hTERT启动子调控的野生型p53基因对膀胱癌细胞的靶向性抑制作用
Inhibitory effect of wild-type p53 gene expression driven by hTERT promoter in targeting gene therapy for human bladder transitional cell carcinoma
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摘要: 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。Abstract: Objective: To investigate the growth inhibition effects of the wild-type p53 gene vector controlled by human telomerase reverse transcriptase (hTERT) promoter on targeting gene therapy of bladder carcinoma. Methods: Human bladder cancer cell line T24 and fetus pulmonary fibroblast was transfected with EGFP as reporter gene and wild-type p53 gene as therapeutic gene under control of hTERT promoter. The gene expression was measured of EGFP by fluorescent microscopy; the cell growth was evaluated by trypan blue dye exclusion method, the morphological changes and apoptosis detection were observed with inverted and transmission electron microscope (TEM), and changes of apoptosis rate and cell cycle were assessed by flow cytometry (FCM), respectively.Results: Expression of EGFP driven by hTERT gene promoter was detected in T24 cells with telomerase activity. Recombinant adenovirus phTERT-p53 could selectively infect T24 cells, and the growth of T24 cells was significantly suppressed(P<0.05), the cell inhibition rate was raised to 48.5% after being transfected with phTERT-p53 for 72h, and the apoptosis was detected. There were increased proportions of cells in the G0/G1 phase of the cell cycle.Conclusion: The growth of the bladder cancer cells could be inhibited by the wild-type p53 target gene expression under control of the hTERT promoter through apoptosis, which may well be one of the ideal strategies for targeted gene therapy for bladder carcinoma.
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