胚胎干细胞来源的巢蛋白阳性细胞向胰岛素分泌细胞分化的实验研究

Experimental study on embryonic stem cell derived nestin positive cells differentiated into insulin-producing cells

目的探讨胚胎干细胞向胰岛素分泌细胞分化的诱导,为糖尿病患者实施细胞移植治疗建立基础。方法将胚胎干细胞(ESC)在成纤维细胞(MEF)饲养层上扩增后脱离饲养层,让ESC自发分化成拟胚体(EB),再将EB诱导为巢蛋白(nestin)阳性的神经前体细胞(NPC),最后将NPC诱导成胰岛素分泌细胞(IPC)。结果ESC在MEF饲养层上扩增4d后,在悬浮培养状态下自发分化为EB。将EB在NPC选择性培养基中做贴壁培养后,ESC先分化为上皮样细胞,再分化为神经细胞样细胞。经过nestin免疫组化检测,在经过NPC培养基诱导4d的细胞,nestin阳性细胞占86.5%。nestin阳性细胞在IPC选择性培养基中诱导5～6d后,可分化成胰岛素分泌细胞。结论ESC来源的nestin阳性细胞既可以分化为NPC,也可以分化为IPC。

Objective To study the induction of embryonic stem cell to insulin-producing cells and establish the basis for cell transplantation therapy in diabetes. Methods Embryonic stem cells （ESC） were amplified for 4 days in mouse embryonic fibroblast feed layer （MEF）,then the amplified ESCs were detached from MEF and cultured in suspension cultures to form embryoid bodies （EB）. The EB were cultivated in neural precursor cell selective media for about 4 to 5 days to differentiate to neural precursor cells. By adding nicotinamide, hetacellulin,activin-A,hepatic growth factor and glucose in serum free insulin-producing cells （IPC） selective medium,the dithezone-positive IPC were harvested. Results The EB differentiated into endotheha-like cells in the fLrst 2 days. The endothelia-like cells differentiated into neuron-like cells at later culture. 86.5 % of the neuron-like cells were nestin positive. The nestin positive cells differentiated into insulin-producing cells when exposed in insulin-producing cell selective media for about 5 to 6 days. Conclusion It is demonstrated that the embryonic stem cell derived nestin positive cells can be induced into neural precumor and insulin-producing cells.