技术领域technical field
本发明涉及脓毒症检测技术领域，特别是涉及检测样本CD72蛋白的试剂或诊断装置在制备或筛选检测脓毒症的试剂或试剂盒中的应用。The invention relates to the technical field of sepsis detection, in particular to the application of a reagent or a diagnostic device for detecting CD72 protein in a sample in the preparation or screening of a reagent or kit for detecting sepsis.
背景技术Background technique
脓毒症是机体对感染反应失调而导致的危及生命的器官功能障碍，具有高发病率和高病死率，已成为一个重要的社会公共卫生问题。Sepsis is a life-threatening organ dysfunction caused by the body's imbalanced response to infection. It has high morbidity and mortality, and has become an important social public health problem.
脓毒症发病期间的宿主免疫应答反应涉及复杂的病理生理学，目前认为是由炎症反应亢进和免疫功能抑制双重机制共同组成。感染会引发最初的细胞因子介导的宿主炎症反应，根据患者的合并症、营养状况、微生物负荷和毒力等因素不同，炎症反应的程度可能强弱不等；与此同时，机体也发生着抗炎反应，表现为免疫细胞代谢功能障碍、细胞无能或死亡等。此外，抗炎环境的持续存在也促进机体持续的免疫抑制状态，导致更易出现继发感染和并发症，加重患者死亡风险。因此，早期诊断和及时治疗对提高脓毒症患者生存率至关重要，寻找快速、有效治疗的新靶点对改善预后具有重要意义。The host immune response during the onset of sepsis involves complex pathophysiology, which is currently considered to be composed of dual mechanisms of hyperinflammatory response and immune function suppression. Infection will trigger the initial cytokine-mediated host inflammatory response, which may vary in intensity depending on the patient's comorbidities, nutritional status, microbial load, and virulence; at the same time, the body also undergoes Anti-inflammatory response, manifested as immune cell metabolic dysfunction, cell incompetence or death, etc. In addition, the persistence of an anti-inflammatory environment also promotes a persistent immunosuppressive state of the body, leading to a higher susceptibility to secondary infections and complications, and aggravating the risk of death in patients. Therefore, early diagnosis and timely treatment are crucial to improving the survival rate of patients with sepsis, and finding new targets for rapid and effective treatment is of great significance for improving prognosis.
目前，临床上主要根据常规感染指标，如降钙素原、白细胞计数、C反应蛋白等，对脓毒症进行诊断、预后评估和治疗，但相关感染指标诊断灵敏度和特异性有限。降钙素原在非脓毒症或感染情况下(如外科手术、严重创伤、烧伤、心源性休克等)也会出现不同程度升高，白细胞计数在感染的早期改变相对滞后且在合并免疫抑制、重症感染、器官移植等患者人群中可能处于持续低水平表达，而C反应蛋白则在区别细菌感染与非细菌感染时以及判断细菌感染程度方面诊断效能低下。针对以上诊断指标的局限性，亟需发明探索一种新的生物标志物用于临床脓毒症的诊断和检测。At present, the diagnosis, prognosis evaluation and treatment of sepsis are mainly based on routine infection indicators, such as procalcitonin, white blood cell count, C-reactive protein, etc., but the diagnostic sensitivity and specificity of relevant infection indicators are limited. Procalcitonin will also increase to varying degrees in non-sepsis or infection conditions (such as surgery, severe trauma, burns, cardiogenic shock, etc.), and the change of white blood cell count in the early stage of infection is relatively lagging and in the combined immune system Inhibition, severe infection, organ transplantation and other patient populations may be continuously expressed at low levels, while C-reactive protein has low diagnostic efficiency in distinguishing bacterial infection from non-bacterial infection and judging the degree of bacterial infection. In view of the limitations of the above diagnostic indicators, it is urgent to invent and explore a new biomarker for the diagnosis and detection of clinical sepsis.
发明内容Contents of the invention
为了弥补现有技术的不足，本发明的目的在于提供一种与脓毒症相关的生物标志物，从而为实现脓毒症的诊断、预后及检测提供一种新的有效选择；也用于解决现用于脓毒症的生物标志物在敏感性或特异性上不足的问题。In order to make up for the deficiencies in the prior art, the purpose of the present invention is to provide a biomarker related to sepsis, thereby providing a new effective option for the diagnosis, prognosis and detection of sepsis; The problem of insufficient sensitivity or specificity of biomarkers currently used for sepsis.
本发明解决技术问题的技术方案是提供了检测样本CD72蛋白的试剂或诊断装置在制备或筛选用于诊断、监测、预测和/或治疗脓毒症的试剂或试剂盒中的应用。The technical solution of the present invention to solve the technical problem is to provide the application of the reagent or diagnostic device for detecting CD72 protein in the sample to prepare or screen the reagent or kit for diagnosing, monitoring, predicting and/or treating sepsis.
其中，上述应用中所述的检测样本中CD72蛋白的试剂或诊断装置用于脓毒症体外诊断和危险分层。Wherein, the reagent or diagnostic device for detecting CD72 protein in a sample described in the above application is used for in vitro diagnosis and risk stratification of sepsis.
其中，上述应用中所述的试剂或试剂用于测定样本中CD72蛋白的表达量和/或浓度。Wherein, the reagents or reagents described in the above applications are used to determine the expression level and/or concentration of CD72 protein in the sample.
其中，上述应用中所述的试剂或试剂通过ELISA(酶联免疫吸附)法检测血浆CD72蛋白的表达量和/或浓度。Wherein, the reagents or reagents described in the above applications are used to detect the expression and/or concentration of plasma CD72 protein by ELISA (enzyme-linked immunosorbent) method.
