类风湿关节炎患者外周血单核细胞诱导转分化为破骨样细胞  

Culture of osteoclast-like cell obtained from peripheral blood mononuclear cells(PBMCs) of patients with rheumatoid arthritis in vitro

作　　者：霍晓聪[1] 张浩[1] 季迎[1] 刘小贤[1] 易斌[1] 刘纪实[1] 

机构地区：[1]中南大学湘雅三医院肾内科,湖南长沙410013

出　　处：《中国现代医学杂志》2010年第7期1063-1066,1072,共5页China Journal of Modern Medicine

摘　　要：目的建立类风湿关节炎(RA)患者外周血单核细胞(PBMCs)转分化的培养体系,并对所培养的破骨样细胞(OLC)进行鉴定。方法抽取RA患者外周静脉血,密度梯度离心法获得PBMCs,分别接种于玻片和骨磨片。随机分为单核细胞集落刺激因子(M-CSF)组(A组),加入25μg/LM-CSF;协同作用组(B组),分别加入不同浓度的核因子κB受体活化子配体(RANKL),5、10、20、25、50μg/L)和25μg/LM-CSF。培养至4、12和20d时,取玻片行HE染色和抗酒石酸磷酸酶染色计数OLC,取骨磨片行甲苯胺兰染色检测OLC骨吸收活性。结果对OLC转分化的影响:各组细胞分别于4、12、20d染色,显示RANKL呈浓度及时间依赖性诱导OLC形成(P<0.05)。对OLC骨吸收活性的影响:RANKL和M-CSF协同诱导骨磨片上吸收陷窝的形成,RANKL呈浓度及时间依赖性增强OLC的吸收活性(P<0.05);单独M-CSF刺激不能形成骨吸收陷窝。结论RANKL和M-CSF可协同诱导RA患者PBMCs转分化为OLC,该培养方法稳定、有效,具有可重复性。【Objective】Establish the system of osteoclast-like cell（OLC） cultural in vitro and appraise the cultured cells were osteoclasts.【Methods】PBMCs were isolated by means of density gradient centrifugation from RA patients and were randomly assigned to 2 groups：group A（macrophage-colony stimulating factor,M-CSF,25 μg/L）,group B（Receptor activator of NF-κB ligand,RANKL,5,10,20,25,50 μg/L,and 25 μg/L M-CSF）.After cultured for 4,12,20 days,the cells on glass coverships were fixed and stained for tartrate resistant acid phosphatase,the bone slices were identified with toluidine blue stain.【Result】 The effect on the differentiation of OLC：RANKL induced a dose-dependent increase in TRAP positive OLC（P0.05）.The effect on the activation of OLC：RANKL and M-CSF induced a dose-dependent increase in the number of bone resorptive lacuna on bone slices（P0.05）,but we can not see bone resorptive lacuna on bone slices in group A.【Conclusion】RANKL and M-CSF can induce the differentiation and activation of osteoclast derived from PBMCs of patient with rheumatoid arthritis,this cultural method is convenient,potent and repretitive for OLC formation in vitro.

关 键 词：类风湿关节炎 破骨样细胞 核因子κB受体活化子配体 

分 类 号：R593.22[医药卫生—内科学]