特异性干扰缝隙连接蛋白40及KV1.5通道对房颤发病机制影响的研究
发布时间：2018-09-01 12:17
【摘要】：目的构建心房颤动(Atrial fibrillation,AF)患者心房肌细胞的细胞模型,并探讨房颤患者心房肌细胞缝隙连接蛋白40(Connexin40,Cx40)和Kv1.5通道蛋白的表达及两者之间的关系。方法1.取房颤患者(行二尖瓣置换+MAZE手术)及窦性心律患者的左心耳标本,并单独使用I型胶原蛋白酶消化法培养房颤患者的心房肌细胞,同时使用胶原蛋白抗体及肌钙蛋白抗体进行免疫组化鉴定。2.利用RNA干扰技术干扰Cx40 mRNA的表达,同时分别使用PCR技术及Western-blot试验分别检测Cx40 mRNA和Cx40蛋白及Kv1.5 mRNA和Kv1.5通道蛋白的表达量。结果1.原代心房细胞分离24 h后可见少量圆形细胞贴壁,72 h后,在显微镜下可见分离的大部分心肌细胞贴壁,贴壁的细胞成团生长,并呈长梭形,5天左右细胞可以长满瓶底,同时使用免疫组化鉴定证实为心肌细胞。2.与对照组相比,房颤患者心房肌细胞Cx40和Kv1.5的mRNA及蛋白表达明显降低;干扰Cx40表达后检测到Cx40的表达量降低,并检测到Kv1.5的mRNA及蛋白表达量同时降低。结论1.使用I型胶原蛋白酶能够成功培养房颤患者的心房肌细胞。2.特异性干预房颤患者心房肌细胞Cx40能够使得Kv1.5的mRNA及蛋白表达明显降低。
[Abstract]:Objective to establish a cell model of atrial myocytes in patients with atrial fibrillation (Atrial fibrillation,AF), and to investigate the expression of gap junction protein 40 (Connexin40,Cx40) and Kv1.5 channel protein in atrial fibrillation patients. Method 1. Left atrial appendage was collected from patients with atrial fibrillation (mitral valve replacement MAZE operation) and sinus rhythm, and atrial myocytes were cultured by type I collagenase digestion alone. At the same time, collagen antibody and cardiac troponin antibody were used for immunohistochemical identification. 2. 2. RNA interference technique was used to interfere the expression of Cx40 mRNA, and PCR and Western-blot tests were used to detect the expression of Cx40 mRNA and Cx40 and Kv1.5 mRNA and Kv1.5 channel proteins respectively. Result 1. After 24 h of primary atrial cell separation, a small number of circular cells adhered to the wall for 72 h. Under the microscope, most of the isolated cardiomyocytes adhered to the wall, the adherent cells grew into clusters, and the cells could grow to the bottom of the bottle for about 5 days in the form of long spindle shape. At the same time, immunohistochemical identification confirmed that cardiomyocytes. 2. 2. Compared with the control group, the expression of mRNA and protein of Cx40 and Kv1.5 in atrial fibrillation patients was significantly decreased, and the expression of Cx40 and mRNA and protein of Kv1.5 were decreased after Cx40 expression was interfered. Conclusion 1. Using type I collagenase can successfully culture atrial myocytes. 2. 2 in patients with atrial fibrillation. Specific intervention of atrial myocyte Cx40 in patients with atrial fibrillation could significantly decrease the expression of mRNA and protein in Kv1.5.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.75
本文编号：2217179
[Abstract]:Objective to establish a cell model of atrial myocytes in patients with atrial fibrillation (Atrial fibrillation,AF), and to investigate the expression of gap junction protein 40 (Connexin40,Cx40) and Kv1.5 channel protein in atrial fibrillation patients. Method 1. Left atrial appendage was collected from patients with atrial fibrillation (mitral valve replacement MAZE operation) and sinus rhythm, and atrial myocytes were cultured by type I collagenase digestion alone. At the same time, collagen antibody and cardiac troponin antibody were used for immunohistochemical identification. 2. 2. RNA interference technique was used to interfere the expression of Cx40 mRNA, and PCR and Western-blot tests were used to detect the expression of Cx40 mRNA and Cx40 and Kv1.5 mRNA and Kv1.5 channel proteins respectively. Result 1. After 24 h of primary atrial cell separation, a small number of circular cells adhered to the wall for 72 h. Under the microscope, most of the isolated cardiomyocytes adhered to the wall, the adherent cells grew into clusters, and the cells could grow to the bottom of the bottle for about 5 days in the form of long spindle shape. At the same time, immunohistochemical identification confirmed that cardiomyocytes. 2. 2. Compared with the control group, the expression of mRNA and protein of Cx40 and Kv1.5 in atrial fibrillation patients was significantly decreased, and the expression of Cx40 and mRNA and protein of Kv1.5 were decreased after Cx40 expression was interfered. Conclusion 1. Using type I collagenase can successfully culture atrial myocytes. 2. 2 in patients with atrial fibrillation. Specific intervention of atrial myocyte Cx40 in patients with atrial fibrillation could significantly decrease the expression of mRNA and protein in Kv1.5.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.75
【参考文献】
相关期刊论文 前6条
1 Bing Sun;Jin-Fa Jiang;Cui-Mei Zhao;Chao-Hui Hu;;Effects of antiarrhythmic peptide 10 on acute ventricular arrhythmia[J];Asian Pacific Journal of Tropical Medicine;2015年03期
2 刘雄涛;苏菲菲;尚福军;杨剑;辛梅;洪昌明;王彬;石苗茜;郑强荪;;人类心房肌细胞的原代培养与鉴定[J];科学技术与工程;2014年02期
3 LI Ying;WU Yang Feng;CHEN Ke Ping;LI Xian;ZHANG Xing;XIE Gao Qiang;WANG Fang Zheng;ZHANG Shu;;Prevalence of Atrial Fibrillation in China and Its Risk Factors[J];Biomedical and Environmental Sciences;2013年09期
4 邓玉莲;高峰;许春萱;张建成;陈林;;心房颤动患者心房组织延迟整流钾通道基因表达的研究[J];中华心律失常学杂志;2005年06期
5 张荣庆,程何祥,王海昌,贾国良,郭文怡,周更须,张殿新,王海燕,李雪;人心房肌细胞的培养与鉴定[J];中华心血管病杂志;2004年09期
6 周自强,胡大一,陈捷,张仁汉,李奎宝,赵秀丽;中国心房颤动现状的流行病学研究[J];中华内科杂志;2004年07期
，本文编号：2217179
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2217179.html
最近更新
教材专著
