大鼠骨髓间充质干细胞在特定培养液条件下向软骨细胞表型定向分化的实验

背景组织工程化软骨的构建为软骨缺损的修复开辟了全新的途径,克服了传统治疗方法的不足.目的探讨分离骨髓间充质干细胞,在特定培养液条件下诱导其向软骨细胞表型转化的体外培养方法.设计完全随机实验.单位广西医科大学第一附属医院创伤骨科、手外科及广西医科大学组织学与胚胎学教研室.方法实验于2002-08/2003-04在广西医科大学完成.实验动物选择新生断奶SD大鼠20只.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间充质干细胞,在含体积分数0.15的胎牛血清的低糖DMEM培养液中,置于37℃,体积分数为0.05的CO2培养箱内培养10～14 d.传代后以体积分数0.15的胎牛血清的高糖DMEM培养液(含转化生长因子β110μg/L,地塞米松10-7mol/L,维生素C 50 mg/L)对其进行诱导培养.主要观察指标观测在体外特定培养条件下间充质干细胞的形态、生长特点和诱导培养后软骨特异性基质的表达情况.结果所有20只SD大鼠全部进行分析观察,无一例脱失.①分离获取了较高纯度的骨髓间充质干细胞,保持了细胞的活性.②骨髓间充质干细胞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,Ⅱ型胶原免疫组化阳性.④诱导培养后软骨特异性基质Ⅱ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骨髓间充质干细胞.②间充质干细胞在体外特定培养条件下能大量增殖,实现数量扩增.③间充质干细胞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理想的种子细胞.

Experimental study of the oriented differentiation of bone marrow derived mesenchymal stem cells into chondrogenic phenotype in a specific culture fluid

BACKGROUND:To construct tissue engineering cartilage would open up a novel way for the repair of cartilage damage in avoidance of the disadvantages of traditional therapeutic method.OBJECTIVE: To probe the techniques for the isolation of mesenchymal stem cells (MSCs) from bone marrow, as well as the in vitro differentiation into chondrocytic phenotype in a specific culture fluid.DESIGN:A complete randomized experimentSETTING:The Department of Traumatic Orthopedics and Hand Surgery of the First Affiliated Hospital of Guangxi Medical University, and Teaching and Research Faculty of Histology and Embryology of Guangxi Medical University.METHODS: The experiment was carried out at Guangxi Medical University between August 2002 and April 2003. Twenty SD neonatal weaning rats were selected. Bone marrow was aspirated from the bones of rat limbs and was isolated by gradient centrifugation in Percoll, and MSCs could be obtained in combination with adherent screening method, which were then cultured in DMEM-LG with 15% fatal bovine serum (FBS) in the incubator of 37℃ with 5% CO2 for 10-14 days. The passage cells were induced in DMEM-HG with 15% FBS (containing TGF-β1 10 μg/L, 10-7 mol/L dexamethasone, 50 mg/L VitC).MAIN OUTCOME MEASURES :The morphology, growth, as well as proliferation and specific expression of chondrogenic matrix of in vitro cultured MSCs due to specific induction.RESULTS: Totally 20 SD rats were observed and analyzed with no loss SCs grew in visible symmetric colonies, displaying a long-spindle shape,and the morphological characteristics of marrow-derived MSCs had no obvious changes during passage-culture, but its proliferation time was found from a shuttle fibroblastic appearance to polygonal shape, displaying posiHC staining of type Ⅱ collagen of cartilage specific matrix.bronectin adherent screening technique is a convenient, effective and practical method to separate and collect MSCs from rat bone marrows in chondrogenic phenotype when induced by a specific medium and can secrete cartilage specific matrix, and they can be the optimal seed cells for cartilage tissue engineering.