摘要
目的研究HIV-1 Tat对细胞周期蛋白(cyclinB1)的影响。方法利用人横纹肌肉瘤细胞(TE671)及已转染tat基因的TE671细胞(TT2)细胞系;通过Western印迹检测辐射前后CyclinB1表达变化;使用Northern-Blot及半定量RT-PCR,分析cyclinB1在mRNA水平表达;放线菌酮阻断蛋白合成实验及免疫共沉淀实验检测cyclinB1的稳定性。结果细胞在接受电离辐射和未接受照射情况下,cyclin B1的表达在转染tat基因细胞中明显增强,Tat蛋白不影响cyclinB1在mRNA水平的表达;放线菌酮阻断蛋白合成实验及Tat蛋白诱导细胞电离辐射后泛素化实验均表明,cyclinB1表达水平的增强主要发生在蛋白质合成后的修饰。结论 HIV-1 Tat蛋白能提高Cyclin B1的稳定性,降低CyclinB1泛素化水平;该项研究也许有助于利用分子生物学手段,干预Tat蛋白的表达以调控CyclinB1及相关蛋白的表达,进而改变肿瘤细胞周期的进程,影响临床的疗效。
OBJECTIVE To investigate the effects of HIV-1 Tat on the the cell cycle-related cyclinB1 protein.METHODS A human rhabdomyosarcoma cell lines TE671 and TT2 cells generated from TE671 cells by transfecting with tat gene of the HIV-1 strain were employed.The expressions of cyclinB1 protein when subjected to ionizing radiation anterior-posterior were detected by Western Blot assay,and the Northern-Blot and semiquantitative RT-PCR were employed to analyse the cyclinB1 protein expression on the level of mRNA.The cycloheximide blocking and co-immunoprecipitation assays were carried out to detect the stability of cyclinB1 protein.RESULTS The expression of cyclinB1 protein was significantly enhanced in TT2 cells by transfecting with tat gene of the HIV-1 strain when subjected to ionizing radiation and unionizing radiation.The cycloheximide blocking and co-immunoprecipitation assays demonstrated that the constitutive overexpression of cyclinB1 protein mainly generate to the protein modification after protein synthesis.CONCLUSION HIV-1 Tat is able to enhance the stability of cyclinB1 protein,and decrease the level of cyclinB1 protein polyubiquitinated.these observations may be help to change the process of tumor cell cycle,and then effect clinical therapy via interfering Tat expression by molecular biology method to regulation the expression of cyclinB1 protein.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2011年第10期1975-1979,共5页
Chinese Journal of Nosocomiology
基金
国际原子能机构合作项目(12510/RO)
国家自然科学基金项目(30400120)
四川省卫生厅科研课题(090175)
