构建携带人GFAP启动子和p27基因的重组腺病毒载体及其转染对星形胶质细胞增殖的影响  

Construction of Recombinant Adenovirus Vector Carrying Human GFAP Promoter and p27 Gene and Its Transfection Effect on Astrocyte Proliferation

作　　者：刘娜[1] 聂璐薇 姚达宝 陈施玲 潘超[1] 徐莉 LIU Na;NIE Luwei;YAO Dabao;CHEN Shiling;PAN Chao;XU Li(Department of Neurology,Tongji Hospital,Tongji Medical College,Huazhong Uni-versity of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院神经内科,武汉430030

出　　处：《神经损伤与功能重建》2023年第10期564-568,573,共6页Neural Injury and Functional Reconstruction

基　　金：湖北省自然科学基金青年基金项目(No.2019CFB113)。

摘　　要：目的:构建携带人GFAP启动子和p27基因的重组腺病毒载体并进行鉴定,研究其转染星形胶质细胞后对细胞生长增殖的影响。方法:对实验室原有的pGFAP-IRES2-EGFP-p27质粒进行转化、扩增、抽提、酶切后进行琼脂糖凝胶电泳鉴定和测序;通过设计引物,进行PCR扩增后分别获取GFAP启动子和p27基因片段,依次交换入线性化表达载体GV269和p DC315-GFAP-EGFP载体,最终构建pDC315-GFAP-p27-EGFP;将其转染HEK293细胞包装成重组病毒颗粒,经在HEK293细胞反复扩增数代后,进行纯化及滴度检测;转染星形胶质细胞后观察细胞生长和荧光表达情况;检测转染后星形胶质细胞细胞周期和凋亡比例、p27的表达情况。结果:经酶切鉴定和测序,成功鉴定真核表达载体pGFAP-IRES2-EGFP-p27;经PCR鉴定和测序,成功构建重组腺病毒过表达载体pDC315-GFAP-p27-EGFP;经包装后,Ad-GFAP-p27-EGFP可见EGFP表达和细胞致病效应,而且能够特异性地在星形胶质细胞中表达绿色荧光蛋白;经流式细胞仪分析,转染星形胶质细胞72 h后,与对照组相比,转染组处于S期的细胞比例下降,G1和G2期的细胞比例增高,凋亡比例增高(P<0.05);转染后第5天,星形胶质细胞中p27蛋白表达水平较对照组有明显升高(P<0.05)。结论:利用腺病毒包装系统AdMax成功构建了携带人GFAP启动子和p27基因的重组腺病毒过表达载体,其可以特异性转染星形胶质细胞过表达p27,对星形胶质细胞的生长增殖有明显抑制作用。Objective:To construct a recombinant adenovirus vector carrying the human GFAP promoter and p27 gene and identify its effects on cell growth and proliferation after transfecting into astrocytes.Methods:The original pGFAP-IRES2-EGFP-p27 plasmid in the laboratory was transformed,amplified,extracted,digested,identified and sequenced by agarose gel electrophoresis.By designing primers and conducting PCR amplification,GFAP promoter and p27 gene fragments were obtained,and then exchanged into linearized expression vectors GV269 and pDC315-GFAP-EGFP,respectively.Finally,pDC315-GFAP-p27-EGFP was constructed.Transfect it into HEK293 cells and package it into recombinant virus particles.After repeated amplification of several generations in HEK293 cells,purify it and detect its titer.Observe cell growth and fluorescence expression after transfecting astrocytes.Detect the cell cycle and apoptosis ratio of astrocytes after transfection,as well as the expression of p27.Result:After enzyme digestion identification and sequencing,the eukaryotic expression vector pGFAP-IRES2-EGFP-p27 was successfully identified.After PCR identification and sequencing,the recombinant adenovirus overexpression vector pDC315-GFAP-p27-EGFP was successfully constructed.After packaging,Ad-GFAP-p27-EGFP showed EGFP expression and cytopathic effects,and was able to specifically express green fluorescent protein in astrocytes.According to flow cytometry analysis,72 hours after transfecting astrocytes,compared with the control group,the proportion of cells in the S phase decreased in the transfection group,while the proportion of cells in the G1 and G2 phases increased and the proportion of apoptosis increased(P<0.05).On the 5th day after transfection,the expression level of p27 protein in astrocytes significantly increased compared to the control group(P<0.05).Conclusion:A recombinant adenovirus overexpression vector carrying the human GFAP promoter and p27 gene was successfully constructed using the adenovirus packaging system AdMax.It can specifically

关 键 词：P27 GFAP启动子 细胞周期 腺病毒载体 星形胶质细胞 

分 类 号：R741[医药卫生—神经病学与精神病学] R741.02[医药卫生—临床医学] R743