技术领域Technical Field
本发明涉及生物医学领域，具体涉及一种糖尿病动脉粥样硬化的生物标记物及其在制备糖尿病动脉粥样硬化药物中的应用。The present invention relates to the field of biomedicine, and in particular to a biomarker of diabetic atherosclerosis and application thereof in preparing a diabetic atherosclerosis drug.
背景技术Background Art
随着生活方式和饮食结构的日益西化，导致糖尿病发病率急剧上升。动脉粥样硬化是糖尿病血管病变的主要病理改变，也是影响糖尿病患者预后的一项主要并发症。动脉粥样硬化是指脂质代谢障碍导致动脉壁增厚变硬、血管腔狭窄，具有早期诊断困难，严重影响患者预后的特点。近年来代谢性心血管疾病的讨论愈演愈烈，动脉粥样硬化的探究也逐渐开拓到代谢性心血管疾病的探索中，作为代谢途径中的重要一环----糖代谢无疑也在心血管疾病的发生发展中起到了举足轻重的作用。既往的研究发现，糖尿病患者处于慢性、低度、全身性的炎症刺激下，炎症刺激对糖尿病患者的心血管疾病的发生发展具有深刻的影响，即血管炎症在早期动脉粥样硬化中起重要作用。With the increasing westernization of lifestyle and diet, the incidence of diabetes has risen sharply. Atherosclerosis is the main pathological change of diabetic vascular disease and a major complication that affects the prognosis of diabetic patients. Atherosclerosis refers to the thickening and hardening of the arterial wall and the narrowing of the vascular lumen caused by lipid metabolism disorders. It is difficult to diagnose early and seriously affects the prognosis of patients. In recent years, the discussion of metabolic cardiovascular diseases has intensified, and the exploration of atherosclerosis has gradually expanded to the exploration of metabolic cardiovascular diseases. As an important link in the metabolic pathway, sugar metabolism undoubtedly plays a pivotal role in the occurrence and development of cardiovascular diseases. Previous studies have found that diabetic patients are under chronic, low-grade, systemic inflammatory stimulation, which has a profound impact on the occurrence and development of cardiovascular diseases in diabetic patients, that is, vascular inflammation plays an important role in early atherosclerosis.
热休克蛋白90（Hsp90）是一种细胞质蛋白质，在生物进化过程中高度保守，约占细胞质蛋白的1%～2%，它参与蛋白质折叠和修饰的分子伴侣，在许多重要的信号通路和转录因子中起着关键作用。通常，它在肿瘤患者体内的含量远远超过健康人，在肿瘤筛查、诊断、判断预后、评价疗效和高危人群随访等方面都具有重要的实用价值。暂时较少报道Hsp90a和糖尿病动脉粥样硬化的关系。细胞外热休克蛋白90 (eHsp90)可以分泌到血清中，参与糖尿病患者血管慢性损伤过程。然而，eHsp90α在早期动脉粥样硬化中的具体机制尚不清楚。Heat shock protein 90 (Hsp90) is a cytoplasmic protein that is highly conserved during biological evolution and accounts for about 1% to 2% of cytoplasmic proteins. It is a molecular chaperone involved in protein folding and modification and plays a key role in many important signaling pathways and transcription factors. Usually, its content in tumor patients is much higher than that in healthy people. It has important practical value in tumor screening, diagnosis, prognosis, efficacy evaluation and follow-up of high-risk populations. There are few reports on the relationship between Hsp90a and diabetic atherosclerosis. Extracellular heat shock protein 90 (eHsp90) can be secreted into serum and participate in the chronic vascular injury process in diabetic patients. However, the specific mechanism of eHsp90α in early atherosclerosis is still unclear.
有鉴于此，特提出本发明。In view of this, the present invention is proposed.
