【作者】 杜娟；

【导师】 郭泽坤；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 胚胎干细胞(Embryonic Stem Cells,ESCs),是从植入前囊胚期胚胎的内细胞团(Inner Cell Mass,ICM)中分离出来的一种具有多向分化潜能的细胞。胚胎干细胞具有自我更新、无限增殖和分化形成机体所需各种类型细胞的能力,是揭示胚胎发育机制和再生医学研究的细胞模型。然而,胚胎干细胞的自我更新和分化机制尚未明确,这显著制约着胚胎干细胞在生物医学领域中的广泛应用。已有研究证实,Tgf-β信号通路能够促进胚胎的分化。但抑制Tgf-β信号通路是否有助于胚胎干细胞多能性的维持目前还存在争议。4-[4-(1,3-苯并二唑-5-基)-5-(2-吡啶基)-1H-咪唑-2-基]-苯酰胺水合物(SB431542)是Tgf-β-Smad2/3通路的特异性抑制剂。SB431542与Tgf-βⅠ型受体Alk4、Alk5和Alk7的ATP结合结构域结合,抑制Smad2/3的磷酸化,并阻止Tgf-β通路信号的转导。研究显示SB431542有助于胚胎干细胞多能性的维持,能够提高诱导性多能干细胞(Induced Pluripotent Stem cells,i PS)的形成效率,但相关机制尚不明确。为揭示SB431542对胚胎干细胞多能性的调控作用,明确Tgf-β信号抑制对胚胎干细胞多能性的影响,本文以小鼠J1胚胎干细胞为研究材料,利用表达谱芯片技术、高通量测序技术和一些分子生物学研究技术探究SB431542对小鼠胚胎干细胞多能性的调控作用。本文的主要研究内容和结果如下:1.无饲养层条件下,检测SB431542能否抑制Smad2/3蛋白质的磷酸化、维持小鼠ES细胞的多能性。蛋白质磷酸化Western blot和CCK-8细胞增殖检测结果显示,SB431542可抑制Smad2/3蛋白质的磷酸化,且对J1细胞的增殖活性无影响。无饲养层条件下,J1 ES细胞倾向于分化,干性促进因子BIO、CHIR99021和SC1都能增强J1 ES细胞的胞间连接作用,使其呈紧密的克隆状生长。用SB431542培养J1细胞时发现:LIF存在的条件下,SB431542能增强J1小鼠胚胎干细胞的胞间连接作用使其呈典型克隆状生长;无LIF的条件下SB431542可也可阻碍J1细胞的分化。对主要干性调控基因和分化促进基因的q PCR检测结果显示:SB431542对核心干性调控基因Oct4、Sox2和Klf4的表达无影响,但可以抑制三胚层分化基因T、Nestin和Gata4的表达。因此,在无饲养层条件下SB431542可以抑制Smad2/3蛋白质磷酸化,也能够维持小鼠ES细胞的未分化状态。2.在基因组水平上研究SB431542对小鼠胚胎干细胞多能性的调控作用。实验用表达谱芯片技术检测了对照和SB431542处理的J1小鼠胚胎干细胞内全基因组基因的表达。基因的差异表达分析结果显示:SB431542未增强主要干性调控基因的表达,但提高了其它干性相关基因如Dppa3、Klf2和Nr5a2等的表达量;SB431542对分化相关基因如T、Fgf8和Gata4等的表达具有抑制作用。对SB431542调控基因的GO和KEGG富集分析结果显示:SB431542可通过基因转录调节、生物粘附和胞内信号传递过程调节胚胎干细胞的多能性。在信号转导过程中SB431542除影响Tgf-β通路外还可通过MAPK通路、Erb B通路、VEGF通路以及细胞粘附通路等调节小鼠胚胎干细胞的多能性。用DLR检测SB431542对不同信号通路活性的影响时发现,SB431542抑制了具有分化促进作用的JNK和MAPK/ERK的活性。此外SB431542还抑制了JAK/STAT通路的活性,这一结果与SB431542对c-Myc表达量的下调、在无LIF条件下不能维持ES细胞的未分化状态一致,说明SB431542可以抑制ES细胞的分化,但它对ES细胞的多能性也有“负作用”,不能在无LIF的条件下维持ES细胞的多能性。3.探究SB431542对分化相关通路活性的调节。SB431542可以降低Smad1/5/8通路的抑制性蛋白Smad7的表达,通过基因的体外克隆、过表达和蛋白质磷酸化Western blot检测实验发现,SB431542在降低Smad2/3蛋白质磷酸化的同时可通过抑制Smad7基因的表达激活Smad1/5/8-Id通路,最终发挥分化抑制作用。另外,SB431542对神经分化相关基因和通路有抑制作用。SB431542下调了RA通路相关基因的表达,体外用RA诱导J1细胞分化时,SB431542的添加能够阻碍J1细胞向神经细胞的分化,同时也可降低RA诱导基因的表达。另外,受SB431542调控的9个Fgf家族基因在SB431542处理的J1细胞中表达量均下调,其中包括对ERK通路具有激活作用的Fgf4。4.通过全基因组mi RNA的表达变化验证SB431542对小鼠ES细胞的分化抑制作用。SB431542处理J1小鼠胚胎干细胞24 h后,用深度测序法检测J1细胞全基因组内mi RNA的表达变化。结果显示SB431542在小鼠胚胎干细胞mi RNA的表达过程中主要发挥表达抑制作用,在SB541542调节的114个mi RNA中仅有4个mi RNA被上调。除去直接受Tgf-β-Smad2/3信号调节的mi RNA,SB431542下调的mi RNA主要是一些能够抑制多能性维持和促进胚胎干细胞分化的mi RNA,说明SB431542主要通过分化抑制维持胚胎干细胞的多能性。总之,本研究显示,LIF存在的条件下SB431542可用于胚胎干细胞的体外无饲养层培养。SB431542可以维持胚胎干细胞的多能性:ES细胞生长过程中SB431542不能增加主要干性基因的表达,但可以抑制分化相关基因、mi RNA和信号通路的活性。SB431542主要通过抑制分化维持胚胎干细胞的未分化状态。更多还原

【Abstract】 Embryonic stem cells(ES cells) are a type of multipotent cells isolated from the inner cell mass of the blastocyst-stage embryos. Because of the characteristics of that can self-renewal indefinitely and differentiate into almost all types of cells, ES cells become a focus in life science research: ES cells are promising models for embryonic development research, and they can also be used to produce regenerative materials. Today, a tremendous progress has been made in ES cells research. However, the exactly mechanisms of self-renewal and differentiation of ES cells remain unknown, which limit the application of ES cells severely.It has been reported that Tgf-β signaling pathway can accelerate the process of embryonic development. However, there is still a controversy on whether inhibition of Tgf-β signaling pathway would contribute to the pluripotent maintenace of ES cells. 