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目的:探讨人端粒酶逆转录酶基因(hTERT)启动子调控的野生型p53基因的靶向性表达,观察对人膀胱癌细胞株T24的选择性杀伤效应及细胞凋亡的影响。方法:采用脂质体转染法,将构建的含hTERT启动子调控表达人野生型p53基因和报告基因绿色荧光蛋白(GFP)的质粒,分别转染膀胱癌细胞株T24和正常人胎肺成纤维细胞,应用荧光显微镜、电镜、台盼蓝拒染法及流式细胞仪等方法,观察基因的靶向性表达及对膀胱癌细胞株T24细胞形态学、生长抑制曲线及细胞周期变化的影响。结果:转染hTERT启动子调控的目的基因可在端粒酶阳性的膀胱肿瘤细胞中靶向性表达发出绿色荧光。靶向转染野生型p53基因能抑制膀胱癌细胞生长,72h后细胞生长抑制率分别为48.5%、高于常规培养组3.6%和阴性对照组2.5%,差异有显著性意义(P<0.05)。电镜可见典型的凋亡细胞。细胞周期分析G0和G1期细胞比例明显增高,并出现凋亡峰。结论:构建的hTERT启动子调控表达的野生型p53基因能通过诱导肿瘤细胞凋亡而发挥靶向性抑制膀胱癌细胞生长作用。
Objective: To investigate the targeted expression of wild type p53 gene regulated by human telomerase reverse transcriptase gene (hTERT) promoter and to observe the selective killing effect and apoptosis on human bladder cancer cell line T24. Methods: The recombinant plasmid containing hTERT promoter and wild type p53 gene and green fluorescent protein (GFP) gene was transfected into bladder cancer cell line T24 and normal human fetal lung Fibroblasts were used to observe the effects of targeted gene expression and morphological changes, growth inhibition curve and cell cycle of bladder cancer cell line T24 by fluorescence microscope, electron microscope, trypan blue exclusion method and flow cytometry . Results: The target gene regulated by hTERT promoter could emit green fluorescence in telomerase positive bladder tumor cells. Targeting transfected wild type p53 gene could inhibit the growth of bladder cancer cells, and the inhibition rate of growth after 72h was 48.5%, which was higher than 3.6% of the control group and 2.5% of the negative control group (P <0.05) . Electron microscopy shows typical apoptotic cells. Cell cycle analysis of G0 and G1 phase cells was significantly increased, and the peak of apoptosis. CONCLUSION: The constructed wild type p53 gene regulated by hTERT promoter can exert a targeted inhibitory effect on the growth of bladder cancer cells by inducing tumor cell apoptosis.