人骨髓间充质干细胞体外诱导分化为角膜缘干细胞的研究

目的探讨体外诱导人骨髓间充质干细胞（human bone mesenchymal stem cells,HBMSCs）分化为角膜缘干细胞（limbal stem cells,LSCs）的可行性。方法用密度梯度离心法体外分离HBMSCs,用消化后组织块培养法分离培养LSCs,培养传代后用流式细胞仪检测细胞的相关抗原。将第4次传代的HBMSCs分成2组：实验组细胞与第2次传代的LSCs共培养于Transwell培养板,加入条件培养基;对照组不加任何诱导剂,以原HBMSCs培养基培养。培养10d,在相差显微镜下进行形态学观察。结果实验组共同培养72h后,贴壁HBMSCs部分呈椭圆形、圆形改变。在体外微环境中诱导10d后大部分细胞呈多边形、圆形、椭圆形,少部分细胞呈梭形。实验组P63弱阳性（7.16±0.56）%,对照组P63阴性（0.74±0.49）%。结论在体外特殊的微环境下,HBMSCs可被其诱导分化为LSCs细胞。

Study on Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Limbal Stem Cells in Vitro

Objective To explore the feasibility of differentiation of human bone marrow mesenchymal stem cell into limbal stem cells in vitro. Methods Human bone marrow derived mesenchymal stem cells were isolated by gradient density centrifugation limbal stem cells were harvested by tissue cultivation after digestion. Then they were cultured and passage. Cell antigen were detected by flow cytometry. Subcultured HBMSCs of the 4th passage were divide into tow groups in the second passage. In the experiment group, HBMSCs and subcultured LSCs of passage 2rd were cocultured in Transwell, using a special medium. On the other hand, none of inductive agents were added in the control group, BMMSCs were cultured in the medium contained of 90% LGDMEM/F12 and 10% FBS. Morphological observations were performed with phase contrast microscope. Results In the experiment group, Part MSCs were shown in ellipse, round and part were fusiform after cocultured for 72 hours. When the induction of HBMSC in exoteric-microen viroment for 10 days, cells were polygon and round ellipse in bulk, and only a small part were fusiform. Flow cytometry analysis show that P63 was weakly positive （7.16±0.56）% in the control group. Cells were fusiform in bulk, and P63 were negative（0.74±0.49） %. Conclusion The HBMSC shows the tendency of induction from HBMSCs into LSCs-like cells in exoteric-microenviroment with LSCs by the intervention of LPS and cytokines.