发明内容Summary of the invention
针对上述问题，本发明的目的是提供一种糖尿病动脉粥样硬化的生物标记物及其在制备糖尿病动脉粥样硬化药物中的应用。In view of the above problems, the object of the present invention is to provide a biomarker for diabetic atherosclerosis and its application in the preparation of diabetic atherosclerosis drugs.
为实现上述目的，本发明解决其技术问题所采用的技术方案如下：To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is as follows:
一种糖尿病动脉粥样硬化的生物标记物，所述生物标志物为eHsp90α。A biomarker for diabetic atherosclerosis, wherein the biomarker is eHsp90α.
本发明还提供所述的生物标志物在制备治疗糖尿病靶向药物方面的应用，所述药物靶向eHsp90α。The present invention also provides the use of the biomarker in preparing a targeted drug for treating diabetes, wherein the drug targets eHsp90α.
本发明所述的糖尿病动脉粥样硬化是指由于胰岛素的不足或抵抗以及血糖增高导致糖尿病患者动脉管壁内膜损伤，脂质沉积在破损处，可使动脉管腔狭窄，甚至闭塞，从而导致动脉粥样硬化。The diabetic atherosclerosis described in the present invention refers to the damage to the intima of the arterial wall of diabetic patients due to insulin deficiency or resistance and increased blood sugar. Lipids are deposited in the damaged areas, which can narrow the arterial lumen or even occlude it, thereby leading to atherosclerosis.
另外，本发明还提供了生物标志物eHsp90α在在早期筛查糖尿病动脉粥样硬化试剂盒中的应用In addition, the present invention also provides the use of the biomarker eHsp90α in a kit for early screening of diabetic atherosclerosis.
进一步地，生物标志物eHsp90α在在早期筛查糖尿病动脉粥样硬化试剂盒中的应用，包括检测eHsp90α蛋白水平表达的试剂。Furthermore, the application of the biomarker eHsp90α in a kit for early screening of diabetic atherosclerosis includes a reagent for detecting the expression level of eHsp90α protein.
进一步地，生物标志物eHsp90α在在早期筛查糖尿病动脉粥样硬化试剂盒中的应用，筛查方法为：Furthermore, the biomarker eHsp90α is used in a kit for early screening of diabetic atherosclerosis, and the screening method is:
（1）采集实验组样品；(1) Collect samples from the experimental group;
（2）通过定量检测eHsp90α的试剂，测定所述实验组样品中的eHsp90α表达水平；(2) determining the expression level of eHsp90α in the samples of the experimental group by using a reagent for quantitatively detecting eHsp90α;
（3）将实验组eHsp90α表达水平与对照组比对，确定实验组是否患有糖尿病动脉粥样硬化。(3) Compare the expression level of eHsp90α in the experimental group with that in the control group to determine whether the experimental group suffers from diabetic atherosclerosis.
与现有技术相比，本发明的有益效果是：Compared with the prior art, the present invention has the following beneficial effects:
1. 本发明提供了一种糖尿病动脉粥样硬化的生物标志物，生物标志物细胞外热休克蛋白90α (eHsp90α)的表达量的高低与糖尿病动脉粥样硬化发病风险存在明显的相关性，可用作糖尿病动脉粥样硬化的生物标记物。1. The present invention provides a biomarker for diabetic atherosclerosis. The expression level of the biomarker extracellular heat shock protein 90α (eHsp90α) is significantly correlated with the risk of developing diabetic atherosclerosis, and can be used as a biomarker for diabetic atherosclerosis.
2.利用eHsp90α抗体可以改善糖尿病患者的全身、慢性、低度炎症状态，减少血管内皮细胞的损伤和单核细胞聚集。本发明为研究治疗糖尿病动脉粥样硬化的新型药物提供了一个新的治疗靶点。2. The use of eHsp90α antibodies can improve the systemic, chronic, low-grade inflammatory state of diabetic patients and reduce the damage of vascular endothelial cells and the aggregation of monocytes. The present invention provides a new therapeutic target for the study of new drugs for the treatment of diabetic atherosclerosis.
