重组腺病毒介导TIMP1基因对类风湿关节炎大鼠滑膜新生血管的影响

Effect of recombinant adenovirus mediating TIMP1 gene on angiogenesis of rats with collagen type II induced arthritis

背景:滑膜血管翳形成后可分泌一系列酶如基质金属蛋白酶等,可促进血管新生和炎症反应,形成软骨和骨的破坏。目的:研究重组腺病毒介导的基质金属蛋白酶组织抑制因子1(TIMP1)基因对类风湿关节炎模型大鼠关节炎症及滑膜血管新生的影响。设计、时间及地点:基因治疗动物体内实验,于2005-07/2007-06在扬州大学完成。材料:HEK-293A细胞系购于中科院上海细胞生物研究所细胞库,ECV-304细胞为自备,带有绿色荧光蛋白(GFP)基因或/和基质金属蛋白酶组织抑制因子1基因的重组腺病毒(rAD-GFP-TIMP1及rAD-GFP)的病毒贮存液由南京师范大学分子医学实验室惠赠。清洁级4～6周龄SD大鼠40只。方法:将rAD-GFP-TIMP1和rAD-GFP扩增、纯化,用紫外分光光度法检测重组腺病毒的颗粒数。用rAD-GFP-TIMP1感染ECV-304细胞检测病毒活力。30只大鼠注射Ⅱ型胶原建立类风湿关节炎大鼠模型,分为:①模型组注射PBS。②模型+rAD-GFP-TIMP1组注射rAD-GFP-TIMP1。③模型+RAD-GFP组注射rAD-GFP,均为膝关节腔内注射;10只大鼠注射生理盐水造模做对照组。1次/周,4周。主要观察指标:①rAD-GFP-TIMP1及rAD-GFP在HEK-293A细胞中扩增、纯化后病毒的颗粒数及GFP蛋白表达。②各组大鼠每周进行1次关节炎指数评分。③于造模第4周处死大鼠取膝关节行滑膜病理免疫组织化学染色及微血管密度检测。④取血清采用WesternBlot检测大鼠体内基质金属蛋白酶组织抑制因子1、血管内皮生长因子、基质金属蛋白酶1,2,3,9的表达。结果:①纯化后的rAD-GFP-TIMP1和rAD-GFP滴度分别为:5.7×1012,4.8×1012pfu/mL;A260nm/A280nm均>1.3;用rAD-GFP-TIMP1感染ECV-304细胞获得成功提示病毒活力正常。②感染rAD-GFP-TIMP1的关节炎大鼠血清中基质金属蛋白酶组织抑制因子1获得了高表达,rAD-GFP-TIMP1治疗组大鼠血清中基质金属蛋白酶组织抑制因子1的相对含量明显高于模型组和rAD-GFP治疗组。③rAD-GFP-TIMP1治疗组的关节炎指数评分明显低于模型组。④感染rAD-GFP-TIMP1可使滑膜新生血管数目明显减少,同时能抑制基质金属蛋白酶1,3,9的表达,而对血管内皮生长因子和基质金属蛋白酶2的抑制作用不明显。结论:①感染rAD-GFP-TIMP1的大鼠体内基质金属蛋白酶组织抑制因子1基因获得了有效的表达。②感染rAD-GFP-TIMP1能明显降低模型大鼠的关节炎指数评分。③血管内皮生长因子,基质金属蛋白酶1,2,3,9共同参与促进关节炎大鼠滑膜血管新生的形成和发展,rAD-GFP-TIMP1可能通过抑制基质金属蛋白酶1,3,9的表达而发挥抗血管新生的作用。

BACKGROUND： The synovial membrane pannus can secrete a series of enzymes such as matrix metalloproteinase（MMP）, which promote angiogenesis and inflammation leading to the formation of cartilage and bone damage. OBJECTIVE： To investigate the effect of recombinant adenovirus mediating tissue inhibitor of metalloproteinases 1 （TIMP1） gene on the angiogenesis of joint synovium in rats with collagen type II induced arthritis （CIA）. DESIGN, TIME AND SETTING： This was an animal experiment of gene treatment in vivo. It was performed in Yangzhou University from July 2005 to June 2007. MATERIALS： HEK-293A cell lines were purchased form cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. ECV-304 cell lines were stored by our private laboratory, rAD-GFP-TIMP1 and rAD-GFP were presented as a gift by Laboratory of Molecular Medicine of Nanjian Normal University. Forty clean SD rats aged 6 weeks were enrolled. METHODS： The rAD-GFP-TIMP1 and rAD-GFP were amplified and purified. The number of recombinant adenoviruses was detected by ultraviolet spectrophotometry. Viral activity was detected by rAD-GFP-TIMP1 infected ECV-304 cells. Thirty rats were treated with injection of collagen type Ⅱ to establish rat models of arthritis. They were divided into three groups： model group injected with PBS, rAD-GFP-TIMP1 group administrated with rAD-GFP-TIMP1, and RAD-GFP group injected with rAD-GFP. Another ten rats were injected with normal saline to be control group. All groups were injected once a week, for 4 weeks. MAIN OUTCOME MEASURES： ①The amount of recombinant adenoviruses and expression of GFP after the rAD-GFP-TIMP1 and rAD-GFP were amplified in HEK-293A cells and purified. ②The general conditions, symptoms of arthritis and arthritis index were assessed every week. ③The rats were executed 4 weeks after modeling for morphology and immunohistochemistry assay of knee joint synovial membrane. ④ The expressions of TIMP1, vascular endothelial growth factor （VEGF） and MMP-1, -2, -3, -9 in serum with Western Blot. RESULTS：①The titers of purified rAD-GFP-TIMP1 and rAD-GFP were 5.7×10^12 pfu/mL and 4.8×10^12 pfu/mL, respectively. Abserbance at 260 nm and 280 nm was less than 1.3. Viral activity was normal because rAD-GFP-ES infected ECV-304 cells successfully. ②There was a high expression of TIMP1 in the rAD-GFP-TIMP1 group, which was significantly higher than that of the model and rAD-GFP group. ③The mean arthritis index of the rAD-GFP-TIMP1 group was lower than the model group significantly. ④The number of new blood vessels in the synovium increased significantly in the rAD-GFP-TIMP1 group. The rAD-GFP-TIMP1 significantly decreased the production of MMP-1, MMP-3 and MMP-9 in serum. There was no significant decrease in VEGF and MMP-2 in serum. CONCLUSION： ①There was an efficient expression of TIMP1 in CIA rats with the therapeutic administration of rAD-GFP-TIMP1. ② The rAD-GFP-TIMP1 had an inhibitory effect on the arthritis index of CIA rats. ③VEGF, MMP-1, MMP-2, MMP-3 and MMP-9 participated in the angiogenesis of synovial tissue in CIA rats. It was probable that the rAD-GFP-TIMP1 had an inhibitory effect on angiogenesis through MMP-1, MMP-3 and MMP-9.