【作者】 蔡广研；

【导师】 陈香美；

【作者基本信息】 军医进修学院 ， 肾脏病学， 2001， 博士

【摘要】 目的：本研究旨在探讨 MRL/1pr自发性狠疮小鼠肾脏明胶酶A、B(MMP-2与MMP-9)的表达变化，明确水蛭的调节作用及机理。方法：8周龄狠疮小鼠60只，随机分为治疗组与对照组。治疗组经饲料服用生水蛭粉3g/kg/d共20周。观察小鼠的存活率、蛋白尿、肾功能与肾脏病理改变。利用免疫组化、明胶酶谱以及原位酶谱法检测肾脏明胶酶A、B的表达及活性变化。同时利用免疫组化与RT-PCR方法检测狼疮小鼠肾脏凝血酶受体PAR-1的表达，以改良的MSB染色法检测纤维蛋白的沉积。为了明确局部沉积的纤维蛋白是否可以调控明胶酶的表达，通过纤维蛋白与系膜细胞体外共培养，利用明胶酶谱法、纤维蛋白酶谱法、RT-PCR以及纤维蛋白平板法现察纤维蛋白诱导活化系膜细胞表达明胶酶A、B以及tPA与uPA的作用，明确明胶酶活化与PA/纤溶酶系统之问的关系。结果：水蛭能够延长狼疮小鼠的存活率(70%.50%)、减少蛋白尿(40%:80%)并减轻肾小球与小血管的病变。与8周龄狼疮小鼠比较，28周龄狼疮小鼠肾小球内明胶酶A、B的表达、活化以及明胶酶的净活性均明显增加，与狼疮性肾炎的病理损害密切相关，水蛭能够抑制明胶酶A、B的表达及活化。8 2 车由进修丰阮博士学仁企父 中又扔要 周龄狼疮小鼠肾脏没有纤维蛋白的沉积，在肾小球内皮细胞、系 膜细胞、上皮细胞与小血管内皮细胞有广泛的凝血酶受体N卜1 的表达。而28周龄狼疮小鼠肾小球系膜区、内皮下与血管内有 广泛的纤维蛋白的沉积，肾小球内皮细胞、系膜细胞与小血管内 皮细胞凝血酶受体 PAR八的表达却明显减少，提示 PARl受到凝 血酶的持续活化。水蛙可以减少肾脏局部纤维蛋白的沉积，抑制 凝血酶受体的活化。体外实验发现，纤维蛋白能够诱导系膜细胞 明胶酶 A、B的表达及活化，纤维蛋白还可以诱导 t PA与 ［JPA的 表达，总 PA活性明显增加。利用抑肽酶阻断 PA／纤溶酶的活性 可以完全抑制酶原形式明胶酶A、B的活化。结论：MRL／lpr 自 发性狠疮小鼠肾脏明胶酶A、B的表达明显增加，在狼疮性肾炎 的发展中扮演重要角色。凝血酶、纤维蛋白与明胶酶A、B的表 达与活化关系密切。水蛀阻断凝血酶的活性，通过抑制PAR习的 活化以及减少纤维蛋白的沉积这两种机制抑制狠疮性小鼠肾脏 明胶酶A、B的表达与活化，这可能是水蛀减轻MRL／l pr自发性 狼疮小鼠’肾脏病理损害的主要作用机制之一。更多还原

【Abstract】 Coagulation and fibrinolytic processes are involved in the pathogenesis of lupus nephritis The presence of thrombin activation and fibrin deposition in the glomeruli may be associated with more severe diseases We have found that hirudo reduce fibrin deposition and ameliorate renal lesions in primary glomerulonephntis In the present study we investigated its effect on inhibiting disease progression in MRL lpr/lpr mice MRL lpr/lpr mice were admimstered daily with hirudo (3g/kgld orally) at the age of 8 weeks After treatment for 20 weeks, a progressive reduction in positive proteinuria number/total mice (40% vs 80%, proteinuria over 300mgIdl as positive) and a prolonged survival rate (70% vs 50%) were found when compared with the control group Histological analysis of kidney tissues indicated that hirudo could inhibit the mesangial proliferation and renal sclerosis that was evident in murine lupus nephntis No significant differences were observed in the tubular and interstitial lesions Using SDS-PAGE gelatin zymography, we have identified increased expressions of both latent and activated form enzymes of MMP-2 and MMP-9 in unne and kidney extraction Immunohistochemical staining showed both MMP-2 and MMP-9 were obviously up-regulated within glomerulus in control group To determine the net gelatinase activities in murine lupus kidney, film in situ zymography was used, we found a markedly increased gelatinase activities in munne lupus nephritis, consistent with the immunohistochemical staining Hirudo suppressed 4 the expression of both latent and activated form of MMP-2 and MMP-9 We compared thrombin receptor PAR-i expression in glomerulus using immunohistochemistry and PAR-i mRNA expression by RT-PCR analysis among the groups With the development of murine lupus nephntis, we observed an increase in thrombin receptor mRNA, while thrombin receptor protein expression was strikingly down regulated, suggesting its continuous activation and degradation Hirudo inhibited PAR-i activation significantly correlated with the reduced fibrin deposition To investigate the effects of covering fibnn on the expression of MMP-2 and MMP-9 in cultured glomerular mesangial cells (GMC) RT-PCR and gelatin zymography were used to detect the changes of MMP-2 and MMP-9 in GMC at levels of mRNA expressions and protein activities The relationship between plasminogen activator/plasmin and the activation of MMP-2 and -9 were also investigated Fibnn treatment induced a dramatic expression of activated MMP-2 and MMP-9 in GMC in a dose and time dependent manner Fibrin zymography showed fibnn also induced the expression of tissue type plasminogen activator (tPA) as well as urokinase type plasminogen activator (uPA) Inhibition of the activities of plasmin by aprotimn can totally block the conversion from pro-MMP to active MMP These in vitro and in vivo results suggested that PAR-i activation and fibnn deposition might play an important role in regulating the MMP-2/-9 expressions in munne lupus nephritis, contributing to the remodeling of extracellular matrix Hirudo delays the development of glomerulonephntis and improves survival in MRL lpr/lpr mice at least by suppressing the expressions of MMP-2/-9 via inhibiting thrombin receptor PAR-i activation and fibrin deposition更多还原