shRNA介导IGF-IR基因沉默抑制鼠心肌肥厚的体内外实验研究

发布时间：2018-03-07 13:26

本文选题：心肌肥厚　切入点：基因治疗　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：评价磁性纳米颗粒作为基因载体介导小发夹结构RNA (shRNA)靶向沉默IGF1R对去甲肾上腺素诱导的鼠心肌肥厚的抑制作用及其可能的机制。方法：检索IGF-1R的基因序列,依照此序列构建同时表达绿色荧光蛋白基因(GFP)和特异性针对小鼠心肌细胞胰岛素样生长因子-1受体(IGF-1R)基因的shRNA的质粒pGFPshIGF-1R,共设计了两条序列,用聚合物作为载体,将质粒pGFPshlGF-1R转染进鼠心肌细胞,筛选转染效率较高的基因序列；根据上述筛选的质粒,转染体外培养的鼠心肌细胞,同时与单纯的脂质体转染方法相比较,通过western blot检测两种转染方法对平滑肌细胞中IGF-1R蛋白表达下调情况；外加磁场作用,筛选转染效率最高的磁场参数。在去甲肾上腺素诱导的鼠心肌肥厚细胞及动物模型中,通过心重/体重比、心肌横截面积、心超检测左室大小及心功能等评价心肌肥厚模型建立情况。转染质粒pGFPshlGF-1R与聚合物的复合物,分子水平用westernblot检测聚合物载体体内转染24h、48h、72h后,组织中IGF-1R蛋白表达受抑情况；注射结束后1周,再次通过心重／体重比、心肌横截面积、心超检测左室大小及心功能等评价聚合物质粒pGFPshlGF-1R体内抑制心肌肥厚程度,并探索IGF1R下游信号通路蛋白活化情况。结果：首先通过测序验证了RNA干扰序列成功插入质粒内,经过western blot和RT-PCR检测IGF1R表达,筛选出序列2为抑制效率较高的序列,对心肌肌细胞IGF-1R在mRNA水平和蛋白水平的抑制率分别达到47-%-1±1.8%,和41%±1.2%。在转染载体筛选方面,不论脂质体转染还是应用聚合物转染方式,都可以将质粒pGFPshlGF-lR转染进心肌细胞内。相对于空白对照组和脂质体转染,聚合物作为载体展现了较高的效率。流式细胞结果表明,绿色荧光蛋白基因(GFP)阳性的细胞数占35.1±3.1%,而在脂质体转染组绿色荧光蛋白基因(GFP)阳性的细胞数只有13.0±5.1%；协同磁场转染,在250mT辐照15min的条件下转染效率最高,可以达到58%左右。在心肌肥厚模型的构建上,通过去甲肾上腺素成功诱导心肌细胞和小鼠心脏肥厚的模型,并检测出IGF1R明显增高、ANP及J3-MHC的niRNA表达也明显增高。在体内转染条件的实验中,经尾静脉注射表达IGFlRshRNA的聚合物,能有效到达心肌组织,转染24h、48h、72h均能明显抑制IGF1R表达,显著减少去甲肾上腺素诱导的心肌肥厚,改善心功能,表明抑制IGF1R可以作为一种有效可行的用于预防或逆转去甲肾上腺素诱导的心肌肥厚,并且与IGF1R下游通路信号蛋白ERK1/1,AKT的磷酸化有关。结论：沉默IGF1R表达能预防或逆转去甲肾上腺素诱导的心肌肥厚,有望为探寻高血压心肌肥厚发病的机制和防治的策略提供新的靶点和思路。
[Abstract]:Aim: to evaluate the inhibitory effect of magnetic nanoparticles on myocardial hypertrophy induced by norepinephrine and its possible mechanism in IGF1R silencing with small hairpin IGF1R mediated by gene vector. Methods: the gene sequence of IGF-1R was searched. According to this sequence, the plasmid pGFPshIGF-1R was constructed, which simultaneously expressed the green fluorescent protein (GFP) gene and the shRNA specific to the insulin-like growth factor-1 receptor (IGF-1R) gene of mouse cardiomyocytes. The plasmid pGFPshlGF-1R was transfected into rat cardiomyocytes to screen the gene sequence with higher transfection efficiency. According to the selected plasmids, the cultured rat cardiomyocytes were transfected in vitro, and the transfection methods were compared with the simple liposome transfection method. The expression of IGF-1R protein in smooth muscle cells was down-regulated by two transfection methods by western blot, and the magnetic field parameters with the highest transfection efficiency were screened under the action of external magnetic field. In the myocardial hypertrophy cells induced by norepinephrine and animal models, the expression of IGF-1R protein was down-regulated by western blot. The establishment of myocardial hypertrophy model was evaluated by heart weight / body weight ratio, myocardial cross-sectional area, left ventricular size by echocardiography and cardiac function. The complex of plasmid pGFPshlGF-1R and polymer was transfected, and the molecular level was detected by westernblot for 24 h, 48 h and 72 h after transfection. The inhibition of IGF-1R protein expression in the tissue, the degree of inhibition of myocardial hypertrophy in polymer plasmid pGFPshlGF-1R was evaluated by cardiac weight / body weight ratio, myocardial cross-sectional area, left ventricular size and cardiac function 1 week after injection. Results: firstly, the RNA interference sequence was successfully inserted into the plasmid by sequencing. IGF1R expression was detected by western blot and RT-PCR. Sequence 2 was selected as the sequence with higher inhibition efficiency. The inhibition rates of IGF-1R at mRNA level and protein level were 47--1 卤1.8 and 41% 卤1.2, respectively. In the selection of transfection vectors, both liposome transfection and polymer transfection were used. Compared with blank control group and liposome transfection, polymer showed high efficiency. Flow cytometry showed that, The number of GFP positive cells was 35.1 卤3.1, while in liposome transfection group, the number of GFP-positive cells was 13.0 卤5.1.The efficiency of co-transfection was the highest at 250mT irradiation for 15min, while the GFP-positive cells were only 13.0 卤5.1cells in liposome transfection group. It can reach about 58%. In the construction of myocardial hypertrophy model, the model of cardiac hypertrophy induced by norepinephrine was successfully used to induce cardiac hypertrophy in mice. The niRNA expression of IGFlRshRNA and J3-MHC were also significantly increased by IGF1R. In the experiment of transfection condition in vivo, the expression of IGF1R could be effectively reached by injection of polymer expressing IGFlRshRNA through tail vein, and the expression of IGF1R could be inhibited significantly at 24 h, 48 h and 72 h after transfection. Myocardial hypertrophy induced by norepinephrine was significantly reduced and cardiac function was improved. It was suggested that inhibition of IGF1R could be used to prevent or reverse norepinephrine induced myocardial hypertrophy. Conclusion: silent expression of IGF1R can prevent or reverse noradrenaline induced myocardial hypertrophy. It is expected to provide new targets and ideas for exploring the pathogenesis and prevention of hypertensive myocardial hypertrophy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.2

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