| Objective: To explore the effect of Tripterygium polyglycosides on VEGF mRNA expression in tissues through developing a real-time fluorescence quantitative PCR method and reproducing collagen-Ⅱinduced arthritis rat model. Methods: 1. A pair primers and TaqMan probe are designed and synthesized, with spanning the splicing site between two exons. The PCR method for detecting VEGF mRNA is established by ABI7500, then its specificity, sensitivity and stability are tested by calibration curve templates, which are constructed by PCR product cloned by TA ligetion. 2.To reproduce a rat model of collagen- II induced arthritis.45 successful rats are randomly divided into 3 groups in average: model group given normal saline, model group with Tripterygium polyglycosides. and model group with thalidomide: ten healthful rats as normal control group. All medicines are given by intragastric administration. The arthritis index (AI) and the edema rate are detected as curative effect every three days, and the level of VEGF mRNA in rat tissues is tested by real-time PCR on the 28th day after medicines administration. Results: DNA sequencing and agarosegel analysis of PCR product identified the speciality. Each diluted 10-fold from 6.6x10~7 to 6.6x10~0 copies/ul plasmid cDNA shows a precise log-linear relationship (r=0.998).The intra-assay cofficients of variation are 2.0～6.7% and inter-assay 3.5～10.6%. After 4 weeks with therapy, Tripterygium polyglycosides significantly reduce the paw edema and AI of CIA rats compared with that of model group (P < 0.01),and decrease the expression level of VEGF mRNA in rats tissues( P < 0.05).Conclusions: The real-time quantitative PCR for detection of VEGF are established successfully with sensitive,specific,repeatable features. Tripterygium polyglycosides can inhibit the development of arthritis in CIA rats. The mechanism may be related to the inhibition effect on the expression of VEGF and angiogenesis in tissues. |
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