【作者】 李建民；

【导师】 周勇；

【作者基本信息】 北京中医药大学 ， 中西医结合基础（免疫学）， 2001， 博士

【摘要】 类风湿性关节炎是一种常见的以关节损伤为主的慢性、多发性炎症性疾病，其病变以滑膜组织炎性细胞浸润、滑膜细胞增殖、血管翳形成和软骨及软骨下骨质破坏为主要免疫病理表现，最终导致受累关节发生畸形而出现关节废用、致残。本实验利用大鼠佐剂性关节炎模型，分别对山茱萸总甙对大鼠佐剂性关节炎模型防治作用、细胞和分子免疫抑制机理进行深入研究，并应用体外细胞培养技术和Cell-ELISA方法研究山茱萸总甙对炎性细胞因子刺激血管内皮细胞表达粘附分子的影响，应用RT-PCR技术研究了山茱萸总甙对正常大鼠腹腔巨噬细胞iNOS和IL-6mRNA表达的影响，从整体、器官、细胞、分子四个不同层次探讨了山茱萸总甙的可能作用环节，以期探讨山茱萸总甙治疗类风湿性关节炎的分子免疫抑制机理，为开发成国家二类中医药新药提供实验依据。1．山茱萸总甙对大鼠佐剂性关节炎的防治作用 实验采用大鼠佐剂性关节炎模型，观察了山茱萸总甙对于大鼠佐剂性关节炎模型原发病变、继发病变和体重的影响。结果显示：山茱萸总甙具有明显对抗大鼠佐剂性关节炎作用，山茱萸总甙的大、中、小三种剂量造模同时给药24天，均能明显抑制大鼠佐剂性关节炎的关节局部的早期原发肿胀和继发肿胀（P＜0.05或＜0.01），并有明显的量效关系，在病理改变方面其作用明显，以中、大剂量为好，山茱萸总甙可以明显抑制佐剂性关节炎大鼠的继发病变侧的炎性细胞浸润、软骨及骨质损伤和血管翳的形成。在大鼠佐剂性关节炎的病变过程中，与正常组比较，模型组和地塞米松组于注射佐剂后，体重逐渐下降，山茱萸总甙大、中、小剂量组的体重具有上升趋势，本实验的结果提示：山茱萸总甙具有明显对抗大鼠佐剂性关节炎作用。2．山茱萸总甙抗佐剂性关节炎分子免疫机理研究 本实验在上述山茱萸总甙对大鼠佐剂性关节炎具有防治作用的基础上，首先观察了山茱萸总甙对大鼠佐剂性关节炎模型T淋巴细胞增殖反应、DTH反应和山茱萸总甙对于正常小鼠T淋巴细胞增殖、T淋巴细胞膜表面CD3、CD4、CD8分子的表达及产生IL-2的影响，结果显示：山茱萸总甙的大、中、小三种剂量造模同时给药24天，山茱萸总甙可以明显抑制佐剂性关节炎大鼠的T淋巴细胞增殖反应和DTH反应（P＜0.01）而抑制细胞免疫，山茱萸总甙的大、中、小三种剂量，连续灌胃7天，山茱萸总甙可以抑制正常小鼠T淋巴细胞增殖（P＜0.01）。T淋巴细胞膜表面CD3分子的表达（P＜0.01），其中，以大剂量抑制作用较强，不同剂量的山茱萸总甙均可抑制T淋巴细胞膜表面CD4、CD8分子的表达（P＜0.05或P＜0.01），中、大剂量还可以提高CD4/CD8的比值，说明山茱萸总甙抑制CD8的作用比抑制CD4的作用更明显，不同剂量的山茱萸总甙还可以抑制T淋巴细胞产生IL-2（P＜0.05或P＜0.01），说明山茱萸总甙具有抑制机体细胞免疫功能的作用。第二观察了山茱萸总甙对AA大鼠炎性细胞因子IL-1、IL-6、TNF-α以及炎性介质PGE₂产生的抑制作用，结果发现：山茱萸总甙可以明显抑制佐剂性关节炎大鼠的腹腔巨噬细胞产生IL-1、IL-6和TNF-α（P＜0.05或P＜0.01），反映本药抑制IL-1、IL-6和TNF-α的炎性顺产生，山莱荚总忒还可以抑制第24天大鼠喇性关节炎血浆中PGE；的产生o功．叮人从而抑制m巳的致炎、渊作用，说明山荣荚总忒账剂性关节炎的作用与对抗上述炎性倾作用有关。为了深A8f究山莱荚总戒的免邮用机理，我们应用RT－PCR 4k7Iv～t究了山莱英总獭于大鼠腻性关节炎模型ILlra和iNOSallA影响，结果显示：山茉英总忒的大、中、小三种剂量造摸同时鞭24天，山莱荚总忒可以明显抑制喇性关节炎大民腹腔巨御胞iNOSlllttnA ＄410诱导ILlraedNA ＆ie．说明山莱荚总忒具有抑制铡性关节炎大邑腹胜巨膨胞ilf0SnlnnA s而抑制炎性介质NO的产生，憎加诱导ILlralnRNA表达而撇炎幽胞因子ILI炎性介质作用，同时捌俐 用体外细鹏躺和R… toftsf究了山某奠总忒对正常大鼠腹腔巨噬细胞表达IL6和iN0haNA影响，结果表明：不同浓度的山莱荚总忒均能抑制IL6和iNOSlllttnA表达，抑制IL4和iNOSallA表达可能是山莱奠总忒治疗猕湿性关节炎的主要分子免删制机理之一． 最后的腑实瓣用 Cel ldsISA方法探讨山莱英总忒对 HIJ’VEC表达 ICdel、CD44、a4①影响，结果发现：r皿 a和 rILl均可诱导的内皮细胞活化而明显增加 IC胁1、CD44、a4p7等粘附分子的表达，地塞米松和山踉总忒均可抑制r TNWTNWa和rILI诱导u＊04鞭I CAeCAe二、C04 4、C4＊ 7等粘跳于＜0．01)。抑制脆内皮细脓ICWel、CD44、a4m等粘附分子而抑制她细胞向关节腔中胭组织内的迁移、积聚可龈山踉总忒主要抗炎和分子免铡制懈之一． 以上台弘良垢示：山菜奠总忒具有抗炎和免铡制作用而防治佐剂性关节炎，其分子刎可能与抑制 胞免疫，涨炎性细胞因子和粘附分子的作用有关。更多还原

