共2次。将第一次灌胃时间记为0h，第二次灌胃时间则为6h。第一次灌胃后12h，立即内眦静脉采血，并检测大鼠的凝血功能，确定高凝血状态大鼠动物模型形成规格(流程图如图2所示)。Twenty male wistar rats were randomly divided into two groups, 10 in each group, and weighed. The rats in the experimental group were given aminocaproic acid (4.2 g/kg) orally, and the rats in the control group were given a corresponding volume of normal saline. Once every 6 hours, a total of 2 times. Record the time of the first gavage as 0h, and the time of the second gavage as 6h. 12 hours after the first gavage, blood was collected from the inner canthus vein immediately, and the coagulation function of the rat was tested to determine the specifications for the formation of a rat animal model in a hypercoagulable state (flow chart shown in Figure 2).
2、实验结果2. Experimental results
通过上述实验步骤，经氨基己酸作用之后，如图2所示的大鼠的活化部分凝血活酶时间(APTT)和凝血时间(CT)检测结果显示，wistar大鼠的APTT和CT均显著缩短(p<0.01)，表明氨基己酸的干预改变了大鼠的凝血功能，血液处于高凝血状态，高凝态模型建模成功。Through the above experimental steps, after the action of aminocaproic acid, the activated partial thromboplastin time (APTT) and coagulation time (CT) detection results of rats shown in Figure 2 show that the APTT and CT of wistar rats are significantly shortened (p<0.01), indicating that the intervention of aminocaproic acid changed the coagulation function of rats, the blood was in a hypercoagulable state, and the hypercoagulable state model was successfully established.
上述方法成功建立了基于不同促凝血药物(酚磺乙胺、氨甲环酸和氨基己酸)的大鼠高凝血状态动物模型，基于此高凝血状态大鼠模型，可开展相关尿液蛋白质组学及相关生物标志物的研究工作，为相关于大鼠高凝血状态相关研究奠定重要的基础。The above method successfully established an animal model of hypercoagulable state in rats based on different procoagulant drugs (ethanolsulfate, tranexamic acid, and aminocaproic acid). Based on this rat model of hypercoagulable state, relevant urine proteomic The research work on biomarkers and related biomarkers has laid an important foundation for the research on the hypercoagulable state of rats.
实施例2、高凝血状态尿液蛋白质组学分析及生物标志物研究Example 2. Urinary proteomics analysis and biomarker research in hypercoagulable state
一、实验方法1. Experimental method
1、乙醇沉淀法提取尿液蛋白质组1. Extraction of urine proteome by ethanol precipitation
取实施例1中基于不同促凝血药物(酚磺乙胺、氨甲环酸和氨基己酸)构建的大鼠高凝血状态动物模型的尿液样品作为实验组样品，同时以各模型中的对照组的尿液样品作为对照组样品，并提取尿液样品中的蛋白质组。具体提取方法如下：取尿液样品8mL于洁净的50mL离心管中，离心：2500g，30min，4℃。转移上清液至洁净的50mL离心管中，离心：12000g，30min，4℃。量取上清液体积，转移至洁净的50mL离心管中，加入三倍体积的-20℃预冷的无水乙醇，摇匀，于-20℃沉淀2小时。沉淀完成后，离心：12000g，30min，4℃。弃上清，室温风干沉淀(约20min)。向沉淀加入适量25mM NH4HCO3(约600μL)复溶沉淀，移液枪反复吹打，使沉淀完全溶解。转移所得溶液至洁净的1.5mL EP管中，超声3min，然后置于旋转混匀仪上4℃旋转混匀2h。之后，离心：12000g，30min，4℃，转移上清于干净的EP管中，即为提取得到的尿液蛋白质组。Get the urine sample of the rat hypercoagulation state animal model based on different coagulation-promoting drugs (acetamine, tranexamic acid and aminocaproic acid) in Example 1 as the experimental group sample, and simultaneously use the control in each model The urine sample of the group was used as the control sample, and the protein group in the urine sample was extracted. The specific extraction method is as follows: Take 8 mL of urine sample in a clean 50 mL centrifuge tube, centrifuge: 2500 g, 30 min, 4 °C. Transfer the supernatant to a clean 50mL centrifuge tube, centrifuge: 12000g, 30min, 4°C. Measure the volume of the supernatant, transfer it to a clean 50mL centrifuge tube, add three times the volume of -20°C pre-cooled absolute ethanol, shake well, and settle at -20°C for 2 hours. After the precipitation is completed, centrifuge: 12000g, 30min, 4°C. The supernatant was discarded, and the precipitate was air-dried at room temperature (about 20 min). Add an appropriate amount of 25mM NH4 HCO3 (about 600 μL) to the precipitate to redissolve the precipitate, and pipette repeatedly to dissolve the precipitate. The resulting solution was transferred to a clean 1.5mL EP tube, sonicated for 3min, and then placed on a rotary mixer at 4°C for 2h. After that, centrifuge: 12000g, 30min, 4°C, transfer the supernatant to a clean EP tube, which is the extracted urine proteome.
2、Bradford法测蛋白浓度2. Bradford method to measure protein concentration
配制1μg/μL牛血清白蛋白(BSA)作为标准蛋白溶液，然后将标准蛋白溶液、Bradford工作液及ddH2O按照表3中的体积混匀，终体积均为300μL，然后于96孔板中用酶标仪检测595nm处吸光度值，绘制标准曲线。Prepare 1 μg/μL bovine serum albumin (BSA) as a standard protein solution, then mix the standard protein solution, Bradford working solution and ddH2 O according to the volumes in Table 3, the final volume is 300 μL, and then put in a 96-well plate The absorbance value at 595 nm was detected with a microplate reader, and a standard curve was drawn.
表3、不同浓度标准蛋白溶液的配制Table 3. Preparation of standard protein solutions with different concentrations
对于尿液样品而言，各取1μL尿液样品，然后与285μL Bradford工作液及14μLddH2O混合均匀，同样检测595nm处吸光度值，根据所得吸光度值，对照标准曲线，得到尿液样品的蛋白质浓度，之后，分装样品，-80℃冻存。For urine samples, take 1 μL of urine samples, and then mix them with 285 μL of Bradford working solution and 14 μL of ddH2 O, and also detect the absorbance at 595 nm, and compare the obtained absorbance with the standard curve to obtain the protein concentration of the urine sample After that, the samples were aliquoted and stored at -80°C.
