多种生长因子诱导小鼠胚胎干细胞分化为胰岛素分泌细胞(英文)  

Differentiation of mouse embryonic stem cells into insulin-producing cells induced by various growth factors

作　　者：冯龄[1] 张宏利[1] 李文毅[1] 张芹[1] 许丽红[1] 赵萸[1] 骆天红[1] 李果[1] 

机构地区：[1]上海市内分泌及代谢病研究所,上海交通大学医学院附属瑞金医院,上海市200025

出　　处：《中国组织工程研究与临床康复》2008年第21期4167-4171,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30571019);全国博士论文作者专项资金(200360);上海市启明星计划(03QC14040)~~

摘　　要：背景：以往研究已证明胚胎干细胞可被诱导分化为胰岛素分泌细胞，但诱导时间较长，大部分需要1个月左右。目的：体外联合应用激活素A、全反式维甲酸、碱性成纤维细胞生长因子和烟酰胺4种生长因子，观察能否在较短的时间内将小鼠胚胎干细胞诱导分化为胰岛素分泌细胞。设计：细胞观察实验。单位：上海市内分泌及代谢病研究所，上海交通大学医学院附属瑞金医院。材料：实验于2004—10／2006—02在上海市内分泌研究所完成。清洁级孕12．5—14．5d龄昆明小白鼠2只，由上海斯莱克实验动物有限公司提供，动物质量合格证号2004A034，实验过程中对动物的处置符合动物伦理学标准。小鼠胚胎干细胞株由法国里昂CNRSUMR5641实验室张昌贤教授提供。激活索A为R＆D公司产品：全反式维甲酸，烟酰胺由Sigma公司提供；碱性成纤维细胞生长因子由Gibco公司提供。方法：取孕鼠胚胎，除去头部和内脏，将剩余组织剪碎，胰酶消化后制备细胞悬液，取上层离心重悬，按3×10^8L^-1接种培养，传2-3代时作为滋养层细胞。将鼠胚胎干细胞接种到新鲜滋养层上，加入含白血病抑制因子的knockout DMEM培养基，常规培养两三天后按1：3-1：6传代，当细胞与细胞之间分开时加入含血清的培养液终止消化，离心弃上清，制成单细胞悬液按2.5×10^4密度接种，加入不含白血病抑制因子的培养液，24～48h后收集形成的胚胎体，铺于Matrigel铺底的培养皿中。胚胎体贴壁后，在含有100μg／L激活素的10％FBs，DMEM中培养24h，再将培液换为10％FBS／DMEM培养6-8h作为间隔，然后把分化的胚胎体在含10^-6mol／L全反式维甲酸的10％FBS／DMEM中培养24h，在含10μg／L碱性成纤维细胞生长因子的10％FBS／DMEM中培养3-5d，在含N2、B27、1μg/L层粘连蛋白、10μg/L碱性成纤维细胞生长因子、10mmol／L烟酰胺的DMEBACKGROUND： Previous studies have confirmed that embryonic stem cells can be induced to differentiate into insulin-producing cells, but the induction process takes a long time. Most of the processes take about one month. OBJECTIVE： Activin A, all-trans retinoic acid （ATRA）, basic fibroblast growth factor （bFGF） and nicotinamide were applied in vitro in combination to observe whether mouse embryonic stem cells could be induct to differentiate into insulin-producing cell in a relatively short time. DESIGN： Cell observation experiment. SETTING： Shanghai Institute of Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. MATERIALS： This study was performed at Shanghai Institute of Endocrine and Metabolic Diseases from October 2004 to February 2006. Two mice of clean grade and of 12.5-14.5 days of gestational age were provided by Shanghai SLAC Laboratory Animal Co., Ltd （Permission No. 2004A034）. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Mouse embryonic stem cell lines were supplied by Dr Changxian Zhang （CNRS UMR5641, France）. Activin A was the product of the R＆D Corporation. ATRA and nicotinamide were supplied by the Sigma Corporation, USA. BFGF was supplied by Gibco Corporaion. METHODS： Head and viscera were removed from embryos of the pregnant mouse. The remaining tissues were cut into pieces and digested with trypsin. Cell suspension was centrifuged and inoculated at 3 × 10^8 L^-1. The cells could be used as mouse feeder layer after 2 3 times of passage. The mouse embryonic stem cells （ESCs） were inoculated onto the feeder layer in knockout Dulbecco＇s modified Eagle medium （DMEM） supplemented with leukemia inhibitory factors （LIF）. ESCs were passaged at 1 ： 3 1 ： 6 after 2 3 days of culture. Culture medium with serum was added into the culture dishes to terminate the digestion. Cell fluid was centrifuged and supernatant was discarded. The s

关 键 词：干细胞 胚胎 生长因子 胰岛素分泌细胞 

分 类 号：R394.2[医药卫生—医学遗传学]