携人ATF6重组腺病毒载体的构建及对C28I2细胞增殖及凋亡的影响  

Construction of Adenovirus Vector Containing Human ATF6 Gene and Its Effect on Proliferation and Apoptosis of C2812 Cells

作　　者：韩晓凤[1,2] 张鹏[1,2] 李美玲[1,2] 夏飞[1,2] 郭风劲[1,2] 

机构地区：[1]重庆医科大学细胞生物学及遗传学教研室,重庆市400016 [2]重庆医科大学发育生物学与模式动物平台,重庆市400016

出　　处：《医学分子生物学杂志》2013年第4期187-192,共6页Journal of Medical Molecular Biology

基　　金：资助项目：国家自然科学基金（N“81171697）,重庆市高等学校优秀人才支持计划（No.渝教人2011-65）,教育部优秀人才支持计划（No.NCET-12-1090）

摘　　要：目的构建携带人活化转录因子6（ activating transcription factor 6, ATF6）的重组腺病毒，观察其对软骨细胞C2812增殖及凋亡的影响。方法将ATF6基因克隆人pAdTrack-cmv质粒，经pme I线性化后的重组穿梭质粒pAdTrack-ATF6通过电转法与腺病毒骨架质粒pAdEasy-1在BJ5183感受态中同源重组．挑选阳性克隆，获得重组腺病毒质粒pad-ATF6。通过脂质体介导转染HEK-293细胞。得到重组腺病毒Ad-ATF6，并采用PCR法对重组腺病毒进行鉴定。Ad-ATF6感染软骨细胞C2812．通过Western印迹检测ATF6的表达，并应用流式细胞仪检测Ad-ATF6对C2812细胞增殖和凋亡的影响。结果成功构建了携带人ATF6基因的重组腺病毒质粒，并成功包装了具有高效感染力的Ad-ATF6腺病毒。Western印迹结果表明．Ad-ATF6感染组ATF6的表达明显增加。FCM检测结果表明，Ad-ATF6处理组G，期的细胞比例分别比Nc组和Ad-GFP组高28．42％和30．50％（P〈0．5）．S期的细胞比例分别比Nc组和Ad-GFP组低22．43％和24．10％（P〈0．5）；凋亡率比Nc组和Ad-GFP组高出21．98％和19．65％（P〈0．5）。结论可高表达ATF6的腺病毒包装成功，感染C2812细胞后。抑制了C2812细胞的增殖并促进其凋亡．为后续研究AqT6基因的生物学功能奠定了基础，为与内质网应激相关的软骨发育疾病提供了一定的参考价值。Objective To construct the adenovirus vector containing human ATF6 gene, and explore its effect on the proliferation and apoptosis of C2812 cells. Methods ATF6 gene was cloned into pAdTrack-cmv. After linearization of pAdTrack-ATF6 shuttle plasmid by pme I digestion, the pAdTrack-ATF6 shuttle plasmid was transformed into E. coli BJ5183 along with skeleton adenovirus plasmid pAdEasy-1 to generate recombinant adenovirus plasmid pAd-ATF6, which was then trans- fected into the 293 packing cells by using lipofectamine to amplify the recombinant adenovirus Ad- ATF6. The sequences of the recombinant adenovirus were identified by PCR. The expression of ATF6 protein was detected by Western blotting after Ad-ATF6 was transfected into C2812 cells. The effects of Ad-ATF6 on the proliferation and apoptosis of C2812 cells were measured by flow cytometry（FCM） . Results The recombinant adenovirns vector pAd-ATF6 was successfully constructed and the adenovirns Ad-ATF6 with high infection was obtained. Western blotting showed that the expres- sion of ATF6 protein was significantly increased in C2812 infected with Ad-ATF6. FCM revealed that the percentage of C2812 cells at G1 phase was increased by 28.42% and 30. 50% in the Ad-ATF6 group as compared with the NC and Ad-GFP groups （P 〈0. 05）, and that of C2812 cells at S phase was decreased by 22.43% and 24. 10% in the Ad-ATF6 group when compared with the NC and Ad- GFP groups （P 〈0.05）. The apoptosis rate was increased by 21.98 % and 19.65% in the Ad- ATF6 group when compared with the NC and Ad-GFP groups （P 〈 0. 05）. Conclusions The ade- novirus Ad-ATF6 with high infection was obtained. The proliferation of Ad-ATF6-infected C2812 cells was suppressed, and the apoptosis of infected cells increased. This study lays a foundation for further study on the biological function of ATF6 gene and provides some clues for cartilage develop- ment diseases related to ERS.

关 键 词：人活化转录因子6 重组腺病毒 C28I2细胞 细胞增殖凋亡 

分 类 号：R373[医药卫生—病原生物学]