3、SDS-PAGE初步检测尿蛋白质组3. Preliminary detection of urine proteome by SDS-PAGE
1)SDS-PAGE检测尿蛋白质组1) SDS-PAGE detection of urine proteome
(1)电泳前样品处理：每个样品上样量为20μg，将样品和5×SDS-PAGE上样缓冲液按照4:1的体积比混合均匀，沸水中加热10min使蛋白质变性。(1) Sample treatment before electrophoresis: the loading amount of each sample is 20 μg, mix the sample and 5×SDS-PAGE loading buffer according to the volume ratio of 4:1, and heat in boiling water for 10 min to denature the protein.
(2)根据说明书安装胶板，向缝隙中注入超纯水，10min后检测是否漏水。(2) Install the rubber sheet according to the instructions, inject ultrapure water into the gap, and check for water leakage after 10 minutes.
(3)按照表4所示，配制12％SDS-PAGE分离胶和5％SDS-PAGE浓缩胶。将分离胶灌入两玻璃板的缝隙中，速度要慢，尽量不要产生气泡，然后立即用无水乙醇覆盖封胶，室温放置20min。待分离胶凝固后倒去无水乙醇，用滤纸吸干残留的液体，稍微晾干，再灌入浓缩胶，插上梳子，室温静置20min，等待浓缩胶聚合。(3) As shown in Table 4, prepare 12% SDS-PAGE separating gel and 5% SDS-PAGE stacking gel. Pour the separating glue into the gap between the two glass plates at a slow speed, try not to generate air bubbles, then immediately cover the sealing glue with absolute ethanol, and place it at room temperature for 20 minutes. After the separation gel is solidified, pour off the absolute ethanol, blot the remaining liquid with filter paper, let it dry slightly, then pour into the stacking gel, insert a comb, and let it stand at room temperature for 20 minutes, waiting for the stacking gel to polymerize.
表4、12％SDS-PAGE分离胶和5％SDS-PAGE浓缩胶Table 4, 12% SDS-PAGE separating gel and 5% SDS-PAGE stacking gel
(4)待浓缩胶聚合完全后，将凝胶板固定于电泳装置上，内外槽分别加入1×SDS电泳缓冲液，小心拔去梳子。(4) After the stacking gel is completely polymerized, fix the gel plate on the electrophoresis device, add 1×SDS electrophoresis buffer into the inner and outer tanks, and carefully pull out the comb.
(5)按照预定顺序上样，其中最左侧孔道加入蛋白Marker。(5) Samples are loaded according to the predetermined order, and the protein marker is added to the leftmost channel.
(6)电泳：在浓缩胶以80V恒压进行电泳，当溴酚蓝指示剂达浓缩胶与分离胶的分界线时，调节电压到120V，在分离胶以120V恒压电泳，待溴酚蓝指示剂达凝胶底部，停止电泳。(6) Electrophoresis: Electrophoresis is performed at a constant voltage of 80V on the stacking gel. When the bromophenol blue indicator reaches the dividing line between the stacking gel and the separation gel, the voltage is adjusted to 120V, and the electrophoresis is performed at a constant voltage of 120V on the separation gel. When the indicator reaches the bottom of the gel, stop the electrophoresis.
(7)电泳结束后，小心取下凝胶，用超纯水反复冲洗，去除凝胶表面的电泳缓冲液。(7) After electrophoresis, carefully remove the gel, rinse repeatedly with ultrapure water, and remove the electrophoresis buffer on the surface of the gel.
2)考马斯亮蓝染色2) Coomassie brilliant blue staining
(1)将凝胶放到玻璃培养皿中，加入适量考马斯亮蓝染液，脱色摇床上室温缓慢振荡2h，染色。(1) Put the gel into a glass petri dish, add an appropriate amount of Coomassie Brilliant Blue staining solution, shake slowly at room temperature for 2 hours on a decolorizing shaker, and stain.
(2)弃染液，超纯水漂洗数次，加入脱色液，脱色摇床上室温缓慢振荡过夜，中途更换脱色液2-3次，脱色致出现清晰蛋白条带，且背景颜色较浅为止。(2) Discard the dye solution, rinse with ultrapure water for several times, add decolorization solution, shake slowly overnight at room temperature on a decolorization shaker, replace the decolorization solution 2-3 times in the middle, until clear protein bands appear after decolorization, and the background color is lighter.
(3)弃脱色液，超纯水漂洗数次，浸入超纯水中，之后凝胶成像仪中成像。(3) Discard the decolorization solution, rinse with ultrapure water for several times, immerse in ultrapure water, and image in a gel imager.
4、蛋白质的膜上辅助酶切4. Membrane-assisted digestion of proteins
1)蛋白质膜上辅助酶切1) Assisted enzyme digestion on the protein membrane
(1)现配UA溶液(8M尿素，0.1M Tris/HCl，pH 8.5)和ABC溶液(25mM NH4HCO3溶液)。(1) Prepare UA solution (8M urea, 0.1M Tris/HCl, pH 8.5) and ABC solution (25mM NH4 HCO3 solution) now.
(2)清洗超滤管：在每个10kD超滤管内加入UA溶液200μL，离心：12 000g，10min，18℃，弃下层滤过液。重复洗涤三次。(2) Clean the ultrafiltration tubes: add 200 μL of UA solution to each 10 kD ultrafiltration tube, centrifuge: 12 000 g, 10 min, 18° C., and discard the filtrate of the lower layer. Repeat the wash three times.
(3)各取蛋白质样品200μg加在滤膜上，然后加入UA溶液，补充总体积至200μL，离心：12 000g，40min，18℃，弃下层滤过液。(3) Add 200 μg of each protein sample to the filter membrane, then add UA solution to make up the total volume to 200 μL, centrifuge: 12 000 g, 40 min, 18 ° C, discard the filtrate of the lower layer.
(4)在滤膜上加入UA溶液200μL，用移液枪反复吹打，离心：12 000g，40min，18℃，弃下层滤过液。此步骤重复三次。(4) Add 200 μL of UA solution to the filter membrane, repeatedly blow and beat with a pipette gun, centrifuge: 12 000 g, 40 min, 18 °C, and discard the filtrate of the lower layer. This step is repeated three times.
(5)在滤膜上加入ABC溶液200μL，涡旋振荡30s。加入DTT至其终浓度为4.5mM(即加入0.9μL的1M DTT)，涡旋振荡30s，充分混匀后，50℃水浴1小时。(5) Add 200 μL of ABC solution to the filter membrane, and vortex for 30 seconds. Add DTT to a final concentration of 4.5 mM (that is, add 0.9 μL of 1M DTT), vortex for 30 seconds, mix well, and place in a 50° C. water bath for 1 hour.
(6)从水浴中取出样品，降温至室温，加入现配的IAA使其终浓度为10mM，涡旋振荡1min，充分混匀后，室温避光作用35min。(6) Take out the sample from the water bath, cool down to room temperature, add freshly prepared IAA to make the final concentration 10mM, vortex for 1min, mix thoroughly, and protect from light at room temperature for 35min.
