兔骨髓基质干细胞体外分离培养及生物学特性研究

目的：研究兔骨髓基质干细胞（bone marrow-derived mesenchymal stem cells,BMSC）体外分离、诱导培养及增殖特点,探讨其生物学特性。方法：抽取兔骨髓,密度梯度离心结合贴壁培养法分离BMSC,诱导培养液定向诱导传代细胞向成骨细胞分化。倒置显微镜下观察细胞生长及增殖情况,流式细胞仪检测细胞表面标志物的表达,免疫组织化学观察Ⅰ型胶原表达,并检测碱性磷酸酶活性及细胞矿化作用。结果：体外培养的兔BMSC贴壁生长,10～14天后形成克隆。细胞形态为均一的长梭形并呈漩涡状排列,流式细胞仪检测CD34,CD45阴性,CD44阳性。免疫组化可检测到Ⅰ型胶原表达,碱性磷酸酶活性明显增高,并且出现矿化结节。结论：密度梯度离心结合贴壁培养BMSC,操作简单,细胞易于成活,诱导条件下成骨能力肯定,适合作为骨组织工程的种子细胞。

Study of the cultured rabbit bone marrow-derived mesenchymal stem cells in vitro and biological characteristics

Objective To study the method of isolation,culture and proliferation of rabbit bone marrow mesenchymal stem cells（BMSC） and observe the main biological characteristics in vitro.Methods Bone marrow cells were derived from rabbit iliac bone and isolated and purified by density gradient centrifugation combined with attachment culture method.The cells were cultured with ordinary culture medium and induced by conditional culture medium.The BMSC morphologic features and growth status were observed under an inverted microscope.The BMSC were identified with HE staining and CD34,CD45/CD44.The biological activity of BMSC were examined by immunohistochemical staining,Von-Kossa staining,ALP staining.Results The BMSC of rabbit attached to the surface of plate and colony formation was seen in 10～14d.The BMSC was big and round exhibiting a fibroblast-like spindle shape and CD34,CD45 negative,CD44-positive by flow cytometry.All showed that the cells have the characteristics and the activity of the osteoblasts by immunohistochemical staining,Von-Kossa staining,ALP staining.Conclusions The method of density gradient centrifugation combined with attachment culture is simple in the culture of BMSC,and the cell is likely to survive.BMSC can be induced to differentiate into osteoblasts in conditional culture medium in vitro BMSC can be optimal seed cell source for bone tissue engineering.