组织蛋白酶S对RA软骨细胞分泌Ⅱ型胶原的研究  

Study of Cathepsin S on type Ⅱ collagen secreted by RA chondrocytes

作　　者：赵进军[1] 黄琴[1] 任昊[1] 欧阳晴晴 毋静[2] 杨敏[1] ZHAO Jinjun;HUANG Qin;REN Hao;OUYANG Qingqing;WU Jing;YANG Min(Department of Rheumatology and Immunology,Nanfang Hospital,Southern Medical University,Guangzhou,Guangdong 510515,China;Department of Rheumatologyand Immunology,Zhujiang Hospital,Southern Medical University,Guangzhou,Guangdong 510515,China)

机构地区：[1]南方医科大学南方医院风湿免疫科,广州510515 [2]南方医科大学珠江医院风湿免疫科,广州510515

出　　处：《重庆医学》2018年第34期4345-4348,共4页Chongqing medicine

基　　金：广东省科技计划项目基金(2017ZC0086);南方医科大学南方医院院长基金(2016B024)

摘　　要：目的通过对滑膜成纤维细胞、滑膜巨噬细胞、肥大细胞和软骨细胞的共培养,以发现对类风湿关节炎(RA)软骨细胞分泌Ⅱ型胶原(CⅡ)影响最大的细胞类型和作用机制。方法选用C57BL/6小鼠进行胶原诱导的关节炎(CIA)造模,原代培养上述4种细胞,并进行共培养。分别加入组织蛋白酶S的特异性抑制剂(LHVS)和非特异性抑制剂(E64),加入肥大细胞的激活剂(C48/80)和膜稳定剂色甘酸二钠(DSCG)等。分别检测培养上清液及软骨细胞中组织蛋白酶S和CⅡ的表达。结果巨噬细胞和肥大细胞表达组织蛋白酶S,而滑膜成纤维细胞不表达。用肿瘤坏死因子-α(TNF-α)激活的滑膜成纤维细胞对软骨细胞CⅡ的表达无影响,巨噬细胞与软骨细胞共培养时可减少CⅡ水平[(8.79±2.79)ng/mL],软骨细胞对照组为(17.75±2.84)ng/mL,但抑制组织蛋白酶S后CⅡ恢复[LHVS组为(16.15±3.05)ng/mL,E64组为(12.55±2.64)ng/mL]。在肥大细胞与软骨细胞共培养时,激活的肥大细胞可明显减少软骨细胞分泌CⅡ[(9.82±0.42)ng/mL],共培养对照组为(26.09±3.34)ng/mL,抑制组织蛋白酶S后,软骨细胞分泌CⅡ水平恢复[分别为(30.21±2.57)ng/mL和(29.68±2.15)ng/mL]。检测各组软骨细胞中CⅡ的mRNA,未发现各组间有差异。结论巨噬细胞和肥大细胞是组织蛋白酶S的两个主要来源,组织蛋白酶S可能是破坏软骨细胞分泌的CⅡ的主要因素。Objective To co-culture synovioblast,synovial macrophages,mastocyte and chondrocyte in order to find the cellular type with greatest impact on secreting typeⅡcollagen in RA chondrocytes and its mechanism.Methods C57BL/6 mice were selected for constructing the CIA animal model,and mouse synovial fibroblasts,peritoneal macrophages,bone marrow-derived mast cells and articular chondrocytes were performed the primary culture and co-culture.Specific inhibitors of Cathepsin S(LHVS)and non-specific inhibitors(E64)were added,and the mast cell activators(C48/80)and membrane stabilizers(DSCG)were added.The supernatant and chondrocytes were collected and the expression of cathepsin S and typeⅡcollagen were detected.Results Cathepsin S was expressed in macrophages and mast cells,but not expressed in synovial fibroblasts.Synovial fibroblasts activated with TNF-alpha had no effect on the expression of typeⅡcollagen in chondrocytes.Macrophages and chondrocytes co-culture could decrease the amount of typeⅡcollagen[(8.79±2.79)ng/mL],while which in the control group was(17.75±2.84)ng/mL,but the amount of typeⅡcollagen recovered after Cathepsin S inhibition[(16.15±3.05 ng/mL in the LHVS group and(12.55±2.64)ng/mL in the E64 group].When chondrocytes and mast cells were co-cultured,the typeⅡcollagen amount secreted by chondrocytes was significantly decreased in activated mastocyte[(9.82±0.42)ng/mL,which in the co-culture control group were[(26.09±3.34)ng/mL),but the typeⅡcollagen amount secreted by chondrocytes was back to original amount after inhibiting Cathepsin S[(30.21±2.57ng/mL and(29.68±2.15)ng/mL respectively).mRNA of typeⅡcollagen in chondrocytes was detected by RT-PCR,but no difference was found.Conclusion Macrophages and mast cells are two major sources of cathepsin S,and Cathepsin S might be the main factor disrupting the typeⅡcollagen secreted by chondrocytes.

关 键 词：肥大细胞 巨噬细胞 软骨细胞 Ⅱ型胶原 组织蛋白酶S 

分 类 号：R593.22[医药卫生—内科学]