Toll 样受体4在成骨细胞共培养体系中对干细胞向破骨细胞分化的影响及机制

摘要

目的：探索 Toll 样受体（Toll like receptor，TLR）-4在成骨细胞共培养体系中对干细胞向破骨细胞分化的影响及机制。方法：Percoll 密度梯度离心法分离原代大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BM-SCs），流式细胞术鉴定细胞表面标志物 CD34、CD44、CD54、CD90。用 Transwell 小室建立干细胞（上室）成骨细胞（下室）共培养体系，在此体系下对 BMSCs 进行破骨细胞诱导培养。将共培养体系分为 TLR-4激活组和空白组，前者以TLR-4激动剂 LPS（10 ng／mL）激活，后者加入等体积 PBS。在诱导培养第6天，对上室细胞行抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）染色，计数并比较各组破骨细胞数。同时，蛋白质印迹检测各组下室细胞悬液中核因子κB 受体活化因子配体（receptor activator for nuclear factor-κB ligand，RANKL）的蛋白表达，观察电泳条带并行定量分析。结果：密度离心法获得原代大鼠 BMSCs，流式细胞分析检测显示 CD34阴性、CD44、CD54和 CD90阳性。TRAP 染色阳性细胞为破骨细胞，细胞计数显示，TLR-4激活组破骨细胞数明显多于空白组（t ＝13．556，P ＜0．05）。蛋白质印迹结果显示，TLR-4激活组 RANKL 的表达明显多于空白组（t ＝17．630，P ＜0．05）。结论：在成骨细胞共培养体系下，激活 TLR-4能够促进 BMSCs 向破骨细胞分化，其机制可能与 TLR-4促进成骨细胞分泌 RANKL有关。%Objective:To investigate the effect of Toll like receptor (TLR)-4 to osteoclastgenesis of stem cells to osteoclasts in co-culture system with osteoblast,and to research the probably mechanism. Methods:Bone marrow mesenchymal stem cells (BMSCs)of rat were abstracted with percoll density gradi-ent centrifugation method.Surface maker CD34,CD44,CD54,and CD90 were identified with flow cytome-try.BMSCs (upper cell)-osteoblasts (lower cell)co-culture system was established with Transwell cells, BMSCs were induced to osteoblasts in this system.Co-culture system were divided into TLR-4 activated group and blank group.Cells in TLR-4 activated group were activated by LPS (1 0 ng/mL),cells in blank group were treated with PBS of the same volume.After 6 days culture,tartrate resistant acid phosphatase (TRAP)staining was performed to examine osteoclasts in the lower cell.Osteoclasts numbers in each group were counted and compared.Meanwhile,western blotting was performed to investigate the expression of re-ceptor activator for nuclear factor-κB ligand (RANKL).Results:BMSCs of rat were abstracted with densi-ty gradient centrifugation method.Surface maker CD34 was negative,while CD44,CD54,and CD90 were positive.TRAP staining positive cell was osteoclasts.Osteoclasts numbers in each group were counted,it was obviously higher in TLR-4 activated group than that in blank group (t =1 3.556,P <0.05).Results of western blotting showed RANKL expression in TLR-4 activated group was higher than that in blank group(t=1 7.630,P <0.05).Conclusion:In “BMSCs-osteoblasts”co-cultured system,activation of TLR-4 is benefit to the osteoclastgenesis of stem cells.The promotion of TLR-4 to the secretion of RANKL may be the mechanism.