其中，上述应用中所述的试剂或试剂通过ELISA双抗夹心法检测血浆CD72蛋白的表达量和/或浓度。Wherein, the reagents or reagents described in the above applications are used to detect the expression and/or concentration of plasma CD72 protein by ELISA double-antibody sandwich method.
其中，上述应用中所述的体液样品中CD72蛋白的表达量和/或浓度与脓毒症的严重程度正相关。Wherein, the expression level and/or concentration of CD72 protein in the body fluid sample described in the above application is positively correlated with the severity of sepsis.
其中，上述应用中所述的样本包含来自受试者的血浆、血清或血液抽取物、刷洗物、活组织检查物或外科手术切除的组织或液体样本。Wherein, the samples described in the above applications include plasma, serum or blood extracts, brushings, biopsies or surgically removed tissue or fluid samples from the subject.
其中，上述应用中所述的样本为离体样本。Wherein, the samples described in the above applications are isolated samples.
其中，上述应用中所述的试剂或试剂用于诊断或监测脓毒症的存在和/或过程和/或严重程度和/或预后。Wherein, the reagents or reagents described in the above applications are used for diagnosing or monitoring the existence and/or process and/or severity and/or prognosis of sepsis.
其中，上述应用中所述的所述预测为预测脓毒症患者的生存率。进一步的，所述的生存率为28天的生存率。Wherein, the prediction mentioned in the above application is to predict the survival rate of patients with sepsis. Further, the survival rate is the survival rate of 28 days.
本发明的CD72蛋白作为生物标志物在制备或筛选脓毒症诊断试剂中的应用，具有以下有益效果：一方面，本发明提供了CD72蛋白可以作为脓毒症的一个诊断指标；另一方面，本发明提供CD72蛋白可以作为评估脓毒症严重程度与预后的一个指标，可根据CD72蛋白的表达水平来进行脓毒症的预后和/或治疗监测，尤其是脓毒症患者28天的生存率。The use of the CD72 protein of the present invention as a biomarker in the preparation or screening of diagnostic reagents for sepsis has the following beneficial effects: on the one hand, the present invention provides that the CD72 protein can be used as a diagnostic index for sepsis; on the other hand, The present invention provides that CD72 protein can be used as an indicator for evaluating the severity and prognosis of sepsis, and the prognosis and/or treatment monitoring of sepsis can be carried out according to the expression level of CD72 protein, especially the 28-day survival rate of sepsis patients .
附图说明Description of drawings
图1、脓毒症患者CD72表达量。图1A显示脓毒症患者和健康对照者的血液中CD72表达量检测结果图。图1B显示为本发明实施例1中伴有休克的脓毒症患者和非休克的脓毒症患者的血液中CD72表达量检测结果图。图1C显示为本发明实施例1中脓毒症死亡者和存活者的血液中CD72表达量检测结果图。Figure 1. The expression level of CD72 in patients with sepsis. Figure 1A shows the detection results of CD72 expression in the blood of patients with sepsis and healthy controls. FIG. 1B is a graph showing the detection results of CD72 expression in the blood of sepsis patients with shock and non-shock sepsis patients in Example 1 of the present invention. FIG. 1C is a graph showing the detection results of CD72 expression in the blood of patients who died of sepsis and survivors in Example 1 of the present invention.
图2为脓毒症患者体内CD72的表达量与临床相关指标相关性分析结果图。Fig. 2 is a graph showing the correlation analysis results of the expression level of CD72 in patients with sepsis and relevant clinical indicators.
图3为ROC曲线，示意CD72与PCT、乳酸、淋巴细胞计数在脓毒症中的诊断效果。Figure 3 is a ROC curve, showing the diagnostic effect of CD72, PCT, lactic acid, and lymphocyte count in sepsis.
图4为Kaplan-Meier曲线分析不同水平CD72蛋白的脓毒症患者预后评估。Figure 4 is the Kaplan-Meier curve analysis of the prognosis of sepsis patients with different levels of CD72 protein.
图5为小鼠血浆CD72蛋白含量检测结果。Fig. 5 is the detection result of CD72 protein content in mouse plasma.
图6为脓毒症模型小鼠检测结果。图6A和图6B分别为CD72蛋白处理组小鼠及对照组小鼠的生存率统计图。Figure 6 is the detection results of sepsis model mice. Figure 6A and Figure 6B are statistical charts of the survival rates of the mice in the CD72 protein treatment group and the mice in the control group, respectively.
图7脓毒症模型小鼠病理检测结果。图7A小鼠的组织器官病理损伤切片照片；图7B为小鼠病理损伤评分结果。Fig. 7 Pathological detection results of sepsis model mice. Figure 7A is a photo of the tissue and organ pathological damage section of the mouse; Figure 7B is the result of the pathological damage score of the mouse.
