MDA-7基因的腺病毒载体构建及对肺腺癌A549细胞凋亡的影响  

Construction of adenovirus vector of MDA7 and its effect on apoptosis of A549 cell

作　　者：李书华[1] 龙捷[1] 王红艳[1] 谢晓斌[1] 张雅洁[1] 

机构地区：[1]广州医科大学,广东广州510182

出　　处：《现代医药卫生》2014年第13期1929-1932,共4页Journal of Modern Medicine & Health

基　　金：教育部博士点基金项目博导类课题(20134423110001);广东省自然科学基金资助面上项目(S2012010010181);广东省医学科研基金项目(A2013244);广州市科技计划项目(2014Y200171);广州市教育系统创新学术团队项目(13C06)

摘　　要：目的构建一种由腺病毒AdMax包装系统表达目的基因——黑色素瘤分化相关基因-7(MDA-7)的复制缺陷型腺病毒,并初步探索其抗肿瘤活性。方法通过基因操作技术将目的基因(MDA-7)插入到5型腺病毒AdMax包装系统的E1A区域,构建复制缺陷型腺病毒Ad.hMDA-7-EGFP;同时构建对照病毒Ad.EGFP。病毒滴度计算按寇化法(Karber法)进行腺病毒感染性滴度(TCID50)测定。应用免疫组化染色法检测MDA-7在感染2种病毒的肺腺癌A549细胞中的表达状态;通过二苯甲亚胺-碘化丙啶(Hoechst33342-PI)双染色法及流式细胞仪检测2种病毒对肿瘤细胞杀伤的差异。结果成功构建携带目的基因的腺病毒载体,经聚合酶链反应(PCR)鉴定正确;MDA-7在感染Ad.hMDA-7-EGFP的肺腺癌A549细胞中表达,以相同感染复数(MOI)值感染Ad.EGFP时肺腺癌A549细胞中未检测到MDA-7表达;Ad.hMDA-7-EGFP对肿瘤细胞株杀伤能力明显高于Ad.EGFP。结论成功构建复制缺陷型腺病毒Ad.hMDA-7-EGFP,并证实MDA-7蛋白在肿瘤细胞中过表达,诱导肺腺癌A549细胞株发生凋亡。Objective To construct replication-deficient recombinant adenovirus of melanoma differentiation associated gene（MDA）-7,which is expressed by adenovirus vector AdMax packing system,and to initially explore its antineoplastic activity.Methods Ad.hMDA-7-EGFP of replication-deficient recombinant adenovirus was constructed by inserting the targeted gene（MDA-7）into E1 A area of type 5 adenovirus vector AdMax packing system with gene manipulation,meanwhile the contrast virus of Ad.EGFP was constructed. Karber′s method was used to calculate virus titer,the tissue culture infective dose 50（TCID50） was adopted to determine infective titer of adenovirus vector,and the immunohistochemical staining was employed to detect the expression status of MDA-7 in A 549 cell lines infected with 2 viruses. The difference of the two viruses on tumor cell was assessed by Hoechst33342-PI staining method and flow cytometry（FCM）. Results Adenovirus vector carrying targeted gene was constructed successfully and identified by polymerase chain reaction（PCR）. MDA-7 only expressed in Ad.hMDA7-EGFP infected lung adenocarcinoma A549 cells but not in Ad.EGFP with the same multiplicity of infection（MOI）. The tumor-killing capacity of Ad.hMDA-7-EGFP was significantly higher than that of Ad.EGFP. Conclusion Replication deficient adenovirus Ad.hMDA7-EGFP is successfully constructed,it also confirms that MDA-T protein overexpresses in tumor cells and induces the apoptosis of lung adenocarcinoma A549 cells line.

关 键 词：病毒复制 基因疗法 肿瘤/治疗 复制缺陷型腺病毒 AdMax包装系统 

分 类 号：R734.2[医药卫生—肿瘤]