【摘要】 为探究生长阻滞和DNA损伤诱导蛋白45γ(Growth arrest and DNA-damage-inducible protein GADD45 gamma,Gadd45g)对小鼠胚胎干细胞(mouse embryonic stem cells,m ESCs)在体外培养条件下自我更新状态的影响,通过设计并构建含有Gadd45g基因的重组质粒,将其导入m ESCs内,过表达目标基因;在含有白血病抑制因子(LIF)的血清培养条件下,通过细胞计数、碱性磷酸酶染色、qRT-PCR以及免疫荧光等实验手段检测m ESCs的生长情况。结果显示,与对照组相比,过表达Gadd45g基因后,m ESCs的生长速度减缓,碱性磷酸酶活性降低,且中内胚层标志基因的表达水平显著上升。进一步研究发现,在添加LIF的有血清或2i无血清培养体系中,过表达Gadd45g均可以降低细胞内STAT3蛋白的磷酸化水平,由此推断上调Gadd45g的表达会抑制STAT3的活性,从而推动m ESCs向中内胚层分化。研究结果扩大了目前人们对于ESCs分化机制的理解,有利于胚胎干细胞未来的基础研究与安全应用。更多还原

【Abstract】 Embryonic stem cells(ESCs) are isolated from the inner cell mass of pre-implantation blastocyst,which can proliferate spontaneously and infinitely under appropriate culture conditions in vitro(selfrenewal),while maintaining the potential to differentiate into various types of cells derived from the three germ layers,and therefore have important significance not only for tissue repair and regeneration,but also can provide a powerful tool for modeling disease and understanding biological development. Growth arrest and DNA damage inducible protein 45 gamma(Gadd45g) is one of the three members of the GADD45 protein family. It is also known as the cytokine response gene 6(CR6),which can affect the cell cycle and regulates cell growth negatively. In addition,as a well-known emergency response gene,Gadd45 g has been studied in neural precursor cell differentiation and in tumors. There are evidences which have shown that Gadd45 g can be methylated in a variety of tumor cells and has become a potential target for clinical therapy of cancers. Because tumor cells and ESCs have similarity in proliferation characteristic,it will be significant to investigate the effects of Gadd45 g on mouse embryonic stem cells(m ESCs). The recombinant plasmid containing Flag tagged Gadd45 g was constructed and transfected into m ESCs to overexpress the target gene. The qRT-PCR and Western blot were then used to determine the expression level of Gadd45 g in transcriptional and translational aspects respectively.Subsequently,the self-renewal status of Gadd45g-overexpressing m ESCs were detected under the serum culture condition,containing leukemia inhibitory factor(LIF),via cell counting,alkaline phosphatase staining,qRTPCR,and immunofluorescence staining. Among these strategies,the cell counting was used to measure the growth rate of the cells,alkaline phosphatase staining was used to detect the self-renewal degree of the cells,qRT-PCR was used to analyze the expression of self-renewal marker genes and differentiation marker genes which represent different germ layers in the cells and immunofluorescence staining was used to demonstrate the self-renewal marker proteins in the cells. The results shown that compared with control group,overexpression of Gadd45 g can make m ESCs growth rate slow down and lead to alkaline phosphatase activity and expression levels of self-renewal marker genes(Oct4,Nanog and Klf2) decline in the cells,meanwhile the expression levels of mesendoderm marker genes,such as Sox17,Foxa2,T,GSC and Mixl1,were up-regulated obviously,which means these cells tend to exit from self-renewal status and differentiate into mesendodermal stage. As LIF/Stat3 signal pathway is important to m ESC maintenance and is able to inhibit mesendoderm formation in m ESCs,subsequent experiments were conducted to examine whether Gadd45 g could regulate the activity of LIF/STAT3 signal pathway,and the result shown that overexpression of Gadd45 g could reduce the phosphorylation level of STAT3 in both the serum condition added LIF and the serum-free condition with 2i containing two small molecules: CHIR99021 and PD0325901,which inhibit glycogen synthase kinase 3(GSK3) and mitogen-activated protein kinase(MAPK)kinases(MEK proteins),respectively. This result suggested that Gadd45 g may induce mesendodermal formation partially via inhibiting STAT3 activity. The overall results will not only extend the understanding of molecular mechanisms of embryonic stem cell fate determination,but also will be beneficial to ESC basic research and safe application in the future.更多还原