体外诱导胚胎干细胞向甲状腺细胞的分化(英文)

Directed differentiation of embryonic stem cells towards thyroid cells in vitro

背景:胚胎干细胞来源于发育早期囊胚的内细胞团,适当条件下能在体外维持未分化状态、正常二倍体核型及无限增殖能力,且具有多向分化潜能,能够发育分化成机体3个胚层的所有类型细胞。目的:观察体外定向诱导胚胎干细胞分化为甲状腺细胞的可行性及相关分子表达检测。设计、时间及地点:以细胞为对象的观察实验,于2004-01/2006-12在中山大学附属第二医院脐血库完成。材料:孕12.5～14.5d的Balb/c孕鼠用于胚成纤维细胞饲养层的制备。E14小鼠胚胎干细胞细胞株由美国哈佛大学Dr.Xu教授惠赠。成年昆明种小鼠用于甲状腺细胞的提取。方法:将制备的鼠胚成纤维饲养层接种于E14小鼠胚胎干细胞进行扩增培养。将脱离饲养层细胞后呈稳定克隆生长的胚胎干细胞诱导发育为胚胎体,再逐步添加促甲状腺素、胰岛素、碘化钾等共培养。以培养的成年昆明种小鼠甲状腺细胞为阳性对照。主要观察指标:①观察分化过程中细胞形态的变化。②免疫荧光法检测分化细胞标记物TSHR,PAX8,TTF-2,TTF-1的表达。③RT-PCR法检测分化细胞相关基因TSHR、PAX8,NIS,TPO,Tg的表达。结果:分化细胞边界清晰,呈圆形、类圆形、梭形或多角形贴壁生长。诱导培养第6天分化细胞中有甲状腺细胞特有基因PAX8,NIS,TPO,Tg,TSHR的表达,第8天检测到分化细胞中甲状腺细胞标记物TSHR,TTF-1,PAX8,TTF-2的表达,阳性细胞形态类似对照甲状腺细胞。结论:胚胎干细胞经胚胎体发育阶段,在特定条件下可定向分化为甲状腺细胞。

BACKGROUND： Embryonic stem cells （ESCs） develop from early blastular inner cell mass. Under proper condition, ESCs can maintain undifferentiated state in vitro, normal diploid nuclear type, and proliferative potential. In addition, ESCs possess multi-directional differentiation capacity and can differentiate into all sorts of cells of three germ layers. OBJECTIVE： To investigate the feasibility of directed differentiation of ESCs towards thyroid cells in vitro as well as related molecular expression. DESIGN, TIME AND SETTING： A cell-based observational experiment was performed at the Bank of Cord Blood, Second Hospital Affiliated to Sun Yat-sen University between January 2004 and December 2006. MATERIALS： Balb/c pregnant mice at pregnancy 12.5-14.5 days were used for preparation of embryonic fihroblast feeder layer. E14 mouse embryonic stem cell （ESC） strains were gifted by professor Xu from Harvard University. METHODS： Murine E14 ESCs were cultured in methylcellulose semisolid medium to form embryoid bodies （EBs）. These EBs were transferred for further inductive culture with the stepwise addition of growth factors- thyrotropin （TSH）, insulin and kalium iodidum （KI）. The cultured thyroid cells of adult Kunming mice were taken as positive control. MAIN OUTCOME MEASURES： ①Cellular morphological change in the process of differentiation. ②Detection of expression levels of TSHR, PAXS, TTF-2, and TTF-1 by immunofluorescence assay. ③ Detection of expression levels of TSHR, PAX8, NIS, TPO, and Tg by reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： The differentiated cells had clear boundary, exhibiting round, oval, shuttle-shaped, or polygon adhesive growth. On day 6 of inductive differentiation, the differentiated cells showed the expression of PAX8, NIS, TPO, Tg, and TSHR, the specific gene of thyroid cells. On day 8 of inductive differentiation, the expression of TSHR, TTF-1, PAXS, and TTF-2 was detectable in the differentiated cells with morphous similar to thyroid cells. CONCLUSION： ESCs can differentiate towards thyroid cells under given inductive conditions.