摘要
目的:观察人截短型凋亡诱导因子(AIF△1-400)对HeLa细胞的促凋亡作用.方法:在AIF△1-120基因克隆成功的基础上进一步改造,构建截去AIF基因线粒体定位信号,FAD结合结构域和NADH结合结构域(1-400位氨基酸)编码序列的AIF△1-400基因,将其克隆入pcDNA3和pIRES2-EGFP真核表达载体,用脂质体法转染HeLa细胞,通过荧光显微镜观察,MTT检测等方法检测该基因在转染细胞中的表达及其对肿瘤细胞的促凋亡活性.结果:经酶切鉴定与测序证实,带有AIF△1-400的真核表达载体构建成功.瞬时转染后出现细胞形态变化,随转染时间的延长转染细胞数减少,荧光变弱.经间接免疫荧光检测可观察到截短型AIF蛋白位于细胞质中,部分已位于细胞核内.MTT法检测发现AIF△1-400转染后对细胞生长有明显的抑制作用.结论:AIF△1-400仍然具有诱导HeLa细胞凋亡的作用.
AIM： To observe the expression of mmcated human apoptosis-inducing factor （AIF） gene and its effect on HeLa cells. METHODS： After AIF△1 -120 gene was successfully cloned, the shorter truncated human AIF gene （AIF△ 1 -400） was constructed by deleting the N-terminal mitochondrial localization sequence （ MLS）, the coding sequence of flavine adenine dinucleotide （FAD） binding domain and nicotinamide adenine dinucleotide （NADH） binding domain of AIF protein. The truncated human AIF gene （AIF △1 -400） was inserted into the plRES2-EGFP and pcDNA3 eukaryotic expression vectors, and the co-expression vectors were then transfected into HeLa cells with LipofectAMINE. The expression of the truncated AIF gene and its effect on HeLa cells were detected by fluorescence microscope analysis and MTT test. RESULTS： The eukaryotic expression vector containing the truncated human AIF （ AIF △ 1 - 400 ） gene was successfully constructed. The AIF protein could be detected in the transfected HeLa cells. After transfection, typical cell apoptotic changes were observed under an electron microscope. MTT test showed that the proliferation of HeLa cells was significantly inhibited. CONCLUSION： The expression of the truncated human AIF （ AIF △ 1 - 400） gene can induce the apoptosis of the transfected human HeLa cells.
出处
《第四军医大学学报》
北大核心
2007年第7期577-580,共4页
Journal of the Fourth Military Medical University
基金
国家"973"计划资助项目(2004CB518805)
国家自然科学基金(30471988
30400403)