(7)离心：12 000g，40min，18℃，使液体充分离干，弃下层滤过液。(7) Centrifuge: 12 000g, 40min, 18°C, fully dry the liquid, and discard the filtrate of the lower layer.
(8)在滤膜上加入ABC溶液200μL，移液枪反复吹打，使膜上蛋白质充分重悬，离心：12 000g，40min，18℃，弃下层滤过液。重复此步骤两次。(8) Add 200 μL of ABC solution to the filter membrane, pipette repeatedly to resuspend the protein on the membrane, centrifuge: 12 000g, 40min, 18°C, discard the filtrate of the lower layer. Repeat this step twice.
(9)在滤膜上加入ABC溶液200μL，按蛋白质与胰酶的质量比为50:1加入胰酶，涡旋1min。然后，放入振荡混匀器中混匀：37℃，400rpm，30min。37℃恒温水浴，过夜酶切14h。(9) Add 200 μL of ABC solution to the filter membrane, add trypsin according to the mass ratio of protein to trypsin at 50:1, and vortex for 1 min. Then, put it into an oscillating mixer and mix well: 37°C, 400rpm, 30min. 37 ℃ constant temperature water bath, overnight enzyme digestion 14h.
(10)取出超滤管，加入甲酸只其终浓度为0.1％，以终止酶切反应。更换新的超滤管外管，离心：12 000g，18℃，至接近完全干燥，转移滤过液至新EP管。(10) Take out the ultrafiltration tube and add formic acid to a final concentration of 0.1% to terminate the enzyme cleavage reaction. Replace the outer tube of a new ultrafiltration tube, centrifuge: 12 000g, 18°C, until almost completely dry, transfer the filtrate to a new EP tube.
(11)再向超滤管中加入ABC溶液200μL，用移液枪反复吹打，涡旋30s，离心：12000g，18℃，离心至完全干燥。将滤过液与(10)中所得滤过液混合，即为蛋白的酶切产物。(11) Add 200 μL of ABC solution to the ultrafiltration tube, pipette repeatedly, vortex for 30 seconds, and centrifuge at 12,000 g at 18°C until completely dry. The filtrate is mixed with the filtrate obtained in (10), which is the product of enzymatic digestion of the protein.
2)酶切产物除盐2) Desalting the digested product
采用Oasis HLB固相萃取柱除盐法进行酶切产物除盐。具体步骤如下：Oasis HLB solid-phase extraction column desalting method was used to desalt the enzyme cleavage product. Specific steps are as follows:
(1)Oasis HLB固相萃取柱的活化：先后用1mL甲醇、1mL乙腈活化柱子。注意使滤液自然滴落。(1) Activation of Oasis HLB solid-phase extraction column: activate the column with 1 mL of methanol and 1 mL of acetonitrile successively. Note that the filtrate drips naturally.
(2)Oasis HLB固相萃取柱的平衡：用1mL 0.l％甲酸/2％乙腈平衡柱子。此步骤重复三次，自然滴落。(2) Equilibration of Oasis HLB solid-phase extraction column: equilibrate the column with 1 mL of 0.1% formic acid/2% acetonitrile. This step is repeated three times and drips naturally.
(3)上样：加入4.4.1中所得酶切后的样品，让其自然滴落。(3) Sample loading: Add the digested sample obtained in 4.4.1 and let it drop naturally.
(4)洗涤除盐：用1mL 0.l％甲酸/2％乙腈洗涤脱盐5次。(4) Washing and desalting: 5 times of washing and desalting with 1 mL of 0.1% formic acid/2% acetonitrile.
(5)肽段洗脱：加入1mL 90％乙腈溶液洗脱萃取肽段，用1.5mL离心管收集洗脱液。样品自然滴落。(5) Peptide elution: Add 1 mL of 90% acetonitrile solution to elute the extracted peptide, and collect the eluate in a 1.5 mL centrifuge tube. The sample drips naturally.
(6)真空抽干样品，-80℃冻存备用。(6) Vacuum-dry the samples, and freeze them at -80°C for later use.
5、尿液蛋白的质谱鉴定5. Mass Spectrometric Identification of Urine Proteins
1)BCA法测肽段浓度1) BCA method to measure peptide concentration
(1)用0.1％甲酸稀释1μg/μL血管紧张素至0.1μg/μL，0.2μg/μL，0.5μg/μL。(1) Dilute 1 μg/μL angiotensin with 0.1% formic acid to 0.1 μg/μL, 0.2 μg/μL, 0.5 μg/μL.
(2)用0.1％甲酸重溶酶切得到的肽段样品，重溶体积为20μL，测量时用0.1％甲酸稀释10倍。(2) Redissolve the peptide sample obtained by enzymatic digestion with 0.1% formic acid, the redissolved volume is 20 μL, and dilute 10 times with 0.1% formic acid for measurement.
(3)配制BCA工作液：A液与B液按照体积比50:1混合均匀。(3) Prepare BCA working solution: Mix A solution and B solution according to the volume ratio of 50:1.
(4)标准曲线制作方法：按照表5所示，将液体加入96孔板中，混匀。(4) Standard curve preparation method: as shown in Table 5, add the liquid into a 96-well plate and mix well.
表5、制作标准曲线所用试剂的浓度与体积Table 5. Concentration and volume of reagents used in making standard curve
(5)样品孔加入10μL稀释后的肽段样品，200μL BCA工作液。(5) Add 10 μL diluted peptide sample and 200 μL BCA working solution to the sample well.
(6)37℃水浴30min。(6) 37°C water bath for 30 minutes.
(7)测定562nm处吸光度值，绘制标准曲线，求出肽段浓度。(7) Measure the absorbance value at 562nm, draw a standard curve, and calculate the peptide concentration.
(8)稀释肽段浓度至0.5μg/μL。稀释后，每个样品各取20μL，离心：12000g，30min，4℃，小心取出，取上清液等待质谱上样。(8) Dilute the peptide concentration to 0.5 μg/μL. After dilution, take 20 μL of each sample, centrifuge: 12000g, 30min, 4°C, take it out carefully, take the supernatant and wait for mass spectrometry loading.
