人p27kip1重组腺病毒载体的构建及其介导的p27kip1表达  

Construction of adenovirus vector for human p27kipl gene and its expression

作　　者：潘晓明[1] 章卫平[2] 吴宗贵[1] 曹雪涛[2] 

机构地区：[1]上海第二军医大学附属长征医院心内科,200003 [2]第二军医大学免疫学教研室

出　　处：《中华肿瘤杂志》2000年第2期120-123,共4页Chinese Journal of Oncology

基　　金：国家杰出青年科学基金!资助项目 (3982 51 2 3)

摘　　要：目的　研究外源性p2 7kip1基因对细胞周期及细胞增殖的影响。方法　构建CMV启动子转录调控的含人p2 7kip1cDNA的E1区替代的腺病毒载体pAxlcw .CIhp2 7kip1,与经EcoT2 2 1酶切的Ad5腺病毒DNA -末端肽复合物共转染 2 93细胞 ,制备p2 7kip1重组腺病毒 ,在体外转染HeLa细胞 ,Westernblot、FACS及MTT检测分析外源性p2 7kip1蛋白在细胞内的表达 ,以及对细胞周期及细胞增殖的影响。结果　获得含人p2 7kip1cDNA的重组腺病毒 ,滴度为 1.7× 10 9pfu/ml。体外转染HeLa细胞2 4h后 ,p2 7kip1蛋白在p2 7kip1转染后的HeLa细胞中高表达 ;FACS检测表明 ,p2 7kip1和LacZ重组腺病毒转染后的HeLa细胞 ,经 10 %血清刺激后处于G1期的细胞分别为 89.93%和 5 9.84% ;MTT法检测表明 ,人p2 7kip1重组腺病毒转染后的HeLa细胞经 2 0 %血清刺激后 ,48及 72h的A值均低于LacZ重组腺病毒转染后的HeLa细胞 (P <0 .0 1)。结论　本研究中所制备的p2 7kip1重组腺病毒能有效介导外源性p2 7kip1基因在体外转染HeLa细胞并高表达p2 7kip1,抑制细胞由G1期向S期过渡 ,从而抑制细胞增殖 ,为进一步研究p2 7kip1基因治疗奠定了基础。Objective To study the effects of exogenous p27kip1 gene on cell cycle and proliferation.Methods E1 substitutive adenovirus vector pAxlcw containing human p27kip1 cDNA (Cihp27kip1) under the transcriptional control of CMV promoter was constructed and then cotransfected with Ad5 adenovirus DNA terminal peptide complex EcoT221 digested into 293 cells for preparing p27kip1 recombinant adenovirus Adhp27kip1. After infecting HeLa cells for 24 h, the exogenous p27kip1 protein expression in HeLa cells and its effects on cell cycle and proliferation were determined by Western blot, FACS and MTT assay.Results The titers of resultant recombinant adenoviruses was 1.7×10 9 pfu/ml. p27kipl protein was over expressed in HeLa cells infected with p27kipl recombinant adenovirus for 24 h, resulting in blockage of cell cycle transition from G 1 phase to S phase and inhibition of cell proliferation.Conclusion p27kipl recombinant adenoviruses prepared in this study can efficiently transfer p27kipl gene into HeLa cells and over express p27kipl protein in the infected cells. Adhp27kipl can be used as an agent for gene therapy. Subject

关 键 词：P27KIP1基因 基因治疗 腺病毒载体 肿瘤 

分 类 号：R73-36[医药卫生—肿瘤]