金雀异黄素对CIA大鼠关节FLS分泌的炎症及血管新生相关因子影响的实验研究
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摘要
目的:
观察金雀异黄素(genistein, gen)对Ⅱ型胶原诱导型关节炎(collagen typeⅡinduced arthritis, CIA)大鼠关节成纤维样滑膜细胞(fibroblast-like synoviocytes, FLS)分泌的白细胞介素-1β(interleukin-1β, IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、基质金属蛋白酶-1、2、3、9(matrix metalloproteinase, MMP-1、2、3、9)的影响。
研究对象:
CIA大鼠关节原代成纤维样滑膜细胞
方法:
首先,采用Ⅱ型胶原和完全弗氏佐剂(complete Freund's adjuvant, CFA)共同建立CIA大鼠模型,对大鼠每周进行一次关节炎指数(arthritis index, AI)评分。于造模第六周摄取关节X线片,然后处死大鼠,选取大鼠关节进行关节滑膜组织病理检测,并取出关节滑膜组织,用胶原酶消化法分离培养原代成纤维样滑膜细胞,流式细胞术(flow cytometry, FCM)检测滑膜细胞血管细胞黏附分子(vascular cell adhesion molecule-1, VCAM-1)的表达。
在CIA大鼠成纤维样滑膜细胞培养中加入用不同浓度的gen(100μM, 200μM, 400μM)及MTX(1mg·L-1),间接酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)法检测大鼠成纤维样滑膜细胞培养上清中IL-1β、TNF-α、VEGF及MMP-1、2、3、9的相对表达量。
结果:
1、Ⅱ型胶原致敏后三天,大鼠开始出现关节肿胀,AI逐渐增高,至造模后第三周最为明显,观察AI评分、关节X线片以及关节滑膜组织病理发现,造模组与空白对照组比较有显著性差异。表明造模成功,可满足本实验需要。
2、CIA大鼠滑膜细胞培养至第4代,FCM检测其VCAM-1的表达高达85.5%,提示所培养的细胞为成纤维样滑膜细胞(FLS),满足本实验要求。
3、不同浓度的gen(100μM, 200μM, 400μM)及MTX(1mg·L-1)作用于FLS后,细胞培养上清中IL-1β、TNF-α、VEGF和MMP-1、2、3、9的分泌受到抑制,且gen的抑制作用随药物浓度增加呈剂量依赖性增强。
结论:
1、本实验成功构建了CIA大鼠模型,并成功培养了原代大鼠成纤维样滑膜细胞。
2、gen能下调成纤维样滑膜细胞分泌的IL-1β、TNF-α,所以gen有可能通过抑制FLS分泌的炎症因子改善关节炎大鼠滑膜炎症。
3、gen能抑制成纤维样滑膜细胞分泌的VEGF和MMP-1、2、3、9,所以gen有可能通过抑制FLS分泌的血管新生相关因子,抗大鼠滑膜血管新生。
Objectives:
To investigate the effect of genistein(gen) on IL-1β(interleukin-1β)、TNF-α(tumor necrosis factor-α)、VEGF(vascular endothelial growth factor) and MMPs(matrix metalloproteinases)(MMP-1、2、3、9) secreted by FLS(fibroblast-like synoviocytes) from rats with CIA(collagen induced arthritis).
Subject:
Primary fibroblast-like synoviocytes from rats with CIA.
Methods:
At first, the rats were scored with the arthritis index(AI)once a week after the CIA was induced with collagen typeⅡand complete Freund’s adjuvant(CFA). At the sixth week, the X-ray of joints were taken out. After the rats were sacrificed, synovial tissue from knee joint were detected by pathological examination. The primary fibroblast-like synoviocytes were separated from the synovial tissue with collagenase digestion and cultured. The expression of VCAM-1 on synovial cell was estimated by flow cytometry.
Finally, different concentration of gen(100μM, 200μM, 400μM) and methotrexate(MTX)(1mg.L-1) were given to the incubation of fibroblast-like synoviocytes. Indirect ELISA(enzyme linked immunosorbent assay) was used to measure the relative contents of IL-1β、TNF-α、VEGF and MMP-1、2、3、9 in the supernatants of FLS.
Results:
1、The rats with CIA were induced with collagen typeⅡand complete Freund’s adjuvant. The rats caught arthrocele apparently three days later. After detected by the arthritis index(AI)、X-ray and pathological detection, it indicated that the CIA was well construceted.
2、After the primary synoviocytes were cultured to 4th generation, expression of VCAM-1 was tested up to 85.5%. It showed that most part of synoviocytes were fibroblast-like synoviocytes. It fits our experimental requirement.
3、With the effect of different concentration of gen(100μM, 200μM, 400μM) and MTX(1mg.L-1)on FLS, the IL-1βand TNF-α, VEGF and MMP-1、2、3、9 in supernatants were apparently restrained and dose-dependent.
Conclusions:
1、The CIA model was successfully constructed, the primary FLS of rats’joint were separated and cultured well.
2、Gen can suppress the secretion of TNF-αand IL-1βby FLS and is dose-dependent. Gen may improve the arthromeningitis of CIA rats by inhibitting the secretion of inflammatory factors.
3、Gen can suppress the secretion of VEGF and MMP-1、2、3、9 in FLS, the supression is dose-dependent. Therefore, gen may resist the angiogenesis by inhibiting angiogenesis-related factors secreted by FLS.