2)液相色谱分离串联质谱鉴定(LC-MS/MS)2) Identification by liquid chromatography tandem mass spectrometry (LC-MS/MS)
用EASY-nLC 1200HPLC系统上样，样品为4.5.1最后所得肽段，每个样品上样量为500ng。自动加样器将样品加入捕获柱(trap column)(75μm×2cm，3μm，C18，)之后，以2.5μL/min的流速冲洗5min进一步去除杂质，之后以0.25μL/min的流速进行反向分析柱洗脱(50μm×150mm，2μm，C18，)。洗脱梯度为5-28％流动相B(流动相A为：0.1％甲酸+99.9％水；流动相B为：0.1％甲酸+79.9％乙腈+20％水)，时间为60min。Use the EASY-nLC 1200HPLC system to load the sample, the sample is the peptide segment finally obtained in 4.5.1, and the loading amount of each sample is 500ng. The autosampler adds the sample to the trap column (75μm×2cm, 3μm, C18, ), wash at a flow rate of 2.5 μL/min for 5 minutes to further remove impurities, and then perform reverse analytical column elution at a flow rate of 0.25 μL/min (50 μm×150 mm, 2 μm, C18, ). The elution gradient was 5-28% mobile phase B (mobile phase A: 0.1% formic acid + 99.9% water; mobile phase B: 0.1% formic acid + 79.9% acetonitrile + 20% water), and the time was 60 min.
洗脱下来的多肽采用Orbitrap Fusion Lumos MS质谱仪检测。喷雾电压2.1kV，离子传输管温度300℃。母离子m/z扫描范围为350-1550，扫描分辨率为120000。以最高速度模式获取MS/MS图谱，离子碎裂模式为HCD子离子m/z扫描范围为250-1800，扫描分辨率为60000，动态排除时间为30s。The eluted peptides were detected by an Orbitrap Fusion Lumos MS mass spectrometer. The spray voltage was 2.1kV, and the ion transfer tube temperature was 300°C. The scanning range of precursor ion m/z is 350-1550, and the scanning resolution is 120000. The MS/MS spectrum was obtained in the highest speed mode, the ion fragmentation mode was HCD product ion m/z scanning range was 250-1800, the scanning resolution was 60000, and the dynamic exclusion time was 30s.
每个样品重复鉴定2次。不同样品之间均相隔两次30min的清洗程序，以减少样品间污染。Each sample was identified twice. Two cleaning procedures of 30 min were separated between different samples to reduce contamination between samples.
3)数据分析3) Data Analysis
(1)数据检索(1) Data retrieval
二级质谱结果用MASCOT 2.4.0软件进行数据库检索。所用数据库为Swiss-protRat Database。检索条件为：胰酶酶切，允许最大2个漏切位点，肽段检索母离子误差为10ppm，子离子容许误差为0.02Da，肽段假阳性率(FDR)<1％，只将鉴定到大于或等于2条肽段的蛋白质纳入分析。The results of MS/MS were searched by MASCOT 2.4.0 software. The database used is the Swiss-protRat Database. The search conditions are: trypsin digestion, a maximum of 2 missed cleavage sites are allowed, the precursor ion error of peptide retrieval is 10ppm, the tolerance error of product ion is 0.02Da, the false positive rate (FDR) of peptides is <1%, only the identification Proteins with more than or equal to 2 peptides were included in the analysis.
(2)差异蛋白筛选(2) Differential protein screening
应用谱图数定量法进行差异蛋白的筛选工作。筛选标准：对照组谱图数平均值和实验组谱图数的平均数进行T-检验，p<0.05；变化倍数1.5倍，即实验组比对照组升高或降低≥1.5倍；升高组中最小谱图数>降低组中最大谱图数。The differential protein screening work was carried out by using the spectral number quantification method. Screening criteria: T-test the average number of spectra in the control group and the average number of spectra in the experimental group, p<0.05; the multiple of change is 1.5 times, that is, the experimental group is ≥1.5 times higher than the control group; Minimum number of spectra in the group > reduce the maximum number of spectra in the group.
二、实验结果2. Experimental results
(一)基于酚磺乙胺构建的大鼠高凝血状态动物模型的蛋白质组学分析及生物标志物(1) Proteomic analysis and biomarkers of rat hypercoagulable state animal model based on etamsulfame
1、酚磺乙胺高凝血状态模型尿液蛋白质的SDS-PAGE分析1. SDS-PAGE analysis of urine protein in hypercoagulable state model
用SDS-PAGE初步分析促凝药物酚磺乙胺干预对尿液蛋白的影响，每个样品的上样量为20μg。SDS-PAGE显示：酚磺乙胺作用后的尿蛋白质样品，与对照组(生理盐水组)相比，绝大部分条带基本相同，没有明显的蛋白质降解(图4)。说明技术重复性良好，蛋白质样品质量良好，可以用于后续实验。SDS-PAGE结果无明显差异，说明变化的蛋白质可能是低丰度蛋白，超出了SDS-PAGE的分辨率，运用常规检测技术、方法无法分辨这种情况下的高凝血状态和对照组的蛋白质差异，需要更加精细的检测手段进行甄别判断。SDS-PAGE was used to preliminarily analyze the effect of procoagulant drug phenetamine intervention on urine protein, and the loading amount of each sample was 20 μg. SDS-PAGE showed that compared with the control group (normal saline group), most of the bands in the urine protein sample treated with phenetamine were basically the same, and there was no obvious protein degradation (Fig. 4). It shows that the technical repeatability is good, and the quality of the protein sample is good, which can be used for subsequent experiments. There is no significant difference in the results of SDS-PAGE, indicating that the changed protein may be a low-abundance protein, which exceeds the resolution of SDS-PAGE. It is impossible to distinguish the hypercoagulation state in this case from the protein difference in the control group by conventional detection techniques and methods. , requires more sophisticated detection methods for screening and judgment.
2、酚磺乙胺高凝血状态模型尿液蛋白质组质谱鉴定结果2. Mass spectrometric identification results of urine proteome of the hypercoagulable state model
本发明共鉴定得到533个蛋白质，其中，生理盐水组(对照组)530个，酚磺乙胺组(实验组)504个，两组共同鉴定到501个。A total of 533 proteins were identified by the present invention, of which 530 were in the normal saline group (control group), 504 were in the etyzamine group (experimental group), and 501 were jointly identified by the two groups.
(1)酚磺乙胺高凝血状态模型差异蛋白种类(1) Differential protein types in the hypercoagulation state model
根据筛选标准，具有显著差异的蛋白质共有20个，具体信息如表6所示。和对照组相比，酚磺乙胺高凝血状态模型的大鼠尿液中的DNA酶-2-β、蛋白聚糖聚合物核心蛋白、高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3、核糖核酸酶4、中性粒细胞明胶酶相关脂质运载蛋白、锌-α-2-糖蛋白和I类组织相容性抗原的含量明显提高，氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂β、谷胱甘肽S-转移酶α-3、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶、载脂蛋白A-IV和血红蛋白β亚基-1的含量明显降低。According to the screening criteria, there are a total of 20 proteins with significant differences, and the specific information is shown in Table 6. Compared with the control group, DNase-2-β, proteoglycan polymer core protein, Golgi protein I, carbonic anhydrase I, phosphatidylinositol The contents of proteoglycan-3, ribonuclease 4, neutrophil gelatinase-associated lipocalin, zinc-α-2-glycoprotein and class I histocompatibility antigen were significantly increased, and chloride intracellular channel protein 1. Biglycan, Rab GDP dissociation inhibitor β, glutathione S-transferase α-3, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triplet nucleus The contents of nucleotide binding protein 1, myosin light chain 6, L-xylulose reductase, apolipoprotein A-IV and hemoglobin β subunit-1 were significantly decreased.
