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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) originate from the inner cell mass of early blastocyst stage. Under suitable conditions, they maintain their undifferentiated state, normal diploid karyotype and immortal proliferative capacity in vitro, and have multi-directional differentiation potential. They can differentiate into body 3 Germ layers of all types of cells. Objective: To observe the feasibility of differentiation of embryonic stem cells into thyrocytes induced by in vitro orientation and the related molecular expression detection. DESIGN, TIME AND SETTING: The observation experiment with cells as the object was performed at the Cord Blood Bank of the Second Affiliated Hospital of Sun Yat-sen University from January 2004 to December 2006. MATERIALS: Balb / c pregnant mice of 12.5-14.5 days pregnant were used to prepare embryonic fibroblast feeder layer. E14 mouse embryonic stem cell line is kindly donated by Dr. Xu of Harvard University. Adult Kunming mice for thyroid cell extraction. Methods: The prepared mouse embryo fibroblast feeder layer was inoculated on E14 mouse embryonic stem cells for expansion culture. Embryonic stem cells stably cloned and growing out of feeder cells were induced to develop into embryoid bodies, and then co-culture of thyrotropin, insulin and potassium iodide was gradually added. Cultured adult Kunming mouse thyroid cells as positive control. MAIN OUTCOME MEASURES: ① observe the changes of cell morphology during differentiation. Immunofluorescence method was used to detect the expression of differentiated cells markers TSHR, PAX8, TTF-2 and TTF-1. ③ RT-PCR was used to detect the expression of TSHR, PAX8, NIS, TPO and Tg in differentiated cells. Results: The differentiated cells had a clear boundary with round, round, fusiform or polygonal adherent growth. The expression of thyroid-specific genes PAX8, NIS, TPO, Tg and TSHR in differentiated cells was induced on day 6, and thyroid cell markers TSHR, TTF-1, PAX8 and TTF-2 in differentiated cells were detected on the 8th day , Positive cells in morphology similar to control thyroid cells. CONCLUSION: Embryonic stem cells can be differentiated into thyroid cells under specific conditions during embryonic development.