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目的构建FOXO1真核表达质粒,明确FOXO1对TNF-α介导的Ⅱ型肺泡上皮细胞凋亡的影响及其可能存在的调控机制。方法根据Gen Bank中人FOXO1 CDS序列设计并合成引物,提取A549细胞总RNA,通过RT-PCR获得FOXO1目的基因片段;通过酶切、连接,构建GV230-FOXO1真核表达质粒并进行测序分析鉴定;经鉴定后的表达载体瞬时转染入A549细胞,荧光显微镜及Western blot法检验FOXO1蛋白表达。体外培养A549细胞,利用脂质体Lipofectamine~(TM) 2000将本次合成的GV230-FOXO1质粒转染入A549细胞,给予10 ng/ml TNF-α刺激24 h,流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白Bim的表达。结果成功构建GV230-FOXO1荧光真核表达质粒;FOXO1组细胞凋亡率明显高于TNF-α组和阴性对照组(P<0.05),FOXO1组蛋白Bim的表达明显高于TNF-α组和阴性对照组(P<0.05)。结论 FOXO1在TNF-α介导的A549细胞损伤中起促进细胞凋亡的作用,其作用机制可能为通过上调促凋亡蛋白Bim的表达,从而促进细胞凋亡。
Objective To construct the FOXO1 eukaryotic expression plasmid and investigate the effect of FOXO1 on TNF-α-mediated apoptosis of type II alveolar epithelial cells and its possible regulatory mechanism. Methods According to GenBank FOXO1 CDS sequence, primers were designed and synthesized. The total RNA of A549 cells was extracted and the FOXO1 gene fragment was obtained by RT-PCR. The eukaryotic expression plasmid of GV230-FOXO1 was constructed and identified by sequencing. The identified expression vector was transiently transfected into A549 cells, and the expression of FOXO1 protein was detected by fluorescence microscopy and Western blot. The A549 cells were cultured in vitro. The GV230-FOXO1 plasmid was transfected into A549 cells with lipofectamine TM 2000 and stimulated with 10 ng / ml TNF-α for 24 h. Flow cytometry was used to detect apoptosis Rate, Western blot detection of apoptosis related protein Bim expression. Results The GV230-FOXO1 fluorescent eukaryotic expression plasmid was successfully constructed. The apoptosis rate of FOXO1 group was significantly higher than that of TNF-α group and negative control group (P <0.05). The expression of FOXO1 histone Bim was significantly higher than that of TNF- Control group (P <0.05). Conclusion FOXO1 plays an important role in the apoptosis of A549 cells induced by TNF-α. The mechanism may be that FOXO1 promotes apoptosis by up-regulating the expression of Bim, a pro-apoptotic protein.