人骨髓间充质干细胞在兔角膜缘干细胞缺损动物模型中的分化

Study of the transdifferentiation of human mesenchymal stem cells in the surface of the cornea of a rabbit model with limbal stem cell deficiency

目的探讨人骨髓间充质干细胞(mesenchymal stem cells,MSCs)分化为角膜上皮细胞的可塑性及可行性。方法20只日本大耳白兔随机分为对照组和实验组,每组10只,右眼为实验眼。实验组:将人MSCs接种在人羊膜上培养4d后移植到兔角膜缘干细胞缺损的动物模型;对照组:仅采用羊膜移植到兔角膜缘干细胞缺损的动物模型。分别于移植后第1、第2、第3、第4和第6周,摘取各组眼球,分别行HE和PAS染色、单克隆抗体AE1和PCNA染色、透射电镜和扫描电镜检查。结果人MSCs接种到羊膜后能在羊膜上生长,与羊膜共培养4d后,人MSCs贴附羊膜生长迅速,组织学特征无明显改变。羊膜负载人MSCs移植到兔角膜基质表面,角膜表层细胞PAS染色呈阴性,AE1和PCNA染色呈阳性,具有典型的上皮细胞形态;而对照组PAS染色呈阳性,AE1和PCNA染色呈阴性。超微结构观察可见,实验组电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构,而对照组未见以上明显特征。结论羊膜负载人MSCs移植到兔眼表角膜基质后,MSCs能存活、增殖并向角膜上皮样细胞分化。

Objective To explore the plasticity and feasibility of the transdifferentiation of human mesenchymal stem cells （MSCs） into corneal epithelial cells.Methods Twenty Japanese white rabbits were randomly divided into 2 groups,10 rabbits for each group;the right eye was the experimental eye.Human MSCs combined with amniotic membrane were transplanted onto the eyes of the experimental animals,and the controls were transplanted with the amniotic membrane only.The MSCs were cultured on human amniotic membrane for 4 days, then the cells were transplanted onto the surface of the cornea of rabbits with limbal stem cell deficiency. The eyeballs were removed after 1, 2, 3, 4 and 6 weeks. The growth and differentiation of human MSCs were observed with HE and PAS staining, monoclonal antibodies with AE1 and PCNA staining, and scanning and transmission electron microscopy. Resuits When the human MSCs cultured on amniotic membrane were transplanted onto the surface of the cornea of the rabbits, the cells can adhere to human amniotic membrane and grow rapidly, showing no obvious change in histology. The cells of the corneal surface layer of the experimental group were negative to PAS staining and positive to AE1 and PCNA staining in the monoclonal antibodies, as revealed by immunohistochemical examination. In contrast, they were positive in PAS staining and negative in AE1 and PCNA staining in the controls. Compared to the control group, typical epithelium cells with large quantities of microvilli, desmosomes and tonofilaments were observed under the electron microscope in the experimental group. Conclusion The data suggests that when human amniotic membrane with MCSs is transplanted into the rabbit model with limbal stem cell deficiency, the MSCs can survive, proliferate and differentiate into corneal epithelium-like cells.