Caspase-8 small hairpin RNA attenuates apoptosis of human bone mar-row mesenchymal stem cells under conditions of serum deprivation and hypoxia
10.3969/j.issn.1000-4718.2014.07.004
- VernacularTitle: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
- Author:
Weiwei YUAN
;
Qiuxiong LIN
;
Jiening ZHU
;
Xiaohong LI
;
Yongheng FU
;
Xiaoying LIU
;
Honghong TAN
;
Chunyu DENG
;
Zhixin SHAN
- Publication Type:Journal Article
- Keywords:
Bone marrow mesenchymal stem cells;
Caspase-8;
Apoptosis;
Small hairpin RNA
- From:
Chinese Journal of Pathophysiology
2014;(7):1172-1178
- CountryChina
- Language:Chinese
-
Abstract:
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 .