具体实施方式Detailed ways
CD72(cluster of differentiation 72)，又称B细胞表面分化抗原，最初被发现是B 淋巴细胞表面上的一种跨膜蛋白，属于钙依赖的C型凝集素超家族成员。CD72蛋白具有两种形式：存在于细胞表面的膜结合型CD72蛋白，以及体液中游离的CD72蛋白(氨基酸序列为 SEQ ID No.1所示)。细胞表面的CD72蛋白主要分布在B淋巴细胞表面，在活化的T细胞、单核巨噬细胞、抗原提呈细胞等表面也有少量表达。游离的CD72蛋白广泛存在人体体液中，包括外周血、关节腔液等，通常只在病理状态下显著升高，其产生机制和生物学作用尚不清楚。然而，脓毒症患者外周血中CD72蛋白表达改变及其生物学作用尚无研究，CD72蛋白在脓毒症病理变化中意义仍是未知。CD72 (cluster of differentiation 72), also known as B cell surface differentiation antigen, was originally discovered as a transmembrane protein on the surface of B lymphocytes, which belongs to the calcium-dependent C-type lectin superfamily member. The CD72 protein has two forms: the membrane-bound CD72 protein present on the cell surface, and the free CD72 protein in body fluid (the amino acid sequence is shown in SEQ ID No.1). The CD72 protein on the cell surface is mainly distributed on the surface of B lymphocytes, and there is also a small amount of expression on the surface of activated T cells, monocyte-macrophages, and antigen-presenting cells. Free CD72 protein widely exists in human body fluids, including peripheral blood, joint cavity fluid, etc., and usually only significantly increases in pathological conditions, and its production mechanism and biological role are still unclear. However, the expression changes of CD72 protein in peripheral blood of patients with sepsis and its biological role have not been studied, and the significance of CD72 protein in the pathological changes of sepsis is still unknown.
本发明通过一系列的探究发现，血浆CD72蛋白参与了脓毒症的发生发展过程，作为脓毒症疾病相关指标具有重要应用价值，是一种良好的脓毒症的诊断、预后和监测的生物标志物，故得到本发明的技术方案。Through a series of investigations, the present invention finds that plasma CD72 protein is involved in the occurrence and development of sepsis, has important application value as a sepsis disease-related index, and is a good biological tool for the diagnosis, prognosis and monitoring of sepsis. Marker, so the technical solution of the present invention is obtained.
以下具体说明CD72蛋白作为生物标志物在脓毒症中的诊断、预后和检测中的作用。前期通过ELISA检测了57例脓毒症患者血浆中CD72蛋白的含量，发现与40例健康体检者相比，脓毒症患者外周血中CD72蛋白浓度明显升高，且在脓毒症致死亡患者体内高于存活患者，在脓毒症病情更重(脓毒症休克)的患者体内高于病情较轻的患者(普通脓毒症)，这提示CD72 蛋白可能参与脓毒症的发生发展，且在患病严重的患者体内含量更高。进一步，将血浆CD72 蛋白浓度与患者危重度评分、相关炎症及感染指标、生化指标等进行相关分析，结果发现CD72 蛋白含量与患者疾病危重度评分(SOFA评分、APACHE II评分)、炎症指标(IL-6)、血乳酸水平显著相关，说明CD72蛋白浓度升高提示患者病情更危重，CD72蛋白在提示患者感染、炎症水平方面具有一定价值，对脓毒症的诊断、病情监测及预后判断具有一定的参考意义。进一步，我们通过ROC曲线评估CD72蛋白以及临床常用的感染指标(PCT、乳酸、淋巴细胞计数)对脓毒症的诊断价值，结果显示CD72蛋白诊断脓毒症效能高于PCT、乳酸及淋巴细胞计数；Kaplan-Meier曲线显示不同浓度的CD72蛋白可评估脓毒症患者预后。因此，CD72蛋白可以作为脓毒症的诊断标准，可以反映患者感染、病情严重程度。The role of CD72 protein as a biomarker in the diagnosis, prognosis and detection of sepsis will be described in detail below. In the early stage, the content of CD72 protein in the plasma of 57 patients with sepsis was detected by ELISA, and it was found that compared with 40 cases of healthy subjects, the concentration of CD72 protein in the peripheral blood of patients with sepsis was significantly higher, and the concentration of CD72 protein in the peripheral blood of patients with sepsis It is higher than that in surviving patients, and it is higher in patients with severe sepsis (septic shock) than in patients with milder conditions (common sepsis), which suggests that CD72 protein may be involved in the occurrence and development of sepsis, and Higher levels are found in severely ill patients. Further, the plasma CD72 protein concentration was correlated with the patient's criticality score, related inflammation and infection indicators, and biochemical indicators. -6) and blood lactic acid level are significantly correlated, indicating that the increase of CD72 protein concentration indicates that the patient's condition is more critical. reference meaning. Furthermore, we evaluated the diagnostic value of CD72 protein and common clinical infection indicators (PCT, lactic acid, lymphocyte count) for sepsis by ROC curve, and the results showed that CD72 protein was more effective in diagnosing sepsis than PCT, lactic acid and lymphocyte count ; Kaplan-Meier curve shows that different concentrations of CD72 protein can evaluate the prognosis of patients with sepsis. Therefore, CD72 protein can be used as a diagnostic criterion for sepsis, which can reflect the infection and severity of the patient's condition.
