兔骨髓间充质干细胞的体外分离、培养及诱导分化

Isolation，cultivation and differentiation of rabbit bone marrow mesenchymal stem cells in vitro

摘要:

目的：研究兔骨髓间充质干细胞（Rabbit bone marrow mesenchymal stem cells，rBMSCs）的体外分离培养，观察其生长特性并探讨其定向诱导分化为成骨细胞和成脂肪细胞的途径，为种子细胞的选择提供一定的实验基础。方法：乌拉坦麻醉下耳缘静脉空气栓塞处死兔子，无菌条件下取出兔股骨，用percoll液密度梯度离心法+全骨髓贴壁培养法相结合进行体外培养、传代，倒置显微镜下观察细胞形态。取第3代细胞向成骨细胞和成脂肪细胞诱导，14 d后成骨细胞诱导组检测碱性磷酸酶，成脂肪细胞诱导组进行油红O染色。结果：percoll密度梯度离心法＋全骨髓贴壁培养法可成功获得rBMSCs，具有活跃的增殖能力。第3代细胞的生物学特征基本一致，并能定向诱导分化为成骨细胞及成脂细胞。成骨细胞诱导组碱性磷酸酶检测表达呈强阳性，成脂肪细胞诱导组油红O染色见胞浆内出现大量红染脂滴。结论：percoll密度梯度离心法+全骨髓贴壁培养法可成功分离和培养rBMSCs，其生长稳定，增殖力强，能定向诱导分化为成骨细胞及成脂细胞，可见rBMSCs是组织工程的优良种子细胞。

Abstract:

Objective：To investigate the isolation and cultivation of rabbit bone marrow mesenchymal stem cells（rBMSCs） in vitro，to observe their biological characteristics and to discuss the paths of inducing the rBMSCs into osteoblasts and lipoblasts in order to pro－vide experimental basis for selecting seed cells. Methods：The rabbit was put to death by air embolism to the ear marginal vein with the anesthesia of urethane. The femur of the rabbits was got under the sterilized condition. rBMSCs were separated by percoll den－sity gradient centrifugation method and the whole bone marrow adherence culture method in vitro. The morphology of cells was ob－served by inverted microscope. At the third passage，rBMSCs were chosen to be induced into osteoblasts and lipoblasts. After 14 d of incubation，alkaline phosphatase detection was used in osteoblast-induced group and oil red O staining was performed in lipoblast-induced group. Results：The rBMSCs with good reproductive activities were obtained successfully by percoll density gradient lentrifu－gation and the whole bone marrow adherence culture method. The biological characteristics of the cells in the third generation were i－dentical. In osteoblast-induced group，alkaline phosphatase test showed strong positive expression. After oil red O staining，there was a large number of intracytoplasmic lipid droplets stained red in lipoblast-induced group. Conclusions：rBMSCs can be isolat－ed and proliferated effectively by percoll density gradient cen－trifugation method and the whole bone marrow adherence culture method.The cultured rBMSCs grow up and reproduce stably and could be induced and differentiated into osteoblasts and lipoblasts. So rBMSCs are excellent seed cells in tissue engineering.