胚胎干细胞向表皮样干细胞分化体外条件的研究

目的　探索体外诱导胚胎干细胞分化为表皮样干细胞的最佳条件 ,为研究其分化的调控机理及寻找新的皮肤组织工程种子细胞奠定基础。方法　将小鼠胚胎干细胞与人羊膜共培养 ,以不同的羊膜铺布方式 ,分 4个实验组 :(1)羊膜上皮面向上铺布全孔底 ,(2 )羊膜基底面向上铺放全孔底 ,(3)羊膜上皮面向上铺布半孔底 ,(4)羊膜基底面向上铺布半孔底 ,以未加羊膜为对照组 ,倒置显微镜下观察细胞的生长和形态分化 ,用抗 β1整合素单克隆抗体免疫组化检测胚胎干细胞向表皮样干细胞的分化。结果　培养 4～ 5d后 ,实验组 (3)中的ES细胞分化为表皮样干细胞 ,细胞呈多边形 ,体积增大 ,排列紧密 ,形成大面积细胞单层 ,而其他各实验组 ,ES细胞虽可分化为表皮样细胞 ,但呈集落生长 ,不形成细胞单层 ;对照组大量细胞死亡 ,形态各异。各实验组的细胞绝大多数呈现 β1整合素阳性 ,而对照组未见 β1整合素阳性细胞。结论　与羊膜共培养可诱导胚胎干细胞分化为表皮样干细胞 ,羊膜上皮面向上铺布半孔底的效果最佳

Reaserch of the culture condition that induce embryonic stem cells differentiate into epidermal like stem cells

Objective To investigate the best condition which can induce embryonic stem(ES) cells to differentiated into epidermal like stem cells, probe the differentiation mechanism of ES cells and to seek new source of seed cells for skin tissue engineering. Methods Mouse ES cells were cocultured with human amnion, and according to the pattern with which amnion was pasted on the bottom of cultural discs, the experimental groups were separated into 4 groups, (1) The epithelial surface upward covered the whole bottom, (2) epithelial surface downward covered the whole bottom, (3) The epithelial surface upward covered the half bottom, and (4) The epithelial surface downward covered the half bottom. The control group was cultured without amnion. The growth and morphological differentiation of cultured cells were observed with microscope, and the committed differentiation of embryonic stem cells into epidermal like stem cells was detected by integrin β 1 immunohistochemistry.Results After coculture with amnion 4～5 days, The ES cells in the group (3) differentiated into epidermal like stem cells and form a continuous single layer in large area, the cells in other experimental groups could also differentiated into epidermal like stem cells clones but didn't form a continuous single layer, most of the cells in control group died , the survival cells were different in morphological shapes. Most of the cells in experimental groups were Integrin β 1 positive, but no Integrin β 1 positive cells were found in control group.Conclusion Human amnion can induce mouse ES cells to differentiate into epidermal like stem cells, and the best culture condition is to paste down amnion with epithelial surface upward and cover the half bottom of disc.