观察金雀异黄素(genistein, gen)对Ⅱ型胶原诱导型关节炎(collagen typeⅡinduced arthritis, CIA)大鼠关节成纤维样滑膜细胞(fibroblast-like synoviocytes, FLS)分泌的白细胞介素-1β(interleukin-1β, IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、基质金属蛋白酶-1、2、3、9(matrix metalloproteinase, MMP-1、2、3、9)的影响。
研究对象:
CIA大鼠关节原代成纤维样滑膜细胞
方法:
首先,采用Ⅱ型胶原和完全弗氏佐剂(complete Freund's adjuvant, CFA)共同建立CIA大鼠模型,对大鼠每周进行一次关节炎指数(arthritis index, AI)评分。于造模第六周摄取关节X线片,然后处死大鼠,选取大鼠关节进行关节滑膜组织病理检测,并取出关节滑膜组织,用胶原酶消化法分离培养原代成纤维样滑膜细胞,流式细胞术(flow cytometry, FCM)检测滑膜细胞血管细胞黏附分子(vascular cell adhesion molecule-1, VCAM-1)的表达。
在CIA大鼠成纤维样滑膜细胞培养中加入用不同浓度的gen(100μM, 200μM, 400μM)及MTX(1mg·L-1),间接酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)法检测大鼠成纤维样滑膜细胞培养上清中IL-1β、TNF-α、VEGF及MMP-1、2、3、9的相对表达量。
结果:
1、Ⅱ型胶原致敏后三天,大鼠开始出现关节肿胀,AI逐渐增高,至造模后第三周最为明显,观察AI评分、关节X线片以及关节滑膜组织病理发现,造模组与空白对照组比较有显著性差异。表明造模成功,可满足本实验需要。
2、CIA大鼠滑膜细胞培养至第4代,FCM检测其VCAM-1的表达高达85.5%,提示所培养的细胞为成纤维样滑膜细胞(FLS),满足本实验要求。
3、不同浓度的gen(100μM, 200μM, 400μM)及MTX(1mg·L-1)作用于FLS后,细胞培养上清中IL-1β、TNF-α、VEGF和MMP-1、2、3、9的分泌受到抑制,且gen的抑制作用随药物浓度增加呈剂量依赖性增强。
结论:
1、本实验成功构建了CIA大鼠模型,并成功培养了原代大鼠成纤维样滑膜细胞。
2、gen能下调成纤维样滑膜细胞分泌的IL-1β、TNF-α,所以gen有可能通过抑制FLS分泌的炎症因子改善关节炎大鼠滑膜炎症。
3、gen能抑制成纤维样滑膜细胞分泌的VEGF和MMP-1、2、3、9,所以gen有可能通过抑制FLS分泌的血管新生相关因子,抗大鼠滑膜血管新生。
Objectives:
To investigate the effect of genistein(gen) on IL-1β(interleukin-1β)、TNF-α(tumor necrosis factor-α)、VEGF(vascular endothelial growth factor) and MMPs(matrix metalloproteinases)(MMP-1、2、3、9) secreted by FLS(fibroblast-like synoviocytes) from rats with CIA(collagen induced arthritis).
Subject:
Primary fibroblast-like synoviocytes from rats with CIA.
Methods:
At first, the rats were scored with the arthritis index(AI)once a week after the CIA was induced with collagen typeⅡand complete Freund’s adjuvant(CFA). At the sixth week, the X-ray of joints were taken out. After the rats were sacrificed, synovial tissue from knee joint were detected by pathological examination. The primary fibroblast-like synoviocytes were separated from the synovial tissue with collagenase digestion and cultured. The expression of VCAM-1 on synovial cell was estimated by flow cytometry.
Finally, different concentration of gen(100μM, 200μM, 400μM) and methotrexate(MTX)(1mg.L-1) were given to the incubation of fibroblast-like synoviocytes. Indirect ELISA(enzyme linked immunosorbent assay) was used to measure the relative contents of IL-1β、TNF-α、VEGF and MMP-1、2、3、9 in the supernatants of FLS.
Results:
1、The rats with CIA were induced with collagen typeⅡand complete Freund’s adjuvant. The rats caught arthrocele apparently three days later. After detected by the arthritis index(AI)、X-ray and pathological detection, it indicated that the CIA was well construceted.
2、After the primary synoviocytes were cultured to 4th generation, expression of VCAM-1 was tested up to 85.5%. It showed that most part of synoviocytes were fibroblast-like synoviocytes. It fits our experimental requirement.
3、With the effect of different concentration of gen(100μM, 200μM, 400μM) and MTX(1mg.L-1)on FLS, the IL-1βand TNF-α, VEGF and MMP-1、2、3、9 in supernatants were apparently restrained and dose-dependent.
Conclusions:
1、The CIA model was successfully constructed, the primary FLS of rats’joint were separated and cultured well.
2、Gen can suppress the secretion of TNF-αand IL-1βby FLS and is dose-dependent. Gen may improve the arthromeningitis of CIA rats by inhibitting the secretion of inflammatory factors.
3、Gen can suppress the secretion of VEGF and MMP-1、2、3、9 in FLS, the supression is dose-dependent. Therefore, gen may resist the angiogenesis by inhibiting angiogenesis-related factors secreted by FLS.
引文
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