表6、酚磺乙胺高凝血状态模型的尿差异蛋白Table 6. Urinary differential protein in the hypercoagulable state model
(2)酚磺乙胺高凝血状态模型尿液差异蛋白的人类同源性分析(2) Human homology analysis of differential proteins in the urine of the hypercoagulable state model
本发明通过构建高凝血状态大鼠模型，来探究血液高凝状态对人类尿蛋白质组的影响，进而为寻找高凝血状态尿液蛋白质组生物标志物提供一定的线索。不同物种的同源蛋白具有相似的结构和功能。通过Uniprot网站(http://www.uniprot.org/blast/)进行序列比对，寻找大鼠高凝血状态差异蛋白的人类同源蛋白。本发明将已被Swiss-prot数据库验证，且同源性≥80％的人类同源蛋白为可信的同源蛋白。比较发现，酚磺乙胺大鼠高凝血状态模型得到的20个差异蛋白中，有13个人类同源蛋白，具体信息如表7所示。将这13个人类同源蛋白与人类核心尿蛋白数据库(Human core urinary proteome)进行比对，发现其中有9个是人类核心尿蛋白质成员，提示高凝血状态下，人类核心尿蛋白质组可能发生变化。The present invention explores the influence of hypercoagulable state on human urine proteome by constructing a rat model of hypercoagulable state, and then provides certain clues for finding urine proteome biomarkers of hypercoagulable state. Homologous proteins from different species have similar structures and functions. Sequence alignment was performed on the Uniprot website (http://www.uniprot.org/blast/) to search for human homologous proteins of rat hypercoagulation state differential proteins. In the present invention, human homologous proteins that have been verified by the Swiss-prot database and have a homology ≥ 80% are credible homologous proteins. By comparison, it was found that among the 20 differential proteins obtained from the rat hypercoagulation state model, there were 13 human homologous proteins, and the specific information is shown in Table 7. Comparing these 13 human homologous proteins with the Human core urinary protein database (Human core urinary proteome), it was found that 9 of them are members of the human core urinary protein, suggesting that the human core urinary proteome may change in a hypercoagulable state .
表7、酚磺乙胺致大鼠高凝血状态尿液差异蛋白的人类同源蛋白Table 7. Human homologous proteins of different proteins in the urine of rats with hypercoagulation state induced by phensulfame
(3)酚磺乙胺致高凝血状态模型差异蛋白的组织表达分析(3) Tissue expression analysis of differential proteins in hypercoagulation state model induced by phensulfame
将步骤(2)中差异蛋白的人类同源蛋白与人类蛋白质表达数据库(Human ProteinAtlas，http://www.proteinatlas.org)进行对比，筛选出表达量为High的组织类型，可以确定，有7个差异蛋白高表达于29个组织中，组织表达分析情况如表8和图5所示。其中，肾脏中高表达的蛋白量最多，这暗示着机体内部，对于血液高凝血状态反应最为敏感的器官是肾脏，肾脏应答高凝血状态变化，在蛋白表达方面进行了最大程度的调整与适应。此外，消化系统和神经系统中高表达的蛋白也较多。Compare the human homologous proteins of the differential proteins in step (2) with the human protein expression database (Human Protein Atlas, http://www.proteinatlas.org), and screen out the tissue types with High expression. It can be determined that there are 7 The differential proteins were highly expressed in 29 tissues, and the tissue expression analysis is shown in Table 8 and Figure 5. Among them, the amount of highly expressed protein in the kidney is the largest, which implies that the kidney is the most sensitive organ in the body to respond to the hypercoagulation state of the blood. In addition, there are more highly expressed proteins in the digestive system and nervous system.
表8、酚磺乙胺高凝血状态模型的尿差异蛋白组织表达分析Table 8. Urinary differential protein tissue expression analysis of the hypercoagulable state model
(二)基于氨甲环酸构建的大鼠高凝血状态动物模型的蛋白质组学分析及生物标志物(2) Proteomic analysis and biomarkers of rat hypercoagulation state animal model based on tranexamic acid
1、氨甲环酸高凝血状态模型尿液蛋白质的SDS-PAGE分析1. SDS-PAGE analysis of urine protein in tranexamic acid hypercoagulable state model
用SDS-PAGE初步检测促凝血药物氨甲环酸干预对尿液蛋白的影响，每个样品的上样量为20μg。SDS-PAGE结果显示：尿液蛋白条带完整，蛋白质没有明显的降解。与对照组相比(生理盐水组)，氨甲环酸组的尿液蛋白在SDS-PAGE凝胶上没有显示出明显变化。可能是因为变化的蛋白质为低丰度蛋白(图4)。SDS-PAGE was used to preliminarily detect the effect of procoagulant drug tranexamic acid on urine protein, and the loading amount of each sample was 20 μg. The results of SDS-PAGE showed that the urine protein bands were complete, and the protein was not degraded significantly. Compared with the control group (normal saline group), the urine protein of the tranexamic acid group showed no significant change on the SDS-PAGE gel. It may be because the changed protein is a low-abundance protein (Fig. 4).
2、氨甲环酸高凝血状态模型尿液蛋白质组质谱鉴定结果2. Mass Spectrometry Identification Results of Urine Proteome of Tranexamic Acid Hypercoagulable State Model
本发明共鉴定得到582个蛋白质。其中，生理盐水组(对照组)568个，氨甲环酸组(实验组)563个，两组共同鉴定到549个。A total of 582 proteins were identified by the present invention. Among them, 568 were in the normal saline group (control group), 563 were in the tranexamic acid group (experimental group), and 549 were jointly identified by the two groups.
