FTY720对聚乙烯颗粒诱导的破骨细胞前体细胞RAW264.7分化的影响  

Influence of FTY720 on differentiation of osteoclast precursor cells RAW264.7 induced by polyethylene particles

作　　者：周园东[1] 王信[1] 李平[1] 万睿[1] 陈婷梅[2] 张健[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,重庆400016 [2]重庆医科大学检验医学院,重庆400016

出　　处：《中国生物制品学杂志》2013年第1期67-71,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81071490)

摘　　要：目的观察FTY720对聚乙烯颗粒诱导的破骨细胞前体细胞RAW264.7分化的影响,探讨其防治人工关节无菌性松动的可能性。方法构建破骨细胞-成骨细胞(RAW264.7-MC3T3)小室共培养体系及破骨细胞-骨片体系,用FTY720干预受聚乙烯磨损颗粒刺激的RAW264.7细胞的分化,倒置显微镜观察分化细胞的形态;抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatas,TRAP)染色法对破骨细胞进行计数;扫描电镜观察破骨细胞的一般形态及骨吸收效应;ELISA法检测共培养体系中肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和白细胞介素-6(Lnterleuk-in-6,IL-6)的分泌水平;RT-PCR检测破骨细胞表面核因子κB受体活化子(Receptor activator of NFκB,RANK)和TRAP基因mRNA的转录水平。结果聚乙烯颗粒组RAW264.7细胞体积增大,胞质丰富,胞体边缘不齐呈云雾状,细胞核较多;FTY720组TRAP(+)细胞数明显低于聚乙烯颗粒组(P<0.01);破骨细胞呈圆形,细胞间可通过纤维样足突连接,骨片上有破骨细胞附着生长,FTY720组骨吸收陷窝和骨吸收面积均明显低于聚乙烯颗粒组(P<0.01);聚乙烯颗粒组TNF-α和IL-6的分泌水平均明显增加(P<0.01),FTY720组TNF-α和IL-6的分泌受到抑制;FTY720组破骨细胞表面TRAP和RANK基因mRNA的转录水平均明显低于颗粒组(P<0.01)。结论 FTY720能有效抑制破骨细胞前体细胞RAW264.7分化成熟,减少破骨细胞的形成及对骨片的溶解吸收,减少TNF-α、IL-6等炎性因子的分泌,下调RANK、TRAP等破骨细胞特异细胞表型和功能基因mRNA的转录水平,有望成为防治人工关节无菌性松动的药物。Objective To observe the influence of FTY720 on differentiation of osteoclast precursor cells（RAW264.7） induced by polyethylene particles,and investigate the possibility of prevention and treatment of aseptic loosening of artificial joint with FTY720.Methods Transwell Millipore co-culture model systems of osteoclasts（RAW264.7）-osteoblasts（MC3T3） and osteoclasts-bone piece were established,then FTY720 was used to intervene the differentiation of RAW264.7 cells irritated by polyethylene wearing particles,and the morphology of differentiated cells was observed under invert microscope.The osteoclasts were counted by tartrate-resistant acid phosphtas（TRAP） staining,and observed for general morphology and bone adsorption effect by scanning electron microscopy（SEM）.The secretion levels of tumor necrosis factor-α（TNF-α） and interleukin-6（IL-6） in co-culture system were determined by ELISA.The transcription levels of receptor activator of NFκB（RANK） and TRAP mRNAs were determined by RT-PCR.Results The RAW264.7 cells treated with polyethylene particles were large in size and rich in cytoplasm and nuclei,of which the border was irregular and in cloudy shape.The count of TRAP positive osteoclasts treated with FTY720 was significantly smaller than that with polyethylene particles（P 0.01）.The osteoclasts were round,which were connected each other by fibroid foot processes.SEM showed attachments of osteoclasts on bone pieces.As compared with those in polyethylene particle group,both the number of absorption trapped concave and area of bone absorption in FTY720 group were significantly small（P 0.01）.The secretions of TNF-α and IL-6 increased significantly in polyethylene particle group,while were inhibited in FTY720 group.Both the transcription levels of TRAP and RANK mRNAs on surface of osteoclasts in FTY720 group were significantly lower than those in polyethylene particle group（P 0.01）.Conclusion FTY720 effectively inhibited the differentiation of osteoclast precursor cells（RA

关 键 词：FTY720 破骨细胞 RAW264 7细胞 聚乙烯颗粒 共培养 

分 类 号：R392.5[医药卫生—免疫学] Q254[医药卫生—基础医学]