【作者】 周迪；

【导师】 郝飞；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2012， 博士

【摘要】 研究背景和目的：系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种典型的自身免疫性疾病。随着SLE近期生存率提高，心脑血管事件发病率显著增加，已形成第二个死亡高峰。研究生本人在从事硕士课题研究阶段，运用了比较蛋白质组学的方法，鉴定并验证了在SLE患者的外周血单个核细胞(peripheral blood mononuclear cells，PBMCs)中存在差异表达的16种蛋白质，并对这些蛋白质进行了相关的生物信息学分析。其中的膜联蛋白A5(annexin A5，AnxA5)在SLE患者中的高表达引起了我们的注意。AnxA5是膜联蛋白家族中少数具有抗凝活性的重要成员，但其在SLE发病中的确切机制目前尚无统一认识。如能明确它在SLE发病中的具体作用，对预防严重血管事件显然具有积极的意义。我们拟主要从SLE患者的临床表现及外周血中的AnxA5实际表达水平出发，明确AnxA5分子在SLE患者的外周血细胞中出现了表达差异，分析AnxA5的表达水平与SLE临床症候群的关联性。同时，筛查AnxA5及几种常见的抗磷脂抗体(anti-phospholipid antibody，aPL)在SLE患者血清中的表达水平，分析这些aPL分子相互之间以及与SLE不同临床表现的关联。最后我们通过外源性重组的AnxA5分子及anti-AnxA5单克隆抗体对SLE患者的数项临床凝血筛查实验进行干预，初步探讨AnxA5分子影响SLE患者血栓形成倾向的作用通路。方法：研究采集了200名女性SLE患者及174名健康对照者的血液标本。1．通过二维电泳(2-DE)、聚丙烯酰胺凝胶电泳(SDS-PAGE)与免疫印迹(Western blot)，验证SLE患者外周血单个核细胞中AnxA5的表达情况，并分析在胞浆、胞膜亚细胞组分中的表达差异。对显影条带进行半定量处理后，分析统计意义及与临床指标的相关性。2．运用酶联免疫吸附实验(ELISA)，测定患者及健康对照者的血清AnxA5浓度，同时测定相关分子anti-AnxA5抗体、β2糖蛋白I (β2GPI)、抗氧化性磷脂(anti-Ox-PLs)和抗心磷脂抗体(aCL IgG)的水平，分析这些分子相互之间及其与各种临床表现的相关性。3．重组AnxA5蛋白和anti-AnxA5单克隆抗体对SLE患者及健康对照的贫血小板血浆进行体外干预，运用自动化凝血分析仪检测外源性物质干预后，凝血酶原时间(PT)、部分凝血活酶时间(APTT)、纤维蛋白原(Fib)和凝血酶时间(TT)等常规凝血筛查实验的结果变化。主要结果：1．Western blot验证了AnxA5在大多数SLE患者的PBMCs中存在高表达，半定量后的统计分析提示其变化有显著性意义(60.7%vs.6.4%，P=0.0004)。2．SLE患者和健康对照者的PBMCs胞膜蛋白中，AnxA5无明显差异，而在胞浆蛋白中患者组的AnxA5高于健康对照组。3．经环磷酰胺(CTX)治疗后的SLE患者，其PBMCs中的AnxA5明显低于未经CTX治疗的SLE患者(17.0%vs.97.4%，P=0.014)，但与CTX累计量不具有剂量依赖关系。4．SLE患者血清中的AnxA5总体上明显低于健康对照者(26.8±3.0ng/mL vs.49.0±3.3ng/mL，P <0.0001)，但存在少数与对照者相近，甚至比对照者更高的SLE患者。5．血清AnxA5升高的SLE患者与血栓形成性疾病的发生有明显相关性(Mann-Whitney Z=-2.084，P=0.037)。6．SLE患者血清中的AnxA5与anti-AnxA5、β2GPI、anti-Ox-PLs、aCL IgG均无统计相关性。7．Anti-AnxA5与血栓形成的相关性不完全明确，但受到血清anti-AnxA5极高值的影响较为明显(Pearson’s (r)=0.340，P <0.0005vs. Mann-Whitney=-1.272，P=0.203)。8．外源性AnxA5及anti-AnxA5的干预都可以延长SLE患者的PT和APTT时间，以及缩短TT时间。9．外源性anti-AnxA5的干预可以提高SLE患者血清中功能性Fib的含量。10．外源性AnxA5干预健康对照的凝血筛查实验，结果的变化趋势与其干预SLE患者时相似，但后者的变化更为显著。结论：1．在SLE的发病过程中，外周血中的AnxA5具有在细胞内升高，在胞外血浆中降低的分布差异。这种分布特点可能发挥了某种保护作用，从而降低SLE患者出现血栓形成性疾病的机率。2．AnxA5在SLE发病过程中，与其他几种主要的aPL没有相关性，而可能是较为独立地参与SLE的发病。3．AnxA5及其抗体在SLE的发病过程中，可能主要通过凝血共同途径发挥作用，从而影响狼疮相关的血栓形成过程。更多还原

