【作者】 陈宏；

【导师】 赵会娟；

【作者基本信息】 天津大学 ， 生物医学工程， 2012， 博士

【摘要】 “自身免疫性疾病”是免疫系统对机体自身的成份发生的免疫反应，造成损害而引发疾病。常见的自身免疫皮肤病有：系统性红斑狼疮（systemic lupus erythematosus， SLE）、大疱性类天疱疮（bullous pemphigoid， BP）、硬皮病、干燥综合征等等。免疫性皮肤病病因病机非常复杂，包括遗传因素、环境因素、免疫调节异常因素和性激素及其受体等多方面，病变侵及全身多系统，且会造成多器官损害。本文以系统性红斑狼疮和大疱性类天疱疮为例，旨在探讨T细胞相关细胞因子对自身免疫性疾病的致病机制。本研究主要研究内容和得出的结果为：通过检测ERα、ERβ、白介素10（IL-10）、B淋巴细胞刺激因子（Blys）在SLE患者外周血中的表达，分析与SLE临床表现及实验室指标的相关性，初步研究雌激素受体（estrogen receptor，ER）在该病发病中的免疫作用机制及其T、B淋巴细胞的协同作用机制。采用反转录-聚合酶链氏反应方法，测定了40例SLE患者和40例正常对照者外周血ER-α、β、IL-10、Blys的表达量，通过Spss13.0统计软件分析这些因子的表达与临床表现及各项化验数据的相关性。结果表明： ER-α、IL-10、Blys mRNA在红斑狼疮患者外周血单核细胞（PBMC）表达水平较正常对照组明显升高（P<0.05或P<0.01），活动期明显高于稳定期（P<0.05或P<0.01），且与病情和红斑狼疮活动指数有相关性；而SLE患者ERβ表达量在合并心血管损害组明显低于无心血管损害组（P＜0.05）。该课题还研究了SLE病人外周血中血管内皮祖细胞水平的变化特征，探讨在内皮损伤过程的机制。采用IL ACL-9000型血液凝固仪测定vW因子抗原含量（vWF:Ag）。用流式细胞仪分析外周血中内皮祖细胞（endothelial progenitor cells，EPCs）和循环内皮细胞（circulating endothelial cell，CEC）的水平。结果显示：SLE活动期患者外周血中CD34+细胞、CD133+细胞、CD34+CD133+双阳性细胞分别高于稳定期（q=7.93，q=8.45，q=10.22）和对照组（q=10.37，q=11.24，q=15.62），差异有统计学意义（P<0.01）。与稳定期患者比较，SLE活动期患者EPCs和vWF:Ag增高，差异有统计学意义（P<0.01）；CEC差异无统计学意义（P>0.05）。稳定期患者各项指标与对照组间无统计学意义（P>0.05）。SLE患者活动期EPCs与vWF:Ag呈显著正相关（P<0.01），与CEC无显著相关性（P>0.05）。稳定期患者和对照组EPCs与vWF:Ag和CEC均无相关性（P>0.05）。研究骨桥蛋白(OPN)、IL-18、IL-17和IL-23在BP的作用机制，探讨血清OPN、IL-18、在BP患者与正常人之间表达的差异及其表达与临床及实验室指标的相关性。研究IL-17和IL-23在BP表达及其之间的相关性，揭示Th1、Th17细胞因子在BP发病中的作用。采用固相夹心法-酶联免疫吸附法（ELISA），测定了30例大疱性类天疱疮患者和30例正常对照者血清中OPN、IL-18、IL-17、和IL-23的浓度，并通过Spss13.0统计软件分析这些因子的表达差异与该病临床症状及各项实验室指标的相关程度。结果显示BP组血清OPN、IL-17、IL-23的表达水平高于正常对照组(P<0.01)，两者之间呈明显的正相关性（r=0.712,P<0.05）且与病情程度和ELISA指数之间呈明显相关性， IL-17、IL-18在合并症者明显增高。以上研究结果得出：ERα、ERβ、IL-10、Blys在SLE发病中发挥一定的作用，具有致病相关性， ER-α、IL-10、Blys与SLE病情活动性有关，可以作为判断SLE疾病活动性的指标。活动期SLE患者由于血管内皮损伤加重，EPCs大量从骨髓动员入外周血中对血管内壁进行修复。外周血中EPCs数量与血管损伤标志物（vWF:Ag）水平密切相关，表明EPCs数量改变可以作为评价病情活动的有效标志物。 OPN、IL-18IL-17、IL-23在BP发生发展中发挥一定致病意义。OPN、IL-17可能与水疱的发生和粘膜的损伤有关，且与抗BP180抗体的表达及BP病情活动性呈正相关。由于粘膜损伤多发生在疾病较重或病程缠绵的患者，因此对于OPN、IL-17水平显著升高的BP患者，临床上要予以重视以防延误病情。IL-18、IL-17在BP合并症的发生发展中发挥重要作用，OPN、IL-18、IL-17之间相互促进、相互影响，在加重BP患者肝肾功能损伤、促进伴有糖尿病或肿瘤的BP患者的病情发展中具有重要意义。总之，Th1\Th2\Th17相关细胞因子相互作用、相互影响、相互制约在自身免疫性疾病发生发展和转归中发挥重要作用。更多还原

