CAJ | 학술논문

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anne-xin 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。
목적：연구caspase-8소발협RNA（Cap8 shRNA）대인골수간충질간세포（hMSCs）조망적조공작용。방법：구건2개파향인caspase-8기인적천사질립pAd-Cap8 shRNA，병감정가유효억제인HEK293세포중caspase-8표체적pAd-Cap8 shRNA질립。구건표체Cap8 shRNA적중조선병독질립，병재HEK293세포중포장、확증중조선병독rAd-Cap8 shRNA。검측rAd-Cap8 shRNA감염적hMSCs중caspase-8 mRNA화단백표체。이용anne-xin V/PI쌍염색법화caspase-8활성검측분석거혈청/결양유도적hMSCs조망변화，이용형광정량PCR검측hMSCs중VEGF、간세포생장인자1（HGF）、이도소양생장인자（IGF-1）、Bcl-2화Bcl-xL mRNA표체。결과：통과정량PCR사선도가유효억제caspase-8표체적pAd-Cap8 shRNA질립。성공구건Cap8 shRNA적중조선병독질립，병포장、확증중조선병독rAd-Cap8 shRNA。형광정량PCR화Western blotting결과증실rAd-Cap8 shRNA가유효억제hMSCs중caspase-8표체。 rAd-Cap8 shRNA능유효억제거혈청/결양유도적hMSCs조망，강저caspase-8활성，증강hMSCs중HGF、IGF-1화Bcl-2표체。결론：Caspase-8 shRNA가이억제거혈청/결양유도적hMSCs조망。
AIM:To investigate the effect of caspase-8 small hairpin RNA ( shRNA) on attenuating apoptosis of human mesenchymal stem cells ( hMSCs ) .METHODS: Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed.Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR.The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid , which was linearized and transfected into HEK 293 cells for packaging and amplification of the recombi-nant adenovirus rAd-Cap8 shRNA.The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting .Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hM-SCs under the conditions of serum deprivation and hypoxia .The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR.RESULTS:The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR.The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant ad-enovirus ( rAd)-Cap8 shRNA successfully .rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 ex-pression in hMSCs .Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the ap-optotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia , with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2.CONCLUSION:Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia .