体外诱导胚胎干细胞分化为甲状腺细胞的实验研究

Primary study on directed differentiation of embryonic stem cells into thyrocyte-like cells in vitro

目的探讨体外定向诱导胚胎干细胞(ESCs)分化为甲状腺细胞的可行性及相关分子表达检测。方法 E14小鼠 ESCs 诱导发育为胚胎体,再逐步添加 TSH、胰岛素、碘化钾(KI)等共同培养。以培养的小鼠甲状腺细胞为阳性对照,观察分化过程中细胞形态变化;用免疫荧光法检测分化标志物 TSH 受体(TSHR)、paired box gene 8(PAX8)、thyroid transcription factor-2(TW-2)、thyroidtranscription factor-1(TTF-1)的表达;RT-PCR 法检测分化相关基因 TSHR、PAX8、钠碘同向转运体(NIS)、过氧化物酶(TPO)、甲状腺球蛋白(Tg)的表达。结果诱导培养第6天分化细胞中存在甲状腺细胞特有基因 PAX8、NIS、TPO、Tg、TSHR 的表达;第8天分化细胞中检测到甲状腺细胞标志物TSHR、TTF-1、PAX8、TTF-2的表达,阳性细胞形态类似对照组甲状腺细胞。结论 ESCs 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。

Objective To investigate the feasibility of directed differentiation of embryonic stem cells （ESCs） into thyrocyte-like cells in vitro. Methods Murine El4 ESCs were cuhured in methylcellulose semisolid medium to form embryoid bodies （ EBs ）. These EBs were transferred for further inductive cuhure with the stepwise addition of growth factors （TSH, insulin and KI） into the cuhure medium. During differentiation, cell morphology was observed through phase contrast microscopy and compared with the normal thyroid cells from mouse. The molecular markers of thyroid cells were performed by indirect immunofluorescent analysis under fluorescent microscopy. Gene expressions of thyroid specific mRNA were analyzed by RT-PCR for molecules TSH receptor （ TSHR）, paired box gene 8 （ PAX8 ）, sodium iodide symporter （NIS）, thyroid peroxidase （TPO） and thyroglobulin （Tg）. Results After EBs formation, on day six of further cuhure added with inductive factors TSH, insulin and KI, ESCs-derived cells expressed thyroid-specific genes such as PAX8, NIS, TPO, Tg and TSHR. On the 8th day, these ESCs-derived thyrocyte-like cells overexpressed TSHR, thyroid transcription factor-1 （TTF-1） whereas PAX8, thyroid transcription factor-2 （TTF-2） remained in a housekeeping level. On the 10th day, all the molecular markers （including TSHR, PAX8, NIS, TPO and Tg） were overexpressed. Morphology of these markers positive differentiated cells was similar to those normal thyroid cells. Conclusions ESCs can differentiate into thyrocyte-like cells under certain inductive conditions and is possibly related to growth factors in vitro. This study suggests that a renewable source of thyroid follicular cells derived from ESCs holds great therapeutic potential for future cell replacement therapy in clinic.