进一步，本发明通过盲肠结扎穿刺术(Cecal Ligation and Puncture，CLP)建立脓毒症动物模型，动态检测脓毒症小鼠在造模后外周血中CD72蛋白表达水平，发现相比较假手术组，脓毒症小鼠在造模后2小时血浆CD72蛋白浓度开始显著升高，并持续到术后24小时，这与在脓毒症患者中发现的CD72蛋白水平升高结果相符，进一步充分说明CD72蛋白参与了脓毒症发生发展过程，在脓毒症中扮演重要意义。在此基础上，给脓毒症小鼠皮下注射不同剂量的小鼠重组CD72蛋白(低剂量、中剂量、高剂量)，以相同条件给假手术组小鼠注射同体积的PBS，发现CD72蛋白处理组小鼠的生存率明显显著对照组小鼠的生存率，且CD72蛋白剂量越高，小鼠预后越差。同时，对假手术组小鼠注射不同剂量重组CD72蛋白，均未引起小鼠死亡率改变，结果排除了由重组蛋白毒性作用等原因引起的小鼠死亡率改变。病理切片 HE染色结果显示CD72蛋白处理组小鼠的组织器官充血水肿、炎症细胞浸润和蛋白审查等更为显著，组织病理评分更高，进一步说CD72蛋白表达水平可以用于检测脓毒症预后。Further, the present invention establishes a sepsis animal model through cecal ligation and puncture (CLP), and dynamically detects the expression level of CD72 protein in the peripheral blood of sepsis mice after modeling, and finds that compared with the sham operation group, The plasma CD72 protein concentration of septic mice began to increase significantly 2 hours after the model was established, and continued until 24 hours after the operation, which was consistent with the increase in the CD72 protein level found in patients with sepsis, further fully explaining that CD72 Proteins are involved in the development of sepsis and play an important role in sepsis. On this basis, different doses of mouse recombinant CD72 protein (low dose, medium dose, high dose) were subcutaneously injected into sepsis mice, and the same volume of PBS was injected into sham-operated mice under the same conditions, and CD72 protein was found The survival rate of the mice in the treatment group was significantly higher than that of the mice in the control group, and the higher the dose of CD72 protein, the worse the prognosis of the mice. At the same time, injecting different doses of recombinant CD72 protein to the mice in the sham operation group did not cause any change in the mortality of the mice, and the results excluded the change in the mortality of the mice caused by the toxic effect of the recombinant protein. The results of HE staining of pathological sections showed that the tissue and organ congestion, edema, inflammatory cell infiltration and protein inspection of mice treated with CD72 protein were more significant, and the histopathological score was higher. Further, the expression level of CD72 protein can be used to detect the prognosis of sepsis.
以下通过特定的具体实例详细说明本发明的。The following describes the present invention in detail through specific examples.
研究人群：收集2021年10月到2022年1月四川大学华西医院重症医学科诊治的脓毒症患者血液为试验组，同期健康体检者为健康对照组，试验组男女分别为37(64.9％)例、20(35.1％)例，对照组男女分别为25(62.5％)、15(37.5％)例。样本置于4℃400×g离心10min，之后冻存在-80℃，通过ELISA检测血浆中的CD72蛋白表达水平。患者均符合国际脓毒症会议中对该病的诊断标准Sepsis 3.0，排除患有自身免疫性疾病、免疫缺陷或长期服用免疫药物、恶性肿瘤或血液系统肿瘤、孕产妇、严重贫血以及住院时间小于24小时等情况，受试者所有研究均经四川大学华西医院科学与伦理委员会批准(伦理编号：2021年审(1287) 号)。Study population: Blood from patients with sepsis who were diagnosed and treated in the Department of Critical Care Medicine, West China Hospital, Sichuan University from October 2021 to January 2022 was collected as the test group, and healthy subjects who underwent physical examination during the same period were the healthy control group, and the test group consisted of 37 men and women (64.9%) 20 (35.1%) cases, 25 (62.5%) and 15 (37.5%) cases of men and women in the control group. The samples were centrifuged at 400×g for 10 min at 4°C, then frozen at -80°C, and the expression level of CD72 protein in plasma was detected by ELISA. All patients met the diagnostic criteria of Sepsis 3.0 in the International Sepsis Conference, excluding those with autoimmune diseases, immunodeficiency or long-term use of immune drugs, malignant tumors or hematologic tumors, pregnant women, severe anemia, and hospitalization time less than 24 hours, etc., all research subjects were approved by the Science and Ethics Committee of West China Hospital, Sichuan University (Ethics Number: 2021 Trial (1287) No.).
实验动物：雄性、野生型C57BL/6小鼠，体重为18-22g，约6-8周龄，购买自北京华阜康生物科技股份有限公司，饲养在四川大学华西医院动物中心无特定病原体级实验室，该实验所用小鼠均为SPF级实验动物。动物实验均严格遵循中华人民共和国科技部颁布的实验室动物照料及使用指南，符合四川大学华西医院实验动物伦理会管理规定。Experimental animals: male, wild-type C57BL/6 mice, weighing 18-22g, about 6-8 weeks old, purchased from Beijing Huafukang Biotechnology Co., Ltd., and raised in the Animal Center of West China Hospital of Sichuan University without specific pathogens In the laboratory, the mice used in this experiment are all SPF grade experimental animals. All animal experiments strictly followed the guidelines for the care and use of laboratory animals promulgated by the Ministry of Science and Technology of the People's Republic of China, and complied with the management regulations of the Experimental Animal Ethics Committee of West China Hospital of Sichuan University.
实施例1脓毒症患者血液中CD72蛋白含量的检测及与临床指标相关性分析Example 1 Detection of CD72 protein content in blood of patients with sepsis and correlation analysis with clinical indicators
收集57例临床脓毒症患者(其中，普通脓毒症患者(不伴有休克)40例，脓毒症休克患者17例；脓毒症幸存者36例，非幸存者21例)及40例健康对组者血浆标本(参见表1)，采用ELISA试剂盒(购自武汉华美生物CUSABIO，货号：CSB-EL004955HU))检测CD72蛋白表达量，操作方法严格按照试剂盒所附说明书进行，用Graphpad Prism 8.0软件进行统计分析和作图。Collect 57 patients with clinical sepsis (among them, 40 patients with normal sepsis (without shock), 17 patients with septic shock; 36 survivors of sepsis, 21 non-survivors) and 40 patients The plasma samples of the healthy control group (see Table 1) were detected by ELISA kit (purchased from Wuhan Huamei Biology CUSABIO, article number: CSB-EL004955HU) to detect the expression of CD72 protein. Prism 8.0 software was used for statistical analysis and graphing.