【Abstract】 "Autoimmune disease" is a constituent of the immune system to the body’s own immuneresponse, which will cause damage and disease. Common autoimmune skin disease includessystemic lupus erythematosus (systemic lupus erythematosus, SLE), bullous pemphigoid(bullous pemphigoid, BP), scleroderma, Sjogren’s syndrome, and so on. The etiology ofautoimmune skin disease is complex and involves the genetic, environmental and immuneregulation abnormalities, sex hormones and other aspects. The autoimmune skin disease has awide range of incidence, and will be accompanied by multi-organ damage.In this paper, with the systemic lupus erythematosus and bullous pemphigoid as theexamples, we aim to explore the pathogenic mechanisms of T cell-associated cytokines onautoimmune diseases.The mechanism of immunity effect from estrogen receptors (ER) on SLE is investigated atfirst, and then the synergistic effect from both the T leukomonocyte and B leukomonocyte inSLE is investigated. The correlation of the mRNA expression of estrogen receptor-α,interleukin-10, B lymphocyte stimulating (Blys) factor with the clinical and laboratory indexesis studied. RT-PCR method is used to analysis the expression quantity of ER-α, ERβ, IL-10,and Blys in SLE patients (40cases) and health adults (40cases), and statistical analysis. Then,according to the diagnostic criteria, we analyze the expression difference of ER-α, βand IL-10,and Blys. The correlation between the mRNA expression of ER-α, βand IL-10, and Blys isanalyzed with the statistical software. The results show that the mRNA expression levelof ER-α, IL-10, and Blys in peripheral blood monocytes (PBMCs) of SLE patients isobviously higher than the normal control（P<0.05or P<0.01），and the active group isobviously higher than the inactive group（P<0.05or P<0.01）, and a positive correlation isobserved with SLEDAI (P<0.05or P<0.01). In the combined group of cardiovascular damageof SLE patients, ERβ expression is lower than those without the cardiovascular damage (P<0.05).We study the variation character of SLE patients with endothelial progenitor cells in peripheral blood and explore the mechanism of endothelial injury. The IL ACL-9000bloodcoagulation is used to measure the vW factor antigen levels (vWF: Ag). Flow cytometry is usedto measure the level of the peripheral blood progenitor cells (EPCs) and the circulatingendothelial cells (CEC). The results show that SLE active patients have high number ofperipheral blood CD34+cells, CD133+cells, and CD34+CD133+double-positive cellsthan these in the stable phase (q=7.93, q=8.45, q=10.22) and the control group (q=10.37, q=11.24, q=15.62), and the difference is statistically significant (P <0.01). Compared with thestable patients, SLE patients are observed with increased active EPCs and vWF: Ag, and thedifference is statistically significant (P <0.01) but has no significant difference to CEC (P>0.05). Patients in stable period show no significance to the control group (P>0.05). EPCs inpatients with active SLE are significantly correlated with vWF: Ag (P <0.01), but CEC has nosignificant correlation (P>0.05). EPCs, vWF: Ag, and CEC for the patients in stable periodand the control group have no correlation (P>0.05).The influence mechanism of osteopontin (OPN), inerleukin-18, inerleukin-17andinerleukin-23on bullous pemphigoid is investigated. The expression difference ofosteopontin, interleukin-18(IL-18), interleukin-17, and interleukin-23in the serums of healthadult and BP patients is also investigated. The difference between the expression of OPN,inerleukin-18, inerleukin-17, inerleukin-23in serums is studied and the correlation of theexpression to the clinical and laboratory indexes is observed. At last, the effect of the Th1,Th17cells on the BP is explored. ELISA is used to analysis the concentration expression ofOPN, inerleukin-18, inerleukin-17, and inerleukin-23in BP patients (30cases, according tothe diagnostic criteria) and health adults (30cases). The consequence is judged using gelformatter and the statistical software Spss13.0is adopted in the statistical analysis. Resultsshow that the concentration expression level of OPN, interleukin-17, and interleukin-23inserums of BP patients is obviously higher than the normal control（P<0.01）, and a positivecorrelation with SLEDAI (P<0.05or P<0.01) is observed. The concentration expression levelof IL-18and IL-17in serums of BP patients with cardiovascular disease is obviously higherthan non-cardiovascular disease（P<0.01）.The above results demonstrate that: the mRNA expression level of ER-α, IL-10, and Blysmay be related with the reactivity of SLE, which suggested that they may take part in the development of SLE and correlate with the severity of SLE. Due to the increased vascularendothelial injury in the active SLE patients, EPCs is mobilized from the bone marrow into theperipheral blood to repair the vascular wall. The numbers of EPCs in peripheral blood areclosely related to the level of vWF: Ag, which indicated that the changes in the number ofEPCs can be used as an effective evaluation of the disease. OPN, interleukin-18, interleukin-17, and interleukin-23in serums of BP patients have certain significance in the pathogenesis ofBP. OPN and interleukin-17primaryly participate in the occurrence of blister and mucosaldamage, and show a positive correlation with antiBP180body and disease activity. IL-18andIL-17are mainly involved in the occurrence and development of the complications of BP.Interaction and mutual influence between OPN, IL-18, ang IL-17could increase the possibilityof the damage in liver and kidney, and correlated to the development of diabetes or tumors inBP patients.In a conclusion, interaction, mutual influence, and mutual restraint of Th1\of Th2\ofTh17cytokines play an important role in the development and outcome of autoimmunedisease.更多还原