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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10～14d。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C50mg/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。
BACKGROUND: The construction of tissue-engineered cartilage has opened a new avenue for the repair of cartilage defects and overcame the deficiencies of traditional treatment methods. OBJECTIVE: To investigate the in vitro culture method of bone marrow mesenchymal stem cells (BMSCs) isolated from chondrocytes and induce their transformation into chondrocytes under the condition of specific culture medium. Design: Completely randomized experiment. SETTING: Department of Orthopedics and Traumatology, First Affiliated Hospital of Guangxi Medical University and Department of Histology and Embryology, Guangxi Medical University. Methods: The experiment was performed in Guangxi Medical University from August 2002 to April 2003. Twenty newborn weanling SD rats were selected as experimental animals. The bone marrow of the extremities of the rats was taken out, and the mesenchymal stem cells were obtained by centrifugation of Percoll gradient centrifugation and adherent screening. The medium was placed in a low glucose DMEM culture medium containing a fetal bovine serum with a volume fraction of 0.15 at 37 ° C. and a volume fraction of 0.05 CO2 Incubation incubator 10 ~ 14d. After passage, the culture medium was induced by high glucose DMEM (containing TGF-β110μg / L, dexamethasone 10-7mol / L, vitamin C50mg / L) with a volume fraction of 0.15% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological and growth characteristics of mesenchymal stem cells and the expression of cartilage-specific matrix after induction culture were observed in vitro. Results: All 20 SD rats were analyzed and observed without any loss. ① isolated higher purity of bone marrow-derived mesenchymal stem cells, maintaining cell activity. ② The primary culture of BMSCs showed uniform distribution of colony-like growth with long fusiform shape. There was no significant change in morphological characteristics of subcultured MSCs, shortened cell proliferation period and improved cell homogeneity. ③ The induced cells changed from fusiform to polygonal, and type Ⅱ collagen was positive for immunohistochemistry. ④ induced cartilage-specific matrix type Ⅱ collagen immunohistochemistry positive. Conclusion: ① Percoll gradient centrifugation combined with adherent screening method can be obtained with high purity and activity of bone marrow mesenchymal stem cells. ② mesenchymal stem cells in vitro culture conditions can be a large number of proliferation, to achieve the number of amplification. ③ The mesenchymal stem cells can transform to chondrocyte phenotype under the induction of specific culture medium and can secrete cartilage-specific matrix, which can be the ideal seed cells for cartilage tissue engineering.