CD59基因突变在肿瘤逃逸中的作用

THE ROLE OF CD59 GENE MUTATION IN TUMOR ESCAPING

目的构建CD59突变的重组体，研究Hela细胞中CD59基因突变引起肿瘤凋亡相关分子caspase-3表达的变化。方法采用重组聚合酶链反应定点诱变技术，将CD59的第39～41位氨基酸突变为色氨酸，克隆入pALTER—MAX质粒，采用阳离子脂质体法将重组质粒转染Hela细胞。G418筛选稳定表达细胞克隆，用免疫荧光、ELISA、流式细胞术筛选出高表达突变CD59的细胞株，用免疫组化法检测转染前后Hela细胞内caspase-3表达的变化。结果酶切鉴定和序列测定均证实成功构建了CD59氨基酸突变的重组质粒。转染突变CD59后Hela细胞内caspase-3表达明显减少，与转染正常CD59的Hela细胞比较差异有显著意义（t=2．846，P〈0．01）。结论caspase-3表达变化可能是CD59引起肿瘤逃逸的另一途径。

Objective To construct mutant CD59 molecular, and study the alleosis in the expression of easpase-3 correlative with tumor apoptosis brought about by CD59 mutation in Hela cells. Methods Using polymerase chain reaction site-directed mutagenesis, the amino acid of the 39th and the 41th mutated to tryptophanes. Mutant CD59 DNA inserted into the vector pALTER-MAX and transfeeted into the Hela cells via lipofectamine method. Stable expression clones were selected by the addition of G418. The G418-resistant clones which expressed relatively high levels of mutant CD59 were sorted by immunofluorescenee method, ELISA and flow eytometry. The expression of easpase-3 in Hela cells before and after transfeetion was tested immunc-histochemically. Results Recombinant plasmids of pALTER-MAX-CD59 had been successfully constructed according to sequence of enzyme digestion analysis and fragment investigation. The amount of easpase-3 in Hela cells which were transfeeted was obviously lower than that in Hela cells which were transfeeted with nomal CD59, and the difference was obvious （t= 2. 846, P〈0.01）. Conclusion Another way by which CD59 leads the tumor escaping may be the expression changing of easpase-3.