中文摘要：

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。

英文摘要：

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.