IRE1α重组腺病毒载体对ATDC5软骨干细胞增殖及凋亡的影响

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＜0.05).Conclusion Infection of recombinant adenovirus containing IRE1α gene may promote the proliferation and apoptosis of ATDC5 cells.%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.