表1脓毒症患者及健康体检者一般临床信息统计Table 1 General clinical information statistics of patients with sepsis and healthy subjects
注：APACHE II：急性生理与慢性健康评分。WBC：白细胞计数。LC：淋巴细胞计数。PCT：降钙素原。 IL-6：白介素-6；CRP：C反应蛋白。Lac：乳酸。a：p>0.5。Note: APACHE II: Acute Physiology and Chronic Health Score. WBC: white blood cell count. LC: lymphocyte count. PCT: Procalcitonin. IL-6: interleukin-6; CRP: C-reactive protein. Lac: lactic acid. a: p>0.5.
图1A显示为脓毒症患者和健康对照者的血浆CD72蛋白表达水平检测结果图，图1B显示为脓毒症休克患者和普通脓毒症患者(不伴有休克)的血液中CD72蛋白表达量检测结果图，图1C显示为脓毒症幸存者和非幸存者的血液中CD72蛋白表达量检测结果图，图2显示为脓毒症患者血浆CD72蛋白的表达量与临床指标相关性分析结果图。结果显示：与健康对照者相比，脓毒症患者血浆CD72蛋白水平显著升高，差异具有统计学意义(P＜0.0001)；脓毒症休克患者血浆CD72蛋白含量显著高于普通脓毒症患者，差异具有统计学意义(P＜0.01)；脓毒症患者中，死亡患者血浆CD72蛋白含量显著高于存活者体内的CD72(P＜0.001)；脓毒症患者血浆CD72蛋白水平与患者疾病危重度评分(SOFA评分、APACHE II评分)、炎症指标(IL-6)、血乳酸水平具有显著正相关性(P<0.01)，并与淋巴细胞计数呈负相关。因此，血浆CD72蛋白可以作为脓毒症诊断指标，提示患者严重程度和预后转归。Figure 1A shows the detection results of plasma CD72 protein expression levels in patients with sepsis and healthy controls, and Figure 1B shows the expression levels of CD72 protein in the blood of patients with septic shock and normal sepsis patients (without shock) Figure 1C shows the detection results of CD72 protein expression in the blood of sepsis survivors and non-survivors, and Figure 2 shows the correlation analysis results of plasma CD72 protein expression and clinical indicators in patients with sepsis . The results showed that: compared with healthy controls, the plasma CD72 protein level in patients with sepsis was significantly higher, and the difference was statistically significant (P<0.0001); the plasma CD72 protein level in patients with septic shock was significantly higher than that in normal sepsis patients , the difference was statistically significant (P<0.01); in patients with sepsis, the plasma CD72 protein content of dead patients was significantly higher than that of survivors (P<0.001); the plasma CD72 protein level of sepsis patients was associated with critical illness The degree score (SOFA score, APACHE II score), inflammatory index (IL-6), and blood lactate level were significantly positively correlated (P<0.01), and negatively correlated with lymphocyte count. Therefore, plasma CD72 protein can be used as a diagnostic indicator of sepsis, indicating the severity and prognosis of patients.
实施例2ROC曲线及Kaplan-Meier曲线比较CD72蛋白、PCT、乳酸及淋巴细胞计数对脓毒症患者预后的预测价值Example 2 ROC curve and Kaplan-Meier curve compare the predictive value of CD72 protein, PCT, lactic acid and lymphocyte count on the prognosis of patients with sepsis
收集实施例1中的脓毒症幸存者及非幸存者的PCT、乳酸、淋巴细胞计数及检测出的CD72 蛋白水平，使用SPSS19.0软件绘制ROC曲线，通过约登指数(灵敏度+特异度-1)计算CD72 蛋白诊断截断值，使用Graphpad Prism 8.0软件绘制Kaplan-Meier生存曲线。Collect PCT, lactic acid, lymphocyte count and detected CD72 protein level of sepsis survivors and non-survivors in Example 1, use SPSS19.0 software to draw ROC curve, by Youden index (sensitivity+specificity- 1) Calculate the diagnostic cut-off value of CD72 protein, and use Graphpad Prism 8.0 software to draw Kaplan-Meier survival curve.
表2Table 2
注：AUC:受试者工作特征曲线下面积；CI：可信区间；Cut-off value：截断值，根据约登指数(灵敏度+特异度-1)计算；Sensitivity：灵敏度；Specificity：特异度；sCD72：血浆可溶性CD72蛋白； LC：Lymphocyte count，淋巴细胞计数(x 109/L)；Lac：乳酸；PCT：降钙素原。P1：每个变量AUC的统计学检验。P2：与CD72诊断价值比较。Note: AUC: area under receiver operating characteristic curve; CI: confidence interval; Cut-off value: cut-off value, calculated according to Youden index (sensitivity + specificity - 1); Sensitivity: sensitivity; Specificity: specificity; sCD72: plasma soluble CD72 protein; LC: Lymphocyte count, lymphocyte count (x 109 /L); Lac: lactic acid; PCT: procalcitonin. P1 : Statistical test of AUC for each variable. P2 : compared with the diagnostic value of CD72.
