MiR-182靶向基因Rac1调控糖尿病心肌病变的研究

发布时间：2018-06-09 13:27

本文选题：糖尿病心肌病 + 心肌肥大　；参考：《上海交通大学》2015年硕士论文

【摘要】：目的:探讨Rac1参与调控糖尿病心肌病变的上游机制,揭示miRNA在糖尿病心肌病发生发展中的作用,为糖尿病心肌病发病机制及临床治疗的研究提供新视角。方法:第一部分利用生物信息学方法预测调控Rac1的候选miRNAs。在糖尿病组db/db小鼠及对照组db/m小鼠的心肌组织中验证所有候选miRNAs的表达;并将糖尿病组小鼠心肌中差异表达的miRNAs与Rac1进行Pearson相关性分析,筛选出与Rac1呈最强负相关的miRNA。第二部分原代培养乳鼠心肌细胞,通过高糖处理心肌细胞,用real-time RT-PCR技术进一步验证Rac1、所选miRNA的表达情况。通过转染所选miRNA的模拟物进行干预,实验分为4组:正常糖组(5mM),高糖组(33mM),正常糖+所选miRNA模拟物组,高糖+所选miRNA模拟物组。通过real-time RT-PCR技术及Western blot技术分别检测各组心肌细胞Rac1、β-MHC、α-SMA的mRNA及蛋白表达情况。第三部分8周龄雄性C57/BL-6J小鼠随机分为正常对照组(n=5),miR-182模拟物对照组(n=5),STZ型糖尿病组(n=15),STZ型糖尿病+所选miRNA模拟物治疗组(n=15)。向C57/BL-6J小鼠一次性腹腔注射链脲佐菌素(STZ)建立STZ型糖尿病动物模型。8周后实验达终点时,记录小鼠的体重及血糖情况;采用小动物用高分辨超声仪检测各组小鼠心脏功能;通过HE染色观察心肌细胞大小,通过Masson染色观察心肌纤维化程度;运用透射电镜观察小鼠心肌组织的超微结构;利用real-time RT-PCR技术检测心肌组织β-肌球蛋白重链(β-MHC),α-肌球蛋白重链(α-MHC),心房钠尿肽(ANP),I型胶原(Col I)和III型胶原(Col III)mRNA,待测mRNA及Rac1的表达情况。结果:第一部分通过生物信息学方法,预测到参与调控Rac1的候选miRNAs共6个:分别是miR-182、miR-142-3p、miR-140、miR-101a、miR-429、miR-200b。与db/m组比较,db/db组糖尿病小鼠心肌组织中miR-142-3p、miR-182的表达显著下调,具有统计学意义(p0.05)。Pearson相关性分析显示miR-182与Rac1表达水平的负相关最显著,r值为-0.89102。第二部分体外实验显示高糖诱导乳鼠心肌细胞肥大时,mir-182表达明显降低,而rac1表达明显升高,两者之间亦呈负相关。与正常糖组比较,高糖组心肌细胞肥大明显。与高糖组相比,高糖+mir-182模拟物组心肌细胞肥大程度明显降低。rt-pcr及westernblot结果均显示与高糖组相比,高糖+mir-182模拟物组心肌细胞rac1及β-mhc的表达均降低,具有统计学意义(p0.05),但α-sma的表达降低不具有统计学意义。第三部分超声心动图检测显示:与糖尿病组相比,糖尿病+mir-182模拟物组小鼠的左室射血分数及左室短轴缩短率明显升高,具有统计学意义(p0.05)。rt-pcr结果显示:与糖尿病组比较,糖尿病+mir-182模拟物组小鼠心肌组织中rac1、anp、coli、coliiimrna的表达及β/α-mhc比值明显下降,具有统计学意义(p0.05)。he染色结果显示:正常对照组及mir-182模拟物对照组心肌细胞排列规则整齐,细胞核大小均一;而糖尿病组小鼠心肌排列紊乱,心肌肥大明显,细胞核增大,形态不规则;糖尿病+mir-182组小鼠心肌肥大程度减低,心肌排列较规则。masson染色后心肌纤维呈现为深红色而心脏间质胶原呈现为深蓝色:正常对照组及mir-182模拟物对照组小鼠心肌间质胶原纤维较少且均匀分布;而糖尿病组小鼠心肌组织胶原纤维明显增多聚集,分布紊乱不均匀;糖尿病+mir-182组小鼠心肌胶原纤维较糖尿病组明显减少。透射电镜结果显示:正常对照组及mir-182模拟物对照组小鼠心肌纤维排列整齐有序,肌小节及明带暗带清晰可见,线粒体大小均一,呈圆形或椭圆形沿心肌纤维整齐排列;糖尿病组小鼠心肌纤维排列紊乱,肌丝扭曲、断裂,心肌线粒体明显异型增大及增多聚集,线粒体可肿胀、嵴消失甚至溶解,可见糖原颗粒及脂滴增多聚集,心脏间质大量胶原纤维聚集,可见较多自噬体;而糖尿病+mir-182模拟物组小鼠心肌超微结构的损伤较糖尿病组明显减轻。结论:(1)利用生物信息学方法预测、通过动物模型体内实验及pearson相关性分析,最终筛选得出rac1的候选mirna为mir-182;同时体外实验进一步验证心肌细胞中mir-182与rac1表达呈负相关。(2)mir-182模拟物降低高糖诱导心肌细胞中rac1的表达,减轻高糖诱导心肌细胞的肥大程度,降低心肌细胞肥大相关表型的表达。(3)mir-182可参与糖尿病心肌病的调控,mir-182模拟物能可减轻糖尿病小鼠心肌组织rac1表达,改善糖尿病小鼠心脏收缩功能,这与其降低糖尿病小鼠心肌肥大及心肌纤维化程度,减轻糖尿病小鼠心肌超微结构损伤有关。
[Abstract]:Objective: To explore the upstream mechanism of Rac1 in the regulation of diabetic cardiomyopathy, to reveal the role of miRNA in the development of diabetic cardiomyopathy, and to provide a new perspective for the pathogenesis and clinical treatment of diabetic cardiomyopathy. Method: the first part was pretested by Bioinformatics Method to regulate the candidate miRNAs. of Rac1 in the diabetes group db/db small The expression of all the candidate miRNAs in the myocardium of the mice and the control group db/m mice was verified, and the miRNAs and Rac1 in the myocardium of the diabetic mice were analyzed with the Pearson correlation, and the myocardial cells of the primary cultured milk rat were screened out with the strongest negative correlation with Rac1, and