RNA干扰大鼠H9c2心肌细胞Hsc70基因条件筛选

Preparation of RNA interference transfection on the Hsc70 gene in H9c2 myocardial cells

为了研究H9c2心肌细胞RNA干扰的转染条件,筛选结构型热休克蛋白70(Hsc70)干扰序列,采用流式细胞仪和Western blot方法,研究不同剂量比例荧光标记的siRNA(FAM-siRNA)和不同浓度脂质体2000(Lipofectamine 2000)对转染效率的影响,进一步筛选Hsc70 siRNA的最佳沉默序列。结果显示:流式细胞仪试验得出的最佳转染条件为80 nmol/L FAM-siRNA+1.5μL Lipofectamine 2000;Western blot结果表明,Oligo 2、Oligo 3和Oligo 4组中的Hsc70蛋白表达极显著降低(P<0.01),其中Oligo 3组的沉默效率最高;转染后48 h收获细胞为最佳转染时间。结果表明:80 nmol/L FAM-siRNA+1.5μL Lipofectamine 2000为最佳转染条件,以Oligo 3为最佳RNA干扰靶基因。

In order to investigate the RNA interference transfection in H9c2 cardiomyocytes and find the Hsc70 interference sequence for the further research on the protection of Hsc70 in cardiomyocytes,the best silencing efficiency of Hsc70 siRNA in H9c2 myocardial cells were examined by flow cytometry and western blotting. Transfected FAM-siRNA fragment into H9c2 cells,the effect of FAM-siRNA and Lipofectamine 2000 in different proportions on the transfection efficiency was examined by flow cytometry. Expression of Hsc70 was determined by western blotting. FACS results showed that the group of 80 nmol/L FAM-siRNA ＋ 1. 5 μL Lipofectamine 2000 had the best transfect efficiency. Western blot results showed that expressions of Hsc70 were significantly decreased in Oligo 2,Oligo 3 and Oligo 4 groups（ P〈0. 01）,and the highest efficiency silencing was Oligo 3 groups. H9c2 cells were harvested after 48 h transfection. In conclusions,80 nmol/L FAM-siRNA ＋ 1. 5 μL Lipofectamine 2000 had the best transfection efficiency,and oligo 3 had the highest interference efficiency on targeting Hsc70 gene.