3. 本发明的生物标志物在在早期筛查糖尿病动脉粥样硬化试剂盒中具有重要作用，有利于患者在早期能够经过筛查及时发现问题。3. The biomarkers of the present invention play an important role in the early screening kit for diabetic atherosclerosis, which is helpful for patients to detect problems in time through screening at an early stage.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例1的糖尿病患者血清中eHsp90α和GRP78水平的分析图；FIG1 is an analysis chart of the levels of eHsp90α and GRP78 in the serum of diabetic patients according to Example 1 of the present invention;
图2为本发明实施例2中eHsp90α单克隆抗体1G6-D7作用以及eHsp90α单独、iHsp90α单独或两者在DAS内皮屏障功能障碍中作用的分析图；FIG2 is an analysis diagram of the effect of the eHsp90α monoclonal antibody 1G6-D7 in Example 2 of the present invention and the effect of eHsp90α alone, iHsp90α alone or both in DAS endothelial barrier dysfunction;
图3为本发明实施例3中LRP1在ehsp90α诱导的内皮屏障功能障碍中的作用；FIG3 shows the role of LRP1 in ehsp90α-induced endothelial barrier dysfunction in Example 3 of the present invention;
图4为本发明实施例4中eHsp90α在DAS中激活内质网应激实验结果；FIG4 is the result of the experiment of activating endoplasmic reticulum stress by eHsp90α in DAS in Example 4 of the present invention;
图5为本发明实施例5中TUDCA处理以及敲低GRP78 对ehsp90α诱导的内皮屏障功能障碍的影响图。FIG. 5 is a diagram showing the effects of TUDCA treatment and GRP78 knockdown on ehsp90α-induced endothelial barrier dysfunction in Example 5 of the present invention.
具体实施方式DETAILED DESCRIPTION
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.
除非特别说明，下述实施例中使用的试剂原料为常规市购或商业途径获得的生化试剂原料，除非特别说明，下述实施例中使用的方法和设备为本领域常规使用的方法和设备。Unless otherwise specified, the reagent raw materials used in the following examples are conventional commercially available or commercially obtained biochemical reagent raw materials. Unless otherwise specified, the methods and equipment used in the following examples are conventionally used methods and equipment in the art.
本发明使用的血清均在无菌条件下从受试者的外周静脉采集血液样本，取上清保存在- 80°C，待用于生化分析。The serum used in the present invention is obtained by collecting blood samples from peripheral veins of subjects under sterile conditions, and the supernatant is stored at -80°C for biochemical analysis.
本发明所述的糖尿病动脉粥样硬化是指由于胰岛素的不足或抵抗以及血糖增高导致糖尿病患者动脉管壁内膜损伤，脂质沉积在破损处，可使动脉管腔狭窄，甚至闭塞，从而导致动脉粥样硬化。The diabetic atherosclerosis described in the present invention refers to the damage to the intima of the arterial wall of diabetic patients due to insulin deficiency or resistance and increased blood sugar. Lipids are deposited in the damaged areas, which can narrow the arterial lumen or even occlude it, thereby leading to atherosclerosis.