图3显示为ROC曲线比较分析CD72与PCT、乳酸及淋巴细胞计数在脓毒症中的诊断效果示意图。结果显示CD72蛋白诊断曲线下面积(Area under the Curve,AUC)为0.7533，乳酸、淋巴细胞计数及PCT的诊断曲线下面积分别为0.6905、0.6905、0。6270，CD72的曲线下面积高于乳酸、淋巴细胞计数及PCE，这表明，与临床常用的PCR、乳酸及淋巴细胞计数指标相比，CD72对脓毒症预后的预测价值更高。进一步，计算CD72诊断预后截断值为81.35 ng/mL(表2)，以此将患者分为高浓度组(CD72浓度>81.35ng/ml)和低浓度组(CD72浓度<81.35ng/ml)，绘制生存曲线如图4，发现高浓度组患者住院28天发生死亡的相对风险是低脓毒症的4.973倍(P<0.0001，1/HR＝4.973)。Fig. 3 is a schematic diagram showing the comparative analysis of CD72, PCT, lactic acid and lymphocyte count in the diagnosis effect of sepsis by ROC curve. The results showed that the area under the curve (AUC) of CD72 protein was 0.7533, and the areas under the diagnostic curve of lactic acid, lymphocyte count and PCT were 0.6905, 0.6905, and 0.6270, respectively. The area under the curve of CD72 was higher than that of lactic acid, Lymphocyte count and PCE, which showed that CD72 had a higher predictive value for the prognosis of sepsis compared with PCR, lactate and lymphocyte count indicators commonly used in clinic. Further, the cut-off value of CD72 diagnosis and prognosis was calculated to be 81.35 ng/mL (Table 2), and patients were divided into high concentration group (CD72 concentration>81.35ng/ml) and low concentration group (CD72 concentration<81.35ng/ml), The survival curve was drawn as shown in Figure 4, and it was found that the relative risk of death within 28 days of hospitalization in the high concentration group was 4.973 times that of the low sepsis group (P<0.0001, 1/HR=4.973).
实施例3脓毒症小鼠血浆CD72蛋白含量检测Example 3 Detection of plasma CD72 protein content in septic mice
通过盲肠结扎穿刺术(cecal ligation and puncture，CLP)建立脓毒症动物模型，具体操作方法如下：选用成年6-8周龄雄性C57BL/6小鼠，术前禁食12小时，以100微升1.5％的戊巴比妥钠腹腔注射麻醉小鼠，固定于操作板，腹部皮肤剃毛，碘伏消毒皮肤，沿腹正中线切开皮肤，长度约1cm，打开腹腔并寻找盲肠，距盲肠尾端0.8cm处结扎并以18G注射器针头贯穿，将盲肠还纳入腹腔，逐层缝合肌肉及皮肤并伤口消毒。其中，假手术组(Sham组)找到盲肠后即放回原位，不结扎及穿刺，其余步骤同手术处理组。术后不同时间点(0-24小时)取小鼠外周静脉血，分离血浆，通过ELISA实验检测小鼠外周血CD72蛋白含量(杭州Sunlong公司，货号：SL0694Mo),操作步骤严格按照试剂盒操作说明进行。用GraphpadPrism8.0软件进行统计分析和作图。Animal models of sepsis were established by cecal ligation and puncture (CLP), and the specific operation method was as follows: adult male C57BL/6 mice aged 6-8 weeks were selected, fasted for 12 hours before the operation, and injected with 100 μl Anesthetize the mouse with 1.5% pentobarbital sodium intraperitoneal injection, fix it on the operation board, shave the abdominal skin, disinfect the skin with iodine, cut the skin along the midline of the abdomen, the length is about 1cm, open the abdominal cavity and look for the cecum, the distance from the tail of the cecum The end was ligated at 0.8cm and penetrated with an 18G syringe needle, the cecum was brought into the abdominal cavity, the muscles and skin were sutured layer by layer and the wound was disinfected. Among them, the sham operation group (Sham group) found the cecum and put it back to its original position without ligation and puncture, and the rest of the steps were the same as those in the operation treatment group. The peripheral venous blood of the mice was collected at different time points (0-24 hours) after the operation, and the plasma was separated, and the CD72 protein content in the peripheral blood of the mice was detected by ELISA experiment (Hangzhou Sunlong Company, article number: SL0694Mo), and the operation steps were strictly in accordance with the kit instructions. conduct. Statistical analysis and graphing were performed with GraphpadPrism8.0 software.
图5显示为脓毒症小鼠模型血浆CD72蛋白水平统计图。结果显示：与正常对照组相比，脓毒症小鼠血浆CD72蛋白水平在术后2小时出现显著升高(P<0.05)并持续至24小时，这与脓毒症患者血浆CD72浓度显著高于健康志愿者结果相一致，说明CD72蛋白参与了脓毒症病理过程。Fig. 5 is a statistical diagram showing the plasma CD72 protein level in a mouse model of sepsis. The results showed that: compared with the normal control group, the plasma CD72 protein level in sepsis mice was significantly increased at 2 hours after operation (P<0.05) and lasted until 24 hours, which is consistent with the significantly higher plasma CD72 concentration in patients with sepsis It is consistent with the results of healthy volunteers, indicating that CD72 protein is involved in the pathological process of sepsis.