【Abstract】 Background and Objective:Systemic lupus erythematosus (SLE) is a classic autoimmune disease. As the short-termsurvival rate of SLE increased in these decades, chronic patients now suffer from increasingthrombosis-related symptoms, which had become a significant lethal factor for long-termprognosis. Our team used comparative proteomic strategies to identify16disregulatedproteins from peripheral blood monouclear cells (PBMCs) of SLE patients. Annexin A5(AnxA5) is one of the upregulated proteins, which we thought we should pay attention to.AnxA5is a distinctive annexin with anticoagulant activity that is rarely observed in theannexin family, but its detailed pathogenesis in SLE remains unclear. If the detailed functionof AnxA5in SLE could be elucidated, it should be very meaningful to help the SLE patientsto avoid the serious vascular events. To provide further insight about AnxA5into this disease,firstly we proposed to start our study about the AnxA5expression in peripheral blood and theclinical manifestation correlation. Secondly, we determined the sera concentrations of serveralanti-phospholipid antibodies, and analyzed their associations between AnxA5sera level ordifferent clinical parameters. At last we applied the recombination AnxA5and anti-AnxA5monoclonal IgG to intervene the progress of several blood coagulation screens, in order toprobe the possible mechanism pathway of the influence of AnxA5on lupus-relatedthrombophilia.Methods:The study involved200SLE patients and174healthy women.1. A proteomic analysis was accomplished using two-dimensional electrophoresis (2-DE)and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) from PBMCslysate. Protein identification and expression were validated using Western blot.Semi-quantitation of bolt film bands were executed with Quantity One software and statisticalcorrelations between clinical manifestation were analyzed with SPSS software or manually, when necessary.2. Sera AnxA5levels and four potentially related molecules, anti-AnxA antibodies, β2glycoprotein I (β2GPI), anti-oxidized phospholipids (anti-Ox-PLs) and anticardiolipin IgG(aCL IgG) were determined using enzyme-linked immunosorbent assays (ELISA). Statisticswere also analyzed with SPSS software.3. Recombination AnxA5and anti-AnxA5monoclonal IgG were used as intervenors andbovine serum albumin (BSA) were use as unrelated control. Preliminary parallel coagulationassays of SLE patients and healthy controls were processed using an automatic analyzer.Results:1. Western blotting confirmed the over-expression of AnxA5, from the PBMCs of themost SLE patients including (SLE vs. control=60.7%vs.6.4%, P=0.0004).2. No significant difference was observed in the results from the screening of thecytomembrane protein lysate. On the other hand, over-expression of AnxA5from theintracelluer protein lysate was observed from the SLE patients comparing to healthy control.3. Reviewing the correlations indicated the cluster without cyclophosphamide (CTX)treatment exhibited much higher AnxA5than the CTX-treated cluster (treated vs. untreated=17.0%vs.97.4%, P=0.014, without dose-dependence).4. ELISAs demonstrated the AnxA5concentrations of the patient sera (26.8±3.0ng/mL)were generally lower than the healthy donors (49.0±3.3ng/mL, P=0.0001), except forseveral patients, whose sera AnxA5were similar with, even higher than the most healthydonors.5. A positive correlation was demonstrated between the manifestation of thrombosis andAnxA5(Mann-Whitney Z=-2.084，P=0.037).6. No significance were found when searching for correlations between AnxA5and theother4molecular levels, anti-AnxA5, β2GPI, anti-Ox-PLs and aCL IgG.7. The correlation between anti-AnxA5and lupus-related thrombophilia was notcompletely certain, but this disputed statistic was mostly contributed the anti-AnxA5extremum values (Pearson’s (r)=0.340，P <0.0005vs. Mann-Whitney=-1.272，P=0.203).8. The coagulation assays using sera from SLE patients revealed that both elevatedAnxA5and anti-AnxA5shortened prothrombin time (PT), activated partial thromboplastintime (APTT) and prolonged thrombin time (TT)(P <0.01). 9. Anti-AnxA5raised functional fibrinogen levels of sera from SLE patients.10. The intervention of AnxA5in healthy control group displayed a similar trend to theSLE groups on PT, APTT and TT, but the alterations were not as numerically significant orstatistically dramatic as these in SLE.Conclusions:1. The heterogeneous transcellular distribution, increased intracellular concentrations anddecreased serum levels of AnxA5represent a protective response to lupus-relatedthrombophilia.2. AnxA5rarely correlated with several aPLs mentioned above, and could probably bean independent factor in the pathogenesis of SLE.3. In the thrombogenesis of SLE, AnxA5and its antibodies, might mostly participate inthe common coagulation pathway, which promoted the hypercoagulation state of SLE.更多还原