【作者】 向导；

【导师】 向阳；

【作者基本信息】 湖北民族学院 ， 中医基础理论， 2012， 硕士

【摘要】 目的：确立祛风清热、活血通络法。培养类风湿关节炎及骨关节炎关节滑液滑膜细胞,评价金边祛风饮对RA滑膜细胞的治疗作用和对炎性细胞因子及金属蛋白酶水平的影响,探讨可能的作用机制,为金边祛风饮在临床上的应用提供实验依据。方法：(1)通过对文献资料的整理,总结及归纳祛风清热、活血通络法运用于类风湿关节炎的理论基础。(2)选择类风湿关节炎(RA)6例及骨关节炎(OA)6例,分为治疗组(RA)与对照组(OA)。患者在无菌条件下行膝关节穿刺抽取关节液,计数细胞数量,加关节液的量按每25cm2培养瓶接种1×107个细胞计算,并加无血清DMEM培养液(含100U/ml青霉素和100mg/L链霉素5-6ml),混匀后置37℃、5%二氧化碳细胞培养箱培养,于倒置显微镜下观察细胞生长情况,用ELISA.PT-PCR法检测祛风清热、活血通络法对类风湿关节炎患者滑膜细胞炎症因子IL-1β,TNF-α和MMP-3水平的表达,MTT法测定滑膜细胞增殖率。结果：(1)滑膜细胞的形态学改变：经反复贴壁及传代后,滑膜细胞逐渐纯化,多以成纤维样滑膜细胞为主。给予金边祛风饮处理72h后,光镜下可见中浓度治疗组及高浓度治疗组RA滑膜细胞细胞密度均一定程度降低,细胞体积明显缩小,在低浓度治疗组,细胞体积变化不明显；(2)ELISA法检测培养上清液中各炎性因子表达：不同浓度金边祛风饮刺激滑膜细胞72h后,培养上清液中的TNF-α.IL-1β、MMP-3均降低,与OA对照组比较有显著差异(P<0.05),与空白对照组相比具有极显著性差异(P<0.01)。组间比较,高剂量组在降低TNF-α、IL-1β和MMP-3水平中有显著性差异(P<0.05)；(3)MTT法检测RA及0A滑膜细胞增殖：不同浓度的金边祛风饮刺激RA、OA滑膜细胞72h后,各浓度治疗组的RA滑膜细胞与空白对照组相比,增殖均受到明显抑制(P<0.05),以高剂量组的抑制率最为明显(P<0.01),RA各治疗组与OA各治疗组比,细胞增殖抑制显著(P<0.05),而OA滑膜细胞在药物各浓度刺激下,细胞增殖没有显著改变(P>0.05)；(4)RT-PCR检测RA滑膜细胞TNF-α、IL-1β及MMP-3表达：结果显示随着金边祛风饮作用浓度的增加,滑膜细胞TNF-α、IL-1β及MMP-3的mRNA表达明显减少,表明金边祛风饮能抑制炎性细胞因子及滑膜细胞基质金属蛋白酶的产生。结论：以祛风清热、活血通络立法指导而组成的金边祛风饮能明显抑制RA滑膜细胞增殖,并呈剂量依赖性减少炎症细胞炎性因子IL-1β、TNF-α、MMP-3分泌,提示本方具有减轻类风湿关节滑膜增殖及炎症反应的理论依据。更多还原

【Abstract】 Objective: To establish a formula Jin Bian Qu Feng Yin (JBY) in treatment ofrheumatoid arthritis based on the theory of traditional Chinense medicine “Qu FengQing Re”-expelling wind and clearing away heat and “Huo Xue Tong Luo”-promoting blood circulation to remove meridian obstruction. To observe the influenceof JBY liquid on production of proinflammatory cytokines and metalloproteinases incultured synovial fibroblasts from patients with rheumatoid arthritis, evaluatingpossible mechanisms of JBY in treatment of rheumatoid arthritis.Methods: The synovial fluid is collected from6patients with rheumatoid arthritis(RA), as well as from6patients with knee osteoarthritis (OA) as control. The cells insynovial fluid are cultured in25cm2bottles and stimulated with low, middle, highdose of JBY, and the level of proinflammatory cytokines IL-1β, TNF-α andmetalloproteinase MMP-3are detected by ELISA and RT-PCR. The proliferation ofthe cells is evaluated by MTT.Results:(1) Synovial cell morphological change: after repeated attached wall andpassaged, synovial cells gradually purified as fibroblast-like cells. After stimulated byJBY72hours, the cells treated with middle or high dose of JBY became small.However, the cells treated with low dose JBY slightly changed as multi-form. Theproliferation of synovial fibroblasts was significantly interfered by JBY with the dosedependent manner；(2) Measured by ELISA the culture supernatant of InflammatoryCytokines: Stimulated72h with different concentration of JBY, culture supernatantsof TNF-ɑ, IL-1β MMP-3were lower, with the OA control group compared significantdifferences (P <0.05), has a very significant difference (P <0.01) compared with thecontrol group,between the two groups, the high dose group in reducing TNF-α, IL-1βand MMP-3level, a significant difference (P <0.05)；(3) MTT assay of RA and OAsynovial cell proliferation:Stimulated72h with different concentration of JBY, thelevel of TNF-α, IL-1β MMP-3in supernatant of synovial cells reduced significantlywith the dose dependent manner, lower in middle-and high-dose JBY-stimulated cellsthan in low-dose stimulated cells.(P <0.05)；(4) RT-PCR show RA synovial cells ofTNF-α, IL-1β and MMP-3expression:In the cells the mRNA expression level ofTNF-α, IL-1β and MMP-3also reduced after co-cultured by JBY. These results weresuggested that JBY could inhibit production of proinflammatory cytokines and MMPsin synovial cells.Conclusion: Based on the theory of traditional Chinese medicine “expelling wind and removing heat and promoting blood circulation to remove meridian obstruction”, theformula JBY significantly suppress RA synovial cell proliferation and reduce IL-1β,TNF-α and MMPs-3production, suggesting JBY may play important role in treatmentof rheumatoid arthritis. The mechanism of the traditional Chinense medical formulashould be further studied.更多还原