论文部分内容阅读
目的 探讨转染缺失突变的人端粒酶逆转录酶 (hTERT)基因对膀胱癌细胞株T2 4端粒酶活性和体外增殖的影响 ,为膀胱肿瘤基因治疗提供新的基因靶点。 方法 采用DNA 磷酸钙共沉淀法 ,将绿色荧光蛋白基因标记的含突变型hTERT真核表达载体 pEGFP hTERT导入人膀胱癌细胞株T2 4中。应用荧光显微镜、端粒酶PCR ELISA法、与衰老相关的 β 半乳糖苷酶染色、软琼脂集落形成试验、裸鼠皮下成瘤试验等方法动态观察转染细胞中端粒酶活性及对细胞恶性表型的影响。 结果 在转染 pEGFP hTERT细胞中可见与突变型hTERT基因融合的绿色荧光蛋白稳定表达于细胞核内 ,转染细胞端粒酶活性降低 ,衰老相关 β 半乳糖苷酶表达增加 ,软琼脂中集落形成减少 ,裸鼠成瘤性降低。与转染空载体组及未转染组细胞相比 ,差别有显著性意义 (P <0 .0 5 )。 结论 转染突变型人端粒酶逆转录酶基因hTERT能抑制膀胱癌细胞T2 4的端粒酶活性 ,促进其衰老并逆转膀胱癌细胞的恶性表型 ,对膀胱肿瘤基因治疗具有潜在的临床应用价值
Objective To investigate the effect of hTERT gene transfected with human telomerase reverse transcriptase (hTERT) gene on the telomerase activity and in vitro proliferation of human bladder cancer cell line T2 4 and to provide a novel gene target for bladder cancer gene therapy. Methods The human telomerase reverse transcriptase (hTERT) plasmid pEGFP hTERT labeled with green fluorescent protein (GFP) gene was transfected into human bladder cancer cell line T2 4 by DNA-calcium phosphate co-precipitation method. Using telomerase PCR ELISA, aging-related β-galactosidase staining, soft agar colony formation assay and subcutaneous tumor formation test in nude mice, we observed the telomerase activity and cell proliferation in malignant cells Phenotype effects. Results The green fluorescent protein fused with mutant hTERT gene was stably expressed in the nucleus of transfected pEGFP hTERT cells, the telomerase activity was decreased, the expression of aging related β-galactosidase increased, and the colony formation in soft agar decreased , Nude mice reduced tumorigenicity. Compared with the cells transfected with empty vector and untransfected cells, the difference was significant (P <0.05). CONCLUSION: Transfection of human telomerase reverse transcriptase gene hTERT can inhibit the telomerase activity of bladder cancer cell line T2 4, promote its senescence and reverse the malignant phenotype of bladder cancer cells, and has potential clinical application in gene therapy of bladder cancer value