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| 血清和无血清诱导方法分化小鼠胚胎干细胞为定形内胚层的比较 |
| 李阳芳1,兰勇2,张亚卓1,王欣3,胡以平4* |
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(1. 北京市神经外科研究所,北京 100050
2. 北京医院普通外科,北京 100730
3. 中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海 200031
4. 第二军医大学基础部细胞生物学教研室,上海 200433
*通信作者) |
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| 摘要: |
| 目的 比较两种方法(血清诱导方法和无血清诱导方法)诱导小鼠胚胎干细胞分化为定形内胚层细胞的效率。方法 利用血清诱导法和无血清诱导法分别诱导分化小鼠胚胎干细胞,根据定形内胚层表面标记蛋白(Cxcr4、c-Kit和E-cadherin)的表达,通过流式细胞术分析定形内胚层诱导的时间及效率, 并利用 RT-PCR检测两种方法诱导的定形内胚层基因表达谱；同时利用荧光定量PCR检测无血清诱导过程中内胚层基因的表达情况；利用流式细胞术分选Cxcr4和 c-Kit双阳性细胞进行定形内胚层基因表达的鉴定。结果 血清诱导法和无血清诱导法都能够将胚胎干细胞诱导分化为定形内胚层,其中无血清组的诱导效率高于血清组,高达74.19%,并且在诱导的第4天就能到达峰值。结论 建立了高效快捷的诱导胚胎干细胞分化为定形内胚层细胞的方法,为进一步向肝、胰等组织细胞诱导打下了基础。 |
| 关键词: 胚胎干细胞 定形内胚层 细胞分化 血清 |
| DOI：10.3724/SP.J.1008.2013.00124 |
| 投稿时间：2012-11-02修订日期：2012-12-31 |
| 基金项目:国家自然科学基金(30671044/C07). |
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| Serum and non-serum method-induced differentiation of mouse embryonic stem cells into definitive endoderm |
| LI Yang-fang1,LAN Yong2,ZHANG Ya-zhuo1,WANG Xin3,HU Yi-ping4* |
(1. Beijing Neurosurgical Institute, Beijing 100050, China
2. Department of General Surgery, Beijing Hospital, Beijing 100730, China
3. Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031, China
4. Department of Cell Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To compare the efficiencies of two methods (serum and non-serum) in inducing mouse embryonic stem cell differentiation into definitive endoderm cells. Methods The serum and non-serum methods were used to induce differentiation of mouse embryonic stem cells into definitive endoderm cells. Fluorescence activated cell sorter (FACS) was used to analyze the inducing time and efficiency of definitive endoderm using their surface protein marks (Cxcr4, c-Kit and E-cadherin). Meanwhile, RT-PCR was used to analyze the gene profile of definitive endoderm induced by the two methods. Real-time PCR was used to analyze the gene expression in definitive endoderm during the induction course in the non-serum group. The Cxcr4 and c-Kit double positive definitive endoderm cells were sorted by flow cytometry and gene profile was characterized by RT-PCR. Results Definitive endoderm cells were induced from mouse embryonic stem cells by both serum and non-serum methods. However, the efficiency of non-serum group (74.19%) was higher than that in the serum group, and the induction outcome reached a climax at the 4th day of induction. Conclusion We have established a highly efficient method to induce differentiation of mouse embryonic stem cells into definitive endoderm, which lays a foundation for further differentiation into liver and pancreatic cells. |
| Key words: embryonic stem cells definitive endoderm cell differentiation serum |
