Vascular Endothelial Growth Factor Receptor Expression In Animal Models Of Collagen-induced Arthritis And Endothelial Endostatin And Angiostatin Expression Intervention

Aim: To establish collagen-induced arthritis(CIA) model in Wistar rats.To explore the expression of VEGFRs in CIA.To explore the possible mechanism of endostatin and angiostatin therapy on CIA.To supply clues to the pathogenesis and therapeutic expectations of rheumatoid arthritis.Materials and Methods: (1) .The immunization complex were made by emulsifying natural type II collagen with Freund's adjuvant. The immunization complex was then injected into Wistar rats intradermally at the base of the tail.The redness and swelling of the joints of the rat were evaluated. The inflammatory index, pathology index, the joint pad thickness, the vessel density, the cartilage Safranin O stain content and the cartilage Safranin O stain index were evaluated .By such means we established a collagen-induced arthritis animal model. The correlation between the titer of anti-type II collagen antibody in CIA with the inflammatory index, pathology index, the joint pad thickness, the vessel density,the cartilage content and the cartilage index were analyzed. The correlation of articular cartilage Safranin O content with articular Safranin O stain index was analyzed. The correlation of vessel density with the inflammatory index, pathology index, articualr cartilage content and the joint pad thickness were analyzed.(2) VEGFR-2 protein expression was detected by immunohistochemistry. We detected VEGFR-2 mRNA expression by in situ hybridization. The VEGFR-2 protein expression by immunohistochemistry and mRNA expression by in situ hybridization with vessel density and articular cartilage were analyzed.The expression of VEGFR-1,VEGFR-2 and VEGFR-3 mRNA expression were detected by RT-PCR.The correlation of VEGFR-1, VEGFR-2 and VEGFR-3 mRNA expression with the inflammatory index, pathology index, the vessel density, the cartilage content and the cartilage index were analyzed.(3) We amplified endostatin cDNA,then inserted it into eukaryotic expression vector pSecTag/HygroB,then it was used to transform the E. coli. JM109.Positive clones were selected .Then the plasmid pSecTag/ ES was amplified .It was emulsified with PVP40.And the emulsified mixture were injected into the pretibial muscle at a dose of 100μg/rat once a week.Each rat accepted three injections. The difference of the pad thickness,inflammatory index, pathology index, the vessel density, the cartilage content and the cartilage index were analyzed between ES treatment group and blank control. The differences of VEGFR mRNA expression were analyzed between ES treatment group and blank control. The difference of VEGFR-2 protein expression was analyzed before and after endostatin therapy.(4) We purified plasminogen from human plasma by lysine affinity chromography. Then the purified plasminogen was digested by pancreatic elastase to generate angiostatin. The...