血管内皮生长因子对佐剂性关节炎滑膜细胞基质金属蛋白酶3及基质金属蛋白酶9表达的影响

Effect of vascular endothelial growth factor on the expression of matrix metalloproteinase-3 and matrix metalloproteinase-9 of synoviocytes in adjuvant arthritis

目的:观察血管内皮生长因子对佐剂性关节炎滑膜细胞质金属蛋白酶3及质金属蛋白酶9表达的影响,并探讨其意义。方法:实验于2006-02/12在桂林医学院实验中心完成。①实验材料:清洁级8周龄雄性Wistar大鼠6只,血管内皮生长因子为Peprotech EC LTD公司产品,基质金属蛋白酶3(扩增369bp)上游引物、下游引物,基质金属蛋白酶9(扩增405bp)上游引物、下游引物均购自晶美公司。②实验干预:先用弗氏完全佐剂造Wistar大鼠模型;造模20d后取Wistar大鼠右后足滑膜细胞进行原代培养。③实验分组:实验分为血管内皮生长因子组:取P2代细胞接种于6孔培养板,分别加入终浓度为5,25,50μg/L血管内皮生长因子;对照组:不加血管内皮生长因子。④实验评估:取病理切片观察滑膜细胞形态学改变;采用半定量反转录聚合酶链反应检测Wistar大鼠佐剂性关节炎滑膜细胞基质金属蛋白酶3及基质金属蛋白酶9的m-RNA表达。结果:①培养细胞的形态学观察:原代培养14d滑膜细胞从组织块边缘逸出,21d密集生长开始传代;传代细胞48h可明显分辨出树突样细胞、巨噬细胞样细胞和成纤维细胞样细胞;传至21代,细胞生长及特性稳定。②病理切片:滑膜组织有中性粒细胞、单核细胞、淋巴细胞浸润,滑膜细胞增生、排列紊乱,纤维素渗出,胶原纤维沉着,纤维素样坏死,呈滑膜炎表现。③基质金属蛋白酶3、基质金属蛋白酶9的mRNA表达:对照组基质金属蛋白酶3mRNA表达相对灰度值与血管内皮生长因子终浓度5,25,50μg/L组比较,差异有显著性意义(0.32±0.03,0.77±0.06,1.12±0.12,1.59±0.02,P<0.05);对照组基质金属蛋白酶9mRNA表达相对灰度值与血管内皮生长因子终浓度5,25,50μg/L组比较,差异有显著性意义(0.47±0.07,0.50±0.10,0.91±0.10,1.31±0.06,P<0.05);基质金属蛋白酶3和基质金属蛋白酶9mRNA表达随血管内皮生长因子浓度的加大表达增加。结论:血管内皮生长因子以剂量递增的方式对体外培养的佐剂性关节炎滑膜细胞基质金属蛋白酶3及基质金属蛋白酶9的表达有促进作用。

AIM： To investigate the effects of vascular endothelial growth factor （VEGF） in the expression of matrix metalloproteinase-3 （MMP-3） and matrix metalloproteinase-9 （MMP-9） of synoviocytes in adjuvant arthritis （AA）. METHODS： The experiment was performed in the Experimental Center of Guilin Medical College from February to December 2006. （1）The materials included six 8-week-old male Wistar rats of clean grade, VEGF and primers of MMP-3 （369 bp） and MMP-9 （405 bp）. （2）AA model in rats was established with Freund＇s complete adjuvant; 20 days later, the synoviocytes in the right hind limbs were harvested and cultivated. P2 cells were seeded in 6-well plate containing VEGF at final concentrations of 0, 5, 25, and 50 μg/L. A semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） was used to investigate the expression of MMP-3 and MMP-9 in synoviocyte of AA. （3）The synovium tissue of AA was observed by histological assay. RESULTS： （1）At 14 days after primary culture, synoviocytes overflowed the edge of synovium tissue; the cells began to accelerate growth and were passaged at day 21. After 48 hours＇ subculture, dendritic cells, macrophage-like cells, and fibroblast-like cells were observed; the cells maintained proliferation till the 21^st passage. （2）In the synovium tissues, neutrophils, mononuclear cells, lymphocyte infiltration, synoviocyte hyperplasia, mess arrangement, fibrin exudation, collagen fiber deposition, and fiber-like necrosis were found, which were manifestations of synovitis. （3）There were significant differences in the gray values of MMP-3 mRNA expression between control group and 5, 25, and 50 μg/L VEGF groups （0.32±0.03, 0.77±0.06, 1.12±0.12, 1.59±0.02, respectively, P 〈 0.05）; so were the gray values of MMP-9 （0.47±0.07, 0.50±0.10, 0.91±0.10, 1.31±0.06, respectively, P 〈 0.05）. MMP-3 and MMP-9 levels in supernatant of cell cultures were elevated with the increase of VEGF concentration. CONCLUSION： VEGF can accelerate the expression of MMP-3 and MMP-9 in synoviocyte of AA cultured in vitro in a dose-dependant manner.