CAJ | 학술논문

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鄄7（MDA鄄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鄄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鄄7鄄EGFP；同时构建对照病毒Ad.EGFP。病毒滴度计算按寇化法（Kar鄄ber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鄄7在感染2种病毒的肺腺癌 A549细胞中的表达状态；通过二苯甲亚胺鄄碘化丙啶（Hoechst33342鄄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鄄7在感染Ad.hMDA鄄7鄄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鄄7表达；Ad.hMDA鄄7鄄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鄄7鄄EGFP，并证实MDA鄄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。
목적：구건일충유선병독AdMax포장계통표체목적기인---흑색소류분화상관기인견7（MDA견7）적복제결함형선병독，병초보탐색기항종류활성。방법통과기인조작기술장목적기인（MDA견7）삽입도5형선병독AdMax포장계통적E1A구역，구건복제결함형선병독Ad.hMDA견7견EGFP；동시구건대조병독Ad.EGFP。병독적도계산안구화법（Kar견ber법）진행선병독감염성적도（TCID50）측정。응용면역조화염색법검측MDA견7재감염2충병독적폐선암 A549세포중적표체상태；통과이분갑아알견전화병정（Hoechst33342견PI）쌍염색법급류식세포의검측2충병독대종류세포살상적차이。결과성공구건휴대목적기인적선병독재체，경취합매련반응（PCR）감정정학；MDA견7재감염Ad.hMDA견7견EGFP적폐선암A549세포중표체，이상동감염복수（MOI）치감염Ad.EGFP시폐선암A549세포중미검측도MDA견7표체；Ad.hMDA견7견EGFP대종류세포주살상능력명현고우Ad.EGFP。결론성공구건복제결함형선병독Ad.hMDA견7견EGFP，병증실MDA견7단백재종류세포중과표체，유도폐선암A549세포주발생조망。
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 E1A 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.