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本研究采用缺氧、血清饥饿(hypoxia and serum deprivation,H/SD)联合处理骨髓间充质干细胞(mesenchymal stem cells,MSCs),模拟MSCs移植的心脏缺血微环境,研究蛋白酶体抑制剂MG-132对H/SD诱导的MSCs凋亡和旁分泌的影响。采用Annexin V/PI流式细胞术检测细胞凋亡,real-time PCR检测白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和白介素-10(IL-10)mRNA表达,免疫荧光染色检测NF-κBp65细胞核转位,Western blot检测IL-1β和TNF-α蛋白表达,ELISA检测IL-10细胞分泌。结果显示,MG-132能够抑制H/SD诱导的MSCs凋亡,并通过抑制NF-κBp65细胞核转位抑制IL-1β和TNF-αmRNA转录;MG-132能够明显增强H/SD诱导的IL-10mRNA转录和IL-10分泌。上述结果提示,MG-132能够抑制缺血诱导的MSCs凋亡和炎症因子IL-1β和TNF-α表达,增强抗炎症因子IL-10的分泌。MG-132预处理有可能成为提高MSCs移植存活率、增强MSCs旁分泌效应的有效策略。
In this study, mesenchymal stem cells (MSCs) were treated with hypoxia and serum deprivation (H / SD) to simulate the ischemic microenvironment of MSCs transplantation and to investigate the effects of proteasome inhibitor MG- 132 on H / SD-induced Apoptosis and Paracrine in MSCs. Cell apoptosis was detected by Annexin V / PI flow cytometry. Real-time PCR was used to detect the mRNA expression of IL-1β, TNF-α and IL-10. The nuclear translocation of NF-κBp65 was detected by immunofluorescence staining, the expressions of IL-1β and TNF-α protein were detected by Western blot, and the secretion of IL-10 was detected by ELISA. The results showed that MG-132 could inhibit the H / SD-induced apoptosis of MSCs and inhibit the transcription of IL-1β and TNF-αmRNA by inhibiting the nuclear translocation of NF-κBp65; MG-132 could significantly enhance H / SD-induced IL-10mRNA Transcription and IL-10 secretion. These results suggest that MG-132 can inhibit the apoptosis of MSCs induced by ischemia and the expression of inflammatory cytokines IL-1β and TNF-α and enhance the secretion of anti-inflammatory cytokine IL-10. MG-132 preconditioning may be an effective strategy to improve the survival rate of MSCs transplantation and enhance the paracrine effect of MSCs.