体外诱导人骨髓间充质干细胞向角膜上皮细胞分化的初步研究  

Study on differentiation of human mesenchymal stem cells into corneal epithelial cells in vitro

作　　者：顾绍峰[1] 付小兵[2] 洪晶[1] 

机构地区：[1]北京大学第三医院北京大学眼科中心,100083 [2]解放军304医院全军烧伤研究所基础部,北京100039

出　　处：《眼科研究》2008年第7期512-516,共5页Chinese Ophthalmic Research

基　　金：国家自然科学基金项目(30471865,30670816);沈阳市科学技术计划重点项目(1053111-3-07)资助

摘　　要：目的探讨在体外损伤微环境下,人骨髓间充质干细胞(MSCs)分化为角膜上皮细胞的可行性。方法体外分离培养人MSCs和人角膜缘干细胞(LSCs),检测细胞CD34、CD44、细胞角蛋白3(CK3)、细胞角蛋白19(CK19)的表达情况。制作人LSCs热损伤模型,加入增强型绿色荧光蛋白基因(EGFP)标记的人MSCs共同培养,2周后检测细胞CK19的表达情况。结果人MSCsCD44表达阳性、CD34表达阴性,流式细胞术显示CD44阳性率为92.59%,CD34阳性率为0.01%;人LSCsCK19表达阳性,CK3表达阴性。共同培养后部分人MSCsCK19表达阳性,并有多核细胞现象。结论在损伤微环境下,人MSCs可以转化为类角膜上皮细胞的细胞,在此过程中存在细胞融合现象。Objective The maintain of the corneal epithelial cell mass is achieved by limbal stem cells （LSCs）. The partial or whole deficiency of the LSCs population may greatly affect the ocular surface integrity and stability. There are still no reliable therapies for severe forms of LSCs dysfunction. It is important to test the suitability of other stem cell lines for the reconstruction of a stem cell-deficient ocular surface. This study was curried out to investigate the differentiation of human mesenchymal stem cells （MSCs） into human corneal epithelial cells in vitro. Methods Human MSCs and human limbal stem cells（LSCs） were isolated from healthy adults and cultured respectively. CD34 and CD44 were used to identify the human MSCs by immunocytochemistry and flow cytometry. Cytokeratins 3 （CK3） , cytokeratinl9 （CK19） were used to identify human LSCs by immunocytochemistry. Human MSCs were transfected .with gene encoding enhanced green fluorescent protein （EGFP） by a retroviral vector. Then a heat-shocked model of human LSCs in vitro was developed and cocultured with human MSCs. The differentiating status of cultured human MSCs and human LSCs were assessed under the fluorescence microscope and immunocytochemistry of CK19. Results The cultured human MSCs showed clonogenic-like cells in shape and were positive re,active for CD44 and absent reaction for CD34. Approximately 92.59% cultured cells showed positive staining for CD44 and 0.01% for CD34. Human LSCs exhibited positive staining for CK19 and absent staining for CK3. Human MSCs transfected by EGFP exhibited strong green fluorescence. After cocultured, a part of human MSCs were positive of CK19 by immunocytochemistry staining,and phenomenon of cell fusion was observed. Conclusion Human MSCs could differentiate into corneal epitheliallike cells in vitro under the microenvironment of injury,and cell fusion is presented during this procedure.

关 键 词：人骨髓间充质干细胞 人角膜缘干细胞 转化 细胞融合 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]