实施例1Example 1
通过收集南方医院60位糖尿病患者的信息进行横断面分析，将糖尿病患者分为单纯糖尿病组（DM）、糖尿病合并动脉粥样硬化组(DAS)，每组30人。分析患者血清中热休克蛋白90(eHsp90α)、内质网应激标志物（GRP78）的表达水平差异。分析结果如图1所示。从图1中的结果发现，DAS患者血清中的eHsp90α和内质网应激标志物GRP78水平显著高于DM患者（图1A，B）。此外，Pearson相关分析显示，eHsp90α与GRP78之间存在显著相关性(r=0.7172,p<0.0001；图1C)。为了进一步评价eHsp90α和GRP78对DAS的诊断效果，采用ROC曲线分析。eHsp90α的曲线下面积(AUC)为0.8107，约登指数为0.5667；GRP78的AUC为0.8450，约登指数为0.6333。综合指标eHsp90α和GRP78，AUC为0.8456，约登指数为0.6334(图1D)。由此可见，糖尿病动脉粥样硬化组的血清eHsp90α、 GRP78水平明显高于单纯糖尿病组并且具有统计学差异，且eHsp90α联合内质网应激标志物GRP78的诊断效能优于单独使用两项指标,即eHsp90α与GRP78 联合使用具有较高的诊断效能。由此，本发明能够提供定量检测eHsp90α的试剂在早期筛查糖尿病动脉粥样硬化试剂盒中的应用，定量检测eHsp90α的试剂检测eHsp90α在蛋白水平上的表达来实现早期筛查。进一步进行中介分析显示GRP78在eHsp90α和DAS之间的关系中起中介作用(图1E)。By collecting information from 60 diabetic patients in Nanfang Hospital for cross-sectional analysis, diabetic patients were divided into simple diabetes group (DM) and diabetes combined with atherosclerosis group (DAS), with 30 patients in each group. The expression levels of heat shock protein 90 (eHsp90α) and endoplasmic reticulum stress marker (GRP78) in the serum of patients were analyzed. The analysis results are shown in Figure 1. From the results in Figure 1, it was found that the levels of eHsp90α and endoplasmic reticulum stress marker GRP78 in the serum of DAS patients were significantly higher than those in DM patients (Figure 1A, B). In addition, Pearson correlation analysis showed that there was a significant correlation between eHsp90α and GRP78 (r=0.7172, p<0.0001; Figure 1C). In order to further evaluate the diagnostic effect of eHsp90α and GRP78 on DAS, ROC curve analysis was used. The area under the curve (AUC) of eHsp90α is 0.8107, and the Youden index is 0.5667; the AUC of GRP78 is 0.8450, and the Youden index is 0.6333. The comprehensive indicators eHsp90α and GRP78, AUC is 0.8456, and the Youden index is 0.6334 (Figure 1D). It can be seen that the serum eHsp90α and GRP78 levels in the diabetic atherosclerosis group are significantly higher than those in the simple diabetes group and have statistical differences, and the diagnostic efficacy of eHsp90α combined with the endoplasmic reticulum stress marker GRP78 is better than the use of the two indicators alone, that is, the combined use of eHsp90α and GRP78 has a higher diagnostic efficacy. Therefore, the present invention can provide the use of a reagent for quantitatively detecting eHsp90α in a kit for early screening of diabetic atherosclerosis, and the reagent for quantitatively detecting eHsp90α detects the expression of eHsp90α at the protein level to achieve early screening. Further mediation analysis showed that GRP78 played a mediating role in the relationship between eHsp90α and DAS ( Figure 1E ).
实施例2Example 2
用人重组eHsp90α(hrHsp90α)预处理单层培养的HUVECs，实验组加用15µg/mL的1G6-D7（1G6-D7选择性识别并强烈中和eHsp90α功能）处理2 h，对照组不做处理，24h后分析FITC-DX通透性。结果表明，与对照组相比，hrHsp90α (10μL/mL)诱导血管通透性增加。1G6-D7抑制hrHsp90α诱导的内皮细胞高通透性。结果：eHsp90α可以提高内皮细胞通透性并加重钙黏蛋白脱位促进内皮细胞损伤（图2A，B）。α-Catenin的免疫染色也显示hrHsp90α处理细胞的脱位和不连续粘附连接。相比之下，1G6-D7恢复了hrhsp90α诱导的β-Catenin的离域(图2B)。Western blot实验发现，粘附连接蛋白在hrHsp90α处理的HUVECs中下调(图2C)；qRT-PCR在基因水平上验证了类似的结果（图2D）。Monolayer cultured HUVECs were pretreated with human recombinant eHsp90α (hrHsp90α), and the experimental group was treated with 15μg/mL 1G6-D7 (1G6-D7 selectively recognizes and strongly neutralizes eHsp90α function) for 2 h, while the control group was not treated. FITC-DX permeability was analyzed 24 h later. The results showed that hrHsp90α (10μL/mL) induced increased vascular permeability compared with the control group. 1G6-D7 inhibited hrHsp90α-induced endothelial cell hyperpermeability. Results: eHsp90α could increase endothelial cell permeability and aggravate cadherin dislocation to promote endothelial cell injury (Figure 2A, B). Immunostaining of α-Catenin also showed dislocation and discontinuous adherens junctions in hrHsp90α-treated cells. In contrast, 1G6-D7 restored the delocalization of β-Catenin induced by hrhsp90α (Figure 2B). Western blot experiments revealed that adhesion junction proteins were downregulated in HUVECs treated with hrHsp90α (Figure 2C); qRT-PCR confirmed similar results at the gene level (Figure 2D).
