转录因子Oct4、Sox2和Nanog在早期胚胎发育过程中的表达调控

胚胎发育的分子机理是现今胚胎工程及发育生物学研究迫切需要了解的。由于胚胎发育的早期阶段就是胚胎干细胞阶段,因此两者在研究上有很好的重合性。通过对调节胚胎干细胞基因转录的三个具有代表性的转录因子Oct4、Sox2和Nanog的综述,展现出胚胎发育分子机理研究概况。     

带有穿膜结构域的转录因子蛋白Oct4和Sox2的融合表达

目的:克隆、表达及纯化带有穿膜结构域的转录因子蛋白Oct4和Sox2。方法:根据GenBank中的Oct4和Sox2基因序列,在其3’端引入穿膜结构域11R,并在其两端引入NdeⅠ和XhoⅠ酶切位点,进行全基因合成;将目的基因克隆至pET41a载体,进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21(DE3),经IPTG诱导表达后,对表达产物进行Western印迹鉴定;最后用Ni-NTA亲和层析柱对所获目的蛋白进行纯化。结果:质粒酶切鉴定结果表明带有目的基因的重组质粒构建成功;SDS-PAGE结果显示有相对分子质量约42×103和38×103的特异性蛋白表达条带,经Western印迹证实为目的蛋白;用Ni-NTA亲和层析柱纯化后,得到均一的Oct4和Sox2目的蛋白。结论:得到带有穿膜结构域的转录因子融合蛋白Oct4和Sox2,为今后安全开展诱导性多能干细胞研究奠定了基础。     

米非司酮对早孕绒毛Oct4、Sox2、Nanog 表达影响*

目的:探讨米非司酮对早孕绒毛组织Oct4、Sox2、Nanog mRNA和蛋白水平表达的影响。方法:要求终止妊娠的正常早孕妇女60例,分为:负压吸宫组及服用米非司酮150mg及米索前列醇联合行药物流产组。运用Real-time PCR方法检测两组早孕绒毛组织中Oct4、Sox2、Nanog mRNA的表达;采用免疫组织化学方法检测两组早孕绒毛组织Oct4、Sox2、Nanog蛋白的定位及半定量表达情况,比较两组差异。结果:Oct4、Nanog、Sox2 mRNA在药物流产组早孕绒毛中相对表达量明显低于负压吸宫组,差异有统计学意义(相对表达量分别为:0.15±0.045;0.37±0.053;0.23±0.040,P值均<0.05);Oct4、Nanog、Sox2在药物流产组早孕绒毛蛋白表达量亦明显低于负压吸宫组,差异有统计学