特发性膜性肾病的致病靶抗原-肌球蛋白9抗原表位的研究

Idiopathic membranous nephropathy (IMN) is the main pathological type in adult nephrotic syndrome. Considerable advances have been made in our understanding of the molecular pathomechanisms of human membranous nephropathy, with the identification of possible antigens. Our preliminary study indicated that myosin9 is one of antigens in IMN.Furthermore,we surprisely found that,rabbits immunized with the b cell epitope of myosin9(synthesized epitope peptide),generated anti-myosin9 antibody and anti-PLA2R antibody in enough period.Inspired by this result,we assume that B-cell epitope spreading, that is the extension of the primary immune response against myosin9 epitope(s) to other epitopes such as PLA2R epitopes, might be relevant to the pathogenesis of membranous nephropathy. On the basis of this hypothesis, we focus on:(1)the classification of epitopes of myosin9 into pathogenic, non-pathogenic and natural epitopes. (2) the relationship of different pathogenic epitopes of myosin9 and PLA2R in IMN patient( including circulating antibodies and renal tissue antigens).(3) figuring out the key pathogenic epitope of myosin9 in the process of b cell spreading and pathogenesis of IMN,by IMN disease-transfer rat models.(4) exploring the mechanism of glomerular subcutaneous immune complex deposition using rats perfused with myosin9 epitope-peptide and anti-myosin9 antibodies in different periods. Our original and groundbreaking study implies the pathogenic antigen myosin 9 may supplement the possible mechanism involved in IMN and could better diagnose IMN, evaluate disease activity or even monitor potential prognostic and therapeutic implications.

特发性膜性肾病（IMN）致病靶抗原的相继发现为研究其发病机制奠定重要基础，但迄今尚无公认解释。在国家自然科学基金81170660/H0509支持下，我们不仅发现抗肌球蛋白蛋白9（myosin9）为IMN致病靶抗原之一，且惊喜发现，将myosin9抗原免疫兔子，最后兔抗血清中抗体成分检测出两种抗体包括PLA2R抗体，为此我们推测：myosin9的抗原表位能扩散至其他靶抗原如PLA2R。为了验证这一推测，我们拟从分析myosin9的抗原表位的致病性着手：（1）明确临床IMN患者体内myosin9不同致病性抗原表位与PLA2R的相互作用（2）通过人类IMN疾病转移大鼠模型及大鼠灌注模型，对比哪一致病性抗原表位在抗原扩散及疾病产生中起关键作用，及观察抗原抗体在肾组织如何沉积而探讨IMN初步发病机制。本研究将从myosin9抗原表位这个新视点为揭示IMN的作用机制奠定基础，为其诊断及治疗提供新思路。

第1部分 离体肾灌注模型鉴定myosin-9致膜性肾病的免疫复合物形成方式.目的：探讨myosin-9致膜性肾病上皮下免疫复合物形成的机制。.方法：通过构建离体大鼠肾灌注模型，将兔抗myosin-9末端B细胞靶位抗原多肽抗血清、兔抗myosin-9多克隆抗体及对照的正常兔血清、正常兔IgG在37℃条件下分别灌注正常大鼠肾组织2小时后，通过免疫荧光的方法观察各组大鼠肾脏中有无兔IgG的沉积及沉积差异。.结果：将兔抗myosin-9末端B细胞靶位抗原多肽抗血清及正常兔血清按照1:2、1:30、1:100的比例进行稀释并分别进行灌注大鼠肾脏，通过免疫荧光观察发现1：100组大鼠肾脏中检测不到兔血清的沉积。1:30可见致敏的兔血清中IgG在肾脏中沉积且非特异性较少，荧光主要沿肾小球基底膜呈颗粒状沉积，部分在系膜区。而1:2稀释的比例中，荧光强度与1:30组相当，但非特异性的沉积较多，而正常兔血清对照组未见有兔IgG的沉积。将兔抗myosin-9多克隆抗体及正常兔IgG按照1:1000、1:500的比例稀释并分别进行大鼠肾灌注，肾组织荧光均为阴性。.第2部分 myosin-9致膜性肾病机制的初步探讨 .目的：探讨myosin-9致膜性肾病的发生机制及观察其对myosin-9、nephrin mRNA的表达的影响。.方法：雄性Sprague-Dawley大鼠分为抗体组、抗原多肽组、抗原多肽+抗体组、对照组。其中抗原多肽+抗体组又分为抗原多肽抗体同时注射组、抗原先注射组、抗体先注射组。然后各组又分为4小时、8小时、24小时、72小时组。采用尾静脉注射的方法将myosin-9抗原多肽、兔抗myosin-9多克隆抗体注射到大鼠体内。通过免疫荧光的方法观察myosin-9抗原多肽及其抗体在肾组织内有无沉积及各组间沉积的差异，并通过RT-PCR的方法观察各组间肾脏nephrin、myosin-9 mRNA表达的差异。.结论: 抗原多肽可于8小时内沉积在肾脏内，说明抗原可以单独种植于肾小球，同时注射抗原和抗体比单独注射抗原较早的进入肾小球，说明抗原抗体复合物较单独的抗原或者抗体更易进入肾小球，单独的抗体较难进入肾小球，尽管在抗体单独注射24小时后在肾小球内能检测到抗体，但不一定是其能够单独进入，可能和内源性的抗原结合后进入肾小球可能性大。