为了继续探究是否eHsp90α单独、iHsp90α单独或两者在DAS内皮屏障功能障碍中起作用，设计三种sirna，并将3种sirna转染到HUVECs中以敲低iHsp90α。western blot结果显示，si-iHsp90α3转染HUVECs后，iHsp90α的相对表达量显著降低(图2E)。先用si-iHsp90α3敲除HUVECs中的iHsp90α，然后用hrHsp90α处理HUVECs细胞。结果显示，iHsp90α敲低组与空白对照组的细胞通透性无显著差异，转染si-iHsp90α3和hrHsp90α处理的HUVECs具有高通透性，粘附体连接蛋白的表达支持FITC-DX通透性结果(图2F，G)。由此可见，eHsp90α可以单独在DAS的内皮屏障功能障碍中起关键作用且eHsp90α单克隆抗体1G6-D7有明显的抑制作用。本实施例也为eHsp90α单克隆抗体1G6-D7及其衍生物在早期预警、改善、治疗或辅助治疗糖尿病动脉粥样硬化制剂中的应用提供了基础。To continue to explore whether eHsp90α alone, iHsp90α alone, or both play a role in DAS endothelial barrier dysfunction, three siRNAs were designed and transfected into HUVECs to knock down iHsp90α. Western blot results showed that the relative expression of iHsp90α was significantly reduced after si-iHsp90α3 was transfected into HUVECs (Figure 2E). iHsp90α in HUVECs was first knocked down with si-iHsp90α3, and then HUVECs cells were treated with hrHsp90α. The results showed that there was no significant difference in cell permeability between the iHsp90α knockdown group and the blank control group, and HUVECs transfected with si-iHsp90α3 and hrHsp90α had high permeability, and the expression of adherens junction proteins supported the FITC-DX permeability results (Figure 2F, G). It can be seen that eHsp90α can play a key role in endothelial barrier dysfunction in DAS alone and the eHsp90α monoclonal antibody 1G6-D7 has a significant inhibitory effect. This example also provides a basis for the use of eHsp90α monoclonal antibody 1G6-D7 and its derivatives in early warning, improvement, treatment or auxiliary treatment of diabetic atherosclerosis.
实施例3Example 3
用hrHsp90α处理HUVECs 24小时；western blot分析显示，LRP1的相对表达量显著升高（图3A）。此外，免疫电镜显示，LRP1受体在质膜附近高表达(图3B)。为了进一步评估LRP1在ehsp90α诱导的内皮屏障功能障碍中的作用，设计了三个sirna并将它们转染到HUVECs中以敲低LRP1。使用qRT-PCR、RT-PCR和western blotting验证了干扰效率并选择si-LRP1 3进行FITC-DX检测细胞通透性。结果表明，LRP1敲低可减轻ehsp90α诱导的高通透性结果（图3C，D）。可以得出结论，LRP1受体接受ehsp90α诱导的内皮屏障功能障碍信号。HUVECs were treated with hrHsp90α for 24 h; western blot analysis showed that the relative expression of LRP1 was significantly increased (Figure 3A). In addition, immunoelectron microscopy showed that the LRP1 receptor was highly expressed near the plasma membrane (Figure 3B). To further evaluate the role of LRP1 in endothelial barrier dysfunction induced by ehsp90α, three siRNAs were designed and transfected into HUVECs to knock down LRP1. The interference efficiency was verified using qRT-PCR, RT-PCR, and western blotting, and si-LRP1 3 was selected for FITC-DX to detect cell permeability. The results showed that LRP1 knockdown could alleviate the high permeability results induced by ehsp90α (Figure 3C, D). It can be concluded that the LRP1 receptor receives endothelial barrier dysfunction signals induced by ehsp90α.
