【作者】 丁晓飞；

【导师】 赵劲民； 杨志； 苏伟；

【作者基本信息】 广西医科大学 ， 骨外科， 2003， 硕士

【摘要】 目的：探讨研究分离骨髓间充质干细胞（MSCs）并诱导其向软骨细胞转化的体外培养方法，同时从组织形态学对MSCs生长特征进行观察，为软骨组织工程的种子细胞来源提供实验依据。 方法：抽取大鼠四肢骨髓，Percoll梯度分离液离心分离结合贴壁筛选法得到MSCs，在含15％FBS的低糖DMEM培养液中，置于37℃，5％CO2培养箱内培养10-14d。传代后以15％FBS的高糖DMEM培养液(含地塞米松10^-7mol／L，VitC50ug／ml)对其进行诱导培养。观测在体外培养条件下MSCs的形态、生长特点和诱导培养后软骨特异性基质的表达情况。 结果：1、分离获取了较高纯度的MSCs，保持了细胞的活性。2、MSCs原代培养呈均匀分布的集落样生长，呈长梭形；传代培养中形态特性无明显变化，细胞均质性明显提高。3、各代培养中碱性磷酸酶染色均呈阴性；诱导后的细胞由梭形向多角形、多边形转变，对甲苯胺蓝呈现强的异染性反应，11型胶原免疫组化阳性。 结论：1、采用Percoll梯度分离液离心分离结合贴壁筛选法可获得较高纯度和活性的MSCS，是简便有效、实用可行的方法。2、MSCS在体外培养条件下能大量增殖。通过离体培养，可使体内环境下低丰度的MSCS实现数量扩增。3、MSCS在特定培养液的诱导下能向软骨细胞表型转化，并能分泌软骨特异性基质，可成为软骨组织工程较理想的种子细胞来源。更多还原

【Abstract】 Objective: To explore a method of isolation, culture and chondrocytic phenotype differentiation of mesenchymal stem cells(MSCs) from the bone marrow of rats in vitro and to observe the biological characteristics of MSCs histomorphologically in order to offer experimental reference for the resources of seeding cells in cartilage tissue engineering.Methods: The bone marrow was aspirated from the bones of limbs of the rats and was isolated by gradient centrifugation in Percoll. The monouclear cells were collected and cultured in DMEM-LG with 15% fatal bovine serum(FBS). Cultures were maintained at 37 in humidified atmosphere chamber containing 5% C02 for10-14d. The higher purity of MSCs were obtained by the repeated removal of nonadherent cells. The passage cells were induced in DMEM-HG with 15% FBS, 10-7 mol/L dexamethasone, 50ug/ml VitC. The changes of morphology, growth and proliferation in vitro and expression of specific chondrogenic matrix after inducing were observed.Results: (1) MSCs, separated from the bone marrow of rats, were collected in higher purity by gradient centrifugation in Percoll and still kept the cells’ activity. (2) Primary MSCs proliferated in visible symmetric colonies with long-spindle shape. The morphological characteristics of marrow-derived MSCs had no change during passage, and its homogeneity rose with passage. (3) The cells had negative staining of ALP during the primary and passage culture. Induced cells changed from a spindle-like fibroblastic appearance to a polygonal shape and showed strong heterochromatic to toluidine blue and positivestaining of collagen II.Conclusion: (1) It is a simple, effective and practical method to separate and obtain higher purity and activity of MSCs from the bone marrow of rats by gradient centrifugation in Percoll and fibronectin ashesion. (2) MSCs that are in low abundance can grow quickly when culturedin vitro. (3) MSCs from the bone marrow of rats can differentiate to be chondrogenic phenotype when cultured in a defined medium. MSCs can likely be served as optimal autogenous cell source for cartilage tissue engineering.更多还原