人RARα基因重组腺病毒表达质粒的构建及其对人白血病NB4细胞的影响研究  

Construction of Recombinant Adenovirus Vector Carrying Human RARα Gene and Its Effect on NB4 Human Promyelocytic Leukemia Cells

作　　者：蒋开玲[1,2] 钟梁[2] 阳小群[2] 王慧[2] 马鹏鹏[2] 朱新瑜[2] 刘北忠[1,2] 

机构地区：[1]重庆医科大学附属永川医院中心实验室,重庆402160 [2]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国细胞生物学学报》2014年第9期1227-1234,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:81171658);重庆市自然科学基金计划重点项目(批准号:2011BA5037)资助的课题~~

摘　　要：利用AdEasy系统构建携带人RARα基因的重组腺病毒表达质粒,感染人白血病NB4细胞后用生理浓度的维甲酸诱导,观察其对NB4细胞增殖和分化的影响。以急性早幼粒细胞株NB4的cDNA为模板,PCR扩增RARα基因,克隆至穿梭质粒pAdTrace-TO4中,构建重组腺病毒穿梭质粒pAdTrace-TO4-RARα。用EcoR V和Sal I双酶切及测序鉴定,然后与骨架质粒AdEasy-1同时转化大肠杆菌BJ5183菌株的感受态,经同源重组获得重组腺病毒质粒Ad-RARα。酶切验证后,Pac I酶线性化后转染AD293细胞,包装出重组腺病毒Ad-RARα,经3轮扩增后,测定病毒滴度,进行PCR鉴定。流式细胞术测定病毒感染效率,Western blot法检测重组腺病毒感染的人NB4细胞中RARα蛋白和凋亡相关蛋白Bax、Bcl-2的表达,CCK-8法检测重组腺病毒感染对NB4细胞增殖的影响,Annexin V/PI双染法测定细胞周期,PI测定细胞凋亡。重组腺病毒穿梭质粒pAdTrace-TO4-RARα经双酶切及测序证明构建正确,病毒感染效率可达70%,与空载体感染及未感染的NB4细胞相比,重组腺病毒感染的NB4细胞内RARα蛋白的表达明显升高(P<0.05),且经生理浓度全反式维甲酸ATRA处理后,能够有效地促进感染重组腺病毒细胞的分化成熟和凋亡。该研究成功构建了携带人RARα基因的重组腺病毒表达质粒,感染NB4细胞后可促进细胞增殖,经生理浓度维甲酸诱导后能促进NB4细胞分化成熟和凋亡,为进一步研究该基因在急性髓细胞白血病发生发展中的作用奠定了基础。In this research, we constructed recombinant adenovirus Ad-RARα and determined its effect on NB4 human promyelocytic leukemia cells. RARα gene was amplified by PCR using cDNA of NB4 cells as a template and inserted into shuttle plsmid pAdTrace-TO4. The constructed recombinant shuttle plasmid pAdTraceTO4-RARα was digested with Pme I, and transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-RARα was digested with Pac I and transfected to AD293 cells for packaging. The obtained recombinant adenovirus Ad-RARα was subjected to three cycles of amplification, then determined titer and identified by PCR and sequencing. NB4 cells were infected by the recombinant adenovirus, with infection rate of 70%. Protein expression, proliferation and differentiation in NB4 cells of RARα were detected by Western blot, CCK-8 and flow cytometry respectively. Cell cycle was measured by Annexin V/PI. Recombinant adenovirus Ad-RARα reached a titer of 5.2×105 pfu/mL after three cycles of amplification and recombinant adenovirus Ad-RARα was constructed correctly as proved by PCR and sequencing, and the RARα gene in Ad-RARα was successfully expressed in NB4 cells. Recombinant adenovirus Ad-RARα was constructed successfully and RARα gene could be expressed in NB4 cells. The proliferation ofinfected NB4 cells was enhanced. Physiological concentration of ATRA（all-trans-retinoic acid） can induce differentiation effectively. All of these results lay the foundation to further study for the role of this gene in acute myeloid leukemia development.

关 键 词：RARα基因 腺病毒 NB4细胞 维甲酸 增殖 

分 类 号：R733.71[医药卫生—肿瘤]