【作者】 杨旭燕；

【导师】 赵小英；

【作者基本信息】 浙江大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 第一部分外周血Th22细胞及白介素-22在系统性红斑狼疮患者中的表达及其临床意义背景和目的T细胞异常活化和T辅助细胞(T helper, Th)异常分泌细胞因子是被认为是SLE发病的重要机制之一。对SLE患者中存在的Th1/Th2细胞比例失衡已有多篇报道[1-3]。尽管研究结果有不同,但总体认为Thl和Th2在系统性红斑狼疮(systemic lupus erythematosus, SLE)相关免疫SLE相关的免疫损伤中均发挥作用,分别在不同阶段占主导作用。Th17细胞可能参与SLE病理机制已在动物实验中得到证实。与SLE组织损伤及自身抗体的形成密切相关。Th22细胞是最近发现新的一群T细胞亚群,Th22能分泌IL-22、IL-26、IL-13等细胞因子,其中IL-22是最主要效应因子。研究发现,Th22参与银屑病、类风湿关节炎和系统性硬化症等慢性炎症性疾病的发病。但Th22在SLE中的作用尚未明确。本研究采用流式细胞仪检测SLE患者外周血Th22、Th17细胞及其IL-17、IL-22等的表达水平及分析其临床意义。研究方法1.资料来源：明确诊断SLE的患者65人,同期年龄性别匹配的正常健康对照者30人。2.诊断标准：疾病诊断和分类根据ACR修订SLE诊断和分类标准及SLE活动指数(SLEDAI):根据2000评分体系评价患者疾病活动性,并按疾病活动性及脏器受累情况进行分组。3.实验方法：采集抗凝外周静脉血20mL。分离抗凝血外周血单个核细胞PBMC。采用流式细胞仪检测Th17、Th22等细胞的表达量,ELISA法检测患者血清IL-17、IL-22的表达水平。4.统计学学方法：使用SPSS19.0软件包进行分析,数据用均数士标准误表示,经正态性检验和方差齐性检验,正态分布资料两组间均数比较采用独立样本t检验或配对t检验,多组间均数比较采用单因素ANOVA方差分析,相关分析采用pearson相关系数检验；非正态分布资料两组间比较采用Mann-Whitney U检验,相关分析采用spearman相关系数检验,P<0.05作为检验差异显著性标准。结果1. SLE患者外周血Th17细胞比例明显高于健康对照组(2.05±1.01%vs.1.13±0.65%,p=0.003),其中活动性SLE患者Th17细胞比例明显高于非活动性SLE患者(2.32±1.00%Vs.1.12±0.37,p=0.001)。其中仅有皮肤损害和有肾脏损害的SLE患者Th17细胞比例均明显高于健康对照组(2.02±0.82%和1.98±0.62%vs.1.13±0.65%,P=0.000和0.000)2. SLE患者外周血Th22、Thl细胞比例与健康对照组比较无显著性差异,但仅有皮肤损害的SLE患者Th22细胞比例高于健康对照组(2.03±1.02%vs.1.28±0.40%,P=0.000),而仅有肾脏损害的SLE患者Th22细胞比例低于健康对照组(0.60±0.19%vs.1.28±0.40%,P=0.020)。Thl细胞比例在两组损害患者间及与健康对照组比较均无显著性差异。其中仅有皮肤损害或仅有肾脏损害的SLE患者Th1细胞比例与健康对照组比较也均无明显差异。3.SLE患者外周血清IL-22水平与健康对照组比较无差异性。但仅有皮肤损害SLE患者血清IL-22水平高于健康对照组[平均值289.34pg/ml(89.70-531.70);P=0.001],仅有肾脏损害的SLE患者血清IL-22水平低于健康对照组[平均值82.44pg/ml(36.80-178.50);P=0.024]。4.SLE患者外周血清IL-17水平高于健康对照组[平均值73.08pg/ml(17.00-176.00)]vs.[平均值46.76pg/ml(16.05-173.20),P=0.001],活动组高于非活动组患者[平均值82.44pg/ml(53.05-176.00)]vs.[平均值41.02pg/ml(17.00-156.70),P=0.002]。仅有皮肤损害和仅有肾脏损害的SLE患者外周血清IL-17水平均高于健康对照组[平均值77.78pg/ml(35.00-172.00);P=0.004]和[平均值85.20pg/ml(23.10-160.40);P=0.000]。5.Th22细胞与血清IL-22水平呈正相关(r=0.855,p=0.000),Th17细胞与血清IL-17水平呈正相关(r=0.771,p=0.000)。6.Th17细胞、血清IL-17水平与SLE活动指数(SLEDAI)呈正相关(r=0.279,p=0.012；r=0.211,p=0.046)。而Th22、Th1和IL-22与SLEDAI指数无相关性(p=0.120,p=0.070和p=0.280)。结论1.活动性SLE患者外周血中伴有Th17细胞比例增高及其细胞因子IL-17水平升高,与SLE活动指数(SLEDAI)相关,可以作为辅助标志物对疾病活动的提供预测。2.外周血Th22细胞及其细胞因子IL-22与SLE患者受累脏器相关,仅有皮肤受损SLE患者Th22细胞比例及其细胞因子IL-22水平升高,而仅有肾脏损害的SLE患者Th22细胞比例及IL-22水平下降,因此,Th22细胞及IL-22可以作为组织受累的辅助标志物。第二部分IL-22及IL-22R在MRL/lpr狼疮鼠中的表达背景和目的系统性红斑狼疮(Systemic lupus erythometasus,SLE)是一种炎症性疾病,其发生发展和种多因素有关,其中细胞因子是一个关键因素。致炎细胞因子和抗炎细胞因子之间失衡的程度决定了炎症严重性和范围。尽管关于细胞因子在SLE中的作用研究很多,但细胞因子之间的相互作用错综复杂,目前仍有尚未阐明各种细胞因子在SLE中作用。IL-22是IL-10细胞因子家族成员之一,其受体为一异源性二聚体,由IL-10受体p链(IL-10R2)、IL-22R1两条链组成。IL-22结合的特异性由IL-22R1决定。IL-22具有独特的作用,它由免疫细胞产生,但并不作用于这些免疫细胞,而是作用于组织细胞。作为一个独特细胞因子,IL-22在炎症性疾病和自身免疫性疾病中的作用越来越多重视。研究表明IL-22在炎症反应中发挥促炎和抗炎两种作用。例如,在银屑病小鼠模型,IL-22被证实介导皮肤炎症；相反,在溃疡性结肠炎动物模型上的研究显示1L-22减轻肠道炎症。在第一部分临床研究中我们发现SLE患者外周血IL-22水平与健康对照组比较没有明显差异,但分层分析后发现皮肤型SLE患者和狼疮肾炎患者血清中IL-22表达出现截然不同的结果,为进一步证实IL-22是否参与SLE发病,是否与受累的组织相关,本部分以SLE的MRL/1pr小鼠为研究对象,检测IL-22及其受体(IL-22R1)在狼疮鼠中的表达。研究方法实验动物为清洁级雌性MRL/1pr转基因小鼠和年龄匹配的雌性C57BL/6J近交系小鼠。分别在6、12、18周龄时麻醉眼眶取血清和摘取肾脏和淋巴组织,进行以下实验：1.对肾组织进行常规HE染色、PAS染色、Masson染色,以评价小鼠肾脏病理学损害程度。2.对腹腔淋巴组织进行了常规HE染色。3.用酶联免疫吸附剂测定(Enzyme liked immunosorbent assay,ELISA)血清抗双链DNA (dsDNA)抗体滴度和血清IL-22水平。4.用逆转录-聚合酶链反应(Reverse transcription-Polymerase chain reactions)法测定小鼠肾脏IL-22、IL-22RmRNA表达水平。