雷公藤多苷对rmMIF诱导大鼠成纤维样滑膜细胞增殖及RANKL／OPG表达的影响

目的观察雷公藤多苷（tripterygiumglycoside，TG）对重组鼠巨噬细胞移动抑制因子（recombinantmousemacrophagemigrationinhibitoryfactor，rmMIF）诱导大鼠成纤维样滑膜细胞（fibroblast—likesynoviocytes，FLS）的增殖及其细胞核因子-KB受体活化因子配体（receptoractivatorofnuclearfactorkappaBligand，RANKL）、骨保护素（osteo—protegerin，OPG）表达的干预作用。方法采用大鼠FLS细胞株RSC-364，常规方法复苏、培养、传代，实验用3～5代FLS。分为4组：空白对照组加入200μlDMEM培养液，另3组分别加入200肛1含rmMIF（200ng／m1）（MIF组）、rm—MIF（200ng／m1）＋TG（20μg／m1）（MIF＋TG组）、rmMIF（200ng／m1）＋MTX（0．5μg／m1）（MIF＋MTX组）的DMEM培养液。置37℃、5％CO2孵箱培养48h。MTT法检测FLS的增殖活性；免疫组化法检测滑膜细胞OPG／RANKL的表达；酶联免疫吸附试验（ELISA）检测FLS上清液OPG、RANKL表达。结果MIF组增殖活性高于空白对照组，MIF＋TG组、MIF＋MTX组增殖活性低于空白对照组和MIF组（P〈0．01）。MIF＋TG组和MIF＋MTX组细胞OPG标记指数均高于空白对照组和MIF组，MIF＋TG组OPG标记指数高于MIF＋MTX组（P〈0．01，P〈0．05）。MIF组细胞RANKL标记指数高于空白对照组，MIF＋TG组、MIF＋MTX组RANKL标记指数均低于MIF组（P〈0．05，P〈0．01）。MIF组上清液RANKL浓度和RANKL／OPG值均高于空白对照组，MIF＋MTX组上清液RANKL浓度低于空白对照组（P〈0．05，P〈0．01）；MIF＋TG组和MIF＋MTX组上清液RANKL浓度和RANKL／OPG值均低于MIF组（P〈0．01）。结论rmMIF可促进体外培养大鼠FLS增殖，上调FLS的RANKL表达和RANKL／OPG值；TG抑制rmMIF诱导的大鼠FLS增殖、降低rmMIF诱导大鼠FLS分泌RANKL、下调FLS的RANKL表达和RANKL／OPG比值，可能是其治疗类风湿关节炎的骨免疫学机制之一。

Effects of Tripterygium Glycosides on Proliferation and the Expressions of RANKL/OPG in Rat's Fibroblast-Like Synoviocytes Induced by rmMIF

Objective To observe the interventional effect of Tripterygium glycosides （TG） on proliferation of fi- broblast-like synoviocytes （FLS） and expressions of nuclear factor-KB receptor ligand （RANKL） and osteoprotegerin （OPG） induced by maerophage migration inhibition factor （rmMIF）. Methods Recovery, culture and subculture were performed with the rat~ FLS （3-5 generation FLS） cell line RSC-364, which were divided into 4 groups： control group was added with 200 μl DMEM culture solution, MIF group with 200 μl containing rmMlF （200 ng/ml） , MIF ＋ TG group with rmMIF （200 ng/ml） and TG （20 μg/ml）, and MIF ＋ MTX group with rmMIF （200 ng/ml） and MTX （0.5 μ/ml） cultured in DMEM. All above cell strains were cultured in incubator at 37℃ and 5% CO2 for 48h. FLS proliferation was detected by MTT method; immunohistochemistr/method was used to detect the expressions of OPG and RANKL in synoviocytes; enzyme-linked immunosorbent test （ELISA） method was used to detect the expressions of OPG and RANKL in FLS supernatant. Results FLS proliferative activity of MIF group was higher than that of control group, and proliferative activity of MIF ＋ TG group and MIF ＋ MTX group were lower than those of MIF group and control group （ P 〈 0. 01 ）. OPG expressions （ OPG labeling index） of MIF ＋ TG group and MIF ＋ MTX group were higher than those of control group and MIF group, and OPG expression of MIF ＋ TG group was higher than that of MIF ＋ MTX group （ P 〈 0. 01, P 〈 0. 05 ）. RANKL labeling index of MIF group was higher than that of control group, and RANKL labeling index of MIF ＋ TG group and MIF ＋ MTX group were significantly lower than that of MIF group （P 〈 0. 05,P 〈 0. 01 ）. The RANKL/OPG concentration in supernatant of MIF group was significantly higher than that of control group, while the concentration of MIF ＋ MTX group was lower than that of control group （P 〈 0.05,P 〈 0. Ol ） ; and the concentrations and RANKL/OPG values of MIF ＋ TG group and MIF ＋ MTX group were lower than those of MIF group （ P 〈0. O1 ）. Conclusion rmMIF may promote the proliferation of cultured rat＇s FLS in vitro, and increased expressions of RANKL and RANKL/OPG values in FLS; TG may inhibit the proliferation of FLS induced by rmMIF, reduce the secretion of RANKL of FLS by rmMIF in rats, down regulate the expressions of RANKL and RANKL/OPG ratio, which may be one of osteoimmunologic mechanisms in treatment of rheumatoid arthritis.