激活Nodal信号通路促进小鼠胚胎干细胞向定型内胚层分化

发布时间：2018-03-22 02:33

本文选题：Nodal信号通路　切入点：小鼠胚胎干细胞　出处：《中国病理生理杂志》2015年11期 　论文类型：期刊论文

【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(P0.05),其中activin A组最高(P0.05)。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(P0.05),其中activin A组的表达最明显(P0.05)。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。
[Abstract]:Objective: to investigate the role of activating Nodal signaling pathway in the differentiation of mouse embryonic stem cells (ESCs) into stereotyped endoderm cells, and to find out the best culture system for the differentiation of mouse ESC into DE. Methods: according to the different culture medium, The experiment was divided into three groups: embryonic stem cells group (basic culture group, natural differentiation group (basic medium) and activin A group (basic medium 50 渭 g / L activin A). Cell culture time was 1 ~ 7 days, collecting cells and cell climbing slices at different time points. The percentage of CXCR4 positive cells was detected by flow cytometry. The expression of CXCR4 protein was detected by immunocytochemistry and the expression of OCT4 and CXCR4 protein was detected by Western blot. Results: the results of flow cytometry showed that the proportion of CXCR4 positive cells in embryonic stem cells group did not change significantly at 1 and 7 days with the extension of culture time. The natural differentiation group and activin A group increased gradually, and reached the peak of P0.05 on the 5th day. The highest level of P0.05 was found in the activin A group. The results of immunohistochemistry showed that the CXCR4 positive cells were brown or brown. Western blot showed that with the extension of culture time. In embryonic stem cells group, the expression of OCT4 and CXCR4 protein was not significantly changed, and the expression of CXCR4 protein in naturally differentiated group and activin A group was gradually increased, and the expression of OCT4 protein decreased gradually. On the 5th day, the expression of P0.05 was the most obvious in activin A group. Conclusion: during the differentiation of ESC in mice, the proportion of DE was highest on the 5th day after induction and culture. Activation of Nodal signaling pathway could promote the differentiation of ESC into DE with CXCR4 markers. It is helpful to obtain more DE cells.
【作者单位】：中山大学孙逸仙纪念医院消化内科;
【基金】：广东省科技社会发展项目(No.2012B031800030)
【分类号】：R321

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