4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate(SB431542) is a specific inhibitor of Tgf-β-Smad2/3 signaling pathway. It can specifically inhibit the phosphorylation of Smad2/3 and block the transduction of Tgf-β signaling pathway through occupying the ATP-binding domain of Alk4, Alk5 and Alk7. As previously reported that SB431542 facilitates the pluripotent maintenance of in vitro cultivated ES cells, however, the specific mechanisms of SB431542 are not well documented. The following studies were conducted in J1 mouse ES cells to investigate the functional role of Tgf-β signaling pathway, as well as the specific mechanism of SB431542 in pluripotent maintenace, the detailed contents and results of this research are as follows:1. The results of CCK-8 assay and phosphorylated protein Western blot indicated that SB431542 could inhibit the phosphorylation of Smad2/3, and had no significant influence on the proliferation ability of J1 ES cells. As previously reported, three small moleculars, namely, BIO, CHIR99021 and SC1 are able to promote self-renewal of ES cells, we also found that these small moleculars could enhance the adhesion of cells and promote ES cells growing in a typical colony morphology. The same phenomenon was observed when ES cells were treated with SB431542. Besides, the alkaline phosphatase activity of ES cells was increased, and the genes refered to differentiation marker of ES cells were down-regulated by SB431542 treatment. These results showed that SB431542 contributes ES cells pluripotency maintenance.2. In order to know the specific mechanism of SB431542 on ES cells pluripotency, we examined the global gene expression changes of ES cells that treated with SB431542 by microarray. We found that SB431542 maintains ES cells pluripotency through multiple mechanisms. First of all, a part of self-renewal genes were up-regulated, and most of differential-related genes were down-regulated by SB431542 treatment. Second, GO analysis showed that SB431542 maintains ES cells pluripotency through regulating cell connection and proliferation. Finally, KEGG analysis showed that SB431542 was involved in regulating MAPK, Erb B, VEGF and cell connection signaling pathways.3. SB431542 enhances ES cells pluripotency by activiating Tgf-β-Smad1/5/8 signaling pathway, as well as inhibiting Fgf/Erk and retinoic acid related signaling pathways. Smad7 is a inhibitor of Tgf-β-Smad1/5/8 signaling pathway. When ES cells were under SB431542 treatment, downregulation of Smad7 could be rescued by overexpression of Smad2/3; and upregulation of phosphorylation of Smad1/5/8 and Id gene expression could be suppressed by overexpression of Smad7. These results indicated that SB431542 could activate Tgf-β-Smad1/5/8 signaling pathway indirectly by decreasing the expression of Smad7. Further, when ES cells were induced to differentiate into neural cells by Retinoic acid(RA) treatment, we found that the addition of SB431542 could not only inhibit the process of RA induced differentiation, but also compromise the downregulation of Hoxb caused by RA inducement.4. We also performed deep-sequencing to detect the expression changes of mi RNA in ES cells treated by SB431542. Among the 114 mi RNAs that affected by SB431542 treatment, only 4 of them were upregulated, while the others were downregulated. Except the mi RNAs involved with Tgf-β-Smad2/3 signaling pathway, mi RNAs that downregulated by SB431542 mainly refer to inhibition of pluripotency and promotion of differentiation. All of these results indicated that SB431542 maintain pluripotency by inhibiton of transcription in ES cells.In conclusion, the results suggest that SB431542 is a pluripotent molecular can be used to maintain the undifferentiated state of mouse ES cells. Besides, SB431542 contributes to mouse ES cells pluripotency maintenance mainly by inhibiting ES cells differential processes.更多还原