p27^(kip1)真核表达载体的构建及其对人胆管癌细胞生长的影响

Transfection of p^(27kip1) Gene and Its Effect on the Growth of Cholangiocarcinoma Cell Lines

目的：构建p27^(kip1)真核表达载体并进行基因转染，研究其对人胆管癌细胞生长特性的影响。方法：将人p^(27kip1) cDNA克隆至真核表达载体pClneo的Not I酶切位点中，然后应用DOTAP脂质体将重组质粒转染人胆管癌QBC939细胞中，用G418筛选抗性细胞克隆，采用PCR、RT－PCR和Western印迹法检测外源性目的基因在靶细胞基因组中的整合及表达，以及转基因细胞对裸鼠致瘤性能力的改变。结果：成功构建p^(27kip1)基因真核表达载体，并将其导入QBC939细胞中。经G418筛选得到稳定表达p^(27kip1)基因的细胞株，p^(27kip1)基因转染细胞生长速度明显减慢，并出现分化的迹象，其裸鼠致瘤性能力明显降低。结论：p^(27kip1)基因具有抑癌基因的作用，在未来胆管癌的基因治疗中可能具有应用前景。

Objective To evaluate the effects of exogenous p^(27kip1) gene on the growth of cholangiocarcinoma cell lines by gene transfection． Methods The eukaryotic expression vector with p^(27kip1) gene were transfected into QBC939 cells by DOTAP liposomal transfected system． The cells resistant to G418 were selected． The p^(27kip1) and neo expression were detected by PCR.RT－PCR and Western blotting． The cell growth and differentiation were observed by MTT method and electronic microscopy． Results The constructed p^(27kip1) eukrayotic expression vector was proved to be the same as that designed by restriction endonuclease analysis． Overexpression of p^(27kip1) in human cholangiocarcinoma cells resulted in cell growth arrest and induced cell differentiation． QBC939 transfected with p^(27kip1) gene showed reversion of tumorgenicity in nude mice． Conclusion The results suggest that p^(27kip1) gene could be a new tumor suppress gene or in tumor gene therapy．