ANO1对心肌梗死后心肌纤维化的抑制作用及其机制研究

发布时间：2018-03-19 08:18

本文选题：ANO1　切入点：TGF-β/smad3信号通路　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：背景心肌梗死后心肌纤维化严重影响氧传输、电信号传导、心脏舒张及收缩功能等,所以有效防治心肌梗死后心肌纤维化的发生发展显得至关重要。然而,即使经过数十年的不懈研究,截至目前为止仍未找到针对心肌纤维化的有效治疗方法。Anoctamin 1(ANO1)于2008年首次被证实为经典钙激活氯离子通道(CaCC)的分子基础,自此引领了世界范围内对ANO1研究的热潮。之后的众多研究表明,ANO1参与了众多细胞的增殖、迁移和分化等生理病理进程,这从而奠定了我们假设ANO1参与了心肌梗死后心肌纤维化的基础。目的研究ANO1对心肌梗死后心肌纤维化是否有抑制作用,并探讨其作用机制。方法和内容b)乳大鼠心肌成纤维细胞的分离和培养从新生1-3天Sprague-Dawley(SD)乳大鼠心脏提取和分离心肌细胞与心肌成纤维细胞,并于DMEM高糖培养基中培养,心肌细胞以及第2-3代心肌成纤维细胞留供后续实验使用。c)构建编码ANO1的重组腺病毒载体及感染乳大鼠心肌成纤维细胞ANO1重组腺病毒载体由上海吉凯基因化学技术有限公司构建的;本课题组检测了 ANO1重组腺病毒载体感染ANO1的效率。d)心肌成纤维细胞的缺氧实验将培养的第2-3代心肌成纤维细胞进行缺氧处理;继而通过MTT检测细胞增殖,qRT-PCR检测ANO1基因表达,蛋白免疫印迹检测缺氧后相关蛋白的表达。e)心肌梗死动物模型的构建以及病毒载体的转染采用8-10周龄C57B1/6J雄性小鼠进行冠状动脉前降支结扎从而构建心肌梗死模型;并于小鼠心肌中注入重组病毒载体从而完成ANO1重组腺病毒载体于小鼠体内的转染。f)过表达ANO1对心肌成纤维细胞缺氧后纤维化指标的影响心肌成纤维细胞过表达ANO1后进行缺氧处理,继之通过MTT检测细胞增殖,qRT-PCR检测ANO1基因表达,蛋白免疫印迹检测纤维化相关蛋白的表达。g)过表达ANO1对小鼠心肌梗死后纤维化指标的影响构建ANO1小鼠模型后,结扎小鼠冠状动脉前降支构建心梗模型;继之通过免疫组化、蛋白免疫印迹检测相关纤维化指标。h)明确TGF-β/smad3通路在ANO1抑制心肌梗死后心肌纤维化中的作用;通过蛋白免疫印迹实验检测缺氧后及过表达ANO1后TGF-β、p-smad3及相关纤维化指标的蛋白表达。i)统计学处理所有数据通过SPSS 17.0系统进行分析,并使用GraphPad Prism软件进行数据的图像呈现。所有的实验都重复至少三次。数据进行了方差分析和t检验,P0.05有统计学意义。结果1.ANO1表达于心肌成纤维细胞通过qRT-PCR、蛋白免疫印迹和细胞免疫荧光检测心肌细胞与心肌成纤维细胞中ANO1表达,得出ANO1较高水平的表达于心肌成纤维细胞中。2.心肌成纤维细胞缺氧后纤维化指标与ANO1表达均升高1)MTT结果显示:心肌成纤维细胞在缺氧0,6,8,10,16和24小时呈逐渐增殖趋势;2)蛋白免疫印迹结果显示:a-SMA的表达于心肌成纤维细胞缺氧0,6,8,10,16和24小时呈逐渐增加趋势;ANO1蛋白表达也随缺氧时间的增加而呈增加趋势,并于缺氧16小时时达到表达高峰;3)qRT-PCR结果显示:ANO1 mRNA表达随心肌成纤维细胞缺氧时间的增加而增加,并于缺氧16小时达到表达高峰。3.心肌梗死后ANO1表达增加蛋白免疫印迹结果显示与假手术组相比心肌梗死后ANO1的表达增加。4.过表达ANO1降低了缺氧后心肌纤维化指标1)首先qRT-PCR和蛋白免疫印迹结果证实心肌成纤维细胞过表达ANO1;2)MTT结果显示:过表达ANO1心肌成纤维细胞于缺氧后细胞的增殖明显降低;3)蛋白免疫印迹结果显示:过表达ANO1心肌成纤维细胞于缺氧后其a-SMA和collagen I蛋白表达明显降低;5.ANO1降低了心肌梗死后心肌纤维化程度1)Masson染色结果表明:过表达ANO1心肌组织于心肌梗死后其纤维化程度明显降低;2)蛋白免疫印迹和免疫荧光结果显示:过表达ANO1心肌组织于心肌梗死后其a-SMA蛋白表达明显降低;6.过表达ANO1抑制了缺氧对TGF-β/smad3通路的激活作用1)蛋白免疫印迹结果显示:心肌成纤维细胞于缺氧0,6,8,10,16和24小时TGF-β、p-smad3蛋白表达逐渐增加,且其增加趋势与ANO1表达相一致;2)心肌成纤维细胞过表达ANO1,其细胞TGF-β、p-smad3表达于缺氧后明显降低(与阴性对照空载腺病毒转染组相比)。结论ANO1通过抑制TGF-β/smad3信号通路活性从而抑制心肌梗死后心肌纤维化。
[Abstract]:Background myocardial infarction after myocardial fibrosis affects oxygen transport, signal transduction, cardiac diastolic and systolic function, so the effective prevention and treatment of myocardial infarction after myocardial fibrosis is crucial. However, even after decades of unremitting study, so far,.Anoctamin has yet to find the effective method for treatment of myocardial fibrosis in 1 (ANO1) for the first time in 2008 was confirmed as the classical calcium activated chloride channel (CaCC) on the molecular basis, since leading the upsurge of ANO1 research in the world. After many studies show that ANO1 is involved in numerous cell proliferation, migration and differentiation of physiological and pathological processes, so as to lay a foundation that we assume that ANO1 is involved in myocardial infarction after the myocardial fibrosis. Objective to study the effect of ANO1 on myocardial fibrosis after myocardial infarction is inhibited, and explore its mechanism. Methods and contents of B ) in neonatal rat myocardial fibroblasts were isolated and cultured from postnatal day 1-3 Sprague-Dawley (SD) rat heart extraction and separation of myocardial cells and cardiac fibroblasts, and cultured in DMEM high glucose, myocardial cells and the 2-3 generation of cardiac fibroblasts left for subsequent experiments using the.C construct encoding ANO1) the myocardial recombinant adenovirus vector and infect neonatal rat fibroblast ANO1 recombinant adenovirus vector was constructed by Shanghai genechem Co. Ltd; the detection efficiency of the.D infected ANO1 ANO1 recombinant adenovirus vector) into myocardial hypoxia experiment fibroblasts cultured in the 2-3 generation of cardiac fibroblasts by hypoxia treatment; then the cell proliferation was detected by MTT, expression of ANO1 gene qRT-PCR, expression of.E related protein determined by Western blot after hypoxia) myocardial infarction animal model construction and disease The transfection of virus vector 8-10 weeks old male C57B1/6J mice of anterior descending coronary artery was ligated to