胃肠道间质瘤c-kit基因突变体的研究

Cell transformation effect of mutant c-kit gene in gastrointestinal stromal tumor

目的:研究突变型c-kit基因对细胞增生及细胞周期的影响从而揭示突变型c-kit基因对人体细胞的恶性转化作用. 方法:将构建于pcDNA3的突变型c-kit cDNA和野生型c-kit cDNA重组质粒及空白pcDNA3质粒(空白对照)通过脂质体法稳定转染人胚胎肾细胞(human embry- onic kidney cell.HEK)细胞.用Western blot方法检测c-kit蛋白的表达情况,观测转染细胞的生长曲线变化。用MTT法检测细胞增生活性的变化,并用流式细胞仪检测c-kit基因突变体对细胞周期的影响. 结果:与阴性对照、空白对照相比,实验组细胞生长速度明显增快,细胞增生活性明显增强,细胞周期检测显示:实验组和阳性对照组处于增生期细胞(S+G2-M) 比例明显高于阴性对照组和空白对照组,各组处于增生期的细胞比例依次为:48.34%(实验组)、48.2496(阳性对照组)、42.03%(阴性对照组)、42.16%(空白对照组). 结论:c-kit基因突变可使人类细胞的增生活性明显增强, 并可以使更多的细胞由静止期进入增生期,提示c-kit基因突变有可能是引起GIST的恶性转化的关键机制之一.

AIM: To investigate the implication in tumorigenesis of a novel c-kit gene mutant, which was identified recently in gastrointestinal stromal tumor (GIST), by examining its effect on cell proliferation and cell cycle. METHODS: Recombinant plasmids, which contained mutant or wile-type okit gene, were stably transfected into human embryonic kidney (HEK) cells. The expression of c-kit protein was detected by Western blot. The proliferation and cell cycle of the transfected cells were detected by MTT colorimetic assay and flow cytometry, respectively. RESULTS: In comparison with the cells transfected with wild-type c-kit cDNA and empty pcDNAS vector, the proliferation of the cells transfected with mutant c-kit cDNA was increased significantly. The percentages of cells in proliferation phase (S+G2+M) were 48.34%, 48.24%, 42.03% and 42.16% in test, positive control, negative control and empty control group, respectively. CONCLUSION: The mutant c-kit gene can promote human cell proliferation, which may play an important role in the malignant transformation of GIST.