【Abstract】 The experimental study of molecular immunological mechanism about TGCO against rheumatoid arthritisAbrstractRheumatoid arthritis is a common systemic disease characterised by chronic joint inflammation, degradation of the extracellular matrix and destruction of the architecture and function of joint. Histoolgicaily, hyperplasia of the synovial lining cells, an inflammatory cell infiltrate composed of lymphocytes and histiocytes with scattered plasma cells and granulocytes and destructive changes of connective tissue and cartilage are seen. In order to provide the experimental basis for adjuvant arthritis (AA) and clinical use of chinese new herb of total glycosides from Cornus Officinalis(TGCO) capsule. We have establisted the niurine model of AA to research the detailed molecular immunological mechanism for TGCO.l.Effects of prevention and cure of TGCO on AAWe have succesefiully establisted the murine model of AA to research effects of prevention and cure of TGCO .The results showed that TGCO had the well effect of prevention and cure on AA. TGCO could inhibit joints of the forepart primary and secondary affect swell. TGCO could inhibit hyperplasia of the synovial lining cells, an inflanunatory cell infiltrate and destructive changes of connective tissue and cartilage of joints of the secondary affect pathology.2.The study of molecular immunological mechanism about TGCO against AAOn the basis of above, we have investigated the molecular immunological mechanism about TGCO against AA. The experimental results showed that TGCO could suppress the proliferation of T lymphocytes stimulated with ConA and DTH reaction in rats adjuvant arthritis. It had the inhibitory effect on IL-i ,IL-6 and TNFa produced in cultured peritoneal macrophages and PEG2 in plasma in rats adjuvant arthritis. It had the inhibitory effect on iNOSmRNA gene expression and the induced effect on IL-ira mRNA gene expression in peritoneal macrophages of SD rats adjuvant arthritis in vivo. It had the inhibitory effect of differed concentration of TGCO on IL-6mRNA gene expression and iNOSmRNA gene expression in cultured rat peritoneal macrophages stimulated with LPS in vitro. The effects of TGCO on T lymphocytes and T lymphocytes subgroup, and the IL-2 production of T lymphocytes were studied in healthy mice in order to explore the immunosuppressive mechanism of TGCO. The results showed that all the different doses of TGCO could inhibit the multiplication of T lymphocytes, CD3 surfaceexpression in T lymphocytes, and CD4 and CD8 surface expression in T lymphocytes: besides, middle and large TGCQ doses could increase the ratio of CD4/CD8 which implies that the TGCO effect of inhibiting CD8 is stronger than that of inhibiting CD4. The results also showed that all the different doses of TGCO could inhibit the IL-2 production of I lymphocytes.TGCO could inhibite the expression of adhesion molecules(ICAM-l and CD~) and integrin (a 4 ~ 7)with cytokines (IL-I and TNF- a )st駈ulated ECV3O4 in vitro that is an immortal endothelial cell line derived from HUVEC.We conclude that total glycosides of comus officinalis (TGCO) were extract from cornus officinalis of an Chinese herb have immunosuppressive effects and against rats adjuvant arthritis. It is the main molecular immunological mechanism of against rats adjuvant arthritis that suppressed function of cell immune and inhibited the expression of adhesion molecules(ICAM-l and CD~) and integrin (a 4 ~ 7) and had inhibitory effect on IL-l,IL--6 and TNF- a produced and iNOSmRNA gene expression and induced effect on IL-ira mRNA gene expression in cultured peritoneal macrophages of SD rats adjuvant arthritis.更多还原