IRE1α重组腺病毒载体对ATDC5软骨干细胞增殖及凋亡的影响  

Effects of recombinant adenoviral vector containing IRE1α gene on proliferation and apoptosis of ATDC5 stem cells

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

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400016 [2]重庆市璧山县人民医院输血科

出　　处：《解放军医学杂志》2013年第8期639-643,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81171697);教育部新世纪优秀人才支持计划(NCET-12-1090);重庆市高等学校优秀人才支持计划(2011-65)~~

摘　　要：目的构建重组人IRE1α腺病毒,观察其对软骨干细胞ATDC5增殖与凋亡的影响。方法应用pAdEasyTM腺病毒载体系统构建包含全长IRE1α基因和RNase+Kinase核心结构域的重组穿梭质粒pAdTrack-IRE1α、pAdTrack-R+K,通过电转法分别与腺病毒骨架质粒pAdEasy-1进行同源重组,获得重组腺病毒pAd-IRE1α、pAd-R+K。随后在HEK-293细胞中包装并扩增重组腺病毒颗粒Ad-IRE1α、Ad-R+K。用直接PCR法鉴定重组腺病毒,后者体外感染软骨干细胞ATDC5,荧光显微镜下观察绿色荧光蛋白(GFP)感染效率,并应用流式细胞仪分别检测在内质网应激(ERS)和非ERS状态时重组腺病毒对ATDC5细胞增殖和凋亡的影响。结果酶切鉴定和PCR证实成功构建了重组腺病毒质粒pAd-IRE1α、pAd-R+K。流式细胞仪检测结果显示,在ERS状态时,重组腺病毒质粒Ad-IRE1α、Ad-R+K感染后ATDC5细胞G1期比例明显降低,S期细胞比例明显升高(P<0.05),细胞凋亡率明显增高(P<0.05)。结论含IRE1α基因的重组腺病毒感染可促进ATDC5细胞的增殖和凋亡。Objective To construct the recombinant adenoviral vector containing human IRE1α（type I transmembrane protein kinase/endoribonuclease） gene,and investigate its effects on proliferation and apoptosis of ATDC5 stem cells.Methods By using pAdEasyTM adenovirus vector system,the recombinant shuttle vectors of IRE1α full-length gene（pAdTrack-IRE1α） and RNase＋Kinase domain（pAdTrack-R＋K） were constructed,and then transferred with pAdEasy-1 to generate recombinant adenovirus plasmid pAd-IRE1α and pAd-R＋K by electroporation method.Subsequently,the plasmids were transfected into HEK-293 cells to pack and amplify the recombinant adenovirus Ad-IRE1α and Ad-R＋K.The expression of recombinant adenovirus was detected by PCR.The ATDC5 cells were infected in vitro by recombinant adenovirus Ad-IRE1α and Ad-R＋K,the infection efficiency of green fluorescent protein（GFP） was observed,and the influence of Ad-IRE1α and Ad-R＋K on the proliferation and apoptosis of ATDC5 cells under endoplasmic reticulum stress（ERS） or non-ERS was detected by flow cytometry（FCM）.Results Restriction endonuclease digestion analysis and PCR indicated that the recombinant adenovirus vector Ad-IRE1α and Ad-R＋K was successfully constructed.FCM detection showed that under ERS conditions,the G1 phase decreased and S phase increased in ATDC5 cells after transfected by Ad-IRE1α and Ad-R＋K,meanwhile the apoptosis rate increased significantly（P＆lt;0.05）.Conclusion Infection of recombinant adenovirus containing IRE1α gene may promote the proliferation and apoptosis of ATDC5 cells.

关 键 词：IRE1α 腺病毒科 ATDC5细胞 细胞增殖 细胞凋亡 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]