【作者】 徐浩；

【导师】 李红莉；

【作者基本信息】 上海交通大学 ， 内科学（心血管病）， 2018， 博士

【摘要】 目的:1.探讨大鼠心肌梗死动模型心肌缺血后心肌细胞中miRNA-145表达水平的变化情况2.通过构建miRNA-145过表达动物模型,探讨miRNA-145对于大鼠心肌缺血损伤后的心肌损伤水平和心功能的影响3.通过培养大鼠心肌细胞中过表达mi R-145,在细胞水平观察miRNA-145对于心肌细胞缺氧/复氧损伤的影响4.通过luciferase试验,探索miRNA-145对于心肌缺血后心肌损伤的细胞功能调控的信号通路方法:1.构建miRNA-145正常表达以及过表达质粒,再通过慢病毒包装系统在HEK-293T细胞中包装出含有miRNA-145(LV-miR-145)和不含有miRNA-145(LV-miR-NC)的慢病毒,并进行扩增2.将40只SD大鼠随机分为四组:空白对照组(sham),心肌梗死缺血损伤组(MI),LV-miR-NC病毒感染的心梗缺血损伤组(LV-NC),LV-miR-145病毒感染的心梗缺血损伤组(LV-miR-145),造模前3天尾静脉注射不同类型的病毒载体3.心肌梗死造模后24小时,通过小动物心超(Vevo-770高频超声系统)检测四组动物模型舒张期左心室前壁的厚度(LVAWd),舒张期左心室后壁的厚度(LVPWd),左心室射血分数(LVEF)以及左心室短轴缩短率(LVFS);心肌梗死模型建成两周后,处死动物,称体重(BW)、脏重量(HW)以及左心室重量(LVW),并且观察心肌梗死的区域面积。心脏组织行HE染色,在光学显微镜下观察心肌纤维化变化、心肌细胞肿胀以及坏死范围的变化。4.利用逆转录聚合酶链式反应(RT-qPCR)的方法检测四组动物模型心肌组织中miRNA-145的表达水平,运用蛋白质免疫印迹法检测四组大鼠心肌组织中bcl-2蛋白,Bax蛋白、活性caspase-3蛋白和非活性的caspase-3蛋白的表达水平。5.取3天左右的SD大鼠乳鼠心肌细胞进行培养,并运用mi R-145和空白的miRNA转染细胞,并构建缺氧/复氧心肌细胞损伤模型,运用流式细胞仪检测细胞凋亡数目、JC-1检测线粒体跨膜电位的改变情况。在HEK 293T细胞中应用双荧光素酶分析法检测miR-145的靶受体。结果:1.相比较于空白对照组,MI组大鼠心肌梗死组织中miR-145的表达水平明显下降。2.相较于空白对照组,MI组大鼠的LVEF和LVFS明显下降,而LVAWd和LVPWd出现明显的上升,HW/BW和LVW/BW的水平要明显高于对照组;在LV-miR-145组大鼠的LVEF和LVFS的水平要显著高于LV-NC组,而LVAWd和LVPWd则明显低于LV-NC组。3.HE染色结果表明在LV-miR-145组中,心肌组织中心肌纤维化程度、心肌细胞肿胀以及坏死区域范围相较于MI组明显减轻;与LV-NC组相比,LV-miR-145组梗死区域范围明显减小;通过蛋白质免疫印迹法检测发现,在LV-miR-145组中与细胞凋亡相关的Bcl-2蛋白/Bax蛋白出现明显的上升,而活性的caspase-3蛋白/非活性的caspase-3蛋白的比率显著下降。4.在缺氧/复氧处理后的心肌细胞中,mi R-145的表达量显著下降;mi R-145处理后的心肌细胞在缺氧/复氧后,细胞凋亡数目显著减少;通过双荧光素酶分析法发现,在大鼠的心肌细胞中发现PDCD4 3’-UTR可以特异性的与mi R-145结合,相比较于mi R-NC组,mi R-145过表达的细胞中PDCD4蛋白的表达量明显降低,此外,PDCD4过表达可以显著降低mi R-145对缺氧/复氧心肌细胞的保护作用。5.相对于控制组,缺氧/复氧处理后的心肌细胞中线粒体去极化水平明显下降,而用miR-145处理的心肌细胞的线粒体去极化水平基本保持不变;此外,心肌细胞给予缺氧/复氧处理后,线粒体释放的细胞色素C的水平明显增加,而miR-145可以降低这一释放水平。结论:1.MI后心肌细胞中miR-145的表达水平明显下降,提示miR-145可能参与调控了MI后心脏功能改变和心肌损伤的病理过程。2.高表达mi R-145可以显著减少MI后心肌细胞的凋亡,同时能够减轻心肌重构;高表达mi R-145大鼠MI后LVEF、LVFS的降低和LVAWd、LVPWd的增加较对照组减轻,提示心功能的改善,因此,miR-145可以作为干预MI的新靶点。3.miR-145可以通过改善心肌细胞中线粒体的去极化水平以及减少线粒体释放的细胞色素C的水平,从而减少MI后心肌细胞的凋亡。4.通过双荧光素酶分析法发现,miR-145通过与PDCD4的特异性结合调控其表达,从而发挥MI后的心肌保护作用。更多还原