实施例4脓毒症小鼠生存率实验Embodiment 4 Survival rate experiment of septic mice
根据实施例3中方法建立脓毒症小鼠模型，将小鼠分别接受不同剂量重组小鼠CD72蛋白体内注射(低剂量：0.25mg/kg，中剂量：0.5mg/kg，高剂量：1.0mg/kg)，并设置假手术组(sham组，接受等量PBS注射)和普通脓毒症组(CLP组，接受等量PBS注射)，观察小鼠生存率改变。进一步的，对假手术组小鼠也分别注射上述不同剂量CD72重组蛋白，观察小鼠生存率。重组小鼠CD72蛋白购自MCE公司，货号HY-P7986。According to the method in Example 3, a sepsis mouse model was established, and the mice were injected with different doses of recombinant mouse CD72 protein in vivo (low dose: 0.25 mg/kg, middle dose: 0.5 mg/kg, high dose: 1.0 mg /kg), and set up a sham operation group (sham group, receiving the same amount of PBS injection) and a common sepsis group (CLP group, receiving the same amount of PBS injection), and observe the changes in the survival rate of the mice. Further, the mice in the sham operation group were also injected with different doses of the above-mentioned CD72 recombinant protein, and the survival rate of the mice was observed. Recombinant mouse CD72 protein was purchased from MCE Company, Cat. No. HY-P7986.
图6A显示CD72蛋白处理组小鼠与假手术组及脓毒症小鼠生存率统计图，结果发现CLP 小鼠存活率为75％，注射低剂量CD72重组蛋白小鼠存活率为70％(二者间无显著差异，P>0.05)，而注射中剂量CD72重组蛋白小鼠存活率显著下降至30％(P<0.01)，注射高剂量CD72重组蛋白小鼠48小时均出现死亡(P<0.0001)。图6B显示，假手术处理小鼠接受不同剂量CD72重组蛋白注射后，其生存率无显著改变(P>0.05)，均为100％。结果说明CD72蛋白可以加重脓毒症小鼠死亡，且剂量越高，脓毒症小鼠死亡率更高，表面CD72可作为脓毒症检测、预后评估的指标。Figure 6A shows the statistics of survival rates of CD72 protein treatment group mice, sham operation group and sepsis mice. It was found that the survival rate of CLP mice was 75%, and the survival rate of mice injected with low-dose CD72 recombinant protein was 70% (2 There was no significant difference among the mice, P>0.05), while the survival rate of mice injected with medium-dose CD72 recombinant protein was significantly reduced to 30% (P<0.01), and the mice injected with high-dose CD72 recombinant protein all died within 48 hours (P<0.0001 ). Figure 6B shows that after the sham-operated mice were injected with different doses of CD72 recombinant protein, the survival rate did not change significantly (P>0.05), all of which were 100%. The results show that CD72 protein can aggravate the death of sepsis mice, and the higher the dose, the higher the death rate of sepsis mice, and the surface CD72 can be used as an index for sepsis detection and prognosis evaluation.
实施例5重组CD72蛋白处理小鼠后组织病理染色和损伤评估Example 5 Histopathological staining and injury assessment of mice treated with recombinant CD72 protein
根据实施例3中方法建立脓毒症小鼠模型，将建模后小鼠分为3组，假手术组和CLP给予PBS缓冲液，处理组给予0.5mg/kg重组CD72蛋白。由于肺脏和肾脏是脓毒症病理变化最早及最重要的器官，因此在术后8小时取不同组小鼠肺脏和肾脏，于4％多聚甲醛中固定48小时，分别进行脱水、浸蜡、包埋、切片，经HE染色后于显微镜下镜检。根据韩丙超等文献记录进行病理评分统计(韩丙超,解立新,赵晓巍,等.全氟化碳汽化吸入对急性肺损伤兔多脏器病理组织学的影响[J].中国呼吸与危重监护杂志,2009,8(2))，进行病理损伤评分。用GraphPad Prism 8.0软件做统计学分析，Excel软件制作表格。数据使用中位数(四分位间距)进行显示。The sepsis mouse model was established according to the method in Example 3, and the mice after modeling were divided into 3 groups, the sham operation group and CLP were given PBS buffer, and the treatment group was given 0.5 mg/kg recombinant CD72 protein. Since the lungs and kidneys are the earliest and most important organs of pathological changes in sepsis, the lungs and kidneys of different groups of mice were taken 8 hours after the operation, fixed in 4% paraformaldehyde for 48 hours, and then dehydrated, paraffin-immersed, and Embedded, sectioned, and examined under a microscope after HE staining. According to the literature records of Han Bingchao et al. (Han Bingchao, Xie Lixin, Zhao Xiaowei, et al. Effect of vaporized inhalation of perfluorocarbons on pathology and histology of multiple organs in rabbits with acute lung injury[J]. Chinese Journal of Respiratory and Critical Care Care, 2009 ,8(2)), the pathological damage score was performed. GraphPad Prism 8.0 software was used for statistical analysis, and Excel software was used to make tables. Data are presented using medians (interquartile ranges).
图7A显示为小鼠的肺和肾脏组织切片病理结果对比图。结果显示与假手术组(Sham)小鼠相比，CLP组小鼠肺脏及肾脏出现明显组织损伤，表现为充血水肿明显加重、炎性细胞浸润增加；而CD72蛋白处理组小鼠肺脏及肾脏损伤加重更为显著，图7B病理评分有统计学差异(P<0.05)。结果说明CD72蛋白加重了脓毒症小鼠组织器官的损伤，可以作为评估脓毒症严重程度及预后检测的指标。Figure 7A shows a comparison of pathological results of lung and kidney tissue sections of mice. The results showed that compared with the sham operation group (Sham) mice, the lungs and kidneys of the mice in the CLP group had obvious tissue damage, manifested as significantly increased congestion and edema, and increased inflammatory cell infiltration; while the mice in the CD72 protein treatment group had lung and kidney damage The aggravation was more significant, and the pathological score in Figure 7B was statistically different (P<0.05). The results showed that the CD72 protein aggravated the tissue and organ damage in mice with sepsis, and could be used as an indicator for evaluating the severity and prognosis of sepsis.
