端粒酶逆转录酶基因hTERT突变表达载体在膀胱癌细胞T24中的表达  

Construction of recombinant vector of a mutant hTERT and its expression in human bladder carcinoma cell line T24

作　　者：符伟军[1] 黄君健[1] 白云秀[1] 严友农[2] 徐兵[1] 邵国兴[3] 王禾[3] 黄翠芬[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]沈阳军区202医院泌尿外科 [3]第四军医大学西京医院泌尿外科

出　　处：《第四军医大学学报》2002年第2期122-125,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金资助项目 ( 39870 783);国家 973资助项目 ( G2 0 0 0 0 5 70 0 1)

摘　　要：目的　构建端粒酶逆转录酶基因 (h TERT)缺失突变的真核表达载体 p EGFP- h TERT,并转入膀胱癌细胞株 T2 4中 ,观察其稳定表达 .探讨其对端粒酶活性调控机制及成为膀胱肿瘤基因治疗新靶点的可能性 .方法　将 PCR扩增的h TERT缺失突变体片段用 Eco RI与 Sal I酶切 ,并连接到真核表达载体 p EGFP- C1上 ,构建成 h TERT缺失突变的真核表达载体 p EGFP- h TERT;利用 DNA-磷酸钙共沉淀法将p EGFP- h TERT导入膀胱癌细胞株 T2 4中 ,应用荧光显微镜、与衰老相关的β-半乳糖苷酶染色等方法观察转染细胞中h TERT- GFP融合蛋白定位表达及对膀胱癌细胞生长的影响 .结果　酶切鉴定证实 h TERT缺失突变体已克隆到p EGFP- C1的 Eco RI与 Sal I位点之间 ,在转染细胞中观察到与其融合的绿色荧光蛋白的稳定表达 ,定位于细胞核内 ;转染细胞 2 wk后与衰老相关 β-半乳糖苷酶表达增加 ,细胞生长受抑制 .结论　构建的端粒酶逆转录酶基因 h TERT缺失突变真核表达载体 p EGFP- h TERT可在膀胱癌细胞 T2 4中稳定表达 .突变型 h TERT蛋白可入细胞核中竞争性影响端粒酶的功能 。AIM To construct a mutant pEGFP hTERT expression vector, to observe its steady expression in transfected human bladder carcinoma cell line T24 and its role in further study of molecular regulatory mechanisms of telomerase and a new target gene for bladder cancer. METHODS PCR amplification was performed by using primers based on the known gene sequence of hTERT, PCR product was cloned into plasmid pGEMT T easy and the sequence of mutant hTERT gene was analyzed. A recombinant mutant hTERT vector (pEGFP hTERT) was constructed at its EcoR I and Sal I sites. After transfecting it into bladder carcinoma cell T24 by the method of calcium phosphate DNA coprecipitation, we had detected the steady expression of GFP hTERT fusion protein by fluorescent light microscopy. The proliferation changes of bladder carcinoma cell line T24 were detected by light microscopy and β galactosidase stain correlated with senescence. RESULTS Identification of pEGFP hTERT by enzyme digestion showed that mutant hTERT fragment had been cloned into Eco RI and Sal I sites of pEGFP C1 vector. The steady expression of GFP hTERT fusion protein was localized in the nuclear of transfected cells. Positive expression senescence associated β galactosidase stain in transfected cells was gradully increased with extended cultured time and their growth was suppressed. CONCLUSION Recombinant mutant vector (pEGFP hTERT) is successfully constructed and expressed steadily in human bladder carcinoma cell T24. The mutant type hTERT gene suppresses the proliferation of bladder carcinoma cell T24 by competitive effect on telomerase activity.

关 键 词：膀胱肿瘤 端粒 转染 HTERT 端粒酶 基因表达 

分 类 号：R737.14[医药卫生—肿瘤]