中文摘要：

目的 对RA成纤维样滑膜细胞（FLS）体外分离培养方法进行优化,观察Dishevelled 2 （Dvl 2）在RA-FLS中对血管内皮生长因子（VEGF）分泌的影响.方法 将行膝关节置换的RA患者滑膜剪碎,利用优化的组织块法进行RA-FLS体外分离培养.构建Dvl 2慢病毒过表达质粒,转染后利用Q-PCR及ELISA检测VEGF mRNA及蛋白表达情况.然后再将转染后RA-FLS予TNF-α重组蛋白10 ng/ml刺激24h,再次检测VEGF mRNA及蛋白表达情况.采用两独立样本t检验进行统计分析.结果 成功将RA-FLS分离并进行体外培养.当感染复数为30并配合使用适当浓度的polybrene促进转染即可满足实验要求,Dvl 2的mRNA较空病毒对照组升高79倍.与空病毒对照组相比,Dvl 2对RA-FLS分泌VEGF有轻度抑制作用;TNF-α刺激后,Dvl 2达表达组VEGF的mRNA[（2.15±0.10）倍,（2.92±0.47）倍,t=-3.924,P=0.003]及蛋白[（285±100） pg/ml,（155±61） pg/ml,t=-2.714,P=0.022]均低于空病毒对照组.结论 Dvl 2可抑制TNF-α对RA-FLS分泌VEGF的促进作用,具体机制有待进一步研究.

英文摘要：

Objective To optimize the culture method for rheumatoid arthritis fibroblast-like synoviocytes in vitro,and observe the effect of Dishevelled （Dvl） 2 on vascular endothelial growth factor （VEGF） in rheumatoid arthritis fibroblast-like synoviocytes （RA-FLS）.Methods Synovium from RA patients who underwent knee arthroplasties were cut into small piece,and RA-FLS were isolated and cultured in vitro using tissue block method.Dvl 2 lentivirus overexpressing plasmid was constructed and transfected into RAFLS.Q-polymerase chain reaction （PCR） and enzyme linked immunosorbent assay （ELISA） were used to detect the mRNA and protein expression levels of VEGF.Then we used 10 ng/ml tumor necrosis factor （TNF）-α recombinant protein to stimulate the transfected RA-FLS.24 h after stimulation,mRNA and protein expression of VEGF were detected again.Student＆#39;s t test was used for two group analyses.Results RA-FLS was successfully isolated and cultured in vitro.The multiplicity of infection was 30 and was in conjunction with appropriate concentration of polybrene to promote transfection.Transfection efficiency could meet the test requirements.The mRNA of Dvl 2 increased for 79-fold than the control group.Compared with the control group,Dvl 2 could mildly inhibit RA-FLS secretion of VEGF.After TNF-α stimulation,Dvl 2 could significantly inhibit the VEGF＆#39;s mRNA （2.15±0.10,2.92±0.47 fold,t=-3.924,P=0.003） and protein [（285±100） pg/ml,（155±61） pg/ml,t=-2.714,P=0.022] expression compared with the control group.Conclusion Dvl 2 can inhibit the effect of TNF-α induced secretion of VEGF in RA-FLS.The specific mechanism needs further study.