摘要
目的观察金雀异黄素（genistein）对Ⅱ型胶原诱导性关节炎（CIA）大鼠关节成纤维样滑膜细胞（FLS）分泌的血管内皮生长因子（VEGF）及基质金属蛋白酶（MMP）-1、2、3、9的影响。方法采用Ⅱ型胶原和完全弗氏佐剂（CFA）建立CIA大鼠模型；每周进行一次关节炎指数（AI）评分；于造模第6周摄取关节X线片，选取大鼠关节进行关节滑膜组织病理检测；取出关节滑膜组织，用胶原酶消化法分离培养原代成纤维样滑膜细胞；流式法检测滑膜细胞血管细胞黏附分子（VCAM）-1的表达；在FLS培养中加入用不同浓度的金雀异黄素（100，200，400μmol／L），间接酶联免疫吸附试验（ELISA）法检测FLS培养上清中VEGF及MMP-1、2、3、9的表达。结果Ⅱ型胶原和CFA共同致敏后3d，大鼠开始出现关节肿胀，AI逐渐增高，于第3周时最明显，观察AI评分、关节X线以及关节滑膜组织病理发现，造模组与空白对照组比较差异有统计学意义。大鼠滑膜细胞培养至第4代，检测其VCAM-1的表达高达85．5％，提示所培养的细胞即为FLS。不同浓度的金雀异黄素（100，200，400μmol／L）和甲氨蝶呤（MTX，1mg／L）作用于FLS后，FLS培养上清中VEGF和MMP-1、2、3、9的分泌受到抑制，且抑制作用强度与药物的浓度有关。结论本实验使用Ⅱ型胶原和完全弗氏佐剂成功构建了CIA大鼠模型，用胶原酶消化法成功地分离并培养了原代大鼠成纤维样滑膜细胞。一定浓度的金雀异黄素能抑制CIA大鼠FLS分泌的VEGF和MMP-1、2、3、9，且抑制作用呈浓度依赖性。
Objective To investigate the effect of genistein （gen） on the secretion of vascular endothelial growth factor （VEGF） and matrix metalloproteinases （MMPs-1, 2, 3, 9） by fibroblast-like synovioeytes （FLS） from rats with collagen induced arthritis （CIA）. Methods The CIA was induced by collagen Ⅱ and complete Freund＇s adjuvant （CFA） into rats. The rats were scored based on the arthritis index（AI） once a week. At the sixth week, the X-ray of joints was taken. The synovial tissues from knee joints were examined pathologically. The primary fibroblast-like synoviocytes were separated from the synovial tissue by eollagenase digestion and cuhured. Then the expression of VCAM-1 was estimated by flow cytometry. After adding gen （100, 200, 400 μmol/L） at different concentrations into the FLS, VEGF and MMP-1, 2, 3, and 9 of the supernatants were tested by indirect ELISA. Results After 3 days of type Ⅱ collagen and CFA injec-tion, the rats started to catch arthrocele and their arthritis index increased gradually. The arthroeele was most remarkable at the 3rd week. The AI, X-ray and pathological examination indicated that the model group were significantly different from the control group. After the synoviocytes were cultured to the 4th generation, the expression of VCAM-1 was as high as about 85.5%. It showed that most synovioeytes were changed to fibroblast-like synoviocytes. Different concentrations of gen （ 100, 200, 400 μmol/L） added to FLS were compared and revealed that the VEGF and MMP-1, 2, 3, and 9 in the supernatants were suppressed evidently and in a dose-dependent manner. Conclusion The CIA model can be successfully constructed by collagen type Ⅱ and CFA; Tthe primary FLS of rats＇ joint can be separated and cultured well by collagenase digestion. Certain levels of gen can suppress the secretion of VEGF and MMP-1, 2, 3 and 9 by FLS. The affect is dose-dependent.
出处
《中华风湿病学杂志》
CAS
CSCD
北大核心
2010年第1期25-29,共5页
Chinese Journal of Rheumatology
基金
江苏省高校自然科学基础研究项目（07K.ID320245）