5.用免疫组化法对IL-22、IL-22R在小鼠肾脏中的表达进行定位。结果1. MRL/lpr小鼠血清抗dsDNA的滴度随着病程的延长而上升,明显高于同龄C57BL/6J小鼠。2.同样,随着病程的延长,MRL/lpr小鼠肾脏的组织病理损害加重。6周龄MRL/lpr小鼠未出现肾脏损害；12周龄出现少量蛋白管型,肾小球系膜细胞和内皮细胞中度增生,间质炎症细胞浸润；18周龄出现大量蛋白管型和重度系膜细胞和内皮细胞增生,并形成新月体。而在任何年龄段的C57BL/6J小鼠中,未见蛋白管型,也均未发现其肾脏组织出现明显病理学改变。3. MRL/lpr小鼠淋巴组织病理也随着病程的延长而改变明显,显示淋巴中心增大。4. MRL/lpr小鼠血清IL-22水平,与同龄的C57BL/6J小鼠相比较无明显差异。5. MRL/lpr小鼠肾脏中IL-22、IL-22RmRNA表达水平明显高于同龄的C57BL/6J小鼠,有显著性差异。6. MRL/lpr小鼠肾脏中IL-22、IL-22R表达呈强阳性,尤其在细胞增生明显的肾小球呈高表达；而在C57BL/6J小鼠肾脏组织不明显。7. MRL/lpr和C57BL/6J小鼠的淋巴组织中均未见有IL-22、IL-22R表达。结论1.MRL/1pr小鼠呈现出狼疮肾具有的血清学和组织病理学改变。2.IL-22/IL-2R在MRL/1pr小鼠肾脏组织中有异常的高达,而在C57BL/6J小鼠不明显,提示IL-22/IL-22R在狼疮性肾炎的发生、发展中起致病作用。更多还原

【Abstract】 Part1:Th22, But Not Th17Might Be a Good Index to Predict the Tissue Involvement of Systemic Lupus ErythematosusBackgroundT helper cells, also called auto-reactive effector CD4+T cells, were first subdivided into2groups, Thl and Th2cells, and Thl cells were believed to a main drive of autoimmune diseases. However, this notion often met challenged with some inconsistencies. The identification of newer effector T-cell subsets in recent years such as Th17and Th22cells brings new insights to these diseases. Th17cells, an independent T cell lineage distinct from classical Thl and Th2cells, which produce IL-17A, play a pro-inflammatory role in autoimmune diseases via the up-regulation of additional proinflammatory and neutrophil-recruiting cytokines and chemokines. Furthermore, increasing evidence both in humans and in mouse models suggesting that Th17cells play a role in the progression of SLE has been noted. Th22subset, a new subset of CD4+T-helper, differentiate from naive T cells in response to TNF-a and IL-6and is characterized by secretion of IL-22but not IL-17or IFN-y. Abnormal expression of Th22cells was seen in peripheral blood of some autoimmune diseases including psoriasis [19], systemic sclerosis (SSc)[20], and rheumatoid arthritis (RA)[21,22]. IL-22-positive CD+T cells were present in high quantities in psoriatic skin [15] and RA synovial tissues [23]. However, the role of Th22cells in systemic lupus erythematosus (SLE) has not been reported yet.In present study, we measured the frequency of Th22cells and their secretion of IL-22in peripheral blood (PB) of patients with SLE and discussed the underlying mechanism of their roles in SLE.Method65patients diagnosed of SLE were recuited.30sex and age matched health Volunteers were accepted as healthy controls(HC).Artti-coagula-ted peripheral blood was collected and peripheral blood mononuclear cells(PBMC)were isolated.PBMC was cultured in RPMI1640complete culture medium with PMA and Ionomycin to stimulate the expression of intracellular cytokine in the presence of protein transport inhibitor-Golgiplug. Th1, Th17and Th22cells were identifiedby the expression of surface CD4and intracellular cytokine INF-r. IL-17and IL-22respectively.Serum concentrations of different cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) kits for IL-22and IL-17Clinical data was collected, organ involvement was asessed and SLEDAI score was evaluated according to SLEDAI2000system.Results1. Th17cells was significantly increased in SLE patients compared