【摘要】 目的:探讨衰老的大鼠骨髓间充质干细胞凋亡抗性是否增加且是否通过凋亡抑制蛋白(IAP)家族介导。方法:用DMEM/F12中加入10%胎牛血清(FBS)的完全培养基培养大鼠骨髓间充质干细胞(BMSC)至第三代,使用D-半乳糖(D-gal)诱导成衰老的BMSC并用β-半乳糖苷酶染色做衰老鉴定。用肿瘤坏死因子α(TNF-α)和环己酰亚胺(CHX)共同处理正常对照组与衰老组的BMSC,通过贴壁细胞计数统计其生存率,并通过蛋白印迹法检测两组细胞中Cleaved PARP、Cleaved Caspase3蛋白、IAP家族成员c-IAP1、c-IAP2、XIAP以及衰老相关标志蛋白的水平。再用IAP抑制剂AT-406处理衰老的细胞,CCK-8检测处理组以及未处理组细胞的细胞活力。最后用凋亡抑制剂z-VAD-fmk和AT-406共同处理正常对照组与衰老组细胞,观察z-VAD-fmk能否减少IAP蛋白被抑制后所引起的细胞死亡。结果:与正常对照组相比,衰老的BMSC中由TNF-α和CHX共同诱导引起的细胞死亡数明显减少,凋亡标志物Cleaved PARP和Cleaved Caspase3蛋白的水平降低,而IAP家族成员c-IAP1、c-IAP2和XIAP的蛋白水平升高。用AT-406处理衰老BMSC后细胞的死亡数明显高于未处理的衰老BMSC组,通过z-VAD-fmk抑制凋亡通路后,AT-406处理的衰老细胞的死亡数减少。结论:衰老BMSC通过升高IAP家族蛋白水平从而提高对凋亡的抵抗能力。更多还原

【Abstract】 Objective: To investigate whether the ability to resist apoptosis in senile bone marrow mesenchymal stem cells is increased, and the mechanism is via increasing apoptosis-inhibiting protein(IAP) family expression. Methods: Rat bone marrow mesenchymal stem cells(BMSC) were cultured in complete medium supplemented with 10% FBS in DMEM/F12 to the third passage,followed by D-galactose(D-gal) to induce BMSC senescence and use β-Galectinase staining was used for aging identification. After normal BMSC and senile BMSC were treated with tumor necrosis factor alpha(TNF-α) and cycloheximide(CHX), the survival rate was analyzed by adherent cell count, and the expressions of Cleaved PARP, Cleaved Caspase3, c-IAP1, c-IAP2, XIAP and senescence-associated marker protein were detected by Western blotting. Then, the senile BMSC were treated with the IAP family genes inhibitor AT-406, and CCK-8 was used to examine the cell viability of treated and non-treated groups. Finally, the apoptosis inhibitor z-VAD-fmk and AT-406 were co-treated normal and senile BMSC to observe whether z-VAD-fmk reduce the number of dead cells caused by inhibiting apoptosis-inhibiting protein(IAP) family. Results: Compared with normal BMSC, the number of dead cells induced by TNF-α and CHX was significantly lower in senile BMSC, and the expressions of Cleaved PARP and Cleaved Caspase3 were also lower. Conversely, the expressions of c-IAP1, c-IAP2 and XIAP in aging BMSC were obviously higher than normal group. In addition,the number of dead senescent cells was significantly higher in AT-406-treated senile BMSC than non-treated senile BMSC. Finally, the number of dead cells in AT-406-treated senile BMSC declined after using z-VAD-fmk. Conclusion: Aging BMSCs can resist apoptosis via increasing the apoptosis-inhibiting protein(IAP) family expression.更多还原