construct the model of myocardial infarction; and injection of recombinant virus vector in mouse myocardium in order to complete the ANO1 recombinant adenovirus vector in vivo transfection of.F) overexpression of ANO1 on myocardial fibrosis effect of fiber cells after hypoxia in cardiac fibroblasts over expression of ANO1 cells after hypoxia treatment, followed by MTT cell proliferation assay, to detect the expression of ANO1 gene qRT-PCR and protein expression of.G related fibrosis determined by Western blot the expression construct) mouse model of ANO1 ANO1 effect on fibrosis of mice after myocardial infarction in mice after ligation of the anterior descending coronary artery following myocardial infarction model construction; by immunohistochemistry, Western blot detection of.H related fibrosis) clear TGF- beta /smad3 pathway in the inhibition of ANO1 after myocardial infarction heart muscle fiber The role of vitamin; by Western blotting assay after hypoxia and after overexpression of ANO1 beta TGF-, p-smad3 expression and fibrosis protein.I) statistical analysis all the data were analyzed by SPSS 17 system, and the data of the image is rendered using the GraphPad Prism software. All experiments were repeated at least three times. The data were analyzed by analysis of variance and t test, P0.05 was statistically significant. Results the expression of 1.ANO1 in cardiac fibroblasts by qRT-PCR, Western blotting and immunofluorescence detection of myocardial cells and ANO1 expression in myofibroblasts, expression of ANO1 reached high level in myocardial.2. into cardiac fibrosis index and ANO1 expression were increased in 1 fiber cells after hypoxia cells) MTT showed that cardiac fibroblasts in hypoxia 0,6,8,10,16 and 24 hours showed a gradual growth trend; 2) Western blot The results showed that the expression of a-SMA in cardiac fibroblasts and 0,6,8,10,16 hypoxia 24 hours increased gradually; the expression of ANO1 protein also increased with the increase of hypoxia was increased, and reached the peak of expression in hypoxia 16 hours; 3) qRT-PCR showed that ANO1 mRNA expression in heart muscle cells to increase the fiber of hypoxic time increased, and reached the peak expression of.3. after myocardial infarction and increase the expression of ANO1 protein by Western blot results showed that compared with the sham operation group, overexpression of ANO1 reduces myocardial fibrosis after hypoxia 1 ANO1 increased expression of.4. after myocardial infarction in the first 16 hours of hypoxia) qRT-PCR and Western blot results showed that cardiac fibroblasts overexpressing ANO1; 2 MTT) results showed that overexpression of ANO1 in cardiac fibroblasts after hypoxia cell proliferation decreased significantly; 3) Western blot results showed that overexpression of ANO1 in cardiac fibroblasts The expression of a-SMA and collagen in cell I protein decreased significantly after hypoxia; 5.ANO1 reduces myocardial fibrosis after myocardial infarction 1) Masson staining results showed that: the expression of ANO1 in myocardial tissue after myocardial infarction, the degree of fibrosis decreased significantly; 2) Western blotting and immunofluorescence results showed that overexpression of ANO1 in myocardial tissue was significantly lower in the protein expression of a-SMA after myocardial infarction; 6. overexpression of ANO1 inhibits the activation of hypoxia on TGF- beta /smad3 pathway 1) Western blot results showed that cardiac fibroblasts in hypoxia 0,6,8,10,16 and 24 hours TGF- beta, p-smad3 protein expression gradually increased, and the increasing trend is consistent with the expression of ANO1; 2) myocardial overexpression of ANO1 fibroblasts, the cells of TGF- beta, p-smad3 expression decreased significantly after hypoxia (compared with negative control) empty adenovirus transfection group. Conclusion ANO1 through inhibition of TGF- beta /smad3 signal. The activity of the road inhibits myocardial fibrosis after myocardial infarction.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R542.22

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