CAJ | 학술논문

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)建寺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(100,200.400 μmol/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3 d,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,1 mg/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.
목적 관찰금작이황소(genistein)대Ⅱ형효원유도성관절염(CIA)대서관절성섬유양활막세포(FLS)분비적혈관내피생장인자(VEGF)급기질금속단백매(MMP)-1、2、3、9적영향.방법 채용Ⅱ형효원화완전불씨좌제(CFA)건사CIA대서모형;매주진행일차관절염지수(AI)평분;우조모제6주섭취관절X선편,선취대서관절진행관절활막조직병리검측:취출관절활막조직,용효원매소화법분리배양원대성섬유양활막세포;류식법검측활막세포혈관세포점부분자(VCAM)-1적표체;재FLS배양중가입용불동농도적금작이황소(100,200.400 μmol/L),간접매련면역흡부시험(ELISA)법검측FLS배양상청중VEGF급MMP-1、2、3、9적표체.결과 Ⅱ형효원화CFA공동치민후3 d,대서개시출현관절종창,AI축점증고,우제3주시최명현,관찰AI평분、관절X선이급관절활막조직병리발현,조모조여공백대조조비교차이유통계학의의.대서활막세포배양지제4대,검측기VCAM-1적표체고체85.5%,제시소배양적세포즉위FLS.불동농도적금작이황소(100,200,400μmol/L)화갑안접령(MTX,1 mg/L)작용우FLS후,FLS배양상청중VEGF화MMP-1、2、3、9적분비수도억제,차억제작용강도여약물적농도유관.결론 본실험사용Ⅱ형효원화완전불씨좌제성공구건료CIA대서모형,용효원매소화법성공지분리병배양료원대대서성섬유양활막세포.일정농도적금작이황소능억제CIA대서FLS분비적VEGF화MMP-1、2、3、9,차억제작용정농도의뢰성.
Objective To investigate the effect of genistein (gen) on the secretion of vascular endo-thelial growth factor (VEGF) and matrix metalloproteinases (MMPs-1, 2, 3, 9) by fibroblast-like synoviocytes (FLS) from rats with collagen Ⅱ 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 synowocytes were separated from the synovial tissue by collagenase digestion and cultured. 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 arthrocele 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 synoviocytes were changed to fibro-blast-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 supematants 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 hy FLS. The affect is dose-depen-dent.