摘要
目的：探讨激活Nodal信号通路在小鼠胚胎干细胞（embryonic stem cells,ESC）向定型内胚层（definitive endoderm,DE）分化中的作用,寻找小鼠ESC向DE分化的最佳培养体系。方法：根据培养基的不同,实验分为3组：胚胎干细胞组（基础培养＋LIF）、自然分化组（基础培养基）和activin A组（基础培养基＋50μg/L activin A）。细胞培养1~7 d,收集不同作用时点（1、3、5、7 d）的细胞及细胞爬片。用流式细胞术检测CXCR4阳性细胞的比例;免疫细胞化学法检测CXCR4蛋白的表达;Western blot检测OCT4及CXCR4蛋白的表达。结果：流式细胞术检测结果提示,随着培养时间的延长,CXCR4阳性细胞的比例,胚胎干细胞组在1~7 d无明显变化,自然分化组和activin A组逐渐增高,第5天达高峰（P〈0.05）,其中activin A组最高（P〈0.05）。细胞免疫组化结果显示CXCR4阳性细胞呈棕褐色或棕黄色。Western blot的结果提示,随着培养时间的延长,胚胎干细胞组OCT4和CXCR4蛋白表达无明显变化;自然分化组和activin A组的CXCR4蛋白的表达逐步增高,OCT4蛋白的表达逐步下降,第5天达高峰（P〈0.05）,其中activin A组的表达最明显（P〈0.05）。结论：在小鼠ESC分化过程中,在诱导培养的第5天,DE的比例最高。激活Nodal信号通路可以促进小鼠ESC向具有CXCR4分子标志的DE分化,有利于获取更多的DE细胞。
AIM： To study the process of promoting mouse embryorfic stem cells （ESC） to specify to definitive endoderul by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells. METHODS： The cells were divided into different groups based on the culture medium： ESC group （serum-free medium ＋ LIF） , nalural differentiation group （serum-free medium） and activin A group （serum- free medium ＋ 50 fxg/L activin A）. The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation. The proportion of CXCR4 ＋ cells was detected by flow cytometry. The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot. RE- SULTS ： The proportion of CXCR4 ＋ cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and aetivin A group and the highest level was ob- served at 5 d. Among the 3 groups, the proportion of CXCR4 ＋ cells at 5 d was the highest in activin A group. The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry, The pro- tein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed. The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CX- CR4 were gradually increased with the highest level at 5 d. It was highest in the cells in activin A group. CONCLUSION ： The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation. Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第11期2070-2075,共6页
Chinese Journal of Pathophysiology
基金
广东省科技社会发展项目(No.2012B031800030)
