腺病毒介导人ATF6基因修饰对体外细胞增殖凋亡的实验研究

Study on cell proliferation and apoptosis by human ATF6 gene adenovirus modification in vitro

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Ad-ATF6和Ad-ATF6 siRNA，并探讨内质网应激（ER Stress，ERS）时其对人软骨肉瘤细胞SW1353增殖凋亡的影响。方法：将ATF6基因序列与靶向ATF6的siRNA序列分别克隆到pAdTrack-cmv和pSES-HUS穿梭质粒上，然后分别与腺病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183感受态中同源重组，得到腺病毒重组质粒pAd-ATF6和pAd-ATF6 siRNA，通过脂质体介导在HEK293细胞中进行包装和扩增。通过RT-PCR和Western blot检测病毒效果。流式细胞仪法（flow cytometry，FCM）、MTT法及Western blot检测ERS状态下ATF6对SW1353细胞增殖凋亡的影响。结果：成功获得了重组腺病毒Ad-ATF6和Ad-ATF6 siRNA。RT-PCR和Western blot结果表明，重组腺病毒能有效的感染细胞。FCM检测结果表明，ERS条件下，ATF6感染组S期细胞比例明显升高，凋亡率明显降低（P=0.000）；Ad-ATF6 siRNA感染组S期细胞比例明显降低，凋亡率明显上升（P=0.000）。MTT与Western blot实验结果与FCM结果一致。结论：ERS状态下，ATF6能促进SW1353细胞的增殖，抑制其凋亡。

Abstract:

Objective：To construct the adenovirus vector containing human activating transcription factor 6（ATF6） gene and to study the effects on the proliferation and apoptosis of SW1353 cells in the stress condition. Methods：The ATF6 gene and the siRNA se－quence targeting ATF6 gene were respectively cloned into the shuttle plasmid pAdTrack-cmv and pSES-HUS，and then were ho－mogenously recombined with the adenovirus backbone plasmid pAdEasy-l in Ecoli BJ5183 to generate the adenovirus vector pAd-ATF6 and pAd-ATF6 siRNA.The recombinant adenovirus vector was transfected into the 293 packing cells by lipofectamine-mediated transfection to amplify the recombinant adenovirus Ad-ATF6 and Ad-ATF6 siRNA. The SW1353 cells were infected with the aden－ovirus and the expression levels of ATF6 mRNA and protein were detected by RT-PCR and Western blot. The effects of the recombi－nant adenovirus on the proliferation and apoptosis of SW1353 in the stress condition were detected by FCM，MTT and Western blot. Results：The recombinant adenovirus Ad-ATF6 and Ad-ATF6 siRNA with high infection were successfully obtained. FCM analysis showed that under stress condition，the S phase of SW1353 cells in the Ad-ATF6 group was increased significantly（P=0.000），and the apoptosis rate was decreased significantly（P=0.000） as compared with those of control group. Meanwhile the results of siATF6 groups were on the control. The MTT results and Western blot results were in accordance with that of FCM. Conclusion：Under stress condition，ATF6 may promote the proliferation of SW1353 cells and suppress its apoptosis.