【作者】 程洁；

【导师】 张野；

【作者基本信息】 安徽医科大学 ， 麻醉学， 2016， 硕士

【摘要】 背景micro RNAs(mi RNAs)是一类新型非编码小RNA,在转录后水平调节靶基因的表达,参与各种病理生理过程。已报道mi RNA可以参与调节心肌细胞凋亡、心肌梗死、心肌重塑、心肌缺血再灌注损伤、药物预处理等过程。本课题组前期利用mi RNA芯片技术发现mi R-133b-5p在心力衰竭大鼠心肌细胞中表达量明显降低,而经历吗啡预处理(morphine preconditioning,MPC)后,mi R-133b-5p水平显著升高。本课题拟在前期研究基础上,进一步研究mi R-133b-5p介导MPC心肌细胞保护效应的作用及机制。方法1.mi R-133b-5p inhibitor转染效率验证。对数期生长的H9c2心肌细胞长至40-50%细胞融合度时转染mi R-133b-5p inhibitor(FAM标记),48 h后荧光显微镜下观察绿色荧光标记的细胞占总细胞的比例,q RT-PCR验证细胞内mi R-133b-5p下降水平。2.mi R-133b-5p在心肌细胞凋亡中的作用及其机制。对数期生长的H9c2心肌细胞随机分为3组:正常对照组(CON)、抑制剂组(mi R-133b-5p inhibitor)、抑制剂对照组(inhibitor-NC)。CON正常培养,mi R-133b-5p inhibitor在细胞长至40-50%融合度时转染mi R-133b-5p inhibitor,inhibitor-NC在细胞长至40-50%融合度时转染mi R-133b-5p inhibitor-NC。48 h后收集培养液上清检测乳酸脱氢酶(lactate dehydrogenase,LDH)活性,收集细胞利用流式细胞仪检测心肌细胞凋亡率,最后利用q RT-PCR和Western Blot检测各组凋亡相关分子Fas的表达情况。3.mi R-133b-5p在吗啡预处理减轻心肌细胞缺氧再给氧损伤中的作用及其机制。对数期生长的H9c2心肌细胞随机分为5组:正常对照组(CON)、缺氧再给氧组(hypoxia/reoxygenation,H/R)、吗啡预处理组(MPC)、吗啡预处理+抑制剂组(MPC+mi R-133b-5p inhibitor)、吗啡预处理+抑制剂对照组(MPC+inhibitor-NC)。CON细胞正常培养。H/R细胞给予缺氧5h、复氧1h处理。MPC细胞在H/R之前给予1mmol/L吗啡预处理10 min。MPC+mi R-133b-5p inhibitor细胞在吗啡预处理前24 h转染mi R-133b-5p inhibitor。MPC+inhibitor-NC细胞在吗啡预处理前24 h转染mi R-133b-5p inhibitor。吸取各组细胞培养液上清检测LDH活性,细胞计数试剂盒8(cell counting kit-8,CCK-8)检测心肌细胞活力,流式细胞仪检测细胞凋亡率,最后利用q RT-PCR和Western Blot检测各组凋亡相关分子Fas的表达情况。结果1.mi R-133b-5p inhibitor转染效率验证。H9c2心肌细胞转染mi R-133b-5p inhibitor(FAM标记)48后,荧光显微镜下观察发现大于80%的细胞带有绿色荧光标记,q RT-PCR检测发现细胞转染mi R-133b-5p inhibitor后mi R-133b-5p表达水平明显降低,即转染效率满意。2.mi R-133b-5p在心肌细胞凋亡中的作用及其机制。与CON组相比,mi R-133b-5p inhibitor组细胞mi R-133b-5p表达明显下降,培养液LDH活性增多,细胞凋亡率升高(P<0.05),而inhibitor-NC组细胞mi R-133b-5p表达、LDH活性、细胞凋亡率变化均无统计学意义。与CON组相比,mi R-133b-5p inhibitor组细胞Fas m RNA与Fas蛋白表达增加(P<0.05),而inhibitor-NC组Fas表达无明显变化。3.mi R-133b-5p在吗啡预处理减轻心肌细胞缺氧再给氧损伤中的作用及其机制。与CON相比,H/R、MPC MPC+mi R-133b-5p inhibitor和MPC+inhibitor-NC组细胞活力下降,LDH水平增加,凋亡率增加,mi R-133b-5p表达下调(P<0.05)。与H/R组相比,MPC组mi R-133b-5p表达明显上调,细胞活力增加,LDH水平减少,细胞凋亡率减少,Fas表达下调(P<0.05)。与MPC组相比,MPC+mi R-133b-5p inhibitor组mi R-133b-5p表达明显下调,细胞活力降低,LDH水平增加,细胞凋亡率增加,Fas表达上调(P<0.05),而MPC+inhibitor-NC组mi R-133b-5p表达、细胞活力、LDH水平、细胞凋亡率、Fas表达变化均无统计学意义。结论吗啡预处理可能通过上调mi R-133b-5p,进一步抑制靶基因Fas m RNA和蛋白表达水平,从而减轻心肌细胞缺血再灌注缺氧再给氧损伤。更多还原