实施例4Example 4
为了明确eHsp90α是否在DAS中激活内质网应激，在不同的时间间隔(3h、6h、12h和24 h)内刺激HUVECs。Western blot分析显示，内质网应激标志物GRP78、p-PERK和p-IRE1α以时间依赖性的方式积累，在12h时显著增加；ATF6表达无明显变化；这些结果经RT-PCR进一步证实(图4A)。通过western blotting和RT-PCR检测发现，hrHsp90α诱导UPRs相关蛋白XBP1s、ATF4和CHOP的表达增加，ER应激下游靶点(图4B)。接下来，用si-LRP1 3转染细胞，随后用hrHsp90α处理细胞。western blot结果显示，LRP1基因的敲低抑制了内质网应激(图4C)。eHsp90α通过LRP1受体激活内质网应激相关的UPR通路。对人主动脉组织切片进行免疫组化处理，发现GRP78在DAS组中的表达水平明显高于AS组(图4D)。To clarify whether eHsp90α activates ER stress in DAS, HUVECs were stimulated at different time intervals (3h, 6h, 12h, and 24h). Western blot analysis showed that ER stress markers GRP78, p-PERK, and p-IRE1α accumulated in a time-dependent manner and increased significantly at 12h; ATF6 expression did not change significantly; these results were further confirmed by RT-PCR (Figure 4A). Western blotting and RT-PCR revealed that hrHsp90α induced increased expression of UPRs-related proteins XBP1s, ATF4, and CHOP, downstream targets of ER stress (Figure 4B). Next, cells were transfected with si-LRP1 3 and subsequently treated with hrHsp90α. Western blot results showed that knockdown of the LRP1 gene inhibited ER stress (Figure 4C). eHsp90α activates the ER stress-related UPR pathway through the LRP1 receptor. Immunohistochemical processing of human aortic tissue sections revealed that the expression level of GRP78 in the DAS group was significantly higher than that in the AS group ( Figure 4D ).
采用雄性小鼠(C57BL/6J，野生型和ApoE−/−)。所有ApoE−/−小鼠均为C57BL/6J背景。将小鼠随机分为两组(每组8只):糖尿病ApoE−/−组和正常ApoE−/−组。糖尿病ApoE−/−组小鼠腹腔注射链脲佐菌素(STZ, 50 mg/kg，溶解于柠檬酸缓冲液中，pH为4.5)5天。注射STZ后，小鼠的空腹血糖水平超过16.67 mM才被认为是成功的糖尿病模型。对照ApoE−/−小鼠给予等量的柠檬酸缓冲液。注射STZ后，饲喂标准鼠饲料12周。安乐死后进行免疫组化检测。免疫组化法检测GRP78在实验动物主动脉窦中的表达,结果表明糖尿病ApoE−/−小鼠中GRP78的表达高于对照ApoE−/−小鼠（图4 E）。综上所述，这些结果表明eHsp90α通过LRP1受体激活内质网应激相关的UPR 通路。Male mice (C57BL/6J, wild type and ApoE−/−) were used. All ApoE−/− mice were of C57BL/6J background. The mice were randomly divided into two groups (8 mice in each group): diabetic ApoE−/− group and normal ApoE−/− group. Mice in the diabetic ApoE−/− group were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg, dissolved in citrate buffer, pH 4.5) for 5 days. After STZ injection, the fasting blood glucose level of mice was considered to be a successful diabetes model only when it exceeded 16.67 mM. Control ApoE−/− mice were given an equal amount of citrate buffer. After STZ injection, they were fed with standard mouse chow for 12 weeks. Immunohistochemistry was performed after euthanasia. Immunohistochemistry was used to detect the expression of GRP78 in the aortic sinus of experimental animals, and the results showed that the expression of GRP78 in diabetic ApoE−/− mice was higher than that in control ApoE−/− mice (Figure 4 E). Taken together, these results indicate that eHsp90α activates the ER stress-related UPR pathway through the LRP1 receptor.
