抗Angptl3单克隆抗体的研制及其干预肾病综合征的研究

Primary nephrotic syndrome is the most common kidney disease. Massive proteinuria as the main manifestation of PNS is also an independent risk factor, Which is mostly related with podocyte dysfunction. We firstly found the expression of Angptl3 was significantly increased in the renal tissue of PNS children. And we further explored that Angptl3 could interact with integrin αVβ3 on the surface of podocyte and endothelial cells through autocrine and paracrine leading to dysfunction of podocyte and high permeability of endotheliocyte respectively. Higher-expression of Angptl3 could aggravate podocyte dysfunction induced by PAN or ADR. Lower-expression or knock-out of Angptl3 could ameliorate podocyte dysfunction induced by PAN or ADR. To illuminate whether podocyte could be protected by monoantibody against Angptl3, We will make monoantibody against Angptl3 with hybridoma technique and observe the effect of antibody on nephrotic syndrome mice model. To confirm which amino sequences involved in proteinuria, we will screen the epitope of Angptl3 using phage display technology. Simultaneously, we will study whether the ILK signal pathway and the Wnt-βcatenin signal pathway involved in the underlying mechanism.

原发性肾病综合征是儿童常见的肾脏病，大量蛋白尿是其主要的临床表现，也是其独立的危险因素，蛋白尿的产生与足细胞的损伤最为相关。本课题组首次发现原发性肾病综合征患儿肾组织血管生成素样蛋白3表达升高。Angptl3可以通过自分泌和旁分泌分别与足细胞和内皮细胞表面整合素αVβ3结合，导致足细胞损伤和内皮通透性增强从而参与蛋白尿的形成。高表达Angptl3或敲除Angptl3分别加重或减轻嘌呤霉素引起的足细胞损伤和阿霉素引起的肾损伤。本项目拟在已有的研究基础上，通过制备针对Angptl3的单克隆抗体，观察外源性拮抗Angptl3是否可保护受损的足细胞。同时将单克隆抗体与噬菌体展示技术结合筛选Angptl3抗原表位，明确Angptl3中参与蛋白尿形成的氨基酸序列。并探讨该氨基酸序列是否通过与整合素αVβ3结合，激活ILK通路，影响 Wnt-βcatenin通路中的GSK3β，从而参与了蛋白尿形成。

原发性肾病综合征是儿童常见的肾脏疾病，大量蛋白尿是其主要的临床表现，蛋白尿的产生与足细胞的损伤最为相关。本课题组首次发现Angptl3可以通过自分泌和旁分泌分别与足细胞和内皮细胞表面整合素αVβ3结合，导致足细胞损伤和内皮通透性增强从而参与蛋白尿的形成。高表达Angptl3或敲除Angptl3分别加重或减轻嘌呤霉素引起的足细胞损伤和阿霉素引起的肾损伤。本项目利用杂交瘤技术制备了针对Angptl3的鼠源单克隆抗体，Western-blot表明该抗体具有较高特异性，SPR提示该抗体具有较强的亲和力，ELISA提示该抗体可阻断Angptl3对其受体整合素αVβ3的激活，体外试验证实抗单克隆抗体可以缓解嘌呤霉素引起的足细胞凋亡，骨架重排。也可以缓解阿霉素肾病小鼠的蛋白尿、低蛋白血症和足细胞病理损伤。