miR-27a介导GLP-1受体激动剂抑制心肌细胞凋亡作用的机制研究

发布时间：2018-06-21 04:27

本文选题：糖尿病心肌病 + GLP-1　；参考：《天津医科大学》2016年硕士论文

【摘要】：目的:糖尿病(DM)是由遗传及环境在内的多种因素共同引起的一组以慢性血糖水平升高为特征的代谢性疾病。心血管疾病是DM的主要并发症之一。近年来研究证实,糖尿病心肌病(DCM)与DM患者心血管疾病的高发病率和高死亡率密切相关。而心肌细胞凋亡是DCM的主要发病机制之一。利拉鲁肽(LR)是人工合成的胰高血糖素样肽-1(GLP-l)受体激动剂,不仅能发挥降糖作用,还具有心血管系统保护作用。微小RNA(miRNA)是一类保守的内源性非编码单链小分子RNA,其在心血管疾病的发生、发展中扮演着重要角色。miR-27a是目前研究较多的与细胞凋亡密切相关的miRNA。而我们的前期研究发现,GLP-1可通过激活AMPK抑制心肌细胞miR-27a的表达。因此本研究旨在探讨GLP-1受体激动剂LR是否通过抑制心肌细胞miR-27a的表达,发挥抗心肌细胞凋亡的作用,从而为其在防治DCM、降低DM患者心血管疾病死亡率中的应用提供理论依据。方法:1.低糖培养H9c2心肌细胞,使用终浓度为150pmol和300pmol的miR-27a mimics以及终浓度为150pmol和300pmol的miR-27a inhibitor分别转染细胞48小时,并设置正常对照组,用荧光定量PCR方法检测各组miR-27a的表达水平,从而检测转染效率。2.低糖培养H9c2心肌细胞,将其分为6组:正常对照组,脂质体lipo2000组(lipo2000组),150pmol低浓度转染miR-27a mimics组(miR-27a 150pmol组),300pmol高浓度转染miR-27a mimics组(miR-27a 300pmol组)及转染其各自阴性对照组(miR-27a N.C 150pmol组、miR-27a N.C 300pmol组)。转染后作用48小时,用TUNEL染色法检测细胞凋亡。3.培养H9c2心肌细胞,分为低糖组(LG组),高糖组(HG组),高糖+miR-27a mimics组(mimics组),高糖+miR-27a inhibitor组(inhibitor组),高糖+miR-27a mimics N.C组(mimics N.C组),高糖+miR-27a inhibitor N.C组(inhibitor N.C组),高糖+脂质体lipo2000组(lipo组),高渗组(HO组),各组转染终浓度均为300pmol,转染后作用48小时,用荧光定量PCR方法检测miR-27a的表达水平,Western Blotting方法检测细胞凋亡相关蛋白Bax、Bcl-2、caspase-3、c IAP-1、Apaf-1蛋白的表达水平,并用TUNEL染色法检测细胞凋亡。4.高糖培养H9c2心肌细胞,分为对照组,利拉鲁肽组(LR组),miR-27a mimics组,LR+miR-27a mimics组,LR+miR-27a mimics N.C组,LR+lipo2000组,利拉鲁肽的干预浓度为1000nmol/L,转染组转染终浓度均为300pmol。作用48小时后,用荧光定量PCR方法检测miR-27a的表达水平,并用TUNEL染色法检测细胞凋亡。结果:1.相较于对照组,低浓度和高浓度转染miR-27a mimics组,其miR-27a的表达水平均升高(P0.05),且高浓度转染组miR-27a的表达水平较低浓度转染组明显升高(P0.05)。与对照组相比,当转染150pmol的miR-27a inhibitor时,miR-27a表达已被明显抑制(P0.05),在转染300pmol miR-27a inhibitor时,miR-27a表达近乎完全被抑制(P0.05)。2.与对照组相比,转染miR-27a mimics组,其心肌细胞凋亡指数呈浓度依赖性升高(P0.05),而lipo2000组、miR-27a N.C 150pmol组、miR-27a N.C 300pmol组的细胞凋亡指数较对照组无明显差异(P0.05)。3.在高糖状态下,转染miR-27a mimics组,其miR-27a表达水平及细胞凋亡指数显著增高(P0.05);转染inhibitor组,其miR-27a表达明显受到抑制且细胞凋亡指数显著降低(P0.05),而lipo2000组、mimics N.C组和inhibitor N.C组与高糖对照组相比均未引起miR-27a表达水平的变化以及细胞凋亡指数的变化(P0.05)。而高渗组miR-27a含量与低糖对照组比较差异无统计学意义(P0.05)。4.Western Blotting实验结果表明,miR-27a mimics组Bax、caspase-3及Apaf-1蛋白表达水平升高,而inhibitor组Bax、caspase-3及Apaf-1蛋白表达水平降低,且两组之间的变化有统计学差异(P0.05)。同时,miR-27a mimics组Bcl-2、c IAP-1蛋白表达水平降低,而inhibitor组Bcl-2、c IAP-1蛋白表达水平升高,且两组之间的变化有统计学差异(P0.05)。5.与对照组比较,单纯给予利拉鲁肽干预组,其miR-27a表达水平及心肌细胞凋亡指数明显降低(P0.05)。而miR-27a mimics组及LR+miR-27a mimics组,其miR-27a表达水平及心肌细胞凋亡指数较对照组明显升高(P0.05),且LR+miR-27a mimics组心肌细胞凋亡指数较miR-27a mimics组降低(P0.05)。结论:1.miR-27a可促进心肌细胞的凋亡,且呈浓度依赖性。2.在高糖状态下,miR-27a可通过上调促凋亡蛋白Apaf-1、caspase-3和Bax的表达,下调凋亡抑制蛋白c IAP-1和Bcl-2的表达促进心肌细胞的凋亡。3.GLP-1受体激动剂通过抑制心肌细胞miR-27a的表达,发挥抗心肌细胞凋亡的作用。
[Abstract]:Objective: diabetes (DM) is a group of metabolic diseases characterized by a variety of factors such as heredity and environment. Cardiovascular disease is one of the major complications of DM. In recent years, it has been confirmed that diabetic cardiomyopathy (DCM) is closely related to the high incidence and high mortality of cardiovascular disease in DM patients. Cardiomyocyte apoptosis is one of the main pathogenesis of DCM. LR is an artificial glucagon like peptide -1 (GLP-l) receptor agonist, which not only plays the hypoglycemic effect, but also has the protective effect of cardiovascular system. Small RNA (miRNA) is a conservative, endogenous, non coding single strand small molecule RNA, which is in the hair of cardiovascular disease. .miR-27a plays an important role in the development of miRNA., which is closely related to apoptosis. Our previous study found that GLP-1 could inhibit the expression of miR-27a in cardiac myocytes by activating AMPK. Therefore, the purpose of this study was to investigate whether the GLP-1 receptor agonist LR has passed the inhibition of the expression of miR-27a in cardiac myocytes and exerts its resistance to the expression of miR-27a. The effect of cardiomyocyte apoptosis provides a theoretical basis for its application in preventing and controlling DCM and reducing the mortality of cardiovascular disease in DM patients. Methods: 1. low sugar cultured H9c2 cardiomyocytes were transfected with 150pmol and 300pmol miR-27a mimics as well as 150pmol and 300pmol miR-27a inhibitor, respectively, for 48 hours, respectively. The normal control group was set up to detect the expression level of miR-27a in each group by fluorescence quantitative PCR, and then the transfection efficiency.2. low sugar cultured H9c2 cardiomyocytes were divided into 6 groups: normal control group, lipo2000 group of liposome (lipo2000 group), 150pmol low concentration transfected miR-27a mimics group (miR-27a 150pmol group), 300pmol high concentration transfection Group s (group miR-27a 300pmol) and its negative control group (group miR-27a N.C 150pmol, miR-27a N.C 300pmol group). After 48 hours of transfection, the apoptotic H9c2 cardiomyocytes were detected by TUNEL staining method, which were divided into low sugar group, high sugar group, high sugar group and high sugar group. Itor group (Group itor), high glucose +miR-27a mimics N.C group (Group mimics N.C), high glucose +miR-27a inhibitor N.C group (inhibitor N.C group), high sugar + liposome lipo2000 group, hypertonic group (Group), the final transfection concentration was all respectively, and the expression level was detected by fluorescein quantitative detection method after 48 hours after transfection. Apoptosis related protein Bax, Bcl-2, Caspase-3, C IAP-1, Apaf-1 protein expression level, and TUNEL staining method was used to detect the apoptotic.4. high sugar culture H9c2 myocardial cells, divided into the control group, the Li La Lu peptide group (LR group), the miR-27a mimics group, the group, the concentration of the intervention group was 1. 000nmol/L, the transfection group was transfected with the final concentration of 300pmol. for 48 hours. The expression level of miR-27a was detected by fluorescence quantitative PCR, and the apoptosis was detected by TUNEL staining. Results: 1. compared with the control group, the expression level of miR-27a in the low concentration and high concentration transfected miR-27a mimics group was all increased (P0.05), and the high concentration transfected group miR-2. The expression level of 7a was significantly higher than that in the low concentration transfection group (P0.05). Compared with the control group, the expression of miR-27a was obviously inhibited when transfected to 150pmol miR-27a inhibitor (P0.05). The miR-27a expression was almost completely suppressed in the transfection of 300pmol miR-27a inhibitor. The death index increased in a concentration dependent manner (P0.05), while the apoptotic index of group lipo2000, miR-27a N.C 150pmol group and miR-27a N.C 300pmol group was not significantly different than that of the control group (P0.05). The expression level and the cell withering index were significantly higher in the miR-27a mimics group. It was obviously inhibited and the apoptosis index decreased significantly (P0.05), while group lipo2000, mimics N.C group and inhibitor N.C group did not cause the change of miR-27a expression and the change of apoptosis index (P0.05), but there was no significant difference between the miR-27a content of the hypertonic group and the low sugar control group (P0.05).4.We. The results of stern Blotting experiment showed that the expression level of Bax, caspase-3 and Apaf-1 protein in group miR-27a mimics increased, while the expression level of Bax, caspase-3 and Apaf-1 protein in inhibitor group decreased, and there was a statistical difference between the two groups. The level of protein expression was increased and the changes between the two groups were statistically different (P0.05).5. was compared with the control group. The expression level of miR-27a and the apoptosis index of myocardial cells were significantly decreased (P0.05). The expression level of miR-27a and the apoptosis index of myocardial cells in the miR-27a mimics group and the LR+miR-27a mimics group were more than that of the control group. The apoptotic index of myocardial cells in group LR+miR-27a mimics was significantly higher than that in group miR-27a mimics (P0.05). Conclusion: 1.miR-27a can promote apoptosis of cardiac myocytes, and the concentration dependent.2. in high glucose state, miR-27a can be regulated by up regulation of apoptotic protein Apaf-1, caspase-3 and Bax, and down regulation of apoptosis inhibitory protein. The expression of Bcl-2 and.3.GLP-1 can promote the apoptosis of cardiomyocytes..3.GLP-1 receptor agonists can inhibit the apoptosis of cardiomyocytes by inhibiting the expression of myocardial cells.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2

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