【作者】 刘志纯；

【导师】 周巧玲；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 研究背景：系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种常见的自身免疫性疾病,狼疮肾炎(Lupus nephritis,LN)是系统性红斑狼疮最常见也是最严重的临床病症之一,其中50%SLE患者有临床肾炎表现,而肾脏病理活检有肾脏病变者几乎达100%。免疫功能紊乱是导致狼疮肾炎发病的核心机制,淋巴细胞活化异常,尤其是T淋巴细胞活化异常是免疫功能紊乱的枢纽。免疫系统的紊乱最终导致B淋巴细胞的多克隆活化,产生多种自身抗体,从而导致多种组织器官功能损害。已有研究证明,某些细胞因子,如MCP-1与IL-10,可刺激淋巴细胞的活化,诱导自身抗体的产生及免疫紊乱,促使炎症细胞肾组织浸润等,参与SLE患者肾损害的发生,SLE患者血清MCP-1与IL-10表达异常提示SLE病情活跃。研究证实,多种信号通路参与二者的调控,如NF-kB信号通路。P38 MAPK (mitogen- activated protein kinase)是NF-kB信号通路的上游炎性调控因子。国内外研究已经证实,狼疮肾炎小鼠肾组织p38 MAPK表达明显增加,p38 MAPK是狼疮肾炎发病的重要信号通路之一。在多种动物模型及细胞的研究中均发现,如人类单核巨噬细胞、树突状细胞以及外周血单一核细胞,糖尿病大鼠及支气管哮喘动物模型等,P38 MAPK参与调控IL-10及MCP-1的表达。最近在研究肌炎的发病机制时发现,p38 MAPK信号通路可被肿瘤坏死因子样凋亡的微弱诱导剂(Tumor Necrosis Factor-Like Weak Inducer of Apoptosis, TWEAK)激活。TWEAK是1997年发现的TNF配体超家族的新成员。TWEAK表达于人体多种组织和细胞,正常情况下弱表达于肾小管上皮细胞和肾小球系膜细胞,其中免疫细胞如单核-巨噬细胞,树状突细胞,NK细胞和活化的T细胞可产生其可溶性的细胞因子形式,特别是在急慢性炎症和损伤的情况下,TWEAK表达明显增加。在既往的研究中,TWEAK与它的同源受体Fn14在免疫系统介导组织损伤和疾病发生的生理病理过程中发挥着重要的作用,可参与血管形成,细胞增殖、凋亡及细胞因子的产生等,并介导免疫所致多种器官损伤,如狼疮性肾炎,实验性自身免疫性脑脊髓炎、免疫性关节炎、多发性硬化症和肌炎等已被学者研究证实。国外有报道,SLE患者外周血T淋巴细胞和单核细胞TWEAK的表达均较正常健康人增高,且尿液TWEAK浓度与狼疮肾炎疾病的严重程度密切相关,在IFN-r刺激下,外周血单核细胞TWEAK表达亦明显增高。而在狼疮肾炎动物模型中,阻断TWEAK表达可明显减少狼疮肾炎小鼠蛋白尿排泄和肾组织炎症介质的产生,免疫球蛋白的沉积和肾组织炎性细胞浸润减少,减轻肾脏病理损害。这些研究均提示TWEAK在SLE发病中起着十分重要的作用。基于上述研究基础及理论支持,本研究拟通过体内、外研究,分子生物学等实验方法,探讨下列几个问题：1.SLE患者外周血PBMC TWEAK表达情况以及TWEAK表达与SLE疾病活动性和肾损害的相关性研究。2.分析体外培养LN患者PBMC的TWEAK的表达及TWEAK-P38 MAPK-MCP-1、IL-10信号通路在LN发病中的作用。3.检测MRL/lpr小鼠模型肾脏组织TWEAK的表达状况,采用TWEAK-siRNA基因沉默技术,探讨TWEAK siRNA对狼疮肾炎小鼠模型肾损害的靶向治疗作用。一-系统性红斑狼疮患者外周血单个核细胞TWEAK表达及其意义方法：研究对象包含SLE患者48例,其中LN患者25例,另选年龄、性别相当的类风湿关节炎(RA)患者20例及健康志愿者15例作对照。SLE和RA的诊断均符合1997年美国风湿病学会的分类标准。LN患者经肾活检证实全部有肾脏损害表现,持续性蛋白尿超过0.5g/24h或有细胞管型。采用RT-PCR、Western印迹及免疫荧光等方法分别在基因及蛋白质表达水平上检测SLE患者PBMC的TWEAK表达,同时对该48例SLE患者疾病活动度进行SLEDAI评分。ELISA方法检测血清IL-10及MCP-1的含量。分析TWEAK表达与血清IL-10、MCP-1和SLE疾病病情活动程度临床指标(血清抗双链DNA抗体,补体C3、C4,免疫球蛋白IgG、IgA、IgM,尿蛋白)的相关性。结果：TWEAK在SLE患者PBMC中mRNA表达明显高于RA患者及正常健康志愿者,(P<0.01), Western印迹显示TWEAK蛋白水平的表达与mRNA水平相似,SLE患者PBMC中TWEAK蛋白表达明显高于RA患者及正常健康志愿者。与SLE无肾损害患者相比,LN患者PBMC中TWEAK mRNA及蛋白表达均明显高于非肾炎组患者(P<0.01)。免疫荧光结果显示TWEAK在SLE患者PBMC胞浆内高表达。SLE患者血清IL-10及MCP-1浓度明显高于RA患者及正常健康志愿者,TWEAK表达与SLEDAI、尿蛋白、血清抗双链DNA抗体、IL-10及MCP-1成正相关,与血清补体C3、C4含量成负相关,与免疫球蛋白水平无明显相关。结论：1.SLE患者PBMC中TWEAK表达升高,其中LN患者升高更明显。2.PBMC中TWEAK表达水平明显升高与SLE活动指标呈正相关,提示TWEAK可作为SLE疾病活动程度指标之一,且可能在SLE肾脏损害中起着重要的作用。二.狼疮肾炎患者外周血单一核细胞TWEAK表达意义及其对P38 MAPK信号通路的影响方法：研究对象包括42例SLE患者和20名健康对照者,将SLE患者分为肾炎组(LN：26例)和非肾炎组(NRSLE:16例)。另选正常健康患者20例做为正常对照。SLE诊断符合美国风湿病学会分类标准,LN患者经肾活检证实全部有肾脏损害表现,持续性蛋白尿超过0.5g/24h或有细胞管型。梯度离心法制备新鲜外周血单一核细胞(PBMC),分成5组。A组：单纯培养组(不加任何刺激物,单纯1640培养基),B组：A+TWEAK-中和性抗体组;C组：A+SB203580组(特异性P38MAPK阻断剂)；D组：PHA/PMA刺激组；E组：D+TWEAK-中和性抗体组；F组：D+SB203580组(特异性P38MAPK阻断剂),每孔加入细胞1×106,加入24孔细胞培养板培养,细胞培养48小时后收集细胞,采取RT-PCR, Western Blot及免疫荧光分别在mRNA及蛋白表达水平上检测TWEAK及P38MAPK的表达。ELISA法检测细胞培养上清液的IL-10, MCP-1和抗双链DNA水平。结果：SLE患者体外培养PBMC的TWEAK和P38 MAPK的蛋白表达均明显高于健康对照组,LN组TWEAK表达明显高于NRSLE。