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目的:探讨体外诱导兔骨髓间充质干细胞(BMSCs)分化为角膜基质细胞的可行性,并观察纤维蛋白胶(FG)作为细胞支架材料的效果。方法:密度梯度法获得BMSCs,体外诱导实验将细胞分为三组:对照组用普通培养皿、BMSCs培养条件并不加角膜基质细胞共培养的条件下培养;非FG共培养组使用普通培养皿并与角膜基质细胞共培养诱导BMSCs分化;FG共培养组使用铺有FG的培养皿并与角膜基质细胞共培养诱导BMSCs分化。培养1w及2w后用Westen Blot法检测三组细胞Keratocan的表达,在相差显微镜下进行形态学观察。结果:原代培养的BMSCs表现出成体干细胞潜能,CD29染色阳性,符合骨髓基质干细胞的特征。诱导培养2周后对照组BMSCs融合成单层、呈条索状生长;非FG共培养组部分细胞体积变小、多突起,局部呈梭形生长;FG共培养组细胞生长状态良好,部分细胞呈梭形或纺锤形,与FG生物相容性好。Westen检测结果:BMSCs细胞在纤维蛋白胶或普通培养皿上特定培养条件下均能诱导表达角膜基质细胞的特异性蛋白Keratocan。结论:骨髓间充质干细胞在条件培养基下可分化为角膜基质细胞,有望作为治疗角膜疾病及角膜组织工程的备选材料,纤维蛋白胶组织相容性好,可为组织工程提供移植细胞片。
Objective: To investigate the feasibility of differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) into corneal stromal cells in vitro and to observe the effect of fibrin glue (FG) as a scaffold material. Methods: BMSCs were obtained by density gradient method. The cells were divided into three groups by in vitro induction experiment: the control group was cultured with ordinary culture dish and BMSCs culture condition without the co-culture of corneal stromal cells; Non-FG co-culture group with ordinary culture dish And co-cultured with corneal stromal cells to induce BMSCs differentiation; FG co-culture group using FG-plated Petri dishes and co-culture with corneal stromal cells induced BMSCs differentiation. After cultured for 1 and 2 weeks, Westen Blot method was used to detect the expression of Keratocan in the three groups of cells, and morphological observation was performed under a phase contrast microscope. Results: Primary cultured BMSCs showed the potential of adult stem cells and positive for CD29, which was in line with the characteristics of bone marrow stromal stem cells. After 2 weeks of induction, the BMSCs in the control group were fused into a single layer and grew in the form of a cord. Part of the cells in the non-FG co-culture group became smaller in volume, more in the process of protrusion and locally in a fusiform shape. The cells in the FG co-culture group grew well and some cells Fusiform or spindle-shaped, biocompatible with FG. Westen test results: BMSCs can induce keratocan, a specific protein expressing corneal stroma cells, under specific culture conditions on fibrin glue or ordinary culture dish. CONCLUSION: Bone marrow mesenchymal stem cells can differentiate into corneal stromal cells under the condition of medium, which is expected to be an alternative material for the treatment of corneal diseases and corneal tissue engineering. Fibrin glue has good histocompatibility and can be used as explants for tissue engineering .