1,25-二羟基维生素D_3协同转染反义端粒酶RNA诱导肝癌细胞凋亡的实验研究

Enhancement of 1,25-dihydroxyvitamin D_3 induced apoptosis through the down regulation of telomerase activity bytransfected antisense telomerase RNA into hepatocarcinoma cells

目的观察在抑制肝癌细胞端粒酶活性表达情况下,1,25-二羟基维生素D3(1,25-VD3)对肝癌细胞凋亡的影响。方法经脂质体介导,将反义端粒酶RNA真核表达载体pBBS212/hTR导入维生素D3受体(VDR)阳性的肝癌细胞株SMMC7721细胞中培养。细胞克隆转移扩大培养,将其分为对照组、药物组、转染组和转染药物组。添加1,25-VD3,分别作用于转染药物组、药物组,用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)和电子显微镜检测肝癌细胞凋亡。结果转染组和对照组细胞的端粒酶活性分别为0.686和1.685,说明反义端粒酶RNA可显著降低肝癌细胞端粒酶活性。对照组、药物组、转染组和转染药物组细胞凋亡率分别为3.4%、12.2%、8.8%、23.6%;差异有显著统计学意义(P<0.01)。超微结构检查也证实存在凋亡发生。1,25-VD3或转染反义端粒酶RNA对肝癌细胞有促细胞凋亡作用,两者协同对肝癌细胞凋亡作用显著增强。结论转染反义端粒酶RNA,可降低肝癌细胞端粒酶活性,可显著增强1,25-VD3对肝癌细胞的促凋亡作用,并且对细胞的增殖也存在明显的抑制作用。

Objective To observe the effect of 1,25-dihydroxyvitamin D3（1,25-VD3） on hepatocarcinoma cell apoptosis after blocaking telomerase activity by antisense telomerase RNA.Methods The pBBS 212/hTR,antisense telomerase RNA gene expression vector was introduced into SMMC7721 cells with vitamin D receptor（VDR） by using lipofectin mediation.After the transfected cells were screened by hygromycin and cloned,divided into control group,drug group,transfected group and drug transfected group.The effects of antisense telomerase RNA on telomerase activity of SMMC7721 cells were observed by TRAP-PCR-ELISA,then the transfected cells were treated with 1,25-VD3.The terminal-deoxynucleotidyl transferase mediated nick end labeling（TUNEL） and electron microscope were adopted to detect the apoptosis in cells.Results The TRAP-PCR-ELISA showed the telomerase activity in transfected cells and control group was 0.686 and 1.685 respectively,suggested that antisense telomerase RNA had a significant inhibition effect on telomerase activity of SMMC7721 cells.The apoptosis rated of control group,drug group,transfected group and drug transfected group were 3.4 %,12.2 %,8.8 %,23.6 %,respectively.The statistically significant difference was found（P 0.01）,and the apoptosis of SMMC7721 cells was further confirmed by ultrastructural examination.On the other hand,the apoptosis by 1,25-VD3-induced was significantly enhancement after inhibition the expression of telomerase activity in SMMC 7721 cells with transfected antisense telomerase RNA.Conclusion It is demonstrated that antisense telomerase RNA can decrease the telomerase activity of hepatocarcinoma cells,and that inhibition of telomerase activity could significant enhance the cell apoptosis and inhibit cell proliferation by 1,25-VD3-induced in hepatoca-rcinoma cells.