Western印迹显示PHA/PMA刺激可上调PBMC中TWEAK和P38 MAPK蛋白表达。TWEAK-中和性抗体明显下调TWEAK和P38 MAPK的表达,使细胞培养上清液抗双链DNA抗体和IL-10水平下调。p38 MAPK的抑制剂SB203580可下调PBMC的p38 MAPK表达,使细胞培养上清液抗双链DNA抗体、IL-10和MCP-1水平下调,但对TWEAK蛋白表达无明显影响。结论：TWEAK可能通过激活P38 MAPK信号通路介导LN患者PBMC中IL-10和MCP-1的表达上调参与狼疮肾炎的发病。三. TWEAK-siRNA对MRL/1pr狼疮小鼠肾保护作用及机制研究方法：12周龄雌性MRL/1pr狼疮小鼠随机分为4组：A组(未干预组,n=9);B组(脂质体组) (n=9)；C组：(阴性对照siRNA组,n=9)；D组(TWEAK -siRNA干扰组,n=9)。另有9只C57BL/6J小鼠被列入为正常对照组(E组)。将siRNA预混于含DOTAP的溶剂中,采取1mg/kgsiRNA尾静脉流体注射法于C、D两组小鼠,每周1次。采用RT-PCR及Western blot技术检测TWEAK和p38 mRNA及蛋白水平,酶联免疫吸附法(ELISA)法检测血清抗dsDNA抗体和抗核抗体(ANA), IgG,IgM水平,留尿测24小时尿蛋白,采用直接免疫荧光法及HE、PAS、Masson染色病理切片检测小鼠肾脏损害情况。RT-PCR方法检测肾组织IL-10和MCP-1的含量。结果：C57BL/6J小鼠肾组织可见肾小管少量TWEAK及P38 MAPK蛋白表达,与C57BL/6J小鼠相比,MRL/1pr各组小鼠24小时尿蛋白、血清抗dsDNA抗体,IgG和IgM水平明显升高,肾组织肾小管、肾小球系膜区及血管内皮可见较多TWEAK及P38 MAPK蛋白表达。与阴性对照siRNA组,脂质体注射组,及未干预组比较,TWEAK-siRNA干预组小鼠肾组织TWEAK、P38 MAPK mRNA及蛋白表达水平下降,血清抗dsDNA抗体水平、IgG和IgM水平均降低,IL-10 mRNA及MCP-1 mRNA水平下调,肾脏病理损害及炎症细胞浸润明显减少,同时,尿蛋白也较前减少。结论：MRL/1pr狼疮肾炎小鼠模型中TWEAK表达明显增高,TWEAK siRNA可有效阻断TWEAK-P38 MAPK-IL-10、MCP-1信号通路,TWEAK有望作为未来靶基因的治疗目标。从以上三部分实验我们总结如下：1.SLE患者TWEAK水平可作为SLE疾病活动程度指标之一。2. PBMC中TWEAK-P38 MAPK-MCP-1、IL-10信号通路在LN发病机制中起着重要的作用。3. TWEAK siRNA阻断P38 MAPK信号通路,下调MCP-1、IL-10表达,提示TWEAK-p38 MAPK信号通路参与MRL-1pr小鼠模型肾损害的发生。更多还原

【Abstract】 Background:Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the presence of autoantibodies directed against nuclearantigens.Renal involvement in SLE, known as lupus nephritis (LN), is a major complication of SLE and is associated with high rates of morbidity and mortality. Although clinical signs of renal involvement appear in only 50-80% of patients, the disease involves the kidney in almost all patients from whom sufficient tissue can be obtained for analysis.Immune dysfunction is the core mechanism of lupus nephritis. It is well known that activation of T and B cells plays a key role in the pathogenesis of this disease in innate immunity and inflammatory responses via production of various cytokines and chemical mediators, such as MCP-l,IL-10. it is intriguing that these cytokines are crucial in the pathogenesis of SLE. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) superfamily (TNFSF) initially described in 1997. The expression of TWEAK is relatively low in normal tissue, however, it is increased in both acute or chronic inflammatory processes in numerous tissues and peritoneal macrophages. TWEAK may be expressed as a membrane-bound protein (mTWEAK) and as a 156-aa, 18kDa soluble protein in mmune cells such as monocytes-macrophages, dendritic cells, NK cells and activated T cells. Many studies showed that TWEAK and its cognate receptor play an important role in angiogenesis, cell proliferation, apoptosis, cytokines, and immune inflammatory diseases, such as lupus nephritis, experimental autoimmune encephalomyelitis, autoimmune arthritis, multiple sclerosis, myositis, lupus nephritis. Cell-surface expression of TWEAK