缺氧状态下人颞下颌关节滑膜成纤维细胞环氧合酶2的表达及意义

The role of hypoxia on expression of cyclooxygenase-2 in synovial fibroblasts derived from human temporomandular joint

目的研究缺氧对人颞下颌关节滑膜成纤维细胞环氧合酶2(COX2)表达的影响,并探讨其在颞下颌关节紊乱病(TMD)中的生物学作用。方法应用逆转录聚合酶链反应(RTPCR)和Western印迹检测缺氧不同时间段滑膜成纤维细胞COX2基因与蛋白质表达水平。采用酶联免疫吸附实验(ELISA)以及明胶酶谱法分别测定缺氧组、缺氧与COX2特异性抑制剂塞来昔布二者联合作用组前列腺素E2(PGE2),金属基质蛋白酶2、9(MMP2、MMP9)分泌量的变化,以常氧组为对照。结果(1)RTPCR测定细胞缺氧4、8hCOX2mRNA分别为1.51±0.34和1.39±0.57,与常氧组0.65±0.24和0.71±0.15比较,差异有统计学意义(均P<0.05)。(2)Western印迹测定细胞COX2蛋白表达,缺氧6h为0.29±0.06,12h为0.51±0.09,24h为0.68±0.11,而常氧组检测不出COX2蛋白(均P<0.01),表明缺氧条件下,细胞COX2蛋白表达呈时间依赖性增加。(3)ELISA测定细胞缺氧12h,释放的PGE2(7.6ng/ml±0.8ng/ml)显著高于常氧组(2.5ng/ml±0.4ng/ml,P<0.01);联合药物处理组(4.3ng/ml±0.4ng/ml)也显著低于缺氧组(P<0.05),表明细胞PGE2释放受抑制。(4)明胶酶谱法测定细胞缺氧12h,分泌的MMP2、MMP9高于常氧组,但在药物联合作用下,分泌能力减弱。结论组织缺氧是导致TMD的一个重要因素;缺氧状况下,人滑膜成纤维细胞可能通过增加COX2的表达来影响缺氧所致颞下颌关节病变进程。

Objective To investigate the effects of hypoxia on expression of COX-2 in synovial fibroblasts derived from human TMJ and to analyze the biological role of COX-2 on TMJ disorders. (Methods)Synovial fibroblasts were induced by hypoxia,COX-2 expression was determined by reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting. Moreover,cells were stimulated by hypoxia or celecoxib or both of them,prostaglandin E2(PGE2)、matrix metalloproteinase 2、9(MMP-2、MMP-9)release was detected by enzyme-linked immunosorbent assay(ELISA)or gelatin zymography. Cells that were placed under normoxic conditons were used as controls. Results (1) In response to hypoxia,at 4 h(1.51±0.34)、8 h(1.39±0.57),significant elevation of COX-2 mRNA expression occurred,compared with data (0.65±0.24)、(0.71±0.15) under normoxic condition(both P<0.05).(2)The elevation of COX-2 protein was detectable at 6 h(0.29±0.06),12 h(0.51±0.09),24 h(0.68±0.11),contrasted with normoxia(all P<0.01). Therefore, it is suggested that COX-2 expression pattern present a time-dependent response to hypoxic conditions.(3)The concentration of PGE2 released by synovial fibroblasts under 12 h hypoxic condition (7.6 ng/ml±0.8 ng/ml) was significantly higher than those under normoxic condition (2.5 ng/ml±0.4 ng/ml,P<0.01)or in response to celecoxib combined with hypoxia (4.3 ng/ml±(0.4 ng/ml),P<0.05).(4) The amount of MMP-2、MMP-9 produced by synovial fibroblasts under 12 h hypoxic condition was upregulated compared with that under normoxia or in response to celecoxib combined with hypoxia. Conclusions Hypoxia is,at least in part,responsible for the pathogenesis of TMJ disorders, probably by promoting COX-2 expression of synovial fibroblasts derived from TMJ.