不同浓度地塞米松对人骨关节炎软骨细胞凋亡及Fas/FasL基因表达的影响  

EFFECT OF DIFFERENT CONCENTRATIONS OF DEXAMETHASONE ON APOPTOSIS AND EXPRESSION OF FAS/FASL IN HUMAN OSTEOARTHRITIS CHONDROCYTES

作　　者：涂意辉[1] 薛华明[1] 夏志道[2] 蔡珉巍[1] 刘晓东[1] 马童[1] 张长青[3] 

机构地区：[1]同济大学医学院附属杨浦医院骨科,上海200090 [2]英国牛津大学临床学院Nuffield骨科和Botnar研究中心 [3]上海交通大学附属第六人民医院骨科

出　　处：《中国修复重建外科杂志》2012年第5期536-541,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：上海市卫生局局级科研课题资助项目(2009218)~~

摘　　要：目的糖皮质激素会破坏软骨,通过观察地塞米松对人骨关节炎软骨细胞(human articular chondrocytes,HACs)凋亡及Fas/FasL基因表达的影响,探讨其促进HACs凋亡的作用机制。方法取行人工膝关节置换的膝骨关节炎患者自愿捐赠软骨组织,体外分离培养HACs,取第2代细胞进行实验。将HACs分别置于含浓度为0.125、1.25、12.5、25及50μg/mL地塞米松培养液中,培养48 h后采用MTT法选择地塞米松最佳工作浓度进行后续实验。将HACs分别采用最佳工作浓度地塞米松(实验组)及不含地塞米松培养液(对照组)培养,0、24、48 h后行TMRE/Hoechst/Annexin V-FITC/7-AAD四重染色检测细胞凋亡,48 h后行实时定量PCR检测Fas/FasL mRNA表达,0、24、48 h后行免疫组织化学染色检测Fas/FasL蛋白表达。结果 25μg/mL浓度组细胞抑制率显著高于50μg/mL浓度组,差异有统计学意义(P<0.05);与其他浓度组比较,差异亦有统计学意义(P<0.05);选择25μg/mL浓度作为后续实验工作浓度。培养0、24、48 h,实验组HACs凋亡细胞百分比分别为5.8%±0.3%、27.0%±2.6%、36.0%±3.1%,呈明显时间依赖性(P<0.05)。培养48 h后实验组及对照组Fas mRNA相对表达量分别为(8.93±1.12)×10—3及(3.31±0.37)×10—3,FasLmRNA相对表达量分别为(5.92±0.66)×10—3及(2.31±0.35)×10—3,两组比较差异均有统计学意义(P<0.05)。随培养时间延长,实验组Fas及FasL蛋白表达逐渐增加,且各时间点均显著高于对照组,差异均有统计学意义(P<0.05)。结论地塞米松可促进HACs细胞凋亡及上调凋亡基因Fas/FasL表达,为进一步探讨Fas/FasL信号通路在地塞米松促HACs凋亡中的作用提供了实验依据。Objective Corticosteroids can destroy the cartilage. To investigate the effect of dexamethasone （Dexa） on the apoptosis and expression of Fas/FasL of human articular chondrocytes （HACs） in vitro so as to explore the mechanism of pro-apoptotic role of Dexa on HACs. Methods Following full agreement of patients, the cartilage specimens were collectedfrom the patients with osteoarthritis undergoing knee replacement. The second passage HACs were incubated in cell culture media containing 0.125, 1.25, 12.5, 25, and 50μg/mL Dexa for 48 hours respectively to determine the optimal concentration of Dexa by MTT. The apoptosis was assessed by TMRE/Hoechst/Annexin V-HTC/7-AAD quadruple staining after culture for 0, 24, and 48 hours. The mRNA expressions of Fas and FasL were determined by real-time quantitative PCR after culture for 48 hours. The protein expressions of Fas and FasL were determined by immunohistochemistry staining analysis after culture for 24 hours and 48 hours. Results The cell inhibitory rate of 25μg/mL Dexa was significantly higher than that of 50 μg/mLDexa （P 〈 0.05）, and there were significant differences when compared with that at other concentrations of Dexa （P 〈 0.05）, so 25 pg/mL Dexa was appropriately selected as an optimal concentration of Dexa. The apoptotic rates of HACs were 5.8% ± 0.3%, 27.0% ± 2.6%, and 36.0% ± 3.1% at 0, 24, and 48 hours, respectively, in a time dependent manner （P 〈 0.05）. The expressions of Fas mRNA were （8.93 ± 1.12） × 10-3 in the experimental group and （3.31 ± 0.37） × 10-3 in the control group, showing significant difference （P 〈 0.05）. The expressions of FasL mRNA were （5.92 ± 0.66）×10-3 in the experimental group and （2.31 ±0.35）× 10-3 in the control group, showing significant difference （P 〈 0.05）. The expressions of Fas and FasL proteins showed an increasingtendency with time in the experimental group and the expressions were significantly higher than those in the control group after culture for 24 hour

关 键 词：地塞米松 骨关节炎 人关节软骨细胞 细胞凋亡 FAS/FASL 

分 类 号：R684.3[医药卫生—骨科学]