Y-27632促进人胚胎干细胞向类神经元细胞的分化  

Y-27632 promotes differentiation of human embryonic stem cells into neuron-like cells

作　　者：何大艳[1] 杨玉红[1] He Da-yan;Yang Yu-hong(Department of Neurology, Chuzhou First People＇s Hospital, Chuzhou 239000, Anhui Province, Chin)

机构地区：[1]滁州市第一人民医院神经内科,安徽省滁州市239000

出　　处：《中国组织工程研究》2018年第13期2063-2067,共5页Chinese Journal of Tissue Engineering Research

摘　　要：背景:Y-27632是Rho相关卷曲螺旋形成的蛋白激酶抑制剂,已被证实具有调节干细胞的自我更新、促进克隆形成和存活的作用。另外,其对神经细胞生长和发育也具有调节和保护作用。而如何提高胚胎干细胞向类神经元细胞分化是神经损伤修复和神经再生领域热点问题。目的:探讨Y-27632对人胚胎干细胞向类神经元细胞分化效率的作用。方法:取16代人胚胎干细胞复苏后,进行形态观察和染色鉴定,采用悬浮培养法制备类胚体,培养8 d后,进行分组贴壁培养:对照组细胞为常规Sato培养基诱导分化;Y-27632组分别在Sato培养基加入5,10,20,40μmol/L Y-27632。培养10 d后进行染色鉴定;10-18 d后,进行免疫荧光染色检测胚胎干细胞分化效率和类神经元细胞突起生长情况。结果与结论:(1)人胚胎干细胞共表达Oct4和SSEA-3干细胞特异蛋白;(2)悬浮分化培养8 d后,进一步贴壁培养10 d,可观察到具有神经形态,且表达Tuj-1的类神经元细胞;(3)经不同浓度的Y-27632诱导后,免疫荧光染色发现贴壁细胞数和Tuj-1阳性细胞数显著增加,提示胚胎干细胞向类神经元细胞分化速率加快,且在浓度为10μmol/L时最为明显;(4)分化培养18 d后,免疫荧光染色显示,与对照组相比,10μmol/L Y-27632组的Tuj-1阳性细胞数及突起数和突起长度显著增加;(5)结果说明,Y-27632不仅能够促进人胚胎干细胞向类神经元细胞分化,同时促进类神经元细胞突起的生长。BACKGROUND： Y-27632 is a Rho-associated coil-formed protein kinase inhibitor that can regulate the self-renewal of stem cells, promote clonal formation and cell survival, and regulate and protect neuronal cell growth and development. How to improve the differentiation efficiency of embryonic stem cells into neuron-like cells is highly important for nerve injury repair and nerve regeneration. OBJECTIVE： To investigate the effect of Y-27632 on the differentiation efficiency and function of human embryonic stem cells into neuron-like cells. METHODS： After resuscitation, human embryonic stem cells at passage 16 were subjected to morphological observation and staining identification. The embryoid bodies were prepared by suspension culture, and after 8 days of incubation, the cells were cultured in Sato medium containing different concentrations of Y-27632 （0, 5, 10, 20, 40 μmol/L） for 10 days and identification by staining. After induction for 18 days, the differentiation efficiency and neurite outgrowth were identified by immunofluorescence staining. RESULTS AND CONCLUSION： The human embryonic stem cells co-expressed Oct4 and SSEA-3 stem cell specific markers. After suspension culture for 8 days and further adherent culture for 10 days, the cells could be differentiated into neuron-like cells with neurogenic morphology and expressing Tuj-1. Y-27632, especially at a concentration of 10 μmol/L, not only promoted cell proliferation （a significant increase in adherent cells an Tuj-1 positive cells）, but also facilitated cell differentiation into neurons. Immunofluorescence staining findings showed that 10 μmol/L Y-27632 significantly increased the number of Tuj-1 positive cells and neurites and the length of neurites after 18 days of differentiation. These results indicate that Y-27632 not only promotes the differentiation of human embryonic stem cells into neuron-like cells, but also accelerates neurite outgrowth.

关 键 词：胚胎干细胞 类胚体 诱导分化 Y-27632 神经突起 Tuj-1 细胞培养 细胞分化 免疫荧光染色 神经损伤 神经修复 神经再生 细胞鉴定 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]