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目的采用RNA干扰技术阻断STAT5基因的表达,观察其对人肝癌SMMC-7721凋亡的影响。方法构建3种靶向STAT5的小干扰RNA(siRNA)真核表达载体,采用LipofectamineTM2000转染肝癌细胞SMMC-7721;分别采用半定量RT-PCR、Western blot技术检测转染前后SMMC-7721细胞STAT5 mRNA和蛋白质表达的变化;采用透射电镜技术观察细胞形态变化;流式细胞术检测细胞凋亡率。结果3条靶向STAT5的序列特异性的siRNA可以有效地抑制SMMC-7721细胞STAT5 mRNA和蛋白质表达,STAT5 mRNA表达抑制率分别为70.43%、43.02%、45.07%,STAT5蛋白表达抑制率分别为67.45%、37.36%、41.86%;转染靶向STAT5的siRNA真核表达载体48h后出现凋亡细胞的典型形态学变化,并诱导25.61%的SMMC-7721细胞发生凋亡。结论靶向STAT5的siRNA真核表达载体可以有效、特异地阻断SMMC-7721细胞STAT5基因的表达,并能够诱导SMMC-7721细胞凋亡,STAT5 siRNA在人肝癌的基因治疗中可能具有潜在的应用价值。
Objective To inhibit the expression of STAT5 gene by RNA interference and observe its effect on the apoptosis of human hepatoma SMMC-7721 cells. Methods Three siRNAs targeting STAT5 were constructed and transfected into SMMC-7721 cells by LipofectamineTM2000. The expression of STAT5 mRNA in SMMC-7721 cells was detected by semi-quantitative RT-PCR and Western blot respectively And the changes of protein expression. The morphological changes of cells were observed by transmission electron microscopy. The apoptosis rate was detected by flow cytometry. Results Three siRNAs targeting STAT5 could effectively inhibit STAT5 mRNA and protein expression in SMMC-7721 cells. The inhibitory rates of STAT5 mRNA expression were 70.43%, 43.02% and 45.07%, respectively, and the inhibitory rates of STAT5 protein expression were 67.45 %, 37.36% and 41.86%, respectively. After transfection with eukaryotic expression vector targeting STAT5 for 48h, typical morphological changes of apoptotic cells were observed and apoptosis was induced in 25.61% of SMMC-7721 cells. Conclusion siRNA eukaryotic expression vector targeting STAT5 can effectively and specifically block STAT5 gene expression in SMMC-7721 cells and induce apoptosis of SMMC-7721 cells. STAT5 siRNA may have potential application in the gene therapy of human hepatocellular carcinoma value.