兔骨髓间充质干细胞的体外培养、鉴定及诱导分化

Cultivation in Vitro,Identification and Induced Differentiation of Rabbits Bone Marrow Mesenchymal Stem Cells

目的建立兔骨髓间充质干细胞的体外分离、培养、鉴定方法。方法通过全骨髓贴壁法体外分离、培养、纯化兔骨髓间充质干细胞,进行形态学观察,绘制生长曲线,检测细胞表面标志物,加入诱导剂诱导细胞向成骨、成脂方向分化,油红O染色,检测细胞内碱性磷酸酶(ALT)、甘油三酯(TG)含量。结果兔骨髓间充质干细胞符合正常细胞生长特性,形状以梭形、纺锤形为主,后期多数细胞呈长梭形;细胞生长较旺盛,可连续传7~10代,以第3代细胞状态最佳;第3代骨髓间充质干细胞CD29表达呈阳性,CD34表达呈阴性;成骨、成脂诱导后,油红O染色出现明显脂滴,ALT、TG含量明显增高。结论全骨髓贴壁法可成功分离和培养出兔骨髓间充质干细胞,具有间充质干细胞的一般生物学特性,且生长状态良好,增殖力强,经诱导后具有向成骨和成脂分化的潜能,该方法获得的骨髓间充质干细胞可能成为组织工程的优良种子细胞。

Objective To establish a method for the isolation,cultivation in vitro,and identification of rabbits bone marrow mesenchymal stem cells(RBMSCs).Methods RBMSCs were isolated,cultured and purified by a whole bone marrow adherence.The morphological changes of cells were observed,curve of growth was drawed,and surface markers were detected.The ossific and adipogenic differentiation of cells were induced,then the cells were treated with oil red O stain,and the triglyceride(TG) content and alkaline phosphatase(ALP) activity in the cells were detected.Results RBMSCs had the growth characteristics of normal cells,fusiform shape or spindle-shaped was dominant,the cells grew well,and could be serially passaged up to 7-10 generations.The growth state of 3rd generation of RBMSCs was the best.The CD29expression in RBMSCs was positive,the CD34expression was negative,some lipid droplets appeared in the cells after treated with oil red O stain,and TG content,ALP activity was increased respectively.Conclusion A whole bone marrow adherence could isolate and cultivate RBMSCs successfully,the cells have common biological characteristics of mesenchymal stem cells,the growth state and proliferative ability of cells were good,and the ossific and adipogenic differentiation of cells could be induced.RBMSCs acquired by the method may be served as optimal cell source for study on osteogenic tissue engineering.