综上所述，本发明发现CD72在临床脓毒症患者血浆中表达水平显著高于健康志愿者，且病情更危重患者表现出更高的表达含量，并与疾病严重程度评分、感染及炎症指标等显著相关，并通过ROC曲线分析发现CD72作为分子生物标志物在脓毒症的诊断上具有较高的效益， Kaplan-Meier生存曲线发现CD72蛋白可用于患者预后检测和评估。进一步，通过动物实验发现脓毒症模型小鼠体内同样出现CD72水平升高的情况，并且与疾病的严重性呈正相关。因此，本发明为评估脓毒症患者病情危重度、住院28天病死率及临床预后提供了一个新的生物标志物CD72及其在制备诊断或辅助诊断脓毒症及其预后的试剂盒中的用途。根据检测指标CD72的表达水平，可以给出患者病情危重度、住院28天病死率以及预后风险的判断，有利于提高医生对患者病情及预后的认识和理解，指导医生临床诊疗措施的选择，有利于与患者家属进行病情沟通交流。本发明只需要患者少量外周血即可进行有效检测，操作简便，检测时间短，诊断效率高。In summary, the present invention found that the expression level of CD72 in the plasma of patients with clinical sepsis was significantly higher than that of healthy volunteers, and patients with more critical conditions showed higher expression levels, which were correlated with disease severity scores, infection and inflammation indicators. etc., and through ROC curve analysis, it was found that CD72 as a molecular biomarker has a high benefit in the diagnosis of sepsis, and the Kaplan-Meier survival curve found that CD72 protein can be used for the detection and evaluation of patient prognosis. Further, through animal experiments, it was found that the level of CD72 also increased in sepsis model mice, and it was positively correlated with the severity of the disease. Therefore, the present invention provides a new biomarker CD72 and its use in the preparation of diagnostic or auxiliary diagnostic kits for sepsis and its prognosis for evaluating the critical condition, 28-day hospitalization mortality and clinical prognosis of patients with sepsis. use. According to the expression level of the detection index CD72, the judgment of the critical condition of the patient, the 28-day mortality rate in hospital and the risk of prognosis can be given, which is conducive to improving the doctor's knowledge and understanding of the patient's condition and prognosis, and guiding the doctor's choice of clinical diagnosis and treatment measures. Facilitate communication with patients' families. The invention only needs a small amount of peripheral blood of the patient to carry out effective detection, and the operation is simple, the detection time is short, and the diagnosis efficiency is high.
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所要求保护的范围。The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, those who have ordinary knowledge in the technical field do not depart from the scope of protection claimed by the present invention.
序列表sequence listing
<110> 四川大学华西医院<110> West China Hospital of Sichuan University
<120> 检测样本CD72蛋白的试剂或诊断装置在制备检测脓毒症的试剂或试剂盒中的应用<120> Application of a reagent or diagnostic device for detecting CD72 protein in a sample in the preparation of a reagent or kit for detecting sepsis
<160> 1<160> 1
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
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<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
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Gln Leu Gly Gln Lys Glu Val Glu Leu Gln Lys Ala Arg Lys Glu LeuGln Leu Gly Gln Lys Glu Val Glu Leu Gln Lys Ala Arg Lys Glu Leu
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Ile Ser Ser Gln Asp Thr Leu Gln Glu Lys Gln Arg Thr His Glu AspIle Ser Ser Gln Asp Thr Leu Gln Glu Lys Gln Arg Thr His Glu Asp
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Ala Glu Gln Gln Leu Gln Ala Cys Gln Ala Glu Arg Ala Lys Thr LysAla Glu Gln Gln Leu Gln Ala Cys Gln Ala Glu Arg Ala Lys Thr Lys
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Glu Asn Leu Lys Thr Glu Glu Glu Arg Arg Arg Asp Leu Asp Gln ArgGlu Asn Leu Lys Thr Glu Glu Glu Arg Arg Arg Asp Leu Asp Gln Arg
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Leu Thr Ser Thr Arg Glu Thr Leu Arg Arg Phe Phe Ser Asp Ser SerLeu Thr Ser Thr Arg Glu Thr Leu Arg Arg Phe Phe Ser Asp Ser Ser
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Asp Thr Cys Cys Pro Cys Gly Trp Ile Pro Tyr Gln Glu Arg Cys PheAsp Thr Cys Cys Pro Cys Gly Trp Ile Pro Tyr Gln Glu Arg Cys Phe
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Tyr Ile Ser His Thr Leu Gly Ser Leu Glu Glu Ser Gln Lys Tyr CysTyr Ile Ser His Thr Leu Gly Ser Leu Glu Glu Ser Gln Lys Tyr Cys
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Thr Ser Leu Ser Ser Lys Leu Ala Ala Phe Asp Glu Pro Ser Lys TyrThr Ser Leu Ser Ser Ser Lys Leu Ala Ala Phe Asp Glu Pro Ser Lys Tyr
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Tyr Tyr Glu Val Ser Leu Pro Ser Gly Leu Glu Glu Leu Leu Asp ArgTyr Tyr Glu Val Ser Leu Pro Ser Gly Leu Glu Glu Leu Leu Asp Arg
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Ser Lys Ser Tyr Trp Ile Gln Met Ser Lys Lys Trp Arg Gln Asp SerSer Lys Ser Tyr Trp Ile Gln Met Ser Lys Lys Trp Arg Gln Asp Ser
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|---|---|---|---|---|
| CN118566510A (en)* | 2024-05-17 | 2024-08-30 | 浙江大学 | A monocyte CD11c detection kit for sepsis identification |
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