【作者】 贺芳；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 妇产科， 2009， 博士

【摘要】 目的:构建含HIV-1 vpr基因的重组腺病毒rAd-vpr,通过重组腺病毒感染,在人T细胞白血病病毒1型转化细胞C8166内源性表达HIV-1 Vpr蛋白,研究表达Vpr蛋白诱导C8166细胞周期阻滞、细胞致死效应等功能;并在建立C8166细胞蛋白质组学研究的核心技术平台基础上,应用蛋白质组学的方法研究HIV-1 Vpr蛋白对C8166细胞蛋白质组的影响,进而由差异蛋白质推测Vpr蛋白对C8166细胞作用可能机制;从而探讨将重组腺病毒rAd-vpr用于成人T细胞白血病基因治疗的可能性。方法:将PCR扩增得到vpr基因片段插入穿梭载体pAdTrack-CMV,产生重组质粒pAdTrack-CMV-vpr,将经PineⅠ线性化后的重组质粒通过电转法导入含腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183进行同源重组,得到重组腺病毒质粒pAdEasy-vpr。用Lipofectamine 2000将pAdEasy-vpr转染到HEK-293A细胞,包装并扩增出大量的腺病毒。重组腺病毒感染C8166细胞,Western blotting检测重组Vpr蛋白在C8166细胞中内表达,重组腺病毒感染C8166细胞效率采用流式细胞术检测和荧光倒置显微镜观察。PI染色流式细胞术分析rAd-vpr感染C8166细胞对其细胞周期的影响;细胞凋亡的形态学检测采用Hoechst 33342/PI染色激光共聚焦显微镜观察,线粒体膜电势采用JC-1染色测定。制备三组(对照组、rAd-vector感染组和rAd-vpr感染组)细胞总蛋白。采用IPG预制胶条(pH4-7,17cm)进行第一维等电聚焦,10%的SDS-聚丙烯酰胺凝胶进行第二维分子量垂直电泳。采用PDQuest 7.4图像分析软件对获得的双向凝胶电泳图谱进行分析。选取12个重组腺病毒rAd-vpr感染后差异表达的蛋白质点,进行胶内酶切、串联飞行时间质谱(QSTARXL)分析,将采集的质谱数据通过MASCOT在线检索NCBInr数据库鉴定蛋白,并通过半定量RT-PCR检测差异表达蛋白质14-3-3和GSTP1在三组细胞中的mRNA水平。结果:成功构建了含HIV-1 wr基因的重组腺病毒rAd-vpr,可高效感染C8166细胞,表达HIV-1 Vpr蛋白,PI染色流式细胞术检测细胞周期结果显示,重组腺病毒rAd-vpr感染48h及72h,C8166细胞晚期凋亡率显著高于rAd-vector组及对照组,重组腺病毒rAd-vpr感染72h,能诱导C8166细胞G2/M期细胞周期阻滞。激光共聚焦显微镜观察Hoechst 33342/PI染色重组腺病毒rAd-vpr感染的C8166细胞出现了凋亡小体,JC-1染色重组腺病毒rAd-vpr感染C8166细胞线粒体膜电势下降。建立并优化了人T细胞白血病病毒1型转化细胞C8166细胞总蛋白的双向凝胶电泳方法,获得了蛋白质分离效果较好的双向凝胶电泳图谱。采用串联飞行时间质谱对选取的12个差异蛋白质点进行质谱分析,获得了相应的肽序列指纹图谱,通过MASCOT在线检索工具检索NCBInr数据库,鉴定这些差异蛋白质为细胞周期调控相关蛋白14-3-3,GSTP1及Galectin-1;肿瘤细胞转移相关的蛋白Rho GDI2;caspase-3激活示意蛋白vimentin和Rho GDI2;线粒体蛋白prohibitin;抗炎蛋白thioredoxin;肿瘤蛋白ProteinDJ-1;增强肿瘤杀伤效应蛋白Nucleoside diphosphate kinase A;与细胞骨架蛋白折叠相关的蛋白Tubulin specific chaperone A等。最后通过半定量RT-PCR在mRNA水平上验证了差异表达蛋白质14-3-3和GSTPl在重组腺病毒rAd-vpr感染的C8166细胞中表达上调。结论:通过细菌内同源重组成功构建了重组腺病毒rAd-vpr,通过重组腺病毒感染,在人T细胞白血病病毒1型转化细胞C8166内表达HIV-1 Vpr蛋白,表达Vpr蛋白能有效致C8166细胞死亡、线粒体丧失功能及细胞周期阻滞。运用蛋白质组学的研究手段发现细胞周期调控相关蛋白14-3-3,GSTP1及Galectin-1;肿瘤细胞转移相关的蛋白Rho GDI2;caspase-3激活示意蛋白vimentin和Rho GDI2;线粒体蛋白prohibitin;抗炎蛋白thioredoxin;肿瘤蛋白Protein DJ-1;增强肿瘤杀伤效应蛋白Nucleoside diphosphate kinase A;与细胞骨架蛋白折叠相关的蛋白Tubulin specificchaperoneA等参与了目的基因HIV-1 vpr导入C8166细胞后,细胞内表达的Vpr诱导细胞周期阻滞、凋亡等过程,描绘了一个更加清晰的Vpr致C8166细胞变化的细胞内蛋白网络图。通过半定量RT-PCR在mRNA水平上确证了两种差异表达蛋白质14-3-3和GSTP1在重组腺病毒rAd-vpr感染细胞中表达上调,可能与Vpr的细胞周期阻滞生物学功能相关。由于能够在诱导细胞发生凋亡的同时激活抗炎因子,使得严重的宿主免疫反应避免发生成为可能,因而重组腺病毒rAd-vpr在成人T细胞白血病的基因治疗中具有诱人的潜力和前景。更多还原

【Abstract】 Aim:To construct the recombinant adenovirus carrying the HIV-1 vpr gene,to express HIV-1 viral protein R（Vpr） endogenously in the recombinant adenovirus rAd-vpr infected HTLV-1transformed C8166 cells.To investigate the effects of expressed HIV-1 Vpr induced cell cycle arrest and cell death.To characterize how intracellular HIV-1 Vpr protein expression modulated the protein expression profile of C8166 cells,and further to speculate the possible mechanisms of Vpr on C8166 cells via differentially expressed proteins identified on the basis of establishing the pivotal technological platform for studying the proteomics of C8166 cells.To evaluate the feasibility of employing the HIV-vpr gene,via targeted gene transfer,as a potential therapy to treat adult T-cell leukemia（ATL）.Methods:The gene fragment of vpr was amplified by PCR and inserted into the shuttle vector pAdTrack-CMV to obtain the recombinant plasmid pAdTrack-CMV-vpr.Then,the pAdTrack-CMV-vpr was linearized with restriction endonuclease Pme I,and transformed into