【Abstract】 Background Micro RNAs(mi RNAs) have emerged as a novel class of endogenous, small, noncoding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target m RNAs, thus regulating all kinds of pathological and physiological process. It has been reported that mi RNAs are involved in cardiomyocytes apoptosis, cardiac infarction, cardiac reconstruction, cardiac ischemic reperfusion injury, medicine precondition, etc. In our previous study, mi RNA microarray was applied to identify the different mi RNA expression in normal rats’ cardiomyocytes(n CON), heart failure rats’ cardiomyocytes(f CON) and morphine preconditioned heart failure rat’s cardiomyocytes(f MPC). We found that mi R-133b-5p was significantly down regulated in f CON, compared with n CON. Meanwhile morphine preconditioning(MPC) obviously increased mi R-133b-5p expression as we compare f MPC with f CON. Based on previous results, in this study we plan to investigate the role of mi R-133b-5p in MPC’s protective effects on cardiomyocytes and its mechanism.Methods 1. To comfirm the transfection efficiency of mi R-133b-5p inhibitor. H9c2 cells in logarithmic phase were transfected with mi R-133b-5p inhibitor(FAM labeled) when they have grown into 40-50% cell confluence. 48 hours later we observed the cells under fluorescence inverted microscope to count the the ratio of cells dots with green fluorescence/all cells and examined the decline of mi R-133b-5p levels by q RT-PCR.2. To explore the role of mi R-133b-5p in myocardial apoptosis and its mechanism H9c2 cells in logarithmic phase were divided into 3 groups: the CON group, mi R-133b-5p inhibitor group and inhibitor-NC group. Cells in CON group were normally cultured. With cells in mi R-133b-5p inhibitor and inhibitor-NC group, mi R-133b-5p inhibitor and inhibitor-NC was transfected into them respectively when they have grown into 40-50% cell confluence. 48 h later, the lactate dehydrogenase(LDH) activity in culture medium, cell apoptosis rate and apoptosis related molecule Fas levels of the cultured cells were examined. 3. To explore the role of mi R-133b-5p in MPC protecting cardiomyocytes from hypoxia-reoxygenation injury and its mechanism H9c2 cells in logarithmic phase were divided into 5 groups: the CON group, the hypoxia/reoxygenation group(H/R), the MPC group, the MPC+mi R-133b-5p inhibitor group and MPC+ inhibitor-NC group. Cells in CON group were normally cultured. The H/R group cells experienced 5 h hypoxia/1 h reoxygenation. The MPC group cells experienced 10 min 1 mmol/L morphine preconditioning before H/R. Cells in MPC+mi R-133b-5p inhibitor group and MPC+ inhibitor-NC group have been transfected with mi R-133b-5p inhibitor and inhibitor-NC for 24 h respectively before MPC. LDH level in culture medium was measured. Cell viability and apoptosis rate were determined by CCK-8 kit and flow cytometry. Apoptosis related molecule Fas levels of the cultured cells were detected by both q RT-PCR and Western Blot.Results 1. To comfirm the transfection efficiency of mi R-133b-5p inhibitor. After 48 h transfection of mi R-133b-5p inhibitor(FAM-labeled), the ratio of cells dots with green fluorescence/all cells under fluorescence inverted microscope was >80%, the mi R-133b-5p levels were aslo obviouly downregulated compared withthe normal cultured H9c2 cells(P<0.05). 2. The role of mi R-133b-5p in myocardial apoptosis and its mechanism Compared with CON, mi R-133b-5p inhibitor group cells showed obviously up-regulated mi R-133b-5p expression, higher apoptosis rate and LDH activity(P<0.05), while the mi R-133b-5p expression, apoptosis rate and LDH activity changes in inhibitor-NC group were not statistically significant. Compared with CON, mi R-133b-5p inhibitor group cells expressed higher level of Fas(P<0.05), while Fas level in inhibitor-NC group didn’t obviously changed. 3. The role of mi R-133b-5p in MPC protecting cardiomyocytes from hypoxiareoxygenation injury and its mechanismCompared with CON, decline of cell viability, increase of LDH activity and apoptosis were observed in the other H/R, MPC, MPC+ mi R-133b-5p inhibitor, MPC+ inhibitor-NC groups(P<0.05), and mi R-133b-5p expression was obviously downregulated in H/R and MPC+ inhibitor-NC(P<0.05). Compared with H/R, MPC cells survived better with higher cell viability, less LDH activity, lower apoptosis rate and decreased Fas expression(P<0.05). MPC+ mi R-133b-5p inhibitor blocked the MPC protection, seen from all the CCK-8, LDH, flow cytometry and Fas m RNA and protein results(P<0.05). While all the indexes didn’t change obviously in MPC+ inhibitor-NC group, compared with MPC.Conclusion MPC protects cardiomyocytes from hypoxia-reoxygenation injury through up-regulating mi R-133b-5p expression. The mechanism might rely on mi R-133b-5p’s regulation of Fas expression at transcription level.更多还原