自噬在TNF-α诱导的RA-FLS增殖和凋亡中的作用及机制研究

Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease , characterized by inflammation,synovial hyperplasia of ioints. Although,excessive proliferation and apoptosis obstacles of fibroblast-like synoviocytes (FLS) induced by tumor necrosis factor-a (TNF-a) played an important role in the pathophysiological process of RA,its underlying mechanism is unknown.Recently, we verified that TNF-a induced autophagy and apoptosis in RA-FLS both. Blockade of autophagy sensitized RA-FLS to TNF-a-induced apoptosis and concurrently abated cell proliferation of RA-FLS.Our results indicated that autophagy might played an important role in regulating cell proliferation and apoptosis of RA-FLS induced by TNF-a in RA.Furthermore, we showed that activation of PI3K/Akt signaling pathway existed in TNF-a-induced cell proliferation of RA-FLS. However,the underlying molecular mechanism by which autophagy regulating the cell proliferation and apotosis of RA-FLS is completely unknown. This project is to investigate the biological role of autophagy in regulating cell proliferation and apoptosis of RA-FLS induced by TNF-α and the potential molecular mechanisms by the application of confocal fluorescence microscope, electron microscope, small interfering RNA（siRNA）,immunoblot and flow cytometry analysis. The study may provide important experimental data or a new theoretical basis for the pathogenesis of RA,which could provide new intervention targets and strategies for the treatment of RA.

TNF-α诱导的RA-FLS肿瘤样异常增殖和凋亡障碍是RA的主要病理特点，但其调控增殖和凋亡的机制尚不完全清楚。近期我们研究发现TNF-α能诱导RA-FLS发生自噬和凋亡。抑制自噬可促进TNF-α诱导的RA-FLS凋亡，阻抑RA-FLS增殖，提示自噬可能参与调控TNF-α诱导的RA-FLS增殖和凋亡。进一步实验发现TNF-α可诱导PI3K/Akt信号通路活化，改变RA-FLS的细胞周期，但自噬调控TNF-α诱导的RA-FLS增殖和凋亡的机制尚不明确。本项目拟在此基础上，通过药物和siRNA上调或下调细胞自噬，应用激光共聚焦显微镜、扫描电镜、免疫印迹、流式细胞术等方法，深入研究自噬在TNF-α诱导的RA-FLS增殖和凋亡中的作用，探讨PI3K/Akt等信号通路介导自噬调控RA-FLS增殖和凋亡的分子机制，从而为RA发病机制提供重要的实验数据和新的理论基础，为RA的治疗提供新的干预靶点和策略。

自噬参与许多疾病的发病，但自噬在类风湿关节炎中的作用及其可能分子机制目前并不完全清楚。我们的研究旨在探讨自噬是能否通过PI3K/AKT 信号通路调控类风湿关节炎滑膜细胞的增殖和凋亡。在体内研究中，我们构建了CIA模型鼠，我们观察了大鼠的一般情况、关节肿胀情况、滑膜组织的炎症反应、细胞增殖和凋亡等情况。另外，在体外研究中，我们培养原代的类风湿关节炎滑膜成纤维细胞（RA-FLS），提前用PI3K抑制剂LY 294002抑制PI3K/AKT 信号通路，然后使RA-FLS暴露于TNF-α，观察细胞增殖、凋亡、细胞因子分泌。细胞周期的情况，并检测检测相关蛋白。我们研究发现自噬的抑制剂氯喹（Chloroquine,CQ)和3-Methyladenine (3-MA)能减轻CIA模型鼠关节的肿胀程度，减轻滑膜组织的炎症反应、滑膜细胞的增殖，同时促进滑膜细胞的凋亡。同时体外实验也发现自噬的抑制剂CQ和3-MA能抑制TNF-α诱导的滑膜成纤维细胞的增殖，并阻断细胞停顿在 G2/M期，减少S 期的细胞数。更重要的是我们还发现提前用LY 294002抑制 PI3K/AKT 信号通路能部分逆转TNF-α诱导的自噬和细胞因子分泌。我们的研究结果提示自噬抑制剂可通过调控PI3K/AKT信号通路减轻类风湿关节炎的炎症反应，抑制类风湿关节炎滑膜成纤维细胞的增殖，促进其凋亡。此项研究对揭示RA新的发病机制有重要意义，并为日后RA治疗提供了新的靶点和理论基础。