摘要
目的探讨RNA干扰技术抑制survivin基因表达对人肝癌细胞株HepG2细胞增殖的影响。方法构建针对人肝癌HepG2细胞survivin基因3个不同靶序列的shRNA真核表达载体pshRNA-survivin-323/387/52,用Lipofectamine 2000脂质体介导转染法转染质粒,采用逆转录聚合酶链反应(RT-PCR)技术检测survivin基因mRNA表达水平。选择对survivin基因表达抑制作用最强的shRNA载体转染HepG2细胞后,免疫细胞化学法(ICC)检测HepG2细胞survivin蛋白的表达情况,四甲基偶氮唑蓝(MTT)法检测各组细胞的增殖情况。结果与对照组比较,构建的3个shRNA质粒中pshRNA-survivin-387转染组survivin mRNA水平下调,差异有显著性(F=18.64,q=4.293～4.925,P<0.05),而pshRNA-survivin-323/52质粒对survivin的表达及mRNA水平的影响差异无显著性(q=0.631～0.126,P>0.05)。转染pshRNA-survivin-387质粒的HepG2细胞survivin蛋白表达较对照组明显下调(F=68.39,q=13.71、14.37,P<0.01),其增殖能力较对照组显著下降(F=20.06～47.65,q=11.186、12.601,P<0.01)。结论构建的针对人survivin基因387位靶序列的shRNA表达载体pshRNA-sur-vivin-387可显著抑制survivin的表达及HepG2细胞增殖。
Objective To study the inhibition of RNA interference targeting the expression of survivin gene, and assess its effects on proliferation of hepatocellular carcinoma cell line HepG2. Methods Three different-surviving-gene sequence specific shRNA vectors pshRNA-survivin-323/387/52 were constructed. Transfection of shRNA vectors was performed using Lipofectmine 2000 liposome. The expression of survivin mRNA was detected by RT-PCR. The vector which could significantly down-regulate the expression of survivin was transfected into HepG2 cells by Lipofectmine 2000 liposome. Immunocytochemistry was employed to detect the expression of survivin protein after transfection. Proliferation of HepG2 cells was assessed by MTT. Results The pshRNA-survivin-387 vector targeting survivin gene significantly down-regulated the expression of its mRNA in HepG2 cells as compared with that of the controls （F=18.64,q=4. 293-4. 925,P〈0.05）. The other vectors targeting survivin gene had no influence on survivin gene expression （P〉0.05）. After transfection with pshRNA-survivin-387 vector, the expression of survivin was obviously down-regulated （F=68.39;q=13.71, 14.37;P〈0. 01） and the proliferation of HepG2 cells inhibited （F=20. 06- 47.65;q=11. 186,12. 601;P〈0.01）. Conelusion The pshRNA-survivin-387 vector targeting survivin gene could specifically suppress its expression and inhibit the proliferation of HepG2 ceils.
出处
《齐鲁医学杂志》
2009年第3期199-201,204,共4页
Medical Journal of Qilu
