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目的:研究caspase-8小发夹RNA(Cap8 shRNA)对人骨髓间充质干细胞(hMSCs)凋亡的调控作用。方法:构建2个靶向人caspase-8基因的穿梭质粒pAd-Cap8 shRNA,并鉴定可有效抑制人HEK293细胞中caspase-8表达的pAd-Cap8 shRNA质粒。构建表达Cap8 shRNA的重组腺病毒质粒,并在HEK293细胞中包装、扩增重组腺病毒rAd-Cap8 shRNA。检测rAd-Cap8 shRNA感染的hMSCs中caspase-8 mRNA和蛋白表达。利用annexin V/PI双染色法和caspase-8活性检测分析去血清/缺氧诱导的hMSCs凋亡变化,利用荧光定量PCR检测hMSCs中VEGF、肝细胞生长因子1(HGF)、胰岛素样生长因子(IGF-1)、Bcl-2和Bcl-xL mRNA表达。结果:通过定量PCR筛选到可有效抑制caspase-8表达的pAd-Cap8 shRNA质粒。成功构建Cap8 shRNA的重组腺病毒质粒,并包装、扩增重组腺病毒rAd-Cap8 shRNA。荧光定量PCR和Western blotting结果证实rAd-Cap8 shRNA可有效抑制hMSCs中caspase-8表达。rAd-Cap8 shRNA能有效抑制去血清/缺氧诱导的hMSCs凋亡,降低caspase-8活性,增强hMSCs中HGF、IGF-1和Bcl-2表达。结论:Caspase-8 shRNA可以抑制去血清/缺氧诱导的hMSCs凋亡。
AIM: To investigate the regulatory effect of Cap8 shRNA on the apoptosis of human bone marrow mesenchymal stem cells (hMSCs). METHODS: Two shuttle plasmid pAd-Cap8 shRNA targeting human caspase-8 gene was constructed and pAd-Cap8 shRNA plasmid, which can effectively inhibit caspase-8 expression in human HEK293 cells, was constructed. The recombinant adenovirus plasmid expressing Cap8 shRNA was constructed and packaged into HEK293 cells to amplify the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 mRNA and protein in hMSCs infected with rAd-Cap8 shRNA was detected. The apoptotic changes of hMSCs induced by de-serum / hypoxia were analyzed by annexin V / PI double staining and caspase-8 activity assay. The levels of VEGF, hepatocyte growth factor 1 (HGF), insulin-like growth factor IGF-1), Bcl-2 and Bcl-xL mRNA expression. Results: The pAd-Cap8 shRNA plasmid that can effectively inhibit the expression of caspase-8 was screened by quantitative PCR. The recombinant adenovirus plasmid of Cap8 shRNA was successfully constructed, and the recombinant adenovirus rAd-Cap8 shRNA was packaged and amplified. Fluorescent quantitative PCR and Western blotting results confirmed that rAd-Cap8 shRNA can effectively inhibit caspase-8 expression in hMSCs. rAd-Cap8 shRNA can effectively inhibit the apoptosis of hMSCs induced by serum / hypoxia, reduce the activity of caspase-8 and enhance the expression of HGF, IGF-1 and Bcl-2 in hMSCs. Conclusion: Caspase-8 shRNA can inhibit the de-serum / hypoxia-induced hMSCs apoptosis.