兔BMSCs复合纤维蛋白胶在体外分化为角膜基质细胞的研究

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WestenBlot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。

Study on the Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells on fibrin glue into corneal stromal Cells in Vitro

Objective： To explore the feasibility of the differentiation of rat bone marrow mesenchymal stem cell （BMSCs） into corneal stromal cells in vitro and the feasibility of fibrin glue （FG） as the scaffold of cell sheet engineering. Methods： BMSCs was obtained by density gradient centrifugation. The cells were divided into three groups： differentiationto keratocyte phenotype was induced in pellet cultures with serum-flee; differentiation of BMSCs was induced in pellet cultures by co-cultured with corneal stromal cell; differentiation of BMSCs was induced in fibrin gels cultures by co-cultured with corneal stromal cell. Keratocyte-specific gene expression was characterized using western blotting. Morphological observations were performed with phpse contrast microscope. Results： BMSCs cul- tured in vitro showed great potential of proliferation. CD29 was positive, indicating that the cultured cells were MSC. Fibrin glue prepared was smooth and transparent with cell growth, after co-cultured with corneal strom al fibroblasts for one week, BMSCs expressed Keratocan, indicating that they had been transdifferentiated into corneal stromal cell. Conclusion： The BMSCs showed the tendency of induction from BMSCs into norneal stromal cell in exoteie-microenviroment. Fibrin glue was well compatible with corneal cells, which could con-struct tissue engineered cell with fibrin glue.