溶瘤腺病毒介导RNA干扰人端粒酶逆转录酶抑制HeLa细胞生长

Inhibitory Effects of Oncolytic Adenovirus with hTERT Small Interference RNA on Proliferation of Human Cervical Cancer HeLa Cells

目的观察表达人端粒酶逆转录酶siRNA(hTERT-siRNA)的溶瘤腺病毒ZD55-TERT-siRNA对体外培养人宫颈癌HeLa细胞生长及hTERT基因表达的影响。方法以溶瘤腺病毒ZD55-EGFP、表达hTERT-siRNA的增殖缺陷腺病毒AD-TERT-siRNA和增殖缺陷腺病毒AD-EGFP作为对照组。不同滴度四种病毒分别感染HeLa细胞,结晶紫法检测细胞毒作用,MTT法测定细胞生长抑制率;RT-PCR、Western blot、免疫细胞化学(ICC)法分别检测hTERT的mRNA、蛋白质及抗原表达,Western blot和ICC法分别测定腺病毒E1A蛋白及抗原表达。结果对HeLa细胞的生长抑制作用和细胞毒作用ZD55-TERT-siRNA>AD-TERT-siRNA和ZD55-EGFP>AD-EGFP;ZD55-TERT-siRNA和ZD55-EG-FP感染的HeLa细胞表达E1A;抑制hTERT表达作用依次为ZD55-TERT-siRNA>AD-TERT-siRNA>ZD55-EGFP、AD-EGFP。结论溶瘤腺病毒介导RNA干扰hTERT能有效抑制HeLa细胞生长及hTERT基因表达。

Objective To study the effects of suppressing cell proliferation and hTERT gene expression on HeLa cells with ZD55-TERT-siRNA,an oncolyic adenovirus associated small interference RNA targeting hTERT gene.Methods ZD55-EGFP,an E1b-55kD gene-deleted oncolytic adenovirus,AD-TERT-siRNA,a replication-deficient adenovirus expressing hTERT-siRNA and AD-EGFP,a replication-deficient adenovirus were used as control groups.Human cervical carcinoma HeLa cells were infected by these four kinds of adenoviruses using different titers respectively.Cytotoxicity was tested by crystal violet staining.Cell proliferation was assayed by MTT method.hTERT expression levels of transfected HeLa cells were detected by RT-PCR,Western blot and immunocytochemistry respectively.The expression of E1A protein was examined by immunocytochemistry and Western blot.Results The cytotoxicity and inhibitory effects of ZD55-TERT-siRNA on HeLa cells were the most significant while ZD55-EGFP and AD-TERT-siRNA were more potent than AD-EGFP.Adenoviral particles could be detected in tumor cells infected by ZD55-TERT-siRNA and ZD55-EGFP,but not in the cells infected with AD-TERT-siRNA and AD-EGFP can not.The most significant inhibition of hTERT expression were observed in lysates from HeLa cells infected with ZD55-TERT-siRNA.Conclusion ZD55-TERT-siRNA has the most significant antitumor effect and can significantly knockdown hTERT expression.