论文部分内容阅读
目的应用特异性siRNA抑制新生SD大鼠心房肌细胞Kir3.1基因表达;筛选出干扰效率最高的siRNA,并应用分子生物学方法进行鉴定。方法设计并化学合成3条针对Kir3.1基因的siRNA,脂质体法转染原代培养的新生SD大鼠心房肌细胞,应用Real-time PCR筛选出抑制效率最高的siRNA,将其转染入细胞;于转染后24h、48h、72h提取细胞总RNA及蛋白质,分别采用Real-time PCR和Western blot检测Kir3.1 mRNA及蛋白质表达情况。结果 Real-time PCR显示siRNA-3干扰效率强于siRNA-1,2,能够显著下调心房肌细胞Kir3.1 mRNA的表达;siRNA-3转染心房肌细胞后,发现其在24,48,72h均能使Kir3.1 mRNA及蛋白表达下降。结论针对Kir3.1 mRNA合成的siRNA能够特异性干扰Kir3.1基因表达和蛋白质的合成,这可能为心律失常性疾病的治疗提供新的实验性依据。
Objective To inhibit the expression of Kir3.1 gene in atrial myocytes of newborn SD rats with specific siRNA. The siRNA with the highest interference efficiency was screened out and identified by molecular biology method. Methods Three siRNAs targeting Kir3.1 gene were designed and synthesized. The primary cultured neonatal SD rat atrial myocytes were transfected with liposome. The siRNA with the highest inhibitory activity was screened by Real-time PCR and transfected into The total RNA and protein of cells were extracted 24h, 48h, 72h after transfection. The expression of Kir3.1 mRNA and protein were detected by Real-time PCR and Western blot respectively. Results Real-time PCR showed that the interference efficiency of siRNA-3 was significantly higher than that of siRNA-1, and significantly down-regulated the expression of Kir3.1 mRNA in atrial myocytes. SiRNA-3 transfected atrial myocytes in 24,48,72h Can make Kir3.1 mRNA and protein expression decreased. Conclusion siRNA targeting Kir3.1 mRNA can specifically interfere with Kir3.1 gene expression and protein synthesis, which may provide a new experimental basis for the treatment of arrhythmic diseases.