【作者】 吴瑾；

【导师】 熊思东；

【作者基本信息】 复旦大学 ， 医学免疫学， 2003， 博士

【摘要】 系统性红斑狼疮（SLE）是多器官、多系统受累的严重的自身免疫病，以产生多种自身抗体为特征，主要包括抗dsDNA、抗Sm、抗RNP等抗核抗体（ANA），其中IgG类抗dsDNA抗体是SLE疾病的致病性抗体，也是SLE发病的重要诊断依据。SLE中关于自身免疫耐受的打破有多种观点，我们实验室自90年代初开始了SLE发病机制的系列研究，通过提取和纯化未活化、活化淋巴细胞的染色质和DNA分别多次免疫同系小鼠，发现只有活化淋巴细胞的染色质及DNA能诱导出高滴度的抗dsDNA、抗组蛋白等多种ANA以及SLE样综合征，而未活化淋巴细胞的染色质、DNA均未诱导出自身抗体，从而提出活化淋巴细胞的DNA（简称ALD-DNA）等核抗原免疫原性改变，是驱动ANA产生的重要原因之一。由于SLE患者体内广泛存在过度活化和凋亡的淋巴细胞，因此由它们释放大量的DNA等自身抗原，从而可能诱导了耐受的打破和自身抗体的产生。血清淀粉样P成分（SAP）作为血清正常糖蛋白，近来被发现在生理状态下可以特异地、以Ca++依赖方式与凋亡细胞释放的DNA、染色质、核小体结合；并且SAP-/-小鼠自发产生抗核抗体及严重的肾小球肾炎，类似于人类的系统性红斑狼疮。因此推测SAP可与凋亡细胞释放的DNA、染色质等自身核抗原相互作用，有助于对它们的清除处理，从而抑制自身抗体的诱生。为证实上述假设，本研究中我们首先分析SAP与SLE疾病的相关性，进而纯化SAP蛋白，从体内、体外两方面具体研究SAP与自身抗原DNA的相互作用以及对抗dsDNA抗体诱生的影响，并进一步观察SAP的基因注射对SLE发病是否具有干预作用，为自身免疫性疾病的防治提供理论基础和实验依据。基于上述考虑，我们进行了以下四个部分的研究：第一部分SLE患者血清SAP水平与SLE疾病相关性研究为了解SAP与SLE疾病的关系，我们首先用ELISA检测了SLE患者血清中SAP水平。结果显示：SLE患者血清SAP含量为（50.35(22.16(g/ml），明显高于正常对照组SAP的含量（32.78(23.48(g/ml），两者存在显著性差异（P<0.01）。进一步以EB-DNA荧光法检测SLE患者血清DNA含量，结果显示：SLE患者血清DNA含量为（76.54(14.40(g/ml），明显高于正常对照组DNA的含量（36.91 (14.40(g/ml），两者存在显著性差异（P<0.01）。通过对SLE患者血清SAP含量与<WP=7>相应的DNA水平进行Pearson相关分析，发现两者呈正相关（相关系数R=0.5014，P<0.01）。在此基础上，进一步比较了活动期、非活动期患者血清中SAP、DNA含量等指标，发现与非活动期相比，活动期患者血清中SAP、DNA含量均显著增加（P<0.01）。以SAP/DNA比值代表与血清DNA水平相比SAP的相对含量，结果显示在SLE患者SAP/DNA比值较正常对照明显下降（P<0.01），提示SLE患者血清SAP含量较DNA水平相对不足。将患者血清SAP/DNA比值与其抗dsDNA抗体进行相关分析，发现两者呈负相关（R=-0.541，P<0.05），提示SAP在SLE疾病中可能发挥一定作用。第二部分 SAP与DNA的结合特性及其后续事件的体外研究一、小鼠血清SAP蛋白的纯化为进一步研究SAP在自身免疫性疾病中的作用，我们以LPS经腹腔注射小鼠，诱导急性时相反应，并在注射24h后获得急性时相血清，通过亲和层析和凝胶过滤分离纯化血清中的SAP蛋白，以SDS-PAGE和Western blot鉴定纯化的蛋白。结果显示获得的蛋白在15%的聚丙烯酰胺凝胶电泳中呈现单一条带，并经兔抗鼠SAP单抗证实所获得的蛋白为SAP蛋白。二、SAP的DNA结合特性为研究SAP是否与DNA结合以及其结合特性，分别提取来自细菌、质粒、酵母、小鼠淋巴细胞等的DNA，用DOT-EIA的方法检测人和小鼠的SAP与它们的结合活性。结果显示，人和小鼠的SAP可以与上述不同来源的DNA结合，二者均与细菌DNA结合活性最强，其次为质粒、酵母DNA，但它们与小鼠淋巴细胞DNA和小牛胸腺DNA的结合较弱，与单链( DNA结合最弱，说明SAP与不同DNA结合活性各异。进一步提取小鼠未活化和活化淋巴细胞的DNA，观察人和小鼠的SAP与它们的结合情况。结果显示，人和小鼠SAP与活化淋巴细胞DNA的结合力显著高于未活化淋巴细胞DNA (P<0.01)。推测SAP与未活化、活化淋巴细胞DNA结合能力的不同可能与SLE的发病机制存在一定的关系。三、SAP与DNA的结合促进巨噬细胞吞噬DNA由于巨噬细胞是体内吞噬清除凋亡细胞及其代谢产物的主要吞噬细胞，为研究SAP与DNA的结合是否会影响巨噬细胞对DNA的吞噬，以常规方法获取小鼠腹腔巨噬细胞，分别将巨噬细胞(5×106/ml)与不同量的EB标记的活化淋巴细胞DNA（简称EB-ALD-DNA）进行孵育，以流式细胞仪检测巨噬细胞吞噬EB-ALD-DNA的情况。结果显示：巨噬细胞可直接吞噬DNA，且吞噬百分数与<WP=8>DNA含量呈一定的剂量依赖关系，当DNA含量为100(g/ml时，吞噬百分数达到饱和。为观察SAP与ALD-DNA的结合是否促进巨噬细胞对DNA的吞噬，我们先将SAP与ALD-DNA孵育2h，然后进行巨噬细胞吞噬试验。结果发现：与巨噬细胞吞噬单纯的ALD-DNA相比，SAP与ALD-DNA的结合可显著增强巨噬细胞对ALD-DNA的吞噬(P<0.01)。按不同比例混合SAP与ALD-DNA，分析SAP促巨噬细胞吞噬DNA的剂量依赖关系，结果提示当SAP与ALD-DNA的比值为1:4或1:2时巨噬细胞对DNA更多还原

