摘要
目的 :探讨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 ,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 1 5 % FBS的低糖DMEM培养液中 ,置于 37℃ ,5 % CO2 培养箱内培养 1 0～ 1 4 d。传代后以 1 5 % FBS的高糖 DMEM培养液 (含 TGF-β1 1 0μg/L,地塞米松 1 0 - 7mol/L,维生素 C5 0 mg/L)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 ,细胞增殖周期缩短 ,细胞均质性明显提高 ;3诱导后的细胞由梭形向多角形转变 , 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;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 ;3MSCs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 。
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 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% CO 2 for 10～14 d. 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, TGF β 1 10ng/ml, 10 -7 mol/L dexamethasone, 50 μg/ml VitC. The changes of morphology, growth and proliferation in vitro and expression of specific chondrogenic matrix after inducing were observed.Result:①MSCs, separated from the bone marrow of rats, were collected in higher purity by gradient centrifugation in Percoll and still kept the cells' activity;②Primary 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 and the proliferation time reduced;③Induced cells changed from a spindle like fibroblastic appearance to a polygonal shape and showed positive staining of collagen Ⅱ.Conclusion:①It 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;②MSCs that are in low abundance can grow quickly when cultured in vitro;③MSCs 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.
出处
《广西医科大学学报》
CAS
2004年第2期179-182,共4页
Journal of Guangxi Medical University
