三水白虎汤抑制类风湿关节炎滑膜成纤维细胞迁移及机制研究

The project is to study the effect and mechanism of San Shui Bai Hu Decoction (SSBH) on inhibiting the migration of human rheumatoid arthritis synovial fibroblasts (RASFs) both in vitro and in vivo. 1.Effects of migration inhibitory: Using transwell method and the scratch assay to test cell mobility of RASFs cultured with SSBH-containing serum in vitro. RASFs-cartilage-SCID mice model is established, intragastrically with SSBH, to observe the degree of erosion of RASFs in cartilage grafts and to find the synovial hyperplasia and cartilage erosion degree in double knee joints in vivo.Injecting marked RASFs intraperitoneal and subcutaneous in SCID mice, to observe the quantity of RASFs in spleen (fluorescence microscope) and blood (flow cytometry).2. Mechanism of inhibiting the RASFs migration : Measuring the RASFs and endothelial cell binding rate to explore the migration mechanism of RASFs in blood. For the exploring of the molecular mechanisms about the cell migration, RASFs cultured with drug-containing serum is collected, joints and implanted cartilage of mouse model is clipped , and the expressions of adhesion molecules (integrinβ1 cadherin -11 ) and cytoskeletal proteins (focal adhesion protein, centrosome protein) are detected. The distribution of cytoskeletal proteins intracellular is observe with laser confocal. Determining FAK, p38MAPK expression and transcription by in situ hybridization and PCR real-time fluorescence quantitative to explore migration signaling pathway. These results will provide a new basis for the definition of pharmacological role SSBH played in the treatment of RA, and for the development of new drugs.

本项目采取体外和体内实验，研究三水白虎汤（SSBH）抑制人类风湿关节炎滑膜成纤维细胞(RASFs)的迁移效果及机制。1.抑制迁移效果：体外实验通过Transwell法、划痕法检测SSBH含药血清培养RASFs后的细胞迁移率。体内实验建立RASFs-软骨-SCID鼠模型，经SSBH连续灌胃后，观察移植软骨、双膝关节中RASFs增生及侵蚀程度；SCID鼠予腹腔和皮下注射标记的RASFs，观察脾脏（荧光镜下）与血液（流式细胞术）中RASFs数量。2.抑制RASFs迁移机制：测定RASFs与内皮细胞结合率，探究其血行迁移机制；收集含药血清培养的RASFs与模型鼠的关节、软骨，测定粘附分子（整合素β1、钙粘蛋白-11）与骨架蛋白（黏着斑蛋白、中心体蛋白）的表达，激光共聚焦观察骨架蛋白的胞内分布；原位杂交、PCR实时荧光定量测定FAK、p38MAPK的表达与转录，探讨抑制迁移的相关分子和信号转导机制。

本项目首先利用经典的体外细胞迁移实验观察分析三水白虎汤及其拆方对RASFs迁移的影响，并以OASFs作为细胞对比，然后采用Western blot和实时定量RT-PCR检测滑膜细胞蛋白表达情况；测定RASFs与内皮细胞结合率，探究其血行迁移机制；同时，体内实验建立RASFs-软骨-SCID鼠模型，观察移植软骨中RASFs侵蚀程度和双膝关节标本滑膜增生及软骨侵蚀程度；此外，SCID鼠予腹腔和皮下注射标记的RASFs，观察脾脏与血液中RASFs数量。收集含药血清培养的RASFs与模型鼠的关节、软骨，测定粘附分子与骨架蛋白的表达，观察骨架蛋白的胞内分布，探讨迁移相关的分子机制；并测定FAK、p38MAPK的表达与转录，探讨迁移相关信号通路的影响。具有以下重要意义：①从细胞形态学角度更直观的明确三水白虎汤抑制RASFs迁移的效果，从细胞迁移角度阐释和完善其治疗RA的药理机制，甚至进一步发现其对OA同样具有一定的疗效，临床上治疗关节炎方面具有较高的的应用价值。②初步探讨三组拆方对滑膜细胞迁移的作用，评价出最有效的拆方组合，为今后SSBH的精减奠定实验基础。③c-Jun氨基末端激酶（JNK）通路与RASFs迁移密切相关，以JNK信号通路的小分子抑制剂sp600125作为阳性药物对照，摸索SSBH抑制细胞迁移的机制与JNK通道的联系。④检测细胞迁移、EMT相关分子的蛋白表达和mRNA转录情况，从蛋白和基因水平探索SSBH抑制细胞迁移可能的机制。⑤高效液色谱-质谱联用(LC-MS/MS)分析技术对大鼠三水白虎汤含药血清、患者服药后血清及关节液的成分进行定性分析，以明确其部分成分并确保基于含药血清进行体外研究的稳定性与可靠性。⑥SSBH抗滑膜成纤维细胞-血管内皮细胞黏附作用可能是其治疗类风湿关节炎的机制之一。⑦激光共聚焦显微镜发现三水白虎汤能下调类风湿关节炎滑膜成纤维细胞中心体蛋白、桩蛋白、黏着斑蛋白等骨架蛋白表达。