靶向 caspase-8小发夹 RNA 减轻去血清／缺氧诱导的人骨髓间充质干细胞凋亡  

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

作　　者：袁伟伟[1] 林秋雄[1] 朱杰宁[1] 李晓红[1] 符永恒[1] 刘晓颖[1] 谭虹虹[1] 邓春玉[1] 单志新[1] 

机构地区：[1]广东省人民医院广东省医学科学院广东省心血管病研究所,广东广州510080

出　　处：《中国病理生理杂志》2014年第7期1172-1178,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30672077;No.81070102;No.81270222);广东省自然科学基金资助项目(No.S2011020005911;No.S2012010009453)

摘　　要：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 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 .

关 键 词：骨髓间充质干细胞 半胱氨酸天冬氨酸蛋白酶8 细胞凋亡 小发夹RNA 

分 类 号：R363[医药卫生—病理学]