the cardiac myocytes were treated with high glucose, and real-time RT was used. -PCR technique further verified the expression of Rac1 and selected miRNA. The experiment was divided into 4 groups: normal sugar group (5mM), high sugar group (33mM), normal sugar +, miRNA mimic group, high sugar + selected miRNA simulation group. The myocardial fines were detected by real-time RT-PCR technique and Western blot technique respectively. The mRNA and protein expression of cell Rac1, beta -MHC, and alpha -SMA. The third part of 8 weeks male C57/BL-6J mice were randomly divided into normal control group (n=5), miR-182 analogue control group (n=5), STZ type diabetes group (n=15), STZ type diabetes + selected miRNA analogue treatment group. The body weight and blood sugar of the mice were recorded at the end of.8 weeks after the experimental diabetic animal model. The cardiac function was detected by the high resolution ultrasonography of small animals, the size of myocardial cells was observed by HE staining, the degree of myocardial fibrosis was observed by Masson staining, and the ultrastructure of the mice was observed by transmission electron microscopy. Real-time RT-PCR technique was used to detect the myocardial tissue beta myosin heavy chain (beta -MHC), alpha myosin heavy chain (alpha -MHC), atrial natriuretic peptide (ANP), I collagen (Col I) and III type collagen (Col III) mRNA. Results: the first part predicts a total of 6 candidates participating in the regulation by bioinformatics. MiR-182, miR-142-3p, miR-140, miR-101a, miR-429, miR-200b. and db/m group compared with the db/m group, the expression of miR-142-3p and miR-182 in the myocardium of the diabetic mice was significantly down, with a statistical significance (P0.05).Pearson correlation analysis showed that the negative correlation was the most significant with the second parts. In the experiment, the expression of mir-182 was obviously decreased and the expression of Rac1 increased obviously. The hypertrophy of myocardial cells in the high glucose group was significantly higher than that in the normal sugar group. Compared with the high glucose group, the degree of myocardial hypertrophy in the high glucose +mir-182 simulated group significantly reduced the results of.Rt-pcr and Westernblot. Compared with the high glucose group, the expression of Rac1 and beta -mhc in the myocardial cells of the high glucose +mir-182 simulated group decreased and had statistical significance (P0.05), but the decrease of the expression of alpha -sma was not statistically significant. The third part echocardiography showed that the left ventricular ejection fraction in the diabetic group was compared with the diabetic group, and the left ventricular ejection fraction of the diabetic mice was compared with the diabetic group. The short axis shortening rate of the left ventricle was significantly increased, with statistical significance (P0.05).Rt-pcr results showed: compared with the diabetic