to healthy controls (P=0.003), and significant difference between active and inactive SLE patients (P=0.000) could be seen. However, Th22cells or Thl cells were unchanged compared to healthy controls.2. Th22cells were significantly elevated in patients with sole lupus skin disease, while they decreased in patients with sole lupus nephritis [(2.03±1.02%) and (0.60±0.19%), respectively versus (1.28±0.40%);(P=0.000,0.020respectively)]3. Thl7cells was significantly increased in both patients with sole lupus skin disease and patients with sole lupus nephritis compared with healthy controls [(2.02±0.82%) and (1.98±0.62%) respectively, versus (1.13±0.65%);(P=0.000,0.000respectively)]. For Thl cells, there was no obvious difference between two groups of patients and healthy controls4. Serum IL-22was unchanged in SLE [median,196.26pg/ml (range36.80-531.70)], compared with healthy controls [median,187.61pg/ml (range56.00-429.00); P=0.725], and no significant difference in serum IL-22between active and inactive SLE patients could be seen, the serum level of IL-22was significantly increased in patients with sole lupus skin disease [median,289.34pg/ml (range89.70-531.70); P=0.001], while it decreased in patients only with sole lupus nephritis [median,82.44pg/ml (range,36.80-178.50); P=0.024]5. Increased level of IL-17was seen in SLE [median,73.08pg/ml (range,17.00.-176.00)], compared to healthy controls [median,46.76pg/ml (range,16.05-173.20); P=0.001]. the level of IL-17in active SLE patients was significantly higher than that in inactive SLE patients P=0.002], and compared to healthy controls, the level of IL-17was increased in both SLE patients with sole lupus skin disease [median,77.78pg/ml (range,35.00-172.00); P=0.004] and SLE patients with sole lupus nephritis [median,85.20pg/ml (range,23.10-160.40); P=0.000]6. Positive correlation both between Th22cells and serum level of IL-22and between Th17cells and serum level of IL-17in patients with SLE(r=0.855, p=0.000; r=0.771,p=0.000).7. Th17cells and serum level of IL-17positively correlated with SLEDAI in SLE patients (r=0.279, p=0.012; r=0.211, p=0.046). Th22, Thl or IL-22failed to show a statistical correlation with SLEDAI (p=0.120, p=0.070and p=0.280respectively). ConclusionAbnomality of T helper subgroups and their cytokines exists in SLE.Patients with active disease had higher expression of Th17cells and increased level of IL-17correlating with the disease activity of SLE. Th17cells could act as biomarker as acticve disease. Expression of Th22cells and IL-22was higher in patients with sole lupus skin disease, and lower in patients with sole lupus nephritis. Therefore, Th22or IL-22might be a more important index to predict the tissue involvement of SLE.To investigate their proprty in SLE,more studies about the expression of Th22or IL-22in tissue are needed. Part2Up-Regulation of Renal IL-22/IL-22R Expression in MRL/lpr MiceBackgroundSystemic lupus erythematosus is a systemic autoimmune disease, characterized by a multitude of autoantibody production, complementactivation, and immune-complex deposition, which causes tissue and organ damage. Many different factors contribute to the pathogenesis of SLE, and cytokine is one