摘要
【目的】研究Wnt3a和ActivinA存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于细胞中,加入25ng/mLWnt3a和100ng/mLActivinA,共同作用不同时间(1~4d),收集不同作用时间点(0,1,2,3,4,5d)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。以看家基阕β-actin作为对照,采用RTPCR方法对原肠作用基因(Goosecoid(GSC)、Mixll)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Braarchyury,中胚层基因Flk-1,外胚层基闪Pax6,胚外内胚层基因Sox7、CDX2)的表达进行检测。【结果]Wnt3a与ActivinA共同作用1~4d,均能获得限定性内胚层细胞,其中二者共同作用1d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。RTPCR结果显示,在Wnt3a与ActivinA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ActivinA共同作用1d,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。
【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.RTPCR 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.
出处
《西北农林科技大学学报(自然科学版)》
CSCD
北大核心
2010年第4期37-41,共5页
Journal of Northwest A&F University(Natural Science Edition)
基金
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)