中文摘要：

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LipofectamineTM 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。

英文摘要：

This study aimed to construct eukaryotic expression plasmid GV230-FOXO1,and identify its effects on TNF-α-mediated apoptosis in alveolar epithelial.According to the sequence of FOXO1 CDS in Genbank,a pair of primers were respectively designed and synthesized,and the total RNA was isolated from A549 cells.After amplification with reverse transcription polymerase chain reaction（RT-PCR）,the product was cloned into GV230 vector,which then identified by PCR,double restrictive edonuclease digestion and sequence analysis.The constructed recombinant expression plasmid was transferred into A549 cells,and the FOXO1 protein expression was identified by Western blotting.By using Lipofectamine~（TM） 2000,GV230-FOXO1 plasmid was transfected to A594 cells,which then stimulated with TNF-α for 24 hours.Subsequently,the apoptosis rates and the relative levels of Bim protein were separately dectected by flow cytometry and Western blotting.Data showed that the GV230-FOXO1 eukaryotic expression plasmid was constructed successfully;the apoptosis rate of FOXO1 group was significantly higher than that of TNF-α group and negative control group（P〈0.05）;and the relative level of Bim protein in FOXO1 group was significant higher than that of TNF-α group and negative control group（P〈0.05）.In conclusion,FOXO1 can promote TNF-α-mediated A549 cell apoptosis by upregulating pro-apoptosis protein Bim.