【Abstract】 Objectives 1.To investigate the expression profile of miR-145 in the myocardial infarction(MI)rat model.2.To investigate the beneficial role of miR-145 overexpression on the cardiac function and myocardial injury of MI rat.3.To determine the protective effects of miR-145 overexpression on the hypoxia/ reoxygenation(H/R)-injured cardiomycocytes.4.To identify the downstream target of miR-145 and further determine its role on MI by dual luciferase reporter assay.Methods: 1.The sequences of miR-145 and sham control were subcloned into plasmid vevtor(LVmiR-145 and LV-miR-NC)and then contransfected into HEK-293 T cells by lentivector packaging system.2.Forty SD rats were randomized into four experiments groups: sham group,MI group,MI rats injected with lentivirus containing miR-145 sequences(LV-miR-145 group)and MI rats injected with lentivirus containing negative control sequences(LV-NC group).3.24 hours after MI,rats were anesthetized and echocardiography was studied.LVAWD,LVPWD,LVEF and LVEFS were evaluated by a Vevo-770 high-frequency ultrasound system.Rats were sacrificed after two weeks.BW,HW and LVW were weighted.Moreover,the infarct area were indentified and expressed by a percentage of the total area of LV.4.The expression level of miR-145 in heart tissues was dertermined by RT-qPCR and the protein expression levels of Bcl-2,Bax and cleaved caspase-3 in heart tissues were evaluated by Western blot.5.The cardiomyocytes isolated from the 1-3-day-old SD ratswere transfected with miR-145 or scrambled control miRNA and subjected to H/R treatment.After that,the cardiomyocytes apoptosis was evaluated by flow cytometry.The cationic dye JC-1 was used to detect mitochondrial membrane potential(MMP)in different cardiomyocyte groups.The dual luciferase reporter assay was performed to explore the potential direct target of miR-145.Results: 1.The BW in different groups was no significant.The expression level of miR-145 was significantly reduced in the infract areas.2.24 hours after anterior descending artery(LAD),the LVEF and LVFS in MI group were decreased,while the LVAWd and LVPWd were increased compared to sham group.The reduction of LVEF and LVFS,and the elevation of LVAWd and LVPWd in LV-mi R-145-treated rats were attenuated.Moreover,the LVW-to-BW ratio and HWto-BW ratio were increased in MI group.However,the LVW-to-BW ratio and HW-toBW ratio were reduced in MI rats transfected with LV-miR-145.3.The structural disorder of myofibril,extensive necrotic lesions and muscle fiber swelling were observed in MI group,and these pathological changes were attenuatedin MI rats transfected with LV-miR-145.Moreover,the infarcted area and cardiomyocyte apoptosis were decreased in MI rats treated with LV-miR-145 compared to MI rats treated with LV-NC.The expression levels of pro-apoptotic proteins cleaved caspase-3 and Bax were decreased and anti-apoptotic protein Bcl-2 was increased in LV-miR-145 group.4.The expression of miR-145 in H/R-treated cardiomyocytes was reduced.The cell apoptosis in H/R treated-cardiomyocytes transfected with miR-145 was significantly decreased.By dual luciferase reporter assay,the PDCD4 was identified as the potential target gene of miR-145.Moreover,the western blot analysis indicated that miR-145 overexpression could reduce the expression of PDCD4 protein.PDCD4 overexpression restored cardiomyocytes apoptosis inhibited by miR-145 after H/R injury.5.The MMP was decreased in H/R-treated cardiomyocytes and this effect was inhibited by miR-145.Moreover,the release level of cytochrome c from mitochondrial was suppressed by miR-145.Conclusion: 1.The reduced expression of miR-145 in MI-injured myocardial tissues suggestted that miR-145 may be implicated in the develpment and progression of MI.2.MiR-145 overexpression attenuates the apoptosis in MI-injured myocardial tissues and improves cardiac function,indicating that miR-145 might be act as a potential therapeutic target for MI.3.MiR-145 overexpression could stabilize the MMP and inhibit cytochrome c release in H/R-treated cardiomyocytes,thus leading to the inhibition of cell apoptosis.4.MiR-145 exerts its protective role partly through regulating its potential direct target PDCD4 in MI.更多还原