group, the expression of Rac1, ANP, coli, coliiimrna and the ratio of beta / alpha -mhc in the myocardium of the diabetic +mir-182 mimics group decreased significantly, and the statistical meaning (P0.05).He staining results showed that the normal control group and mir-182 simulation were shown. The myocardial cells in the control group were arranged regularly and the nucleus size was uniform, but the myocardium of the diabetic mice was arranged in disorder, the myocardial hypertrophy was obvious, the nucleus enlarged and the shape was irregular. The degree of myocardial hypertrophy in the diabetic +mir-182 group was reduced, the myocardial arrangement was deep red after the regular.Masson staining, and the cardiac collagen was found in the heart. The collagen fibers in the myocardium of the normal control group and the mir-182 mimic control group were less and evenly distributed, while the collagen fibers in the diabetic group were significantly increased, and the distribution disorder was unevenly distributed. The collagen fibers in the diabetic group +mir-182 mice were significantly lower than that in the sugar urine group. Transmission electron microscopy showed that: The myocardial fibers in the control group and the control group of mir-182 were arranged orderly and orderly. The muscle segments and the dark bands were clearly visible. The size of the mitochondria was uniform, and the size of the mitochondria was round or oval in order. The myocardial fibers in the diabetic mice were arranged in disorder, the muscle filament was twisted, and the myocardial mitochondria were obviously abnormal and increased. The mitochondria can be swollen, the crista disappears or even dissolve, the accumulation of glycogen particles and lipid droplets, the aggregation of mass collagen fibers in the cardiac interstitium and more autophagic bodies, and the damage of the ultrastructure of the diabetic +mir-182 mimics mice is obviously less than that in the diabetic group. Conclusion: (1) the bioinformatics method is used to predict and pass the animal model body. The internal experiment and Pearson correlation analysis showed that the candidate miRNA of Rac1 was mir-182, and in vitro experiments further verified that the expression of mir-182 and Rac1 in cardiac myocytes was negatively correlated. (2) mir-182 mimics reduced the expression of Rac1 in cardiac myocytes induced by high glucose, alleviated the hypertrophy of hyperglycemia induced cardiomyocytes and reduced the hypertrophy of cardiomyocytes. The expression of related phenotypes. (3) mir-182 can be involved in the regulation of diabetic cardiomyopathy. Mir-182 mimics can reduce the expression of Rac1 in myocardial tissue of diabetic mice and improve cardiac contractility in diabetic mice, which is related to reducing myocardial hypertrophy and myocardial fibrosis in diabetic mice and reducing myocardial ultrastructural damage in diabetic mice.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

【参考文献】