胃肠道间质瘤中C-kit原癌基因突变体的构建及功能研究

Construction of recombinant plasmids with mutant C-kit cDNA in gastrointestinal stromal tumor

目的:构建含突变C-kit基因cDNA的真核表达质粒并行进一步的功能分析。方法:用RTPCR方法从人胎脑组织克隆野生型C-kit基因蛋白编码区cDNA。根据胃肠道间质瘤中检测出的Ckit基因突变序列,体外突变野生型CkitcDNA得到突变型CkitcDNA,与pcDNA3重组成真核表达质粒,并用脂质体法稳定转染人胚胎肾细胞(humanembryonickidneycell,HEK)293细胞系,G418加压筛选出阳性克隆;用Western印迹法检测转染细胞KIT蛋白表达,绘制细胞生长曲线,MTT法检测细胞增殖活性,流式细胞仪检测转染HEK细胞后细胞周期改变,并观察稳定转染突变型重组质粒人胚肾细胞在裸鼠内的成瘤性。分别设转染pcDNA3空白质粒和野生型的CkitcDNA真核表达质粒的HEK细胞做为对照。结果:构建了突变型C-kitcDNA与pcDNA3的真核表达质粒;功能检测分析表明:细胞生长曲线显示与对照组相比,实验组的生长速度明显增快;MTT比色显示,与对照组相比实验组细胞增殖活性显著增强;细胞周期检测结果显示,处于增殖期细胞(S+G2-M)比例显著高于对照组;体内结果显示转染突变型C-kit基因的HEK细胞在裸鼠体内成瘤。结论:C-kit原癌基因突变体可促使人类细胞生长加快,增殖活性明显增强,并可以使更多的细胞由静止期进入增殖期,并可使人胚胎肾细胞发生恶性转化,提示C-kit基因突变有可?

Objective:To construct the recombinant eukaryotic expression vector plasmids with mutant C-kit cDNA and to study the effect of the mutant C-kit gene on cell proliferation and cell cycle in gastrointestinal stromal tumor (GIST). Methods: Wild-type C-kit cDNA was cloned from human embryonic brain tissue by RT-PCR technique.Site-directed mutagenesis of the wild type C-kit cDNA was performed according to the C-kit mutations we cloned. Recombinant plasmids were stably transfected into human embryonic kidney cell line and the cells expressing mutant C-kit were selected by special cell culture medium containing G418.Expressions of C-kit protein of the transfectants were detected by Western blot.Cell proliferation and cell cycle of the transfectants were detected by MTT clolorimetic assay and flow cytometry,respectively.Whether HEK cell with mutated C-kit cDNA could grow autonomously in nude mice or not was also detected. pcDNA3 vector transfected and recombinant plasmids with wild-type C-kit cDNA transfected HEK cell were used as the control groups. Results: The mutant C-kit cDNA was obtained by site-directed mutagenesis of the wild type C-kit cDNA. Compared with the 2 control groups ,the growth rate and proliferative activity of the HEK cells with mutant C-kit cDNA were increased significantly.The analysis of cell cycle showed that more HEK cells with mutanted C-kit cDNA remained in proliferation phase (S+G 2-M)than the groups without mutated C-kit cDNA.HEK cells with the mutated C-kit also grew autonomously in nude mice.Conclusion: Mutation of C-kit gene can increase proliferation of human cells,causing malignant transformation of human normal cells,which may play an important role in the malignant transformation of GIST.