Xaf1诱导XIAP-/-成纤维细胞凋亡机制的研究

Investigation of Mechanism of Xaf1 Induction Apoptosis in XIAP -/- Fibroblasts

【目的】利用XIAP-/-成纤维细胞,检测在剔除XIAP的情况下Xaf1诱导细胞凋亡的作用,探索Xaf1诱导细胞凋亡的机制。【方法】Xaf1诱导的XIAP-/-成纤维细胞凋亡由流式细胞DNA含量来表示,免疫荧光显微镜检测XIAP-/-成纤维细胞中Xaf1的亚细胞分布,共转染并细胞周期DNA含量流式细胞术检测Bcl2对Xaf1诱导的细胞凋亡的影响,细胞色素C流式细胞术检测Xaf1对细胞色素C释放的调节。【结果】Xaf1与TNFα显著协同诱导XIAP-/-成纤维细胞凋亡,凋亡峰值27%。Bcl2能抑制Xaf1诱导的XIAP-/-成纤维细胞凋亡,凋亡峰值由27%下降至6%。在剔除XIAP的情况下,Xaf1仍可诱导细胞色素C释放,峰值为21%。【结论】Xaf1通过胱冬肽酶非依赖机制释放细胞色素C而诱导肿瘤细胞凋亡。

[Objective] XIAP-/- fibroblasts were used to detect the effect of Xafl on cell apoptosis without XIAP and to investigate the mechanism of Xafl induces cell apoptosis. [Methods] The effect of Xafl on cell apoptosis were measured by flow cytometry. The sub-cellular localization of Xaf1 in XIAP-/- fibroblasts was estimated by immunofluoresces assay. The effect of Bcl2 on Xafl induced apoptosis were measured by DNA content flow cytometry after co-transfection. Regulation cytochrome C releasing by Xafl was measured by cytochrome C flow cytometry. [Results] Flow cytometry demonstrated that Xafl dramatically cooperated with TNFα to induce in XIAP-/- fibroblasts apoptosis, the peak was 27%. Introduction of Bcl2 in XIAP-/- fibroblasts inhibited Xaf1 induced apoptosis, the peak decreased from 28% to 6%. Xaf1 released cytochrome C without XIAP, the peak was 21%. [Conclusions] Xafl releases cytochrome C and induces cell apoptosis by a caspase independent mechanism.