(1)氨甲环酸高凝血状态模型差异蛋白(1) Tranexamic acid hypercoagulation model differential protein
根据筛选标准，具有显著差异的蛋白质共有28个，具体信息如表9所示。和对照组相比，氨甲环酸高凝血状态模型的大鼠尿液中的胞液氨肽酶、SMR1蛋白、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、CD302抗原、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基、血管紧张素转换酶2同系物、白细胞介素-4受体α亚基、巢蛋白-2和尿蛋白1的含量明显提高，三叶因子1、WAP四硫化物核心结构域蛋白2、超氧化物歧化酶[Cu-Zn]、D-多巴胺脱羧酶、淀粉样蛋白2、普罗沙星(硫酸化糖蛋白1)、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1、胰石蛋白和钙调蛋白(CaM)的含量明显降低。According to the screening criteria, there are 28 proteins with significant differences, and the specific information is shown in Table 9. Compared with the control group, the cytosolic aminopeptidase, SMR1 protein, nucleoside triphosphate diphosphate hydrolase 5, guanine nucleotide binding protein G (I )/G(S)/G(T)β-1 subunit, actin-like protein, guanine nucleotide binding protein G(k)α subunit, CD302 antigen, guanine nucleotide binding protein G( Significantly increased levels of I)/G(S)/G(T) β-2 subunit, angiotensin-converting enzyme 2 homologue, interleukin-4 receptor α subunit, nestin-2, and urinary protein 1 , trefoil factor 1, WAP tetrasulfide core domain protein 2, superoxide dismutase [Cu-Zn], D-dopamine decarboxylase, amyloid 2, profloxacin (sulfated glycoprotein 1), calcium Binding protein, fumaryl acetoacetase, myosin light chain 6, Frizzled 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1, pancreatic stone protein, and calmodulin (CaM) content was significantly reduced.
表9、氨甲环酸高凝血状态模型的尿差异蛋白Table 9. Urine differential protein in tranexamic acid hypercoagulable state model
(2)氨甲环酸高凝血状态模型尿液差异蛋白的人类同源性分析(2) Human homology analysis of differential proteins in urine of tranexamic acid hypercoagulable state model
按照步骤(一)的2中的(2)中的方法分析差异蛋白的人类同源蛋白。氨甲环酸致大鼠高凝血状态，鉴定到的28个差异蛋白中，有18个有人类同源蛋白，其中，有10个是人类核心尿蛋白质组的成员，具体信息如表10所示。其中，凝血功能相关蛋白载脂蛋白M也属于人类核心尿蛋白质组。According to the method in (2) of step (1) of 2, analyze the human homologous protein of the differential protein. Tranexamic acid induced hypercoagulation in rats. Of the 28 differential proteins identified, 18 had human homologous proteins, and 10 of them were members of the human core urine proteome. The specific information is shown in Table 10 . Among them, coagulation-related protein apolipoprotein M also belongs to the human core urine proteome.
表10、氨甲环酸致大鼠高凝血状态尿液差异蛋白的人类同源蛋白Table 10. Human homologous proteins of urine differential proteins in hypercoagulable state of rats induced by tranexamic acid
(3)氨甲环酸高凝血状态模型差异蛋白的组织表达分析(3) Tissue expression analysis of differential proteins in tranexamic acid hypercoagulation state model
按照步骤(一)的2中的(3)中的方法确定差异蛋白的人类同源蛋白的组织表达。最终确定，有8个差异蛋白高表达于19个组织中，具体如表11和图5所示。可以看到，这些蛋白质大部分是在泌尿系统和消化系统中高表达。Determine the tissue expression of the human homologous protein of the differential protein according to the method in (3) of step (1) of 2. It was finally determined that 8 differential proteins were highly expressed in 19 tissues, as shown in Table 11 and Figure 5. It can be seen that most of these proteins are highly expressed in the urinary system and digestive system.
表11、氨甲环酸高凝血状态模型的尿差异蛋白组织表达分析Table 11. Urinary differential protein tissue expression analysis of tranexamic acid hypercoagulable state model
(三)基于氨基己酸构建的大鼠高凝血状态动物模型的蛋白质组学分析及生物标志物(3) Proteomic analysis and biomarkers of rat hypercoagulation state animal model based on aminocaproic acid
1、氨基己酸高凝血状态模型尿液蛋白质的SDS-PAGE分析1. SDS-PAGE analysis of aminocaproic acid hypercoagulation state model urine protein
用SDS-PAGE初步检测促凝药物氨基己酸的干预对尿液蛋白质组的影响，每个样品的上样量为20μg。在SDS-PAGE凝胶上，可以看到，与对照组相比(生理盐水组)，氨基己酸组的尿液蛋白显示出一定的变化。分子量约为55kDa的蛋白质，氨基己酸组比生理盐水组表达量要少一些，但分子量约为60kDa左右的蛋白质，氨基己酸组比生理盐水组表达量要多一些。分子量为15kDa上下的两条蛋白质条带，氨基己酸组比生理盐水组的表达量要略少，但分子量为15kDa的蛋白质条带，氨基己酸组比生理盐水组表达量略高。两组尿液蛋白条带均完整，蛋白质没有明显的降解(图4)。SDS-PAGE was used to preliminarily detect the effect of the procoagulant drug aminocaproic acid on the urine proteome, and the loading amount of each sample was 20 μg. On the SDS-PAGE gel, it can be seen that compared with the control group (normal saline group), the urine protein of the aminocaproic acid group showed certain changes. For proteins with a molecular weight of about 55kDa, the expression level of the aminocaproic acid group is lower than that of the normal saline group, but for proteins with a molecular weight of about 60kDa, the expression level of the aminocaproic acid group is higher than that of the normal saline group. For the two protein bands with a molecular weight of 15kDa, the expression level of the aminocaproic acid group was slightly lower than that of the normal saline group, but the expression level of the protein band with a molecular weight of 15kDa was slightly higher in the aminocaproic acid group than that of the normal saline group. The urine protein bands of the two groups were intact, and the protein was not significantly degraded (Figure 4).
2、氨基己酸高凝血状态模型尿液蛋白质组质谱鉴定结果2. Identification results of aminocaproic acid hypercoagulable state model urine proteome mass spectrometry
本发明共鉴定得到423个蛋白质。其中，生理盐水组(对照组)417个，氨基己酸组(实验组)291个，两组共同鉴定到285个。A total of 423 proteins were identified by the present invention. Among them, 417 were in the normal saline group (control group), 291 were in the aminocaproic acid group (experimental group), and 285 were jointly identified by the two groups.