E.coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1 for homologous recombination by electroporation.This generated recombinant plasmids, which were digested by Pac I and transfected into HEK293 cells with Lipofectamine 2000 to package and amplify the recombinant adenovirus rAd-vpr.The intracellular expression of HIV-1 Vpr in recombinant adenovirus rAd-vpr infected C8166 cells were detected by Western blotting.The efficiency of C8166 cell infected with recombinant adenovirus was detected by flow cytometry and observed under fluorescence microscopy.C8166 cell membrane integrity change,cell cycle arrest,apoptosis and mitochondrial membrane potential loss that induced by the recombinant adenovirus rAd-vpr were checked by FACS and observed by laser confocal microscopy.The whole cell proteins were prepared from mock-,rAd-vpr and rAd-vector infected C8166 cells respectively,and applied to two dimension gel electrophoresis（2-DE）.The linear IPG dry strip（pH4-7,17cm） was used for isoelectric focusing of the first dimension and 10%SDS-PAGE was performed for the second dimension.The 2-DE patterns of the whole cell proteins from 3 group cells were analyzed with PDQuest 7.4 software.12 differentially expressed protein spots were selected for trypsin digestion,and the resulted peptides were subjected to LC/MS/MS analysis.MASCOT was employed to identify proteins from acquired MS spectra by searching against NCBInr database,and the mRNA levels of 14-3-3 and GSTP1 were detected by semi-quantitative RT-PCR.Results:The recombinant adenovirus rAd-vpr containing vpr gene was successfully constructed,which could effectively infect C8166 cells.The result of Western blotting confirmed Vpr expression endogenously in recombinant adenovirus rAd-vpr infected C8166 cells.G₂/M phase cell cycle arrest was observed and typical characteristics of apoptosis were detected in rAd-vpr infected cells,including sub-diploid peak exhibition in DNA content assay,Hoechst 33342 accumulation,apoptotic body formation,mitochondrial membrane potential and plasma membrane integrity loss.The 2-DE patterns of mock-, rAd-vpr and rAd-vector infected C8166 cells with high resolution and reproducibility were obtained.12 differentially expressed protein spots were successfully identified by liquid chromatography-tandem mass spectrometry（LC/MS/MS）.By proteomic assay,the apoptosis mechanism was confirmed,exhibiting the regulation of caspase-3 activity indicator proteins（vimentin and Rho GDP-dissociation inhibitor 2）,mitochondrial protein （prohibitin） and other regulatory proteins.In addition,the up-regulation of anti-inflammatory redox protein,thioredoxin,was identified in the rAd-vpr infected group. Finally,the mRNA levels of 14-3-3 and GSTP1 were higher the recombinant adenovirus rAd-vpr infected C8166 cells than those in the control and rAd-vector infected groups.Conclusion:The recombinant adenovirus rAd-vpr was successfully constructed by homologous recombination in bacteria.C8166 cells infected with the recombinant adenovirus expressed Vpr.Intracellular expressed HIV-1 Vpr can kill HTLV-1transformed cells effectively,and may avoid the risks of inducing severe host inflammatory responses through apoptosis-inducing and anti-inflammatory activities.These properties of recombinant adenovirus rAd-vpr shown in C8166 cells demonstrated its potential benefits in ATL therapy.更多还原