反义抑制Survivin表达对槲皮素诱导肝癌SSMC-7721细胞凋亡的影响

Effect of survivin expression inhibition with antisense oligodeoxynucleotides compound on apoptosis of hepatocellular carcinoma cell line SSMC-7721 induced with quercetin

目的:采用Survivin反义寡核苷酸(ASODN)复合物抑制survivin的表达,研究其对槲皮素诱导肝癌SSMC-7721细胞凋亡的影响.方法:体外培养人肝癌SSMC-7721细胞,取对数生长期细胞进行实验.实验分Ⅰ,Ⅱ两部分:Ⅰ分为空白对照组(设平行组)、等量脂质体LipofectamineTM2000对照组、400μmol/L SODN复合物组、400μmol/L ASODN复合物组(设平行组),转染48h;Ⅱ弃掉Ⅰ部分各组培养液,换新指定培养液,分为空白对照组、40μmol/L槲皮素组、脂质体Lipo-fectamineTM2000组、SODN复合物组、ASODN复合物组、ASODN复合物+40μmol/L槲皮素组(联合组),孵育细胞24h后检测.Ⅰ结束时采用RT-PCR、细胞免疫组化染色检测各组SSMC-7721细胞survivin表达变化;Ⅱ结束时用吖啶橙染色法观察细胞凋亡时形态变化,MTT法检测SSMC-7721细胞生长抑制率,Annexi-V/PI双染流式细胞仪检测SSMC-7721细胞凋亡率.结果:ASODN复合物作用肝癌SSMC-7721细胞48h后能下调survivin mRNA及Survivin蛋白的表达(P<0.01);与其他组比较,联合组AO染色图片可观察到凋亡小体等典型的细胞凋亡形态特征变化且凋亡细胞数量明显增多,并用MTT法、Annexin-V/PI双染流式细胞仪检测显示联合组细胞的生长抑制率增高(P<0.01)、细胞凋亡率提高(P<0.01).结论:ASODN复合物可有效抑制肝癌SSMC-7721细胞sur-vivin基因的表达;ASODN复合物能增强槲皮素对SSMC-7721细胞增殖抑制及诱导凋亡的作用即二者具有协同作用,该作用可能与抑制survivin基因的表达有关.

AIM ： To inhibit the expression of survivin with antisense oligodeoxynucleotides （ASODN） compound, and then to study its effect on apoptosis of hepatocellular carcinoma cell line SSMC-7721 induced with quercetin. METHODS： Human hepatocellular carcinoma cell line SSMC-7721 was cultured in vitro, and the cells in logarithmic growth phase were used. The whole experiment was divided into two parts - Ⅰ and Ⅱ. In part Ⅰ , the cells were divided into blank control group, Lipofectamine^TM2000 group, 400 μmol/L SODN compound group, 400 μmol/L ASODN compound group, and the ODN compound was transfected into SSMC-7721 cells mediated by Lipofectamine^TM2000 for 48 h; In part Ⅱ, the cells were divided into blank control group, 40 μmol/ L quercetin group, Lipofectamine^TM2000 group, SODN compound group, ASODN compound group, ASODN compound combined with quercetin group. The changes of survivin protein and mRNA expression at the end of part Ⅰ were assessed by cell immunohistochemistry and reverse transcription-polymerase chain reaction （RT-PCR） method. At the end of part Ⅱ, the changes of morphology of SSMC-7721 cells were observed with AO staining, and the growth inhibition rate was measured by MTT, and the apoptosis rate of SSMC-7721 cells was measured by the flow cytometry with Annexi-V/PI staining. RESULTS： The expressions of survivin mRNA and its protein were significantly down-regulated by transfecting ASODN compound for 48 h （ P 〈 0.01 ）. In ASODN compound combined with quercetin group： the typical morphological changes of cell apoptosis were observed; and the apoptotic cell number was increased significantly; The growth inhibition rate and the apoptosis rate of SSMC-7721 cells were higher significantly than that in other groups （P 〈0.01 ）. CONCLUSION： ASODN compound can efficiently inhibit expression of survivin gene. Inhibiting the expression of survivin in SSMC-7721 cells with ASODN compound could promote the growth inhibition and apoptosis of the cells induced with quercetin, which may be related to its inhibitory effect on expression of survivin gene.