(1)氨基己酸高凝血状态模型差异蛋白(1) Aminocaproic acid hypercoagulation model differential protein
根据筛选标准，具有显著差异的蛋白质共有65个，具体信息如表12所示。和对照组相比，氨基己酸高凝血状态模型的大鼠尿液中的细胞粘附分子3、Thy-1膜糖蛋白、丝氨酸蛋白酶抑制剂A3M、补体C9、组织蛋白酶S、前胶原C-内肽酶增强剂1、铜蓝蛋白、补体因子D、前列腺碱性蛋白、硫氧还蛋白、CD59糖蛋白、血清白蛋白、CD44抗原、分泌磷蛋白24、β-2微球蛋白和中性粒细胞明胶酶相关脂质运载蛋白的含量明显提高，α-1抑制剂3、肽基脯氨酰顺反异构酶A、γ-谷氨酰转肽酶、ABHD14B蛋白、甲基多巴Aβ亚基、补体C3、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、半胱氨酸蛋白酶抑制剂相关蛋白2、连接粘附分子A、卷曲蛋白2、巯基氧化酶1、吸引素、单核细胞分化抗原CD14、Neuroplastin、CD166抗原、丛生蛋白、谷胱甘肽S-转移酶P、蛋白FAM151A、碱性磷酸酶、谷胱甘肽S-转移酶α-3、14-3-3蛋白ζ/δ、前列腺蛋白、β-氨基己糖苷酶β亚基、喹诺酮氧化还原酶、D-多巴胺脱羧酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、运钴胺素蛋白-2、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、足糖萼蛋白、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、抑制素-βC链、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1、犬尿氨酸/α-氨基己酸转氨酶(线粒体)、硫酸软骨素蛋白多糖4和Na(+)/H(+)交换调节辅因子NHE-RF3的含量明显降低。According to the screening criteria, there are 65 proteins with significant differences, and the specific information is shown in Table 12. Compared with the control group, cell adhesion molecule 3, Thy-1 membrane glycoprotein, serine protease inhibitor A3M, complement C9, cathepsin S, procollagen C- Endopeptidase enhancer 1, ceruloplasmin, complement factor D, prostatic basic protein, thioredoxin, CD59 glycoprotein, serum albumin, CD44 antigen, secreted phosphoprotein 24, beta-2 microglobulin, and neutral The content of granulocyte gelatinase-associated lipocalin was significantly increased, α-1 inhibitor 3, peptidylprolyl cis-trans isomerase A, γ-glutamyl transpeptidase, ABHD14B protein, methyldopa Aβ Subunits, complement C3, Frizzled 4, α-enolase, anthrax toxin receptor 1, cystatin-associated protein 2, junctional adhesion molecule A, Frizzled 2, sulfhydryl oxidase 1, attractin , monocyte differentiation antigen CD14, Neuroplastin, CD166 antigen, clusterin, glutathione S-transferase P, protein FAM151A, alkaline phosphatase, glutathione S-transferase α-3, 14-3- 3 protein ζ/δ, prostaglandin, β-hexosaminidase β subunit, quinolone oxidoreductase, D-dopamine decarboxylase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, transcobalamin Inosin-2, polyphosphoinositide phosphatase 1, glucose-6-phosphate isomerase, podocalyxin, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel Protein 4, histidine triad nucleotide binding protein 1, inhibin-βC chain, linker adhesion molecule C, stanniocalcin-1, glutamate-cysteine ligase catalytic subunit, N(G ), N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetylgalactosaminidase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5- Methylthiopentene dioxygenase, selenium binding protein 1, kynurenine/α-aminocaproic acid transaminase (mitochondrial), chondroitin sulfate proteoglycan 4, and Na(+)/H(+) exchange regulatory cofactors The content of NHE-RF3 was significantly reduced.
表12、氨基己酸高凝血状态模型的尿差异蛋白Table 12. Urinary differential protein of aminocaproic acid hypercoagulable state model
(2)氨基己酸高凝血状态模型尿液差异蛋白的人类同源性分析(2) Human homology analysis of aminocaproic acid hypercoagulation state model urine differential proteins
按照步骤(一)的2中的(2)中的方法分析差异蛋白的人类同源蛋白。氨基己酸致大鼠高凝血状态，鉴定到的65个差异蛋白中，有34个有人类同源蛋白，其中，有16个是人类核心尿蛋白质组的成员，具体信息如表13所示。According to the method in (2) of step (1) of 2, analyze the human homologous protein of the differential protein. Aminocaproic acid induced hypercoagulability in rats. Among the 65 differential proteins identified, 34 had human homologous proteins, and 16 of them were members of the human core urine proteome. The specific information is shown in Table 13.
表13、氨基己酸致大鼠高凝血状态尿液差异蛋白的人类同源蛋白Table 13. Human homologous proteins of aminocaproic acid-induced hypercoagulable urine proteins in rats
(3)氨基己酸高凝血状态模型差异蛋白的组织表达分析(3) Tissue expression analysis of differential proteins in aminocaproic acid hypercoagulation state model
按照步骤(一)的2中的(3)中的方法确定差异蛋白的人类同源蛋白的组织表达。分析后确定，有9个差异蛋白高表达于36个组织中，具体如表14和图5所示。可以看到，这些蛋白质在泌尿系统、消化系统和呼吸系统均有较高表达。Determine the tissue expression of the human homologous protein of the differential protein according to the method in (3) of step (1) of 2. After analysis, it was determined that 9 differential proteins were highly expressed in 36 tissues, as shown in Table 14 and Figure 5. It can be seen that these proteins are highly expressed in the urinary system, digestive system and respiratory system.
表14、氨基己酸高凝血状态模型的尿差异蛋白组织表达分析Table 14. Urinary differential protein tissue expression analysis of aminocaproic acid hypercoagulable state model
因此，实际应用中可以根据上述各个模型筛选到的高凝血状态标志物按照如下(1)-(3)中任一种方法来判断受试者是否处于高凝血状态：Therefore, in practical application, it can be judged whether the subject is in a hypercoagulable state according to any of the following methods (1)-(3) based on the hypercoagulable state markers screened by the above models:
(1)若受试者尿液的高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3和核糖核酸酶4的含量均高于健康人至少一倍，且氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于酚磺乙胺诱导所致高凝血状态；(1) If the content of Golgi protein I, carbonic anhydrase I, glypican-3 and ribonuclease 4 in the urine of the subject is at least twice that of healthy people, and the chloride ion intracellular Channel protein 1, biglycan, Rab GDP dissociation inhibitor, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triad nucleotide binding protein 1, myosin light chain 6. If the content of L-xylulose reductase and hemoglobin β subunit is at least one time lower than that of healthy people, the subject is in or is expected to be in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to phenol Ethylamine-induced hypercoagulable state;
若受试者尿液的高尔基体蛋白I、碳酸酐酶I、磷脂酰肌醇蛋白聚糖-3和核糖核酸酶4的含量未均高于健康人至少一倍，或氯离子胞内通道蛋白1、双糖链蛋白多糖、Rab GDP解离抑制剂、磷酸甘油酸激酶1、黄曲霉毒素B1醛还原酶成员2、组氨酸三联体核苷酸结合蛋白1、肌球蛋白轻链6、L-木酮糖还原酶和血红蛋白β亚基的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于酚磺乙胺诱导所致高凝血状态。If the content of Golgi protein I, carbonic anhydrase I, glypican-3 and ribonuclease 4 in the urine of the subject is not at least double that of healthy people, or the chloride ion intracellular channel protein 1. Biglycan, Rab GDP dissociation inhibitor, phosphoglycerate kinase 1, aflatoxin B1 aldehyde reductase member 2, histidine triplet nucleotide binding protein 1, myosin light chain 6, The contents of L-xylulose reductase and hemoglobin β subunit are not at least one times lower than that of healthy people, then the subject is not in or candidate is not in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to phenol Hypercoagulable state induced by sulphonethylamine.
