PADI4调控巨噬细胞极化和增殖在RA关节滑膜病变中的作用及分子机制

The peptidylarginine deiminase IV (PADI4) is an important co-regulator of gene transcription, which converts arginine residues at histone tails and other associated protein to citrulline. Citrullination can take place at hematopoietic stem cells, tumor cells and other cell nucleus, regulation of cell differentiation, proliferation or apoptosis. Macrophages (Mø) and fibroblast-like cells (FLS) highly expressed PADI4 are two key effector cells in the RA synovium (RA-ST). Our previous study found that PADI4 promote RA-FLS proliferation, secretion of inflammatory cytokines and inhibition of apoptosis. This study will continue to explore the following research work: 1) Using immunohistochemistry and fluorescent antibody technique, to clarify M1/M2 Macrophages polarization mode in the different course of RA-SM. 2) To explore its role of PADI4 overexpressed in synovium Macrophages to promote M1 and M2, osteoclasts and osteoblasts disequilibrium. 3) In the cell co-culture system using a confocal laser dynamic analysis, flow cytometry technology to clarify that the interactions between M1, M2 Macrophages and FLS, and the effect on Macrophages polarization, proliferation and secretion of inflammatory cytokines. 4) Construction of shRNA-PADI4 nano-targeting vectors, at the level of RA models, to explore the precise role of therapeutic strategies, separately or combined with targeted inhibition of FLS, Mø, M1 and osteoclasts expression PADI4 in the prevention and treatment of RA.

肽精氨酸脱亚氨基酶Ⅳ（PADI4）是一个重要的基因转录共调节因子，通过对造血干细胞、肿瘤等多种细胞核内组蛋白及其相关蛋白的瓜氨酸化修饰，调控细胞的分化、增殖或凋亡。巨噬细胞（Mø）和成纤维样细胞（FLS）是RA滑膜（RA-ST）病变的两个关键效应细胞，均高表达PADI4。项目组前期已阐明PADI4能促进RA-FLS增殖、抑制凋亡并促进分泌炎症因子。本项目聚焦于RA-ST Mø，采用免疫组化技术阐明不同病程Mø极化模式及增殖状况，分析Mø内PADI4过表达促进M1/M2、破骨细胞/成骨细胞失平衡的作用;在细胞共培养体系中采用激光共聚焦动态分析Mø、FCM等研究M1、M2细胞与FLS间相互调控对Mø极化、增殖及分泌炎症因子的影响。构建shRNA-PADI4壳聚糖纳米靶向载体，在RA模型中探索分别或联合靶向抑制FLS、Mø、M1、破骨细胞表达PADI4的精准策略在RA防治中的作用。

肽精氨酸脱亚氨基酶Ⅳ（PADI4）是一个重要的基因转录共调节因子，通过对造血干细胞 、肿瘤等多种细胞核内组蛋白及其相关蛋白的瓜氨酸化修饰，调控细胞的分化、增殖或凋亡。 巨噬细胞（Mø）和成纤维样细胞（FLS）是RA滑膜（RA-ST）病变的两个关键效应细胞。项目组前期已阐明RA-FLS中 PADI4异常高表达，PADI4能促进RA-FLS增殖、抑制凋亡并促进分泌炎症因子。本项目聚焦于PADI4调控RA-ST Mø极化和破骨细胞（OC）分化展开。首先采用免疫组化和双标记免疫荧光分析明确RA-ST中Mø细胞浸润程度高于OA，存在M1、M2 Mø极化增加、且以M1极化为主的现象。进一步研究发现RA关节腔特征性低氧是促进M1、M2共极化的主要原因。低氧环境诱导Mø表达PADI4增加，PADI4促进M1极化、抑制其凋亡，进一步的PADI4促进M1向OC分化、增强其骨吸收能力。同时在缺氧环境中RA-FLS凋亡减少并向成脂细胞分化。细胞共培养研究发现，低氧诱导RA-FLS和Mø表达PADI4增加、并出现以M1极化为主的M1和M2共极化现象，抑制PADI4表达则显著抑制M1极化、抑制M2趋化因子表达。上述研究结果阐明了低氧和PADI4异常高表达是RA关节滑膜慢性炎症性增生、关节骨质破坏增加的关键致病因素。在此研究的基础上，项目组成功制备PADI4siRNA-MSN纳米载体，该纳米载体具有siRNA载量高、转染效率高且稳定、对细胞无毒性、对机体无免疫原性等特点，细胞学实验显示其能有效抑制低氧环境诱导的PADI4表达、抑制RA-FLS增殖和M1、OC分化，CIA动物实验显示能有效减缓模型鼠关节症状，抑制关节炎症、减少骨质损伤。本项目成功制备PADI4siRNA-MSN纳米载体有望成为新的RA生物治疗药物，在后续研究中将标化制备流程、扩大动物模型研究，争取成功转化。