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本研究旨在探讨心肌肌浆网Ca2+-ATP酶2a(sarcoplasmic reticulum Ca2+-ATPase2a,SERCA2a)基因过表达对正常犬离体心肌细胞收缩功能的影响。通过胶原酶消化法分离犬心肌细胞,将分离的心肌细胞分为未转染组、空载体组、SERCA2a转染组,转染载体为含绿色荧光蛋白基因的重组腺病毒载体,转染后均培养48h。利用免疫印迹法检测各组心肌细胞SERCA2a蛋白表达水平,并通过单细胞收缩动态边缘检测系统测定各组心肌细胞收缩功能的改变。结果显示,与未转染组相比,SERCA2a转染组SERCA2a蛋白表达水平明显升高,基础状态心肌收缩百分比和药物刺激条件下心肌最大收缩百分比均明显升高,达到峰值收缩时间(TTP)和50%舒张时间(R50)亦明显延长,差异具有显著性;而空载体组各项指标均无显著变化。以上结果提示,SERCA2a基因转导过表达能够增强正常犬心肌细胞的收缩功能。
This study aimed to investigate the effect of over-expression of sarcoplasmic reticulum Ca2 + -ATPase2a (SERCA2a) on contractile function of isolated canine cardiomyocytes in vitro. Canine cardiomyocytes were isolated by collagenase digestion, and the isolated cardiomyocytes were divided into untransfected group, empty vector group and SERCA2a transfected group. The transfected vector was a recombinant adenovirus vector containing green fluorescent protein gene. After transfection, Cultivation 48h. The expression of SERCA2a protein in cardiomyocytes of each group was detected by Western blotting, and the change of systolic function of cardiomyocytes in each group was determined by single-cell contraction dynamic edge detection system. The results showed that the expression of SERCA2a protein in SERCA2a transfected group was significantly higher than that in untransfected group, and the percentages of basal myocardial contractile percentage and maximal myocardial contractile percentage in drug-stimulated group were significantly increased, reaching the peak systolic time (TTP) and 50% diastolic time (R50) also significantly prolonged, the difference was significant; and no significant changes in the indicators of the empty vector group. The above results suggest that SERCA2a gene transduction overexpression can enhance the contractile function of normal canine cardiomyocytes.