(2)若受试者尿液的胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基和血管紧张素转换酶2同系物的含量均高于健康人至少一倍，且超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于氨甲环酸诱导所致高凝血状态。(2) If the test subject's urine contains cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, and guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 Subunit, actin-like protein, guanine nucleotide-binding protein G(k)α subunit, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunit and angiotensin-converting enzyme 2 homologues are at least twice as high as those in healthy people, and superoxide dismutase [Cu-Zn], amyloid 2, calcium binding protein, fumaryl acetoacetase, myosin The content of light chain 6, Frizzled protein 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1 and calmodulin (CaM) is at least one time lower than that of healthy people, then the subject is in or The candidate is in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by tranexamic acid.
若受试者尿液的胞液氨肽酶、核苷三磷酸二磷酸水解酶5、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-1亚基、肌动蛋白样蛋白、鸟嘌呤核苷酸结合蛋白G(k)α亚基、鸟嘌呤核苷酸结合蛋白G(I)/G(S)/G(T)β-2亚基和血管紧张素转换酶2同系物的含量未均高于健康人至少一倍，或超氧化物歧化酶[Cu-Zn]、淀粉样蛋白2、钙结合蛋白、延胡索酰乙酰乙酸酶、肌球蛋白轻链6、卷曲蛋白2、载脂蛋白M、肌红蛋白、肝配蛋白B1、硒结合蛋白1和钙调蛋白(CaM)的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于氨甲环酸诱导所致高凝血状态。If the subject's urine contains cytosolic aminopeptidase, nucleoside triphosphate diphosphate hydrolase 5, guanine nucleotide binding protein G(I)/G(S)/G(T)β-1 subunit, Actin-like proteins, guanine nucleotide-binding protein G(k)α subunits, guanine nucleotide-binding protein G(I)/G(S)/G(T)β-2 subunits, and angiotensin The content of homologues of protein converting enzyme 2 was at least double that of healthy people, or superoxide dismutase [Cu-Zn], amyloid 2, calcium binding protein, fumaryl acetoacetase, myosin light chain 6. If the content of Frizzled protein 2, apolipoprotein M, myoglobin, ephrin B1, selenium binding protein 1 and calmodulin (CaM) is not at least one time lower than that of healthy people, the subject is not in or The candidate is not in a hypercoagulable state; and the mechanism of this hypercoagulable state is related to the hypercoagulable state induced by tranexamic acid.
(3)若受试者尿液的细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白和硫氧还蛋白(Trx)的含量均高于健康人至少一倍，且肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4的含量均低于健康人至少一倍，则受试者处于或候选处于高凝血状态；且这种高凝血状态的形成机制相关于氨基己酸诱导所致高凝血状态。(3) If the content of cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin and thioredoxin (Trx) in the subject's urine is at least twice that of healthy people, and Peptidylprolyl cis-trans isomerase A, ABHD14B protein, Frizzled 4, α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transfer Enzyme P, alkaline phosphatase, 14-3-3 protein ζ/δ, quinolone oxidoreductase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, polyphosphate inositol phosphatase 1, glucose- 6-phosphate isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triad nucleotide binding protein 1, linker adhesion molecule C , stanniocalcin-1, glutamate-cysteine ligase catalytic subunit, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetyl The contents of galactosaminidase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase, selenium binding protein 1 and chondroitin sulfate proteoglycan 4 were all lower than At least twice that of healthy people, the subject is or is expected to be in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by aminocaproic acid.
若受试者尿液的细胞粘附分子3、前胶原C-内肽酶增强剂1、铜蓝蛋白和硫氧还蛋白(Trx)的含量未均高于健康人至少一倍，或肽基脯氨酰顺反异构酶A、ABHD14B蛋白、卷曲蛋白4、α-烯醇化酶、炭疽毒素受体1、卷曲蛋白2、吸引素、Neuroplastin、CD166抗原、谷胱甘肽S-转移酶P、碱性磷酸酶、14-3-3蛋白ζ/δ、喹诺酮氧化还原酶、Rho GDP-解离抑制剂1、过氧化物还原酶-6、多磷酸肌醇磷酸酶1、葡萄糖-6-磷酸异构酶、E3泛素-蛋白连接酶UBR4、前列腺素F2受体负调节因子、氯离子胞内通道蛋白4、组氨酸三联体核苷酸结合蛋白1、连接粘附分子C、斯钙素-1、谷氨酸-半胱氨酸连接酶催化亚基、N(G),N(G)-二甲基精氨酸二甲基氨基水解酶1、α-N-乙酰半乳糖胺酶、延胡索酰乙酰乙酸酶、1,2-二羟基-3-酮-5-甲硫基戊烯双加氧酶、硒结合蛋白1和硫酸软骨素蛋白多糖4的含量未均低于健康人至少一倍，则受试者不处于或候选不处于高凝血状态；且这种高凝血状态的形成机制相关于氨基己酸诱导所致高凝血状态。If the content of cell adhesion molecule 3, procollagen C-endopeptidase enhancer 1, ceruloplasmin and thioredoxin (Trx) in the urine of the subject is not at least double that of healthy people, or the peptide group Prolyl cis-trans isomerase A, ABHD14B protein, Frizzled 4, α-enolase, anthrax toxin receptor 1, Frizzled 2, attractin, Neuroplastin, CD166 antigen, glutathione S-transferase P , alkaline phosphatase, 14-3-3 protein ζ/δ, quinolone oxidoreductase, Rho GDP-dissociation inhibitor 1, peroxide reductase-6, polyphosphoinositide phosphatase 1, glucose-6- Phosphate isomerase, E3 ubiquitin-protein ligase UBR4, prostaglandin F2 receptor negative regulator, chloride intracellular channel protein 4, histidine triplet nucleotide binding protein 1, junctional adhesion molecule C, statin Calcein-1, glutamate-cysteine ligase catalytic subunit, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1, α-N-acetylgalactose The contents of aminase, fumaryl acetoacetase, 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase, selenium-binding protein 1 and chondroitin sulfate proteoglycan 4 were not lower than those in healthy The subject is not or is not in a hypercoagulable state; and the formation mechanism of this hypercoagulable state is related to the hypercoagulable state induced by aminocaproic acid.
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