YB-1腺病毒载体的构建及其对乳腺癌MCF-7细胞耐药性的影响  

Construction of hYB1 gene recombinant adenovirus vector and its increasing in the drug resistance of breast cancer MCF-7 cells strain

作　　者：冷加燕[1] 许文林[1] 沈慧玲[1] 龙璐璐[1] 吴晓君[1] 陈巧云[1] 姚俊[1] 

机构地区：[1]江苏大学附属人民医院中心实验室,江苏镇江212002

出　　处：《江苏大学学报（医学版）》2011年第4期303-307,F0002,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(81070423);江苏省自然科学基金资助项目(BK2010332);江苏省卫生厅重大资助项目(K2005017)

摘　　要：目的:构建携带人YB1基因的重组腺病毒载体,并观察其对乳腺癌细胞株MCF-7药物敏感性的影响,并分析其可能的机制。方法:设计含有BglⅡ和HindⅢ酶切位点的引物,PCR克隆人YB1基因,插入穿梭载体pAdTrack-CMV,与腺病毒骨架载体pAdEasy-1在大肠埃希菌BJ5183内同源重组,获得重组子pAdEasy-1-hYB1,转染293A细胞,包装获得含hYB1的重组腺病毒(AdYB1),体外转染MCF-7细胞。采用MTT法分析在As2O3作用下YB1对MCF-7细胞半数抑制浓度(IC50)的影响;采用Annexin V/PI双染法,经流式细胞仪分析在As2O3作用下YB1对MCF-7细胞凋亡的影响。结果:成功构建了含有hYB1基因的腺病毒载体AdYB1,该重组腺病毒能显著提高MCF-7细胞的YB1mRNA及蛋白的表达水平,转染YB1组能够增加MCF-7细胞对AS2O3的IC50值;通过Annexin V/PI检测证实YB1能够抑制早期细胞凋亡。结论:成功构建含有hYB1基因的腺病毒载体AdYB1,能高效感染MCF-7,并通过抑制早期细胞凋亡提高MCF-7细胞对As2O3的耐药性。Objective： To construct the recombinant adenovirus vector containing human Y-box binding protein-1（hYB1） gene,and observe whether the sensitivity to anticancer agent could be affected after the adenovirus mediated-hYB1 into the MCF-7 cells,and analyze the possible mechanism.Methods： The amplification products of hYB1 by PCR with a pair of primers with BglⅡand HindⅢ restriction endonuclease sites was subcloned into shuttle plasmid pAdTrack-CMV.The recombinant adenovirus vector AdYB1 was obtained by homologous recombination with pAdTrack-CMV and pAdEasy-1 in bacteria BJ5183.The Recombined adenovirus was packaged and propagated in 293A cells and subsequently infected MCF-7 cells.MTT were used to discover how does YB1 affect the IC50 of MCF-7 under the arsenic trioxide,and using Annexin V/PI double staining,analysis the affection of YB1 on MCF-7 cell apoptosis under the As2O3 by flow cytometry.Results： The adenovirus vector expressing hYB1 gene could specifically increase both levels of mRNA and protein of the hYB1 in MCF-7 cells.The introduction of exogenous YB1 gene also resulted in the increased IC50 value of MCF-7 cells under the As2O3,and Annexin V/PI test confirmed that YB1 can suppress early apoptosis.Conclusion： The recombinant adenovirus vector was constructed and could infect the MCF-7 cells successfully,which increase the MCF-7 cells resistance to As2O3 by inhibiting which early apoptosis.

关 键 词：hYB1基因 MCF-7细胞 腺病毒载体 耐药 

分 类 号：R155.3[医药卫生—营养与食品卫生学]