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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。
The AdEasy system was used to construct the recombinant adenovirus expression vector carrying human RARα gene. After being infected into NB4 cells, the cells were induced with physiological retinoic acid to observe its effect on the proliferation and differentiation of NB4 cells. The recombinant adenoviral shuttle plasmid pAdTrace-TO4-RARα was constructed by PCR amplification of the RARα gene using the cDNA of the acute promyelocytic strain NB4 as a template and cloning into the shuttle plasmid pAdTrace-TO4. EcoR V and Sal I double enzyme digestion and sequencing identification, and then with the backbone plasmid AdEasy-1 transformed E. coli BJ5183 strains of competent cells by homologous recombination to obtain the recombinant adenovirus plasmid Ad-RARα. After restriction enzyme digestion, Pac I enzyme was linearized and then transfected into AD293 cells to package the recombinant adenovirus Ad-RARα. After 3 rounds of amplification, the virus titer was determined and PCR identification was performed. Flow cytometry was used to determine the efficiency of virus infection. Western blot was used to detect the expression of RARα and Bax, Bcl-2 in NB4 cells infected by recombinant adenovirus. CCK-8 was used to detect the proliferation of NB4 cells The cell cycle was determined by Annexin V / PI double staining and apoptosis was determined by PI. The recombinant adenovirus shuttle plasmid pAdTrace-TO4-RARα was confirmed by double enzyme digestion and sequencing, and the virus infection efficiency was up to 70%. Compared with the empty vector-infected and uninfected NB4 cells, the recombinant adenovirus-infected NB4 cell RARα (P <0.05). After all-trans retinoic acid ATRA treatment, the ATRA could effectively promote the differentiation, maturation and apoptosis of infected adenovirus. In this study, a recombinant adenovirus plasmid carrying human RARα gene was successfully constructed. After infected with NB4 cells, cell proliferation can be promoted. After induced by physiological tretinoin, NB4 cells can promote differentiation and apoptosis. To further study the expression of RARα gene in acute myeloid leukemia The role of cell leukemia in the development and laid the foundation.