实施例5Example 5
用TUDCA (50 μM)预处理HUVECs 6 h，然后用hrHsp90α预处理24 h，免疫荧光结果显示eHsp90α促进VE-Cadherin的脱位和GRP78的显著上调，而TUDCA逆转了这些作用(图5A)。FITC-DX渗透性实验的结果也表明，TUDCA显著降低了ehsp90α诱导的高渗透性(图5B)。Western blot分析结果也显示，TUDCA恢复了eHsp90α诱导的粘附连接蛋白表达下降的现象(图5C)。然后敲除HUVECs中的GRP78，并进行FITC-DX通透性实验。结果表明，GRP78 基因敲低可显著恢复细胞通透性至正常水平（图5D）。结果：抑制内质网应激和敲低GRP78可消除ehsp90α诱导的内皮屏障功能障碍。HUVECs were pretreated with TUDCA (50 μM) for 6 h and then with hrHsp90α for 24 h. Immunofluorescence results showed that eHsp90α promoted the dislocation of VE-Cadherin and the significant upregulation of GRP78, while TUDCA reversed these effects (Figure 5A). The results of FITC-DX permeability assay also showed that TUDCA significantly reduced the hyperpermeability induced by ehsp90α (Figure 5B). Western blot analysis also showed that TUDCA restored the decreased expression of adherens junction proteins induced by eHsp90α (Figure 5C). GRP78 was then knocked out in HUVECs, and FITC-DX permeability assay was performed. The results showed that GRP78 gene knockdown could significantly restore cell permeability to normal levels (Figure 5D). Results: Inhibition of ER stress and knockdown of GRP78 can eliminate ehsp90α-induced endothelial barrier dysfunction.
以上各实施例仅用以说明本申请的技术方案，而非对其限制；本技术领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；这些修改或者替换也应视为本发明的保护范围。The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. A person skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features may be replaced by equivalents. These modifications or replacements should also be regarded as within the scope of protection of the present invention.
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| CN202411155894.1ACN118858657A (en) | 2024-08-22 | 2024-08-22 | eHsp90α as a biomarker for diabetic atherosclerosis and its application in the preparation of targeted drugs |
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| CN101889205A (en)* | 2007-07-27 | 2010-11-17 | 卡瓦迪斯有限责任公司 | The protein markers that is used for cardiovascular event |
| CN107095867A (en)* | 2017-04-01 | 2017-08-29 | 上海长海医院 | A kind of HSP90 inhibitor is preparing the purposes in preventing and treating arotic disease medicine |
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20100075894A1 (en)* | 2004-09-15 | 2010-03-25 | Harvard University | Reducing er stress in the treatment of obesity and diabetes |
| CN101889205A (en)* | 2007-07-27 | 2010-11-17 | 卡瓦迪斯有限责任公司 | The protein markers that is used for cardiovascular event |
| CN107095867A (en)* | 2017-04-01 | 2017-08-29 | 上海长海医院 | A kind of HSP90 inhibitor is preparing the purposes in preventing and treating arotic disease medicine |
| Title |
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| XINYI DING等: "Extracellular Hsp90α, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes", 《BMJ OPEN DIABETES RESEARCH & CARE》, 31 December 2022 (2022-12-31), pages 2* |
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