| 摘要: |
| 【目的】研究Wnt3a和Activin A在限定性内胚层诱导中共同作用的最佳时间窗。【方法】 在无饲养层体系培养的人胚胎干细胞中，加入25 ng/mL Wnt3a和 100 ng/mL Activin A，共同作用不同时间（1～4 d），收集不同作用时间点（0，1，2，3，4，5 d）细胞，对原条、内中胚层前体标记Brachyury及限定性内胚层标记Sox17分别进行免疫荧光染色，统计阳性细胞总数，计算阳性细胞率。以看家基因β-actin作为对照，采用RT-PCR方法对原肠作用基因（Goosecoid(GSC)、Mixl1）及三胚层发育相关基因（内胚层基因Sox17、Foxa2，内中胚层前体基因Brachyury，中胚层基因Flk1，外胚层基因Pax6，胚外内胚层基因Sox7、CDX2）的表达进行检测。【结果】 Wnt3a与Activin A共同作用1～4 d，均能获得限定性内胚层细胞,其中二者共同作用1 d分别能获得(78.9±7.3)% Brachyury阳性细胞和(85.2±3.8)%的Sox17阳性细胞。RT-PCR结果显示，在Wnt3a与Activin A共同作用下，原肠作用基因及三胚层发育相关基因表达的时间有差异。【结论】 Wnt3a和Activin A共同作用1 d，是有效促进人胚胎干细胞向限定性内胚层细胞分化的最佳作用时间窗。 |
| 关键词: 人胚胎干细胞 限定性内胚层 Wnt3a Activin A |
| DOI： |
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| 基金项目:国家“973”项目(2005CB522705，2007CB948103);国家“863”计划项目(2006AA02A102)；国家自然科学基金项目（30800659） |
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| Wnt3a synergized with Activin A promote differentiation of human embryonic stem cells into definitive endoderm |
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| Abstract: |
| 【Objective】 The study is done to confirm the effective time window of Wnt3a and Activin A in endoderm induction. 【Method】 Human embryonic stem cells cultured on feeder free system are treated with 25 ng/mL Wnt3a and 100 ng/mL Activin A for 4 days.Cells on different time points (0，1,2,3,4,5 d) are collected to perform immunofluorescence staining of Brachyury (primitive streak and endomesodermal marker) and Sox17 (definitive endodermal marker) and genes analysis of early developmental genes (primitive streak marker (Goosecoid(GSC),Mixl1),endodermal marker (Sox17,Foxa2),mesoendodermal marker Brachyury,mesodermal marker Flk1,ectodermal marker Pax6,extra endodermal marker Sox7,CDX2) expression.【Result】 Among the different durations of combination of Wnt3a and Activin A all can induce the definitive endoderm cells.When the duration is 1 day,the highest percentage of Brachyury and Sox17 positive cells are gained (78.9±7.3)% and (85.2±3.8)% respectively.RT PCR results indicate the gastrulation and germ lines relating genes expression modes are different.【Conclusion】 It is the optimal endoderm inducing protocol to combine Wnt3a with Activin A for the 1st day. |
| Key words: human embryonic stem cell definitive endoderm Wnt3a Activin A |