has also been reported in human monocytes after IFN-rexposure and in T cells from patients with systemic lupus erythematosus. Treatment with an anti-TWEAK Ab or deficiency of the TWEAK receptor Fn14 can decrease renal damage of cGVH-induced lupus mice as indicated by significantly reduced proteinuria, glomerular Ig deposition and kidney cytokine expression.Recent study showed that biological effects of TWEAK may be mainly through the induction of downstream signaling pathways, including prolonged of NF-KB activation and activating the canonical (or alternative) pathway of NF-KB. p38 MAPK (mitogen-activated protein kinase) is an important upstream regulatory factors of NF-KB, some scholars have found that TWEAK can activate p38 MAPK signaling pathway involved in the pathogenesis of myositis. Studies have found that P38 MAPK expression increased significantly, its specific inhibitor SB203580 could significantly reduce the renal tissue T cells macrophages infiltration, reduce the immunoglobulin deposition, and decrease renal pathological damage, anti-dsDNA antibodies inflammatory mediators and chemokine also decreased significantly. P38 MAPK signaling pathway plays an important role in the pathogenesis of LN.Thus, We carried out serious research to explore the following questions:(1) To detect the expression of TWEAK in peripheral blood mononuclear cells and analyze the correlation between TWEAK and disease activity and renal damage of SLE.(2) To support a possible role of TWEAK in PBMCs in the pathogenesis of LN and to find whether TWEAK influences the activation of p38 MAPK signalling in activated PBMCs.(3) To study the expression of TWEAK in renal tissue in MRL/lpr mice and analysis its possible mechanism. 1. The expression of TWEAK in peripheral blood mononuclear cells and its correlation to disease activity and renal damage of SLE.Methods:Subjects comprised 48 patients with SLE including 25 patients with ranal damage and 23 without,20 patients with rheumatoid arthrithis(RA) and 15 healthy controls.The expression of TWEAK in PBMCs was determined by RT-PCR and western blot. SLE disease activity was evaluated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) 2000 score. Next were analyzed the correlations of TWEAK mRNA and protein to serum IL-10, MCP-1 and some laboratory parameters of SLE disease activity.Results:The results showed that TWEAK expressions in PBMCs from SLE patients were significantly higher than that in RA patients or healthy controls, especially higher in those patients with renal disease. Elevated production of TWEAK is correlated positively and significantly with SLEDAI, proteinuria, serum anti-dsDNA,IL-10 and MCP-1,but inversely associated with serum complements.Conclusion:Our results suggested that TWEAK in PBMCs is positively related to SLE disease activity and might be involved in the pathogenesis of LN. 2. Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) mediates p38 Mitogen-Activated Protein Kinase Activation (P38 MAPK) Signal Transduction in Peripheral Blood Mononuclear Cells from Patients with Lupus nephritisMethods:42 patients with SLE including 26 patients with ranal damage and 16 without, and 20 healthy controls were included in. The isolated PBMCs were treated with p38 inhibitor, SB203580 or anti-TWEAK mAb, with or without PHA/PMA stimulation. Immunofluorescence technique and western blot experiments were uesd to evaluate the protein expression of TWEAK in PBMCs and to identify the subcellular distribution of TWEAK and P38 MAPK.Next the contents of Interleukin (IL-10) monocyte chemoattractant protein (MCP-1) and anti-double stranded (DNA) (anti-dsDNA) in the supernatant were measured by ELISA.Results:The results showed that expression of TWEAK protein in PBMCs from LN patients was significantly higher than that from healthy controls. PHA/PMA simulation could up-regulate the productions of TWEAK and p38MAPK in PBMCs from LN. Anti-TWEAK mAb treatment down-regulated both TWEAK and p38 MAPK expression in PBMCs,as well as anti-dsDNA,IL-10 and MCP-1 in the supernatant, SB203580 had the same effect on cytokine production in PBMC, but have no effect in the expression of TWEAK.Conclusion:TWEAK-p38 MAPK-IL-10、MCP-1 signaling pathway in PBMC played an important pathogenic role in lupus nephritis.3. Study of the therapeutic efficacy and mechanism of TWEAK-siRNA in the MRL/lpr murine model of lupus nephritis Methods:12-week-old female MRL/lpr mice were divided into 4 groups: group A (MRL/lpr mice without intervention)(n=9); (2)group B (liposome intraperitoneal injection)(n=9); (3) group C (Negative control siRNA)(n=9); (4) group D (TWEAK-siRNA group),(n=9); Another 9 C57BL/6J were included in as the normal group(group E). 1mg/kg synthetic siRNAs dissolved in liposomes or empty liposomes were intravenous injected per week in 1000 ul volume. Urine protein, serum anti-dsDNA,IgQIgM were determined;RT-PCR, Immunofluores-cence stain, Western blotting were used to detect TWEAK, P38 MAPK expression. RT-PCR was used to detect IL-10 and MCP-1.Results:Urine protein excretion,serum anti-dsDNA,IgG,IgM increased and renal pathology showed pathological changes in MRL/lpr mice.Compared with the C57BL/6J group, there was significant increase in local TWEAK and P38 MAPK level in kidney tissue. TWEAK-siRNA significantly down-regulated of TWEAK、P38 MAPK and IL-10、MCP-1 expression in MRL/lpr mice. Urine protein excretion,serum anti-dsDNA,IgG,IgM were also significantly down-regulated. Injection with TWEAK-siRNA could improve the damage of vessels and nephric tubules, and decrease immune complex deposit in glomerulus.Conclusion:TWEAK take part in the renal damage of MRL/lpr lupus model through P38 MAPK signalling pathway, TWEAK-siRNA is expected to become one of the therapeutic targets of LN.更多还原