人骨髓间充质干细胞体外分化为结膜上皮样细胞  

Differentiation of human bone marrow mesenchymal stem calls into conjunctival epithelium-like cells in vitro

作　　者：陈国玲[1] 刘执玉[2] 

机构地区：[1]山东大学眼科中心/第二医院眼科,济南250033 [2]山东大学医学院人体解剖与组织胚胎学研究所,济南250012

出　　处：《解剖学报》2012年第1期34-37,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30872523);山东省优秀中青年科学家科研奖励基金资助项目(2008BS03035)

摘　　要：目的探讨体外诱导人骨髓基质干细胞(hMSCs)向结膜上皮样细胞(hCjEs)定向分化的可能性。方法体外分离、培养hMSCs和人hCjEs,分别从细胞形态学和免疫细胞化学的方法对细胞进行鉴定;Transwell非接触共培养法诱导hMSCs向结膜上皮样细胞定向分化;倒置相差显微镜观察分化细胞的形态学改变;免疫细胞化学和反转录聚合酶链反应分别对分化细胞角蛋白13(CK13)的蛋白和mRNA表达进行检测;并用单独培养的hMSCs和hCjEs做对照。结果实验获取的hMSCs以长梭形为主,融合呈漩涡状;hCjEs呈不规则圆形或多边形,融合的hCjEs成铺路石样;hMSCs阳性表达CD44和CD29,hCjEs阳性表达CK13;非接触共培养使hMSCs的形状由长梭形逐渐变为不规则多边形;免疫细胞化学和RT-PCR分别在蛋白和基因水平证实分化细胞表达CK13,与单独培养的结膜上皮细胞表达相似。结论在一定条件下,可以诱导体外培养的hMSCs形态发生变化、向具有结膜上皮细胞表型的上皮样细胞定向分化。Objective To investigate whether human bone marrow stroma stem cells（hMSCs） differentiate into conjunctival epithelium-like cells. Methods The hMSCs and human conjuoctival epithelial cells（hCjEs） were isolaed and cultured. The morphological characteristics of the hMSCs and hCjEs were observed under a phase contrast mic.roscope, amt the cells were identified according to their morphology and cell-surface antigen profiles by immunoc＋ytnchemieal analysis. The hMSCs were co-cultured with hCjEs in Trans-well. The differentiated cells were identified according to their morphological characteristics,and the expression of cytokeratinl3 （CKI3） protein was detected by imnmnocytochemistry. The abundance of the mRNAs for CK13 in hCjEs, differentiated cells and hMSCs without co-cultured with hCjEs were determined by reverse transcriptasepolymerase chain reaction （RT-PCR）. Results The hMSCs showed spindle-like morphology and the adherent cells showed whirlpool-like shape. The hCjEs were shown in shapc of irregular round or polygon, and the adherent cells showed paving stones-like shape. The results of immunocytochemistry showcd that there was positive expression of CD44 and CD29 in hMSCs. The hCjEs had positive expression of CKI3. After induced by conjunctival epithelium, the shape of differentiated cells became wide and irregularly round accompanied by losing its spindle-shape. The results of immunocytochemistry showed that there was positive expression of CKI3 in diftk;rentiated ceils, and the expression of CK13 mRNA was detected by RT-PCR. No mRNA products were detected in the hMSCs without co-culturing with conjunctival epithelium. Conclusion The hMSCs and hCjEs are successfully cultured and expanded with high purity in vitro. Conjunctival epithelium-like cells may be differentiate from the hMSCs under a given conditions in vitro.

关 键 词：干细胞 分化 细胞培养 上皮细胞 免疫组织化学 

分 类 号：R777.3[医药卫生—眼科]