软骨中硫化氢含量及其对白介素1β诱导的软骨细胞基质金属蛋白酶13表达的抑制作用  

Hydrogen sulfide in cartilage and its inhibitory effect on matrix metalloproteinase 13 expression in chondrocytes induced by interlukin-1β

作　　者：潘利平[1] 曹永平[1] 文立成[1] 柴卫兵[1] 杜军保[2] 金红芳[2] 柳佳[2] 杨昕[1] 孟志超[1] 刘恒[1] 崔云鹏[1] 王瑞[1] 吴浩[1] 周星彤[1] 李翔[1] 李卓扬 塔拉提百克.买买提居马 

机构地区：[1]北京大学第一医院骨科,北京100034 [2]北京大学第一医院儿科,北京100034

出　　处：《北京大学学报（医学版）》2016年第2期194-202,共9页Journal of Peking University:Health Sciences

摘　　要：目的:研究关节液及关节软骨中硫化氢(hydrogen sulfide,H2S)及其合成酶的含量和变化,以及H2S对白介素1β(interlukin-1β,IL-1β)诱导的软骨细胞人基质金属蛋白酶-13(matrix metalloproteinase 13,MMP-13)表达的影响。方法:收集在北京大学第一医院行膝关节手术(关节镜手术或膝关节置换术)患者的关节液及废弃的关节软骨,用亚甲基蓝法检测关节液H2S的含量;从废弃的关节软骨中分离出相对正常的软骨细胞,用H2S分子探针方法检测软骨细胞中的内源性H2S,用免疫细胞化学方法检测软骨细胞中H2S合成酶胱硫醚-β-合成酶(cystathionine beta-synthase,CBS)、胱硫醚-γ-裂解酶(cystathionine gamma-lyase,CSE)、巯基丙酮酸硫转移酶(3-mercaptopyruvate sulfurtransferase,MPST);用Western blot法检测H2S合成酶CBS、CSE、MPST在不同退变程度的软骨组织中的含量。培养相对正常的人软骨细胞至第三代,分为4组:(1)对照组:不加任何药物;(2)IL-β组:单加5μg/L的IL-1β;(3)IL-1β+H2S组:提前0.5 h加200μmol/L的Na HS,再加5μg/L的IL-1β;(4)H2S组:单加200μmol/L的Na HS。Western blot法检测各组细胞MMP-13蛋白、总核因子κB p65(nuclear factorκB p65,NF-κB p65)及磷酸化NF-κB p65蛋白的含量,Real-time PCR检测各组细胞MMP-13基因的转录情况。结果:退变膝关节的关节液中H2S含量为(14.3±3.3)μmol/L,Outerbridge分级3级软骨组织中CSE表达量显著高于Outerbridge分级1级软骨组织中的表达量(1.67±0.09 vs.1.26±0.11,P<0.01),而CBS与MPST的表达量差异无统计学意义(P>0.05)。IL-1β组MMP-13的表达较正常对照组明显升高,且差异具有统计学意义(1.87±0.67 vs.0.22±0.10,P<0.05),IL-1β+H2S组MMP-13的表达较IL-1β组明显降低,且差异具有统计学意义(0.55±0.11 vs.1.87±0.67,P<0.05),H2S组MMP-13的表达较正常对照组差异无统计学意义。Real-time PCR方法测量了药物干预实验中各组细胞MMP-13基因转录情况,结果显示IL-1β组MMP-13基因转录较�Objective： To investigate whether endogenous hydrogen sulfide（ H2S） was involved in the pathogenesis of osteoarthritis（ OA） and its underlying mechanism,to detect H2 S and its synthases expression in knee cartilage in patients diagnosed with different severity of OA,and to explore the transcription and expression of gene MMP-13 in chondrocytes treated with IL-1β or H2 S. Methods： Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2 S content using methylene blue assay. Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes. The chondrocytes were cultured to the P3 generation and H2 S molecular probes were used for detection of endogenous H2 S generation in the chondrocytes. Immunocytochemistry was used to detect the localization of H2 S synthases including cystathionine β-synthase（ CBS）,cystathionine-γ-lyase（ CSE）,and mercaptopyruvate sulfurtransferase（ MPST） in OA chondrocytes. Western blot was used to quantify the protein expressions of CSE,MPST,and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty. The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments：（ 1） the normal control group,no reagent was added;（ 2） the IL-1β group,5 μg /L of IL-1β was added;（ 3） the IL-1β ＋ H2 S group,200 μmol / L of Na HS was added 30 min before adding 5 μg / L of IL-1β;（ 4） the H2 S group,200 μmol / L of Na HS was added.The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot,respectively. And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot. Results： The content of H2 S in the synovial fluid of degenerative knee was（ 14. 3 ± 3. 3） μmol / L. Expressions o

关 键 词：硫化氢 骨关节炎 基质金属蛋白酶13 软骨疾病 

分 类 号：R681.3[医药卫生—骨科学]