安徽医科大学学报2019，Vol.54Issue(1)：64-68,5.DOI:10.19405/j.cnki.issn1000-1492.2019.01.013
siRNA可降低IL-1β刺激椎间盘细胞所致凋亡敏感性
siRNA can reduce the apoptotic sensitivity of the rat intervertebral disc cells stimulated by IL-1β
摘要
Abstract
Objective To identify whether siRNA can reduce the apoptotic sensitivity of the rat intervertebral disc (IVD) cells stimulated by IL-1βin vitro, and its potential mechanism. Methods The cells transfected with negative control siRNA cocultured with 10 ng/ml IL-1βin 1％fetal bovine serum (FBS) medium for 4 h, 8 h, 16 h, 32 h respectively.Then the expression of Fas was evaluated by RT-PCR.Forty-eight hours after rat IVD cells transfected with negative control siRNA or Fas-siRNA, the cells were divided into 5 group:N, N-20 ng and N-IL were transfected with negative control siRNA;Si-20 ng and Si-IL were transfected with Fas-siRNA.N set as control, cultured in 1％FBS medium without IL-1β;N-20 ng and Si-20 ng, cocultured in 1％FBS medium for 8 h, then replaced the medium with 1％FBS medium containing 20 ng/ml recombinant rat Fas ligand (FasL);N-IL and SiIL, cocultured in 1％FBS medium containing 10 ng/ml IL-1βfor 8 h, then the medium was replaced with 1％FBS medium containing 20 ng/ml recombinant rat FasL;all the cells were cultured for 24 hours in the condition of37℃, 5％CO2.Apoptosis morphous of the cells was observed by microscop with Hochest33258 dyeing, apoptosis incidence was evaluated by flow cytometry with double stained with annexin V-FITC and propidium iodide, gene or protein expression levels of Fas was analyzed by RT-PCR or Western blot respectively.Results (1) The Fas mRNA expression of negative control siRNA transfected rat IVD cells began to increase cocultured with 10 ng/ml IL-1βin1％FBS medium for 4 h, but there was no statistical difference in the increased expression of Fas protein among the3 time point (4 h, 8 h, 16 h).It was until 32 h cocultured with 10 ng/ml IL-1βthat appeared the statistical difference. (2) The cell apoptosis incidence of N-IL and Si-IL increased, compared with N-20 ng and Si-20 ng respectively.The cell apoptosis incidence of Si-IL decreased significantly compared with that of N-IL. (3) The cell apoptosis incidence was corelated with the Fas expression. Conclusion siRNA can reduce the apoptotic sensitivity of the rat intervertebral disc cells stimulated by IL-1β, but its exact mechanism still needs further study.关键词
椎间盘细胞/凋亡/siRNA/IL-1β/RNA干扰Key words
intervertebral disc cell/apoptosis/small interference RNA/interleukin-1 beta/RNA interference分类
医药卫生引用本文复制引用
韩敦富,尹荷珊,王延,王鹏云,汪震,魏超,时明..siRNA可降低IL-1β刺激椎间盘细胞所致凋亡敏感性[J].安徽医科大学学报,2019,54(1):64-68,5.基金项目
山东省医药卫生科技发展计划项目(编号:2011HW102) （编号:2011HW102）
