Ad—XBP15重组腺病毒对ATDC5细胞周期与凋亡的效应研究  

Study on ATDC5 Cells Proliferation and Apoptosis by Ad-XBP1S

作　　者：刘艳娜[1,2] 周菁华[1,2] 张鹏[1,2] 韩晓凤[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》2012年第5期326-332,共7页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81171697）,重庆市高等学校优秀人才支持计划（No.渝教人2011-65）

摘　　要：目的构建重组人剪接型X盒结合蛋白1（X—boxbindingprotein1spliced，XBP1S）并探讨其对软骨祖细胞ATDC5周期和凋亡的影响。方法应用pAdEasy“腺病毒载体系统，构建得到XBP1S基因全长的重组穿梭质粒pAdTrack—XBP1S，通过电转法同腺病毒骨架质粒pAdEasy—1进行同源重组，获得重组腺病毒pad．XBP1S。随后在HEK-93细胞中包装并扩增重组腺病毒颗粒Ad—XBP1S。用直接PCR法鉴定重组腺病毒。体外感染软骨祖细胞ATDC5，观察绿色荧光蛋白（GFP）感染效率，并应用流式细胞仪（FCM）分别在加入毒胡萝卜素（tunicamycin，TM）和不加入TM的条件下，检测重组腺病毒Ad—XBP1S对ATDC5细胞周期和凋亡的影响。结果酶切鉴定和PCR证实成功构建了重组腺病毒质粒pAd．XBP1S。FCM检测结果表明，在ERS条件下，Ad—XBP1S实验组ATDC5细胞G，（60．70％）期细胞比例低于TM（87．78％）、Ad。GFP对照组（85．24％）（P〈0．05），Ad—XBP1S实验组ATDC5细胞S期（26．64％）高于TM（6．69％）、Ad—GFP对照组（7．35％）（P〈0．05）；TM刺激诱导ERS条件下，感染Ad—XBP1S腺病毒上清后，ATDC5细胞的凋亡率为7．81％，与TM（33．32％）、Ad—GFP对照组（25．95％）比较，差异具有显著性（P〈0．05）。结论含XBP1S基因的重组腺病毒感染ATDC5细胞后，可加速ATDC5细胞周期的转化并抑制其凋亡。为后续研究XBP1S生物学功能奠定了基础。Objective To construct the recombinant adenoviral vector containing human X-box binding protein 1 spliced （XBP1 S） , and to study the effect of the recombinant adenovirns on the proliferation and apoptosis of ATDCS. Methods By using pAdEasyTM adenovirus vector system, XBP1S full-length IRElct full-length was structured the reeombined shuttle vector of pAdTrack- XBP1S which was transferred with pAdEasy-1 to generate recombined adenovirus plasmid pAd- XBP1S through electroporation, respectively. Subsequently, the plasmid were transfected into HEK- 293 cell line to pack and amplify the recombined adenovirus Ad-XBP1S. The expression of recom- bined adenovirus was detected by PCR. With the unicamycin （TM） stimulate or not, the influence of the recombinant adenovirns on the proliferation and apoptosis of ATDC5 ceils was checked by FCM. Results Restriction endonuclease digestion analysis and PCR indicated that the recombined adenovirus vector Ad-XBP1S had been successfully constructed . FCM analysis showed that on the endoplasmie reticulum stress （ERS） conditions, the G1 phase of ATDC5 cells in the Ad-XBP1S group as compared with the control groups were decrease, S phase were increased. The apoptosis rate of the Ad-XBP1 S group wasT. 81% , while the control group of Ad-GFP was 25.95 %. Con- clusion After infected ATDC5 cells with the recombinant adenovirus containing XBP1 S, it can be promoted the proliferation and inhabit the apoptosis of ATDC5 cellsin vitro.

关 键 词：X盒结合蛋白1 重组腺病毒 ATDC5细胞 细胞凋亡 增殖 

分 类 号：R373[医药卫生—病原生物学]