2018-08-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/664685摘要：原發性膜性腎病變是一種自體免疫抗體攻擊足細胞上抗原的一種疾病，磷脂酶A2受體是其中一個重要抗原。但是，並沒有直接證據證實注射磷脂酶抗體會造成膜性腎病變，自體免疫抗體產生的機轉也並不清楚。B細胞與follicular helper T細胞(Tfh) 的交互作用對於體液免疫反應的調控，扮演了重要的角色。這個交互作用包含數個共同刺激分子，如IL-21-IL21R, B7–CD28, CD40–CD40L, ICOSL- ICOS及PDL1- PD1 ，這個作用維持B細胞的增生及影響基因表現方向。有證據顯示：follicular helper T上的ICOS(+) /PD-1(+)比率增加與原發性膜性腎病變的產生有關，我們的研究結果也發現膜性腎病變病患B細胞上PD-1及PDL-1的表現也顯著增加。我們假設：在膜性腎病變患者，T細胞及B細胞上的共同刺激分子表現會顯著改變，加強了Tfh與B細胞交互作用，影響膜性腎病變的發展。我們將網羅100個膜性腎病變病患及40個正常人，將以ELISA檢測他們的血中磷脂酶A2受體抗體力價，並以螢光抗體標定上述共同刺激分子，以流式細胞儀分析他們在T細胞及B細胞的變化，這些共同刺激分子的變化將與疾病活性做關聯性分析，並探討是否為膜性腎病變的預後因子。我們也將生產足細胞專一性可引發人類磷脂酶A2受體蛋白過度表現基因鼠，在誘發磷脂酶A2受體蛋白表現後，注入磷脂酶A2受體蛋白抗體陽性病患血漿，檢視是否產生蛋白尿、形成足細胞下的免役複合體沈積。我們也將投與對抗磷脂酶A2受體蛋白抗體的decoy peptide或蛋白，拮抗自體免疫抗體的作用，檢視是否減輕蛋白尿。同時，也將注射利用磷脂酶A2受體蛋白（或合併抗PD-1藥物）於此基因鼠，刺激產生自體抗磷脂酶A2受體抗體，檢視是否產生膜性腎病變。這個計劃將提供磷脂酶A2受體抗體造成膜性腎病變的直接證據，我們產生的足細胞專一性可引發人類磷脂酶A2受體蛋白過度表現基因鼠也可以作為治療磷脂酶A2受體抗體相關膜性腎病變藥物療效的測試平台，並可研究免疫異常造成膜性腎病變的機轉。了解膜性腎病變中T細胞與B細胞的交互作用，探討共同刺激分子在膜性腎病變的臨床意義及機轉，也有助於發展標靶治療，以最少副作用達到最大治療效果的目的。<br> Abstract: Idiopathic membranous nephropathy (MN) is considered to be auto-antibody against podocyte antigen, such as phospholipase A2 receptor (PLA2R). However, there is no direct evidence showing infusion of anti-PLA2R anybody causes the development of MN. The detail mechanism of the generation of auto-antibody is still unknown. The interaction between follicular helper T cell (Tfh) and the B cell plays an important role in the formation and maintenance of germinal center that controls the humoral immune response. The interaction includes several costimulatory molecules, such as IL-21-IL21R, B7–CD28, CD40–CD40L, ICOSL- ICOS and PDL1- PD1 which sustain B cells proliferation and gene expression programs. There were evidences showing that increased ratio of ICOS(+) /PD-1(+) follicular helper T cells positively correlates with the development of human idiopathic membranous nephropathy. Our preliminary results also revealed the expression of PD-1/PDL-1 in B cells was enhanced in MN patients. We hypothesize that these costimulatory molecules are up-regulated enhancing the interaction between Tfh and B cells in idiopathic MN patients. This study is to provide the direct evidence that anti-PLA2R antibody is the pathogenic mediator in idiopathic MN. We will also explore the expression of costimulatory molecules and their clinical significance in idiopathic MN patients. We will include 100 MN patients and 40 normal subjects. The presence and titer of anti-PLA2R antibody will be evaluated with ELISA kit. The costimulatory molecules, such as IL-21-IL21R, B7–CD28, CD40–CD40L, ICOSL- ICOS and PDL1- PD1, will be evaluated with flow cytometry after labeling with suitable fluorescence conjugated antibody. The expression of costimulatory molecules will be correlated to disease activity and anti-PLA2R antibody. These expressions will also be analyzed to examine the prediction power of remission in MN patients.We will also generate podocyte specific inducible human PLA2R over expression mice. After induction of PLA2R protein at podocyte in these mice, plasma derived from anti-PLA2R antibody positive patients will be infused into podocyte human PLA2R antigen carrying mice. The proteinuria and subepithelial deposit will be examined to demonstrate the development of MN. We expect neutralizing anti-PLA2R antibody specifically with decoy peptide or protein will attenuate proteinuria. Besides, we will also try to induce active MN mice model. The inducible podocyte human PLA2R over expression mice will be challenged with human PLA2R protein with/without anti-PD-1 agents. Then proteinuria will be measured and the pathological findings of MN, including subepithelial deposit will be examined. This project will provide direct evidence that anti-PLA2R antibody is the pathogenic mediator in idiopathic MN. The podocyte specific inducible human PLA2R over expression mouse strain could be a platform to test any novel drug for treating anti-PLA2R antibody associated MN. The active PLA2R MN model could be used to study the immune dysfunction leading to MN. Besides, understanding the interaction between Tfh and B cells is helpful realizing the mechanism of auto-antibody generation in MN patients. This knowledge helps us developing a new target treatment strategy for MN patients.膜性腎病變磷脂酶A2受體免疫細胞共同刺激分子membranous nephropathyphospholipase receptor A2 receptorimmune cellcostimulatory molecules