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

作　　者：韩敦富[1] 尹荷珊 王延[1] 王鹏云[1] 汪震[1] 魏超[1] 时明 Han Dunfu;Yin Heshan;Wang Yan(Dept of Spine Surgery, The Central Hospital of Zibo, Zibo 255036)

机构地区：[1]山东省淄博市中心医院脊柱外科,淄博255036

出　　处：《安徽医科大学学报》2019年第1期64-68,共5页Acta Universitatis Medicinalis Anhui

基　　金：山东省医药卫生科技发展计划项目(编号:2011HW102)

摘　　要：目的明确siRNA是否可降低大鼠椎间盘细胞对IL-1β预刺激导致的凋亡敏感性,并初步探讨其机制。方法用阴性对照siRNA转染原代大鼠腰椎间盘细胞后,用含10ng/ml IL-1β的1%胎牛血清(FBS)培养基和被转染椎间盘细胞共培养,分别在4、8、16、32 h检测其对Fas受体(Fas)表达的影响。利用Fas-siRNA和阴性对照siRNA分别对原代大鼠椎间盘细胞进行转染48 h后,实验分组和处理:共分5组,N、N-20 ng、N-IL为阴性对照siRNA转染细胞,Si-20 ng、Si-IL为Fas-siRNA2转染的细胞。N为对照组,只加1%FBS培养基; N-20 ng和Si-20 ng在1%FBS培养基培养8 h后,更换为含20 ng/ml Fas配体(Fas L)的1%FBS培养基; NIL和Si-IL:与10 ng/ml IL-1β在1%FBS培养基中培养8 h后,更换为含20 ng/ml Fas L的1%FBS培养基;以上各组加入20 ng/ml Fas L后均继续培养24 h,观察细胞的凋亡情况,Annexin V-FITC/PI双染流式细胞仪检测各组细胞凋亡率,RT-PCR、Western blot分别检测Fas mRNA和蛋白表达水平。结果 (1)与10 ng/ml IL-1β共培养的阴性对照siRNA转染细胞的Fas mRNA在4 h时开始升高,但32 h时才出现统计学差异;而蛋白检测各时间点均未出现表达差异。(2)经IL-1β预处理的Si-IL、N-IL细胞组较相应的未经IL-1β预处理的Si-20 ng、N-20 ng细胞组,细胞凋亡率均升高; Fas-siRNA转染的Si-IL细胞组较阴性siRNA转染的N-IL细胞组的细胞凋亡率明显下降。(3)细胞凋亡率与Fas的表达具有相关性。结论 siRNA可降低大鼠椎间盘细胞对IL-1β预刺激导致的凋亡敏感性,但其确切机制尚需进一步研究。Objective To identify whether siRNA can reduce the apoptotic sensitivity of the rat intervertebral disc( IVD) cells stimulated by IL-1β in vitro,and its potential mechanism. Methods The cells transfected with negative control siRNA cocultured with 10 ng/ml IL-1β in 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-1β for 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 of 37 ℃,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-1β in1% FBS medium for 4 h,but there was no statistical difference in the increased expression of Fas protein among the 3 time point( 4 h,8 h,16 h). It was until 32 h cocultured with 10 ng/ml IL-1β that 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 red

关 键 词：椎间盘细胞 凋亡 SIRNA IL-1Β RNA干扰 

分 类 号：R318.04[医药卫生—生物医学工程]