of the most important factors. IL-22belongs to the IL-10family of cytokine. Special immune cell populations including Th cells secreated IL-22. The main biological role of IL-22includes the increase of innate immunity, protection from damage, and enhancement of regeneration. Recent studies have showed in the serum of patients with SLE, IL-22levels were significantly decreased compared with normal controls. However, our results in part I have demonstrated unchanged serum IL-22levels in patients with SLE. In the study, to invesitage the role of IL-22in SLE, we detect the expression of IL-22/IL-22R in MRL/lpr mice.Materials and MethodsSerum samples and kidney tissue were obtained from MRL/1pr mice and age-matched C57BL/6J(control) mice at age6weeks,12weeks and18weeks. The following were carried out:1. Anti-dsDNA antibodies levels and IL-221evels in serum were determined by enzyme linked immunosorbent Assay(ELISA).2. Renal and lymph node morphologic features were examined by light microscopy3. The mRN A expression of IL-22/IL-22R were invesitaged by RT-PCR. 4. Immunohistologic analyses were employed to examine the localization of IL-22/IL-22R in kidneys and lymph tissues.Reslut1. MRL/lpr mice showed characteristic alterations of serum immune parameters, with progressive increases in the levels of antibodies with age, compared with age-matched C57BL/6J(control) mice. The levels of anti-dsDNA antibodies in MRL/lpr mice were significantly higher than what in C57BL/6J mice at ages12weeks and18weeks2. Conventional histologic staining of kidneys and lymph node demonstrated minimal abnormalities in MRL/lpr mice at ages6weeks. MRL/lpr mice showed progressive development of renal damage, and obvious follicle proliferation and germinal center of lymph node, which is noticeable at ages12weeks and reaches significance at ages18weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. A pareallel increase in mesangial matrix was also noted. Prominent interstitial mononuclear cell infiltrating was also observed, with predominantly perivascular localization in the cortex and medulla of the kindey. Crescents were identified in the later phases. In contrast, no histopathologic abnormalities can be observed in C57BL/6J mice.3. Serum IL-22levels in MRL/lpr mice were unchanged, in compared with what in C57BL/6J mice.4. Higher expression of IL-22, IL-22RmRNA was found in MRL/lpr mice than what in C57BL/6J mice.5. Strong expression of IL-22, IL-22R protein was found mainly located in glomeruli of MRL/lpr mice, especially in the enlarged hypercellular glomeruli, whereas weak and negative expression was found in glomeruli of C57BL/6J mice. 6. No expression of IL-22, IL-22R protein was found in lymph node in MRL/lpr mice and C57BL/6J mice.ConculationSerum IL-22levels did not differ between MRL/1pr mice and C57BL/6J mice.Whereas higher expression of IL-22, IL-22RmRNA were prominent in the kindey of MRL/lpr mice. IL-22, IL-22R proteins were more distinguished in the enlarged hypercellular glomeruli. The results the present study demonstrated that IL-22/IL-22R axis may be over-activated in murine lupus nephritis, suggesting that IL-22/IL-22R may contribute to the pathogenesis of lupus nephritis更多还原