【Abstract】 Systemic lupus erythematosis (SLE) is a severe autoimmune disease with multiorgan and multisystem lesioned, characterized by production of various antinuclear antibodies (ANA), including anti-dsDNA, anti-Sm, anti-RNP antibody, et al. The spontaneous occurrence of antibodies against the dsDNA is a highly specific marker for the diagnosis of SLE and proved to be pathogenetic. There are many different opinions about the mechanisms of tolerance-broken. Once we extracted and purified chromatin and DNA from unactivated lymphocytes and activated lymphocytes respectively, immunized syngeneic mice separately. We found that chromatin and DNA from activated lymphocytes induced many kinds of ANA, such as anti-dsDNA, anti-histone antibodies, while none of the mice immunized by chromatin and DNA of unactivated lymphocytes produced autoantibodies. It indicated that DNA derived from activated lymphocytes (ALD-DNA), which changed its immunogenicity, was one of the important initiators to trigger the production of ANA. Since the extensive lymphocytes activation and excessive apoptosis could be observed in the SLE patients, the large amount of released DNA may comprise the major source of such kind of autoantigen to induced the production of autoantibodies.As one of the normal glycoprotein in the serum, Serum amyloid P component (SAP) was found to have the capacity of binding DNA, chromatin and nucleosome specifically and calcium-dependently under the physiological conditions, and the SAP gene knock-out mice could produce ANAs spontaneously and suffered from severe glomerulonephritis, just resembled the SLE syndrome. We postulated that SAP interplayed with the nuclear antigen released from apoptotic cells and was involved in the disposal of the DNA and chromatin, which would inhibit the induction of autoantibody. In this article we try to analyse the relationship between SAP and SLE, then purify the<WP=13>SAP protein and study its role in the process of the DNA clearance and its effect on the induction of anti-dsDNA antibody in vivo and in vitro, further observe the possibility of the inhibition of SLE-like syndrome based on the gene injection of SAP. This research may provide experimental and theoretic basis for the new prevention strategy of SLE.PART I Research on the correlation between SAP level and systemic lupus erythematosus (SLE)In order to analyse the relationship between SAP and SLE, we detected the serum SAP levels of the patients and normal controls by ELISA firstly, it showed that the SAP concentration of SLE patients was (50.35(22.16(g/ml), which was much higher than that of the normal controls (32.78(23.48(g/ml), there was significantly statistic difference between them (P<0.01). Then we detected the concentration of DNA in both serum by DNA-EB fluorescent assay, it showed that the DNA level of SLE patients was (76.54(14.40(g/ml), which was much higher than that of the normal controls (36.91(14.40(g/ml), there was significantly statistic difference between them (P<0.01). Pearson correlation analysis showed that SAP concentration correlated positively with DNA level (R=0.5014，P<0.01). Subsequently we compared the DNA and SAP level between the patients in active and inactive phase, it showed that both the SAP and DNA concentrations of the patients in active phase increased significantly than those of the patients in inactive phase (P<0.01). The SAP/DNA ratios decreased obviously in SLE patients compared to the normal persons (P<0.01), which suggested that the SAP level were relatively insufficient in SLE patients. We also found that the SAP/DNA ratios were negative-correlation with the anti-dsDNA antibodies level(r=-0.541，P<0.05), what mentioned above suggested that SAP plays certain role in the etiology of SLE. PATR II The in vitro study on the binding characteristics of SAP to DNA and the later events1. Purification of murine SAPWe induced the acute phase response of BALB/c mice by injection of LPS through peritoneal, then collected the serum after 24 hours, the SAP protein w更多还原