过表达FAF1基因慢病毒构建及对人胃癌细胞凋亡影响

目的：构建过表达FAs相关因子1（fas-associatedfactorl，FAF1）基因的慢病毒载体，探讨其对胃癌细胞凋亡的影响。方法：RT-PCR法扩增FAF1cDNA，亚克隆至GV218质粒中，经酶切及DNA测序鉴定正确以后，把含有FAF1基因的重组质粒与pHelper1．0和pHelper2．0载体质粒转染至293T细胞包装慢病毒。用包装好的慢病毒感染人胃癌细胞HGD27，RealtimePCR和蛋白质印迹法检测转染前后FAF1基因和蛋白的表达，流式细胞仪检测细胞凋亡的改变。结果：FAF1基因扩增产物长度为1996bp，酶切及测序结果鉴定GV218-FAF1质粒构建正确，成功包装的慢病毒滴度为2×10^8TU／mL。HGC-27细胞转染FAF1过表达慢病毒48h后，细胞形态由长梭形变得不规则。过表达FAF1慢病毒转染组FAF1基因mRNA表达水平2．436±0．538，是阴性对照组1．086±0．226的2．24倍，P〈0．001；是空载转染组1．182±0．381的2．06倍，P〈0．001。过表达FAF1慢病毒转染组FAF1蛋白相对表达水平1.515±0．377，显著高于阴性对照组的0，P〈0．001；也显著高于空载转染组的0，P〈0．001。过表达FAF1慢病毒转染组凋亡率（84．66±5．92）％显著高于阴性对照组的（4．60±3．80）％，P〈0．001；也显著高于空载转染组的（7．32±3．82）％，P〈0．001。结论：成功构建并鉴定FAF1基因过表达慢病毒载体，FAF1基因表达上调使胃癌细胞凋亡增加。

Construction of overexpression lentivirus of Fas associated factor 1 and its effects on apoptosis of human gastric cancer cell line

OBJECTIVE：To construct the overexpression lentivirus of Fas-associated factorl and explore its effect on apoptosis of human gastric cancer cell line. METHODS：FAF1 cDNA was amplified by RT-PCR, and subcloned into GV218 plasmid. After the constructed plasmid was identified by restriction analysis and sequencing, the G218 plasmid containing FAF1 gene and pHelperl. 0 plasmid and pHelper2.0 plasmid transfected to 293T cells for packing lentivirus. Then FAF1 overexpression lentivirus transfected in human gastric cancer cells HGC-27,FAF1 gene and protein expression levels were detected by Real time PCR and western blot before and after transfection, and flow cytometry was used to detect apoptosis rate. RESULTS：The amplification product length of FAF1 gene was 1 996 bp, the GV218-FAF1 plasmid was constructed correctly identified by restriction analysis and sequencing. Lentivirus was successfully packaged and the titer was 2 x 10^8 TU/mL. The morphology of HGC-27 ceils changed from long fusiform to irregular after transfected with overexpression FAF1 lentivirus for 48 h. The expression levels of FAF1 mRNA in overexpression FAF1 group and negative control group and vector control group were 2. 436±0. 538 and 1. 086±0. 226 and 1. 182±0. 381 respectively,The expression levels of FAF1 mRNA in overexpression FAF1 group was 2. 24 times higher than that in negative control group（P〈0. 001） ,and 2.06 times than that in vector control group（P〈0. 001）. The expression of FAF1 protein in overexpression group was 1. 515±0. 377 ,which was significantly higher than that in negative and vector control group （0, P〈0. 001）. Comparing with the negative control group（4.60±3.80） and vector control group（7.32±3.82）% ,the apoptosis rate ofoverexpression FAF1 group（84.66 ±5.92） % was significantly higher（P〈0. 001）. CONCLUSIONS： Overexpression lentivirus of FAF1 was successfully constructed and identified,the up-regulation of FAF1 expression can enhance gastric cancer cell＇s apoptosis capability.