PIMT对类风湿性关节炎滑膜成纤维样细胞增殖/凋亡的调控

Recent studies have confirmed that fibroblast-like synoviocytes (FLS) play a key role in pathogenesis of rheumatoid arthritis (RA). The imbalance between proliferation and apoptosis of RA-FLS is contributed to inflammation of synovium and degradation of joint. Our previous study found that the expression of protein aspartylmethyltransferase (PIMT) in RA-FLS is decreased, and PIMT can identify and repair the abnormal l-aspartyl residues in protein. The loss of PIMT gene can lead the loss of this repair function and lead to cell proliferation and apoptosis resistant. But the specific relationship between depression of PIMT in RA-FLS and imbalance between proliferation and apoptosis of RA-FLS is still unknown. In this study we will transfect the PIMT-SiRNA and PIMT expression plasmid or vectors to the RA-FLS cells and RA rats, and determine the regulation of PIMT on proliferation/apoptosis of RA-FLS by detecting the proteins and cytokines related to proliferation and apototis, such as L-isoAsp, MEK1/2, ERK,JUK, Raf, Bcl-2, Bcl-Xl, Bax, Bak and Bim. This study will clarify that the regulation of PIMT on proliferation/apoptosis of fibroblast-like synoviocytes of rheumatoid arthritis from cell levels and animal animal levels, and it provided a new experimental evidence for investigating the precise role of FLS in RA.

新近研究已证实关节滑膜成纤维样细胞（FLS）的增殖与凋亡失调是类风湿性关节炎（RA）滑膜炎性增生，关节破坏的关键原因之一。我们前期研究发现蛋白异天冬氨酰甲基转移酶（PIMT）在RA-FLS中表达下降，PIMT具有识别并修复蛋白质中异常的L-天冬酰胺残基作用，而PIMT基因缺失会使这种修复作用丧失从而引起细胞增殖和抵御凋亡，但PIMT在RA-FLS中表达下降与RA-FLS增殖/凋亡失调之间的具体机制仍然未知。本研究将在前期研究基础上，利用特异性PIMT-SiRNA和PIMT表达载体，分别作用于原代培养的RA-FLS细胞和RA模型小鼠，通过检测L-isoAsp,MEK1/2,ERK,Raf,JUK,Bcl-2,Bcl-xL,Bax,Bak,Bim等增殖凋亡相关蛋白和细胞因子，在细胞和动物水平上确定PIMT对RA-FLS增殖/凋亡的具体影响及调控机制，为研究RA的发病机制提供实验依据。

前期研究发现蛋白异天冬氨酰甲基转移酶（protein isoaspartyl- methyltransferase, PIMT）在类风湿性关节炎（rheumatoid arthritis, RA）滑膜成纤维样细胞（fibroblast-like synoviocytes，FLS）中表达显著下降，而PIMT是一个与细胞增殖、凋亡相关的甲基转移酶。本项目以原代培养的人和大鼠RA-FLS细胞为研究对象，探讨PIMT对于RA-FLS细胞增值凋亡的影响，主要完成了以下研究内容：.1.确定了RA患者体内PIMT表达水平降低：与正常体检者比较，PIMT mRNA在RA患者的外周血单个核细胞内的表达明显降低（p<0.01）；对RA和OA患者关节滑膜组织的免疫组化结果显示PIMT在RA患者关节滑膜组织中表达降低；.2.确认了PIMT通过Bax/Bcl-2通路调控RA-FLS凋亡:特异性SiRNA降低PIMT的表达，RA-FLS增值增加，凋亡减少；在RA-FLS中过表达PIMT，导致RA-FLS凋亡降低；检测凋亡相关蛋白，发现PIMT是通过Bax/Bcl-2凋亡通路调控RA-FLS的凋亡；.3.确认了PIMT通过MAPK信号通路调控RA-FLS细胞的增值：在RA-FLS中过表达PIMT质粒，测定MAPK信号通路中相关蛋白，发现PIMT通过MAPK信号通路调控RA-FLS细胞的增值；.4.确认了PIMT对RA-FLS细胞中细胞因子的影响：过表达PIMT后，RA-FLS细胞中IL-1b，IL-6，IL-8，IL-17，TNF-α和VEGF表达下降，IL-10表达增高；特异性SiRNA下调PIMT的表达后，RA-FLS细胞中的IL-10表达降低；.6.在II型胶原诱导的大鼠RA模型的RAT-FLS中验证了PIMT能通过Bax/Bcl-2凋亡通路调控RAT-FLS的凋亡和通过MAPK信号通路调控RA-FLS细胞的增值。.本项目在细胞水平和动物水平上确定了PIMT对于RA-FLS细胞增殖凋亡的调控机制，为阐明RA 发病机制和寻找治疗靶点提供实验依据。