青藤碱联合甲氨蝶呤对类风湿关节炎成纤维样滑膜细胞的影响

目的 观察青藤碱（sinomenine,SIN）联合甲氨蝶呤（methotrexate, MTX）对体外培养类风湿关节炎（rheumatoid arthritis,RA）患者成纤维样滑膜细胞（fibroblast like synoviocytes，FLS）增殖、凋亡及其破骨细胞分化因子表达的影响。方法 从RA患者滑膜组织分离培养FLS，分为SIN1、2、3、4组，MTX1、2、3、4组，联合1、2、3、4组，分别加入0.001、0.010、0.100、1.000 mg/mL SIN，0.001、0.010、0.100、1.000 mg/mL MTX，0.001 mg/mL SIN+0.001 mg/mL MTX，0.010 mg/mL SIN+0.010 mg/mL MTX，0.100 mg/mL SIN+0.100 mg/mL MTX，1.000 mg/mL SIN+1.000 mg/mL MTX的含药培养基，并以未加药物的培养基作为对照组，共13组，每组设3个复孔。采用MTT法检测细胞增殖情况；流式细胞术检测细胞凋亡情况；半定量RT PCR检测FLS NF-κB受体活化因子配体（receptor activator of nuclear factor kappa B ligand,RANKL）、骨保护素（osteoprotegerin,OPG）mRNA表达水平。结果 与对照组比较，各药物组RA FLS增殖OD值均降低, 联合3组RA FLS凋亡OD值、OPG mRNA表达升高，RANKL mRNA表达降低，差异均有统计学意义（P<0.05）;与联合3组比较，SIN 3组及MTX 3组RA FLS增殖OD值升高（P<0.05）。结论 SIN与MTX联合对RA FLS具有协同抑制效应，这可能是二者联用抑制RA骨破坏的机制之一。

Effects of Sinomenine and Methotrexate on Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Objective To investigate the effects of sinomenine(SIN) and methotrexate(MTX) on the proliferation and apoptosis of in vitro cultured fibroblast-like synoviocytes(FLS) in rheumatoid arthritis(RA) patients,as well as the expression of osteoclast differentiation factor in FLS.Methods FLS were isolated from the synovium of RA patients and cultured in vitro.FLS were incubated with different concentrations of SIN and MTX respectively or combined:0.001,0.010,0.100,1.000 mg/mL SIN;0.001,0.010,0.100,1.000 mg/mL MTX;0.001 mg/mL SIN+0.001 mg/mL MTX,0.010 mg/mL SIN+0.010 mg/mL MTX,0.100 mg/mL SIN+0.100 mg/mL MTX,1.000 mg/mL SIN+1.000 mg/mL MTX,namely SIN1,2,3,4 groups;MTX1,2,3,4 groups and the combination1,2,3,4 groups.The medium without drugs was used as a control group.There was a total of 13 groups,each group with 3 complex holes.MTT was applied to detect the growth of FLS.The flow cytometry was applied to detect the apoptosis of FLS.The expressions of FLS receptor activator of nuclear factor kappa B ligand(RANKL) mRNA and osteoprotegerin(OPG) mRNA were observed by semi-quantitative RT-PCR.Results Compared with the control group,RA FLS proliferation OD values of all the drug groups were lower(P<0.05).The RA FLS apoptosis OD value of the combination 3 group increased,the OPG mRNA expression increased,the expression of RANKL mRNA decreased with statistical difference(P<0.05).The RA proliferation OD values of the SIN3 group and the MTX3 group increased when compared with the combination3 group(P<0.05).Conclusions SIN and MTX had synergistic effects in inhibiting FLS.This might be one of the mechanisms for inhibiting RA bone damage.