【作者】 唐勇；

【导师】 张亚臣；

【作者基本信息】 上海交通大学 ， 内科学（心血管病）， 2016， 博士

【摘要】 背景动脉粥样硬化是心肌梗死等心血管事件发病的共同病理基础和致死的主要原因。肿瘤坏死因子α(TNF-α)等炎性因子破坏内皮系统这一天然屏障,是引起动脉粥样硬化的始动环节。目前已发现有多种miRNA在调节内皮细胞功能、促进血管新生和内皮化的过程中发挥着重要的作用。有研究表明miR-17-92基因簇在动脉粥样硬化(AS)患者外周血中表达明显减低,提示miR-17-92基因簇可能与AS患者血管内皮功能相关。然而miR-17-92基因簇在血管内皮细胞的损伤修复过程中的作用机制仍有待研究。目的本研究拟通过体外与在体双层面探讨miR-17-92基因簇对TNF-α诱导的内皮细胞损伤作用及机制研究,为动脉粥样硬化疾病的诊治提供新的思路和手段。方法和结果(1)入选我院心内科诊断为冠心病(CAD)的患者12例和同等数量的健康对照组,依次抽取外周血10 ml,利用qRT-PCR技术进行miR-17-92基因簇表达差异验证。结果发现,miR-17-92基因簇中miR-19b、miR-92a在CAD患者外周血中表达明显减低,而miR-17、miR-18a、miR-19a和miR-20a未检测到明显差异。(2)原代HUVECs细胞培养。分离、培养HUVECs细胞,通过形态学观察和细胞表面CD31、vWF因子鉴定。成功分离培养出表面CD31、vWF因子阳性的HUVECs细胞,并选取3至5代的细胞进行后续试验。(3)TNF-α诱导内皮细胞损伤模型构建。分别用0、1、10、50、100 ng/ml的TNF-α处理HUVECs 24h,利用TUNEL法和流式细胞术验证各组细胞凋亡情况。结果显示,随着TNF-α浓度升高,细胞凋亡比例增加,且在TNF-α浓度大于等于10 ng/ml时可显著诱导HUVECs凋亡。(4)采用AgilentmiRNA microarray芯片技术,将3例TNF-α10 ng/ml处理过的HUVECs与3例空白对照组进行差异miRNA筛选。结果发现两组细胞中有18种miRNA表达存在显著差异,其中TNF-α处理组有6种miRNA表达上调、12种miRNA表达水平明显下降,包括miR-17-92基因簇中miR-19b在TNF-α10 ng/ml处理后表达明显下降。(5)利用miR-19b的mimics和inhibitors以及各自对照物分别转染HUVECs24h,qRT-PCR检测各组miR-19b的表达水平,TUNEL法和流式细胞术检测细胞凋亡水平。结果发现转染miR-19b的mimics和inhibitors后可明显升高或抑制细胞内miR-19b的表达水平。与对照组相比,转染miR-19b mimics可显著减低内皮细胞的凋亡比例,而miR-19b inhibitors则可增加内皮细胞凋亡比例。(6)利用TargetScan,miRanda和miRBase等几个miRNAs靶点预测软件,并结合相关文献查询显示,凋亡相关蛋白Apaf-1、PTEN为miR-19b的潜在作用靶点。双荧光素酶报告载体进一步证实,Apaf-1基因和PTEN基因为miR-19b的直接作用靶点。(7)体外TNF-α诱导内皮细胞凋亡模型中,共转染miR-19b inhibitors和Apaf1-siRNA或者共转染miR-19b inhibitors和PTEN-siRNA至HUVECs中,验证miR-19b、Apaf-1或者PTEN蛋白在TNF-α诱导内皮细胞凋亡中的作用。结果显示,miR-19b可通过直接作用于Apaf1/Caspase通路或者PTEN/Caspase通路发挥缓解TNF-α诱导内皮细胞损伤的作用。结论综上所述,冠心病患者外周血中miR-19b的表达减低是导致血管内皮损伤的重要因素之一。miR-19b可通过直接作用于Apaf1/Caspase通路或者PTEN/Caspase通路发挥缓解TNF-α诱导内皮细胞损伤的作用。更多还原

