【作者】 任文英；

【导师】 陈扬荣；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2003， 博士

【摘要】 系统性红斑狼疮(SLE)是严重危害人类健康的自身免疫性疾病。肾脏是该病最常受累的器官(称为狼疮性肾炎,LN)和主要死亡原因。目前该病的发病机制尚未明了,从中医理论出发,结合现代医学探讨 LN 的发病机制及治疗方法已成为该病研究的一个重要方向。本课题参照 Bergjk 报道的方法成功诱导了慢性移植物抗宿主病(cGVHD)狼疮样小鼠模型,在此基础上,观察临床上证实有较好疗效的中药方剂补肾清热毒方对 cGVHD 模型的作用,并进一步从细胞凋亡及 Th 亚群功能失衡方面探讨 LN 的发病机制和补肾清热毒方发挥作用的分子机制。本课题分四部分进行:1. cGVHD 狼疮样小鼠模型的诱导选用 6-8 周龄的(DBA/2×C57BL/6J)F1 小鼠,通过尾静脉注射雌性 DBA/2 小鼠的淋巴细胞制作模型。取雌性 DBA/2 小鼠的脾、胸腺、淋巴结制成淋巴细胞混悬液,注射时间是 0、3、7、10 天,每次注射 50×106个活细胞。于注射后 0、2、4、8、10、12 周检测尿蛋白浓度并处死部分小鼠,分别取血及肾脏标本,测定抗 dsDNA 自身抗体、血肌酐、尿素氮、血脂和观察肾脏病理改变。尿蛋白浓度用考马斯亮蓝法,自身抗体用间接免疫荧光法,肾组织制成石蜡切片后,经 PAS、Masson、PAM 染色光镜下观察病理改变,同时用透射电镜观察肾组织超微结构变化。结果 F1 小鼠注射母鼠淋巴细胞后 2 周产生抗 dsDNA 自身抗体,出现蛋白尿,4 周血脂、血肌酐、尿素氮轻度升高(P<0.05),肾脏病理仅有系膜细胞轻度增生,未见间质损害。8 周血肌酐等血生化指标明显改变,肾脏病理示肾小球系膜细胞中度增生,间质炎细胞浸润和肾小管大量蛋白管型,10-12 周尿蛋白浓度、抗 dsDNA 自身抗体、血肌酐、尿素氮、胆固醇、甘油三脂明显升高(P<0.05 或 P<0.01),白蛋白明显降低(P<0.01),病理出现局灶或弥漫肾小球硬化,免疫荧光示 IgG、IgM、C3 沿毛细血管壁及系膜区沉积。模型组电镜下可见系膜区免疫复合物沉积。实验结果表明 cGVHD 狼疮样小鼠模型类似人类狼疮性肾炎,是良好的狼疮性肾炎模型。2.补肾清热毒方对 cGVHD 狼疮样小鼠模型的疗效观察模型雌鼠于诱导后第 4 周,随机分为补肾清热毒方组(10 只),强的松组(10 只),模型组(6 只)及未诱导的 F1 对照组(6 只),用药 8 周后处死,观察小鼠血清抗 dsDNA抗体、血肌酐、尿素氮、血脂及肾脏病理改变等指标。结果显示,经补肾清热毒方和强的松干预后,尿蛋白浓度、抗 dsDNA 抗体、血肌酐、尿素氮均有降低(P<0.05 或 P<0.01),白蛋白升高(P<0.05),而补肾清热毒方尚有降低胆固醇作用(P<0.05),两药对系膜细胞增生及间质炎细胞浸润有减轻作用(P<0.05),表明补肾清热毒方对 cGVHD 狼疮样小鼠模型具有良好的的治疗作用。3.补肾清热毒方对 cGVHD 狼疮样小鼠模型肾组织细胞凋亡的调节作用<WP=5>2 补肾清热毒方治疗狼疮性肾炎的疗效与机理的实验研究 按实验二方法进行分组并用药,采用原位末端标记法(TUNEL)染色观察肾组织细胞凋亡;免疫组化、western blot 和 RT-PCR 技术检测 Fas、FasL 基因转录及蛋白表达情况。结果表明补肾清热毒方可增加肾组织 TUNEL 阳性积分,上调 Fas、FasLmRNA 和蛋白水平,提示补肾清热毒方治疗 LN 的作用可能是通过上调 Fas、FasL,促进细胞凋亡实现的。 4.补肾清热毒方对 cGVHD 狼疮样小鼠模型 Th1/Th2 细胞因子的影响 按实验二方法进行分组并用药,采用放射免疫法检测外周血 IL-2、TNF-α、IL-6 水平,结果补肾清热毒方对促进Th1细胞分化,从而加重免疫反应的IL-2,及前炎症因子IL-6、TNF-α 均有抑制作用,表明补肾清热毒方对细胞因子引起的炎症反应有抑制作用。采用免疫组化和 RT-PCR 方法检测肾组织 IL-4、INF-γ 蛋白及 mRNA 的表达,以观察补肾清热毒方对肾组织 Th1/Th2 的影响,结果补肾清热毒方可下调肾组织 IL-4 水平,且 INF-γ / IL-4比值与正常对照组比值相近,表明补肾清热毒方具有调节 Th1/Th2 细胞因子平衡的作用。 以上研究结果表明,cGVHD 狼疮样小鼠模型肾组织细胞凋亡异常及 Th1/Th2 细胞因子失衡,在狼疮性肾炎的发病中起重要作用。补肾清热毒方通过影响肾组织细胞凋亡,调节凋亡调控基因 Fas、FasL 以及 Th1/Th2 细胞因子平衡两种机制而减轻肾脏病理损伤,这可能是该方取得疗效的分子机制之一。更多还原

