人软骨细胞培养上清诱导脐血间充质干细胞向软骨细胞分化

背景：人脐血间充质干细胞作为软骨缺损修复的种子细胞日益受到关注,现有常用诱导方法无论是应用诱导培养基还是诱导因子,因其价格昂贵、用量大等因素限制了实际应用。 目的：验证人软骨细胞培养上清诱导人脐血间充质干细胞向软骨细胞分化的可行性。 方法：利用密度梯度离心法和贴壁培养法分离新生儿脐血,获取并培养人脐血间充质干细胞,流式细胞仪鉴定细胞表面抗原。切取股骨头置换或全髋关节置换患者透明软骨组织,体外分离培养软骨细胞,利用其培养上清培养人脐血间充质干细胞,培养2周后观察细胞表型外观变化,免疫组化染色检测Ⅱ型胶原表达结果。 结果与结论：密度梯度离心法与贴壁培养法可以分离获取人脐血间充质干细胞,流式细胞仪鉴定表面标记CD90,CD105高表达,不表达CD34,CD45。软骨细胞培养上清诱导2周后,人脐血间充质干细胞向多角形、圆形转变,Ⅱ型胶原免疫组化检测表达阳性。提示软骨细胞培养上清可诱导人脐血间充质干细胞表达Ⅱ型胶原,向软骨细胞分化。

Differentiation of human umbilical cord blood derived-mesenchymal stem cells into chondrocytes induced by supernatants of human chondrocytes

BACKGROUND:Human umbilical cord blood derived-mesenchymal stem cells have been used as seed cells to repair cartilage defects. Their application has been greatly limited by high cost and sufficient amount although they could be cultured in induced medium or induction factors. OBJECTIVE:To verity the feasibility to induce human umbilical cord blood derived-mesenchymal stem cells to differentiate into the chondrocytes using supernatants of human chondrocytes. METHODS:Human umbilical cord blood derived-mesenchymal stem cells were separated with density gradient centrifugation and adherent method from human umbilical cord blood. Surface markers of human umbilical cord blood derived-mesenchymal stem cells were identified by flow cytometry. Human hyaline cartilage was dissected, digested, and chondrocytes were cultured in vitro. The human umbilical cord blood derived-mesenchymal stem cells were cultured with supernatant from chondrocytes. After 2 weeks, the changes of phenotype of the cells were identified, and type II collagen expression was detected using immunohistochemistry. RESULTS AND CONCLUSION:Human umbilical cord blood derived-mesenchymal stem cells were isolated from umbilical cord blood using density gradient centrifugation and adherent method. Flow cytometry demonstrated that the human umbilical cord blood derived-mesenchymal stem cells expressed CD90 and CD105 positively, failed to express CD34 or CD45. After 2 weeks of induction, human umbilical cord blood    derived-mesenchymal stem cells turned to polygon and round shaped. Immunohistochemistry staining showed some human umbilical cord blood derived-mesenchymal stem cells expressed type II collagen. Results indicate that culture medium of chondrocytes can induce human umbilical cord blood derived-mesenchymal stem cells to express type II collagen, and differentiate into the chondrocytes.