中文摘要：

目的探讨原癌基因c-jun在毒胡萝卜素内酯（TG）诱导的小鼠胚胎成纤维细胞凋亡中的作用及机制。方法应用TG处理野生型（c-jun＋/＋）,基因敲除型（c-jun-/-）和基因重组型（c-jun Re）小鼠胚胎成纤维细胞后观察细胞生存率;流式细胞术（FACS）和DNA染色进行细胞凋亡分析;蛋白免疫印迹技术分析蛋白c-jun、caspase-12及内质网应激反应分子伴侣Grp78、Gadd153和α-tubulin的表达;共聚焦显微镜观察内质网染色及形态。结果不同浓度TG处理3组细胞4 h后观察细胞生存率,TG在50 nmol/L及100 nmol/L时3组细胞生存率出现差异,c-jun＋/＋与c-jun-/-组较c-jun Re组生存率减低（P〈0.05）;TG未处理与处理均未影响c-jun蛋白的表达,c-jun-/-无c-jun蛋白表达,c-jun Re高表达c-jun蛋白;FACS与DNA染色结果显示,TG处理后c-jun-/-组较c-jun Re组DNA含量增高（P〈0.05）,并在c-jun-/-组出现典型的凋亡细胞亚二倍体DNA;荧光显微镜下观察细胞DAPI染色,与c-jun Re组比较c-jun-/-组DNA染色弱且模糊紊乱;在c-jun＋/＋与c-jun-/-组中12 h比24 h caspase-12蛋白表达低（P〈0.05）,与c-jun＋/＋组比较,c-jun-/-组caspase-12蛋白表达明显增高（P〈0.05）,而在c-jun Re组几乎无caspase-12蛋白表达;经TG处理后,内质网应激反应分子伴侣Grp78和Gadd153,c-jun Re组此两种标识物的表达明显低于其在c-jun-/-组中的表达,并发现了内质网构造在两种细胞中的不同样式,c-jun Re组相比c-jun-/-组内质网结构更加完整丰富宽广。结论 TG可以引起小鼠成纤维细胞出现细胞凋亡。c-jun基因表达的不同改变了细胞凋亡的程度,这种改变是通过细胞对内质网应激反应的介导。c-jun基因的表达在TG诱导的小鼠成纤维细胞凋亡中起保护作用。

英文摘要：

Objective To examine the impact of c-jun on thapsigargin-induced fibroblast cell apoptosis and address the mechanisms. Methods The thapsigargin was used to treat c-jun- /- mouse fibroblast cells,wild-type mouse fibroblasts and reconstitution of cjun expression in c-jun- /- cells（ c-jun Re）. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was detected by fluorescence activated cell sorting（ FACS） and DNA staining. c-jun,caspase-12,Grp78,Gadd153 and α-tubulin were analyzed by immunoblotting.Confocal images of cells loaded with the ER-Tracker and visualized by two-photon microscopy. Results Wild type（ c-jun ＋ / ＋）,c-jun- /-and c-jun Re cells exhibited dramatic differences in sensitivity to TG. The c-jun- /- cells were the most sensitive,while c-jun Re cells were relatively resistant,to TG-induced cell death（ P〈0. 05）. TG treatment led to the activation of caspase-12,as indicated by the cleavage of procaspase-12,in c-jun- /- cells in a time-dependent manner. In contrast,caspase-12 activation was significantly attenuated in wild type fibroblast cells and abrogated in c-jun Re cells in response to TG treatment（ P〈0. 05）. c-jun- /- cells exhibited a large degree of apoptosis after treatment with TG,while c-jun Re cells demonstrated relative resistance to TG-induced apoptosis. c-jun- /- mouse fibroblast cells were more sensitive to TG-induced cell death compared to wild-type mouse fibroblasts,while reconstitution of c-jun expression in c-jun- /- cells（ cJun Re） enhanced resistance to TG-induced cell death（ P〈0. 05）. The expression levels of ER chaperones Grp78 and Gadd153 induced by TG were lower in c-jun Re than those in c-jun- /- cells. ER-tracker found different patterns in the ER structure between c-jun- /- and c-jun Re cells.Conclusions Thapsigargin could cause mouse fibroblast cells apoptosis. c-jun gene expression changes of apoptosis degree,this change is mediated by endoplasmic reticulum stress. c-jun gene expression plays a protective role in thapsigar