【Abstract】 Background:Atherosclerosis is the common pathologic basis of cardiovascular diseases such as myocardial infarction and the main cause of death.Tumor necrosis factor-α(TNF-α),an important inflammatory factor,induces endothelial cell injury,which results in endothelial dysfunction,initiating the atherosclerosis progress.Recently,lots of miRNA have been proved playing important role in regulating endothelial cell function,enhancing angiogenesis and endothelium.Researchers have noted that the miR-17-92 cluster is significantly downregulated among patients with atherosclerotic diseases.This indicates that the miR-17-92 cluster may be involved in the functions of vascular endothelium of patients with atherosclerosis.However,the mechanism of the miR-17-92 cluster in repairing endothelial cel injury remains unclear.Objective:In this research,we investigate the effect and mechanism of the miR-17-92 cluster on TNF-α induced endothelial cell dysfunction.We hope that the results of our search could help to find new ideas and methods for atherosclerotic diseases diagnosis and treatment.Methods and Results:(1)Twelve patients diagnosed with coronary arteary disease(CAD)by our Division ofCardiology and twelve healthy donors were recruited.A 10 ml sample of peripheral blood was collected in an EDTA-containing vacutainer tube from eachindividual to detect the miR-17-92 cluster expression by qRT-PCR.Among the miR-17-92 family of miRNAs,the levels of miR-19 b and miR-92 a were significantlylower in patients with CAD than in healthy subjects,.However,nodifferences were observed with respect to the levels of miR-17、miR-18a、miR-19 a and miR-20 a between the two groups.(2)Isolation and identification of HUVECs.Primary human umbilical vein endothelial cells(HUVECs)were isolated and cultured following a previously described protocol.Through the morphological observation and the factors(CD31 and vWf)in cell surface identification,endothelial cel s passagesfrom three to five were used for this study.(3)Apoptosis of HUVECs is induced by TNF-α.Apoptosis of endothelial cells treated with 0,1,10,50 or 100 ng/ml of TNF-α for 24 h was measured by flow cytometry and TUNEL/DAPI-stained cell photomicrography.Both results showed that TNF-α induces endothelial cell apoptosis in a dose-dependent manner and that the lowest effective dose of 24 h TNF-α treatment was 10 ng/ml.(4)We used a microarray chip to analyze changes in miRNA expression between 3 TNF-αtreated groups and 3 control groups.The results in TNF-αtreated groups revealed that 18 miRNAs(6 of which were upregulatedand 12 of which were downregulated)exhibited a significant change compared with the control group.The expression of miR-19 b among the miR-17-92 cluster was significantly decreased following 24 h of TNF-α treatment.(5)Changes in miRNA-19 b levels of the HUVECs at 24 h following transfection with the vehicle,the miR-19 b mimic,the mimic control,the miR-19 b inhibitor or the inhibitor control were detected by qRT-PCR.The miR-19 b mimic upregulated,whereas the miR-19 b inhibitor downregulated miR-19 b expression.Flow cytometry and TUNEL/DAPI-stained cell photomicrography were used to detect cell apoptosis levels.In comparison with the control group,the miR-19 b mimic could significantly decrease endothelial cell apoptosis,whereas the miR-19 b inhibitor increase endothelial cell apoptosis.(6)According to several publicly available bioinformaticsweb sites(TargetScan,miRanda and miRBase),both Apaf-1,PTEN and Casp7 may be direct targets ofmiR-19 b.The luciferase reporter plasmids and function experiments further demonstrated that Apaf-1,PTEN and Casp7 were direct target genes of miR-19 b.(7)In TNF-α induced endothelial cell apoptosis model,the miR-19 b inhibitors and Apaf1-siRNA or PTEN-siRNA were cotransfected into HUVECs in order to test the role that miR-19 b,Apaf-1 and PTEN may play in.The results showed that miR-19 b could attenuate TNF-α induced endothelial cell dysfunction via the Apaf1/Caspase pathway and the PTEN/Caspase pathway.Conclusion:In conclusion,the downregulated expression levels of miR-19 b in the CAD patients may be one factor which causes endothelial cell dysfunction.miR-19 b could attenuate TNF-α induced endothelial cell injury via the Apaf1/Caspase pathway and the PTEN/Caspase pathway.更多还原