人骨髓间充质干细胞与成骨细胞共培养诱导破骨细胞方法的建立和鉴定

目的：建立人骨髓间充质干细胞（hMSCs）与人成骨细胞共培养体系，为人破骨细胞体外培养提供更完善的方法。方法：采用酶消化法分离流产胎儿头盖骨，获取人成骨细胞，对在体外培养的成骨细胞进行碱性磷酸酶染色（ALP染色）；利用Percoll 梯度分离法和贴壁筛选法培养hMSCs,应用流式细胞术检测hMSCs 免疫学表型，对其进行鉴定；将获得的成骨细胞和hMSCs在体外共培养作为实验组，单独培养的成骨细胞作为对照组；对诱导后的细胞应用抗酒石酸酸性磷酸酶染色（TRAP）进行鉴定。结果：体外分离、培养的hMSCs具有独特的细胞免疫学表型, 即CD73和CD105阳性, CD34和CD45 阴性。hMSCs与成骨细胞共同培养5 d后,即可见单个核细胞融合成多核细胞，TRAP 染色可见多数细胞胞浆呈酒红色，为诱导成功的破骨细胞。结论：hMSCs与人成骨细胞共同培养能诱导为具有典型的破骨细胞表型的多核细胞。

Establishment of method to induce osteoclasts through co-culture of human bone marrow mesenchymal stem cells and osteoblasts

Objective To establish a co-culture system of human bone marrow mesenchymal stem cells(hMSCs)and osteoblasts to get human osteoclasts.Methods The osteoblasts were obtained with collagenase and the alkaline phosphatase staining was used to test the form of osteoblasts;hMSCs were isolated,cultivated and proliferated by Percoll gradient centrifugation.Co-culture hMSCs and osteoblasts were cultivated together,and osteoclasts were identified with tartrate-resistant acid phosphatase(TRAP)staining.Results High homogenous hMSCs were obtained and after co-culture for 5 d with osteoblasts,they fused to form multinuclear cell clusters in micrograph and were postive in TRAP staining.Conclusion The co-culture system method is successful.With this method osteoclasts can be obtained from co-culture of hMSCs and osteoblasts.