体外诱导小鼠月不胎干细胞分化为T淋巴细胞

Induction of T cell development from mouse embryonic stem cells differentiated in vitro

目的体外诱导小鼠胚胎干细胞（ESC）分化为T淋巴细胞。方法借助小鼠胚胎干细胞体外自然分化形成的类胚体（EB）中含有三胚层细胞的独特细胞环境，加入多种细胞因子和胸腺肽αl，体外诱导小鼠ESC向T淋巴细胞分化。利用流式细胞仪在不同时间点检测诱导细胞表面T淋巴细胞发育相关的表面标志CD25、CD44、CD3、CD4、CD8以及T细胞受体（TCR）αβ和TCRγδ分子的表达水平。结果诱导3d后，出现CD44^＋细胞；第6天出现CD44^＋、CD44^＋CD25^＋和CD25^＋细胞群；第15天开始表达CD3分子；1周后开始出现CD4^＋CD8^＋双阳性细胞。随着诱导时间的延长，CD3^＋细胞和CD4^＋CD8^＋细胞的百分比不断升高。培养1个月后，出现少量的CD4^＋和CD8^＋的单阳性细胞。诱导细胞早期表达TCRαβ和TCRγδ。随着诱导时间的延长，T细胞进一步成熟，诱导细胞以TCRαβ细胞为主。结论细胞网子和胸腺肽αl的共同作用可以在体外诱导小鼠胚胎干细胞分化成具有T淋巴细胞表型的细胞，且细胞的表型变化与T细胞体内正常的胸腺发育过程中的表型变化一致。

Objective To establish the system that induces differentiation of T lymphocytes from mouse embryonic stem cells （ESCs） in vitro. Methods ESCs can be spontaneously differentiated into embryoid bodies （EBs） which contain a variety of cells derived from ectoderm, mesoderm and endoderm. Some cytokines and thymosin αl were added to induce cells in EBs differentiation into T cells. Then the phenotypic change of the induced cells which were relative to T lymphocyte development was observed by flow cytometry at different time points, such as CD25, CD44,CD3, CD4, CD8,T cell receptor （TCR）αβ and TCRγδ. Results At the third day after induction, CD44^ ＋ cells appeared. At the sixth day after induction, CD44^ ＋, CD44^ ＋ CD25^ ＋ and CD25^ ＋ cell population were detected. At the fifteenth day, cells begun to express CD3 molecule. One week later, CD4 ^ ＋ CD8 ^ ＋ double positive cells could be found. When the induction time was prolonged, more CD3^ ＋ cells and CD4^ ＋ CD8 ^ ＋ cells appeared. One month later, few CD4 ^ ＋ or CD8 ^ ＋ single positive cells appeared. Induced T cells in earlier stage of development expressed both TCRαβ and TCRγδ. In the later period of induction, T cells were more mature, and most of induced T cells mainly expressed TCRαβ molecule. Couclusion Some cytokines and thymosin αl can induce mouse ESCs to differentiate into T lymphocytes, and the phenotypic change of induced cells is followed the normal pattern of development as observed in the thymus.