【Abstract】 Systemic lupus erythematosus(SLE) is a autoimmune disease which severitly harm tohuman healthy. Kidney is the most sensitive organ which can be involved in this disease andnamed with lupus nephritis(LN).At present , the mechanism of SLE is not clear.Exploring themechanism and therapeutic methodes from the direction of Tranditional Chinese Medicine andbinding Western Medicine has been one important pathway to investigate the disease. This topicrefer to the methods of Bergjk and induce successfully the lupus nephritis murine models ofchronic graft-versus-host disease(cGVHD),At the same time, Observing the effects ofBushenQingredu Recipe(BQR)on the models. BQR has been clinically confirmed to havebetter effects on the patients with LN.Additionally ,the topic investigae the pathologicalmechanisms of LN by apoptosis in kidney and the disequilibrium of Th1/Th2 in cGVHD mice. The topic include four parts: 1. Induction of lupus nephritis models of chronic graft-versus-host disease To induce murine lupus nephritis models,we select 6-8 weeks (DBA/2×C57BL/6J)F1hybrids mice and inject parental DBA/2 mice lymphoid cells by caudal vein.Spleen、thymiandlymph nodes(inguinal,axillary,cervical and mesenteric)were removed from female donor miceunder aseptic condition .Single-cell suspensions were prepared with sterile 0.9% NaClsolution(NS).At days 0,3,7and 10, female recipients were injected intravenously(iv)with 50×106viable lymphocytes in 0.3ml NS..At weeks 2,4,8,10and12 after first injection oflyphocytes,urines were collected from all experimental mice.Albuminuria were determined ByCoomassie brilliant blue methods.At the same time ,6 mice were sacrificed to detectedantibody,serum creatinine,blood urea nitrogen and pathological damage of kidneytissue.Antibody(dsDNA) were detected by indirect immunofluoresence,pathological damage ofkidney tissue were observed by the staining PAS、Masson、PAM and electron microscopy.Theresults diplay that two week after injection of lymphocytes,antibody and proteinuria wereproduced ,serum creatinine and blood urea nitrogenthe increased after four week,glomerularmesangial cells increased significantly,interstitial without damaging .At eight week,All theindexes changed significantly,the pathological damage of kidney tissue was glomerularmesangial cells diffuse increased,protein casts appeard .From ten to twelve week,glomerularappeared focal or diffuse hyalinosis and glomeorulosclerosis . immunofluorescence showeddeposits of immunoglobulins IgG、IgM、C3 were observed along the glomerular capillary andmesangial。All the results suggested that the murine models is comparable to the lesions<WP=7>characterizing human LN and offer an excellent model for studies on LN. 2. Effects of BQR on murine lupus nephritis models of chronic graft-versus-hostdisease 4 weeks after first injection of lymphocytes,34 female murine were randomly divided intofour groups ,BQR group(10)、prednisone group(10)、model group(6) and control group(6).8 weeks later,all the mice were killed and observed the index as part one. The results diplay thatthe levels of albuminuria、antibody、serum creatinine and blood urea nitrogen were decreased,albumin were increased in both BQR group and prednisone group. In addition,BQR coulddecrease cholesterol.BQR group and prednisone group could decrease glomerular mesangialcell and infiltrating leucocyte . This result suggested that BQR has better therapeutic effects oncGVHD mice. 3. Effects of BQR on apoptosis in kidney of lupus nephritis models 32 female murine were randomly divided into 4 groups as part two .Immunohistochemistryand western blot was used to detect the protein level of Fas、FasL.the transcription of Fas、FasLmRNA in kidney were detected by RT-PCR, apoptosis was observed by TUNEL assay. Theresults diplay that apoptosis in kidney increased in BQR group and prednisone group, theprotein and mRNA level of Fas and FasL increased in BQR group and predison group. Thissuggested that BQ更多还原