人羊膜上皮细胞干细胞特性及向胰岛素分泌细胞分化的研究  

Stem cell characteristics and islet differentiation potential of human amniotic epithelial cells

作　　者：王建[1] 彭琳[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所/人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《南方医科大学学报》2011年第9期1484-1487,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30700272);教育部新教师基金(422201017);湖南省科技计划项目(2009FJ3180)~~

摘　　要：目的建立人羊膜上皮细胞(hAECs)的分离及培养方法,探讨其向胰岛素分泌细胞分化的能力。方法观察不同培养方法对hAECs生长倍增时间的影响以优化其培养体系;通过细胞免疫荧光法检测不同培养代数细胞多能干细胞的表面标记;通过体外添加诱导因子的方法,探讨诱导hAECs向胰岛素分泌细胞分化的潜能。结果在10 ng表皮生长因子培养下,hAECs可长期传代。hAECs表达胚胎干细胞表面标记如阶段特异性胚胎抗原4等;采用悬浮培养法诱导分化,可检测到胰岛发育、胰岛功能基因如胰腺十二指肠同源盒、神经源素3、同源异形盒转录因子6、胰岛素、胰高糖素的表达,免疫组化检测细胞可以表达胰岛素,并且随着外界葡萄糖浓度升高,胰岛素分泌显著增加。结论建立了人羊膜上皮细胞的分离和培养方法(不添加动物血清);在体外可诱导分化为胰岛素分泌细胞。Objective To establish a method for isolating and culturing human amniotic epithelial cells（hAECs） in a serum-free medium and investigate their transdifferentiation ability into islet-like cells.Methods The culture condition of hAECs was optimized using DMEM with different supplements.The genetic stability of the tenth-passage cells was assessed by chromosome analysis and G-banding method.The stem cell characteristics of the cells were identified by examination of the surface markers using immunofluorescence methods.The endocrine-related genes and hormones of the cells were tested after induced differentiation into islet-like cells.Results The hAECs allow stable passaging in the presence of 10 ng epidermal growth factor（EGF） in the culture medium.After 10 passages,the cells maintained a normal karyotype and G-banding profile.The hAECs expressed many multi-potent stem cell markers,including SSEA4,TRA-1-60,and TRA-1-81.After induced differentiation,the endocrine-related genes were expressed in the islet-like cells,including PDX1,ngn3,insulin and glucagon.Insulin secretion increased in the differentiated islet-like cells in response to high glucose exposure.Conclusion We established a method for isolating and expanding the hAECs in a serum-free medium.hAECs possess stem cell characteristics and can be induced to differentiate into islet-like cells in vitro.

关 键 词：人羊膜上皮细胞 胰岛素分泌细胞 横向分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]