SMAD2介导的Activin A信号对人胚胎干细胞向限定性内胚层细胞诱导分化的促进作用

【目的】研究SMAD2在Activin A促进人胚胎干细胞向限定性内胚层诱导分化中的作用。【方法】在人胚胎干细胞中转染SMAD2基因siRNA或者过表达质粒后,收集经Activin A分别诱导0,6,12,24,48,72,96和120 h的细胞各100 ng/mL,采用实时定量PCR检测SMAD2 与内中胚层共同前体标记Brachyury和内胚层标记Sox17的表达,进一步通过Western-blot分析Activin A诱导中SMAD2和磷酸化SMAD2（p-SMAD2）表达的变化。【结果】在Activin A诱导人胚胎干细胞向限定性内胚层细胞分化的过程中,干扰SMAD2 后48 h时才检测到Brachyury强表达,而单纯Activin A处理组24 h就检测到强表达;Sox17的表达始终较单纯Activin A处理组明显降低,因此,干扰SMAD2直接抑制了Activin A的诱导作用。而过表达SMAD2,Brachyury和Sox17的表达水平较单纯Activin A处理组明显增加,促进了限定性内胚层的发生;并且在Activin A诱导过程中,p-SMAD2的表达水平明显提高,而SMAD2的表达没有明显改变。【结论】SMAD2作为关键因子,介导了Activin A诱导人胚胎干细胞向限定性内胚层的分化,并转录调控Brachyury和Sox17的表达;SMAD2的磷酸化,激活并介导了Activin A诱导的信号转导通路。     

胚胎干细胞体外分化研究进展

胚胎干细胞是来源于胚胎的、内细胞团的、具有多向分化潜能的细胞。随着胚胎干细胞,特别是分化机制研究的不断深入,胚胎干细胞分化为各种类型细胞的研究也取得了较大进展,从而为细胞移植、细胞治疗和组织工程等提供了一个新的途径。主要综述了小鼠和人胚胎干细胞向内、中、外三个胚层细胞类型分化的技术,以期为胚胎干细胞定向诱导分化的研究提供一些参考。     

Promoting effects of activated Nodal signal pathway on definitive endoderm induction from mouse embryonic stem cells

AIM: To study the process of promoting mouse embryonic stem cells (ESC) to specify to definitive endoderm 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), natural differentiation group (serum-free medium) and activin A group (serum-free medium +50μg/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. RESULTS: 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 activin A group and the highest level was observed 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 protein 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 CXCR4 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.     

Wnt3a和Activin A共同促进人胚胎干细胞向限定性内胚层细胞分化

【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Flk-1,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。     

Activin A特异性对人胚胎干细胞向限定性内胚层诱导分化的促进作用

【目的】研究Activin A对人胚胎干细胞（hESCs）向限定性内胚层（DE）诱导分化的促进作用及其信号通路分子,为hESCs向DE诱导分化体系的优化提供参考。【方法】在人饲养层体系培养的hESCs中,收集100 ng/mL Activin A分别诱导0,6,12,24,48,72,96,120 h的细胞,用实时荧光定量RT-PCR检测原条标记Gsc和Mixl1、中内胚层共同前体标记Brachyury、内胚层标记Foxa2和Sox17、中胚层标记Flk1、外胚层基因Pax6、多能性相关基因Oct4与Nanog表达水平的变化,用细胞免疫荧光检测Brachyury和Sox17蛋白表达水平的变化。【结果】在人饲养层（HEF）培养体系上,高浓度Activin A能更快地促进中内胚层基因的表达并提高其表达水平;Brachyury和Sox17蛋白的细胞免疫荧光检测表明,Activin A诱导12和48 h就可检测二者的表达明显增加,且二者的表达水平分别在诱导48和96 h时达到高峰;hESCs高效分化为限定性内胚层细胞,DE细胞分化率为（81.7±5.4）%,并且体外的内胚层分化过程遵循从原条开始、经过中内胚层共同前体阶段、再到内胚层的发育过程,与体内发育规律相似。【结论】Activin A能特异性地诱导人胚胎干细胞向限定性内胚层分化,转录调控Brachyury和Sox17蛋白的表达。     

The effects of cisapride on the restoration of gut motility after surgery of the small intestine in horses; A clinical trial

Clinical and pathological data concerning the degree of leg weakness and severity of osteochondral lesions in gilts and barrows have been compared. It became clear that the pathological condition of the distal part of the ulna and the medial femoral condyle of gilts was less severe than that of barrows. The clinical symptoms of leg weakness in gilts also tended to be less severe than those in barrows.     

小麦品种与小麦白粉菌的相互作用——Ⅱ.小麦品种的限定性基因型分析

按生物间遗传学的原理和方法,用 9个小麦白粉菌株接种28个小麦品种,得到的侵染型资料,推导小麦品种的限定性基因型.结果表明,在7个推广品种与菌株的相互作用中,只存在2个相应基因对显示限定性表现型,而部分区试品种和抗源品种则含有丰富的限定性基因.讨论了如何把限定性基因转移到感病品种中去,和根据侵染型推导小麦品种的限定性基因的可行性及实用意义.