PIAS3绿色荧光蛋白表达载体构建及其对胶质瘤U251细胞增殖和凋亡的影响

目的：构建PIAS3绿色荧光蛋白真核表达载体,探讨外源性PIAS3对胶质瘤细胞U251周期和凋亡的影响。方法：利用RT-PCR扩增人全长PIAS3编码序列,并将其克隆到带有绿色荧光蛋白的真核表达载体pEGFP-N1中,得到重组质粒pEGFP-N1-PIAS3。应用脂质体法转染体外培养的人胶质瘤U251细胞株。荧光显微镜观察蛋白表达,用RT-PCR和Westernblot检测PIAS3的表达,Annexin V2FITC和PI双染流式细胞术检测细胞凋亡,流式细胞术检测细胞增殖周期变化。结果：转染PIAS3基因的细胞株外源目的基因和蛋白表达增强,瞬时转染48 h后,光镜下可见部分细胞变圆,部分细胞脱落死亡;对照组和未转染组无明显变化。流式细胞仪分析显示,pEGFP-N1-PIAS3转染组凋亡细胞增多,细胞凋亡率显著高于未转染组和pEGFP-N1转染组,P=0.0073;U251细胞转染pEGFP-N1-PIAS3后细胞周期阻滞于S期,S期细胞比例增高,P=0.025,G2期细胞比例降低。结论：外源性PIAS3使U251阻滞在S期,诱导胶质瘤细胞凋亡。

Construction of PIAS3/EGFP eukaryotic expression plasmid and its effects on cell cycle and apoptosis of glioma cell line U251

OBJECTIVE: To construct PIAS3/EGFP(enhanced green flurescent p rotein) eukaryotic expression plasmid,and investigate the effects of exogenous PIAS3 on cell cycle and apoptosis in human glioma cell line U251.METHODS: Standard RT-PCR was used to amplify the full-length coding sequence of PIAS3.The amplified PIAS3 gene was cloned into the eukaryotic expression vector pEGFP-N1,generating pEGFP-N1-PIAS3.Human glioma cell line U251 was transfected with pEGFP-N1-PIAS3 or mock pEGFP-N1 with lipofectamine.The protein expression was observed by a fluorescence microscope.RT-PCR and Western blot were used to determine target gene expression.Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI.Flow cytometry was also used to assay the cell cycle of the transfected cells by pEGFP-N1-PIAS3 and pEGFP-N1.RESULTS: The expression of PIAS3 mRNA and protein was increased in the cells transfected by pEGFP-N1-PIAS3.The rounding and floating cells were observed by a microscope after transient transfection 48 h,while there were no changes in the mock-transfected and non-transfected cells.After transfection,the cells of apoptosis increased.The transfected cells showed an increase in the rate of apoptosis,compared with non-transfected and mock-trasfected cells(P=0.007 3).The transfected cells were blocked at S phase of cell cycle.The percentage of G2 phase in the PIAS3-transfected cells was lower than that in the mock-transfected and non-transfected cells,while the percentage of S phase was higher,and the difference was significant(P=0.025).CONCLUSION: Exogenous PIAS3 can block U251 cell cycle at the S phase,induce the apoptosis of glioma cells.