【摘要】：目的構(gòu)建心房顫動(dòng)(Atrial fibrillation,AF)患者心房肌細(xì)胞的細(xì)胞模型,并探討房顫患者心房肌細(xì)胞縫隙連接蛋白40(Connexin40,Cx40)和Kv1.5通道蛋白的表達(dá)及兩者之間的關(guān)系。方法1.取房顫患者(行二尖瓣置換+MAZE手術(shù))及竇性心律患者的左心耳標(biāo)本,并單獨(dú)使用I型膠原蛋白酶消化法培養(yǎng)房顫患者的心房肌細(xì)胞,同時(shí)使用膠原蛋白抗體及肌鈣蛋白抗體進(jìn)行免疫組化鑒定。2.利用RNA干擾技術(shù)干擾Cx40 mRNA的表達(dá),同時(shí)分別使用PCR技術(shù)及Western-blot試驗(yàn)分別檢測Cx40 mRNA和Cx40蛋白及Kv1.5 mRNA和Kv1.5通道蛋白的表達(dá)量。結(jié)果1.原代心房細(xì)胞分離24 h后可見少量圓形細(xì)胞貼壁,72 h后,在顯微鏡下可見分離的大部分心肌細(xì)胞貼壁,貼壁的細(xì)胞成團(tuán)生長,并呈長梭形,5天左右細(xì)胞可以長滿瓶底,同時(shí)使用免疫組化鑒定證實(shí)為心肌細(xì)胞。2.與對照組相比,房顫患者心房肌細(xì)胞Cx40和Kv1.5的mRNA及蛋白表達(dá)明顯降低;干擾Cx40表達(dá)后檢測到Cx40的表達(dá)量降低,并檢測到Kv1.5的mRNA及蛋白表達(dá)量同時(shí)降低。結(jié)論1.使用I型膠原蛋白酶能夠成功培養(yǎng)房顫患者的心房肌細(xì)胞。2.特異性干預(yù)房顫患者心房肌細(xì)胞Cx40能夠使得Kv1.5的mRNA及蛋白表達(dá)明顯降低。
[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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.75

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