羊膜上皮细胞体外培养条件的优化及其干细胞标志的表达  

Optimization of In Vitro Culture Conditions for Human Amniotic Epithelial Cells and Expression of Stem Cell Markers

作　　者：陈宥艺[1,2] 陆琰 王科 王琰[1,2] 吴东颖 刘兵[5] 杨瑛[2] 吕双红[5] 

机构地区：[1]第四军医大学唐都医院妇产科,陕西西安710038 [2]中国人民解放军307医院妇产科,北京100071 [3]广州市妇女儿童医疗中心,广东广州510623 [4]307医院青藤转化医学中心,北京100071 [5]中国人民解放军307医院肿瘤学研究室,北京100071

出　　处：《中国实验血液学杂志》2011年第2期464-468,共5页Journal of Experimental Hematology

基　　金：国家863计划;编号2008AA8070099

摘　　要：本研究优化人足月胎盘羊膜上皮细胞(human amniotic epithelium cells,hAEC)的体外培养方法并观察hAEC的干细胞标志的表达情况。取健康产妇足月剖宫产术后的羊膜,采用胰酶多次消化获取hAEC,分别应用10%FBS的DMEM、类似胚胎干细胞的培养条件及添加表皮细胞生长因子(epidermal growth factor,EGF)的类似胚胎干细胞的培养条件对其进行原代和传代培养,观察培养后细胞形态,并通过流式细胞术检测、细胞免疫荧光染色等方法对培养细胞的干细胞多能性标志进行鉴定。结果表明:在类似胚胎干细胞培养条件的基础上添加10 ng/ml EGF可提高hAEC原代细胞的贴壁率和增殖能力,与广泛应用的10%FBS的DMEM培养条件相比,在此培养条件下细胞传代能力显著增强,传至5代仍保持上皮细胞表型,细胞形态在传代过程中改变不明显,并且表达胚胎干细胞全能性的表面标志SSEA-4,同时细胞免疫荧光染色显示培养后的hAEC表达波形蛋白(vimentin)。结论:采用类似胚胎干细胞的培养条件有利于hAEC在体外的扩增和传代,EGF对其增殖和传代有促进作用,培养的hAEC表达胚胎干细胞多能性标志。This study was purposed to optimize the culture conditions of the human amniotic epithelium cells（hAEC） in vitro,and detect the expression of hAEC pluripotent markers.Amnion tissues were separated from the underlying chorion through the spongy layer immediately after elective cesarean section of healthy pregnancy women at term.After the subsequent exposure to trypsin digestion,hAEC were cultured in DMEM with different supplements.The growth and proliferation potential of hAEC was evaluated,and the expression of cultured hAEC pluripotent markers was detected by using flow cytometry and immunohistochemistry methods.The results indicated that when being cultured in the mediums similar to that of embryonic stem cell culture supplemented with 10 ng/ml EGF,the hAEC grew better and the time for passage was shortened.In addition,compared to other culture conditions,under this condition,the cells could be passaged up to 5 times as much without obvious morphological changes,and the pluripotent marker SSEA-4 was detected in the cultured cells by flow cytometry.Meanwhile,the detection of immunofluorescence showed the expression of vimentin in cultured hAEC was strengthened as compared with primary cells.It is concluded that the culture condition similar to that for embryonic stem cells supplemented with EGF facilitates the proliferation and passage of hAEC in vitro.

关 键 词：羊膜上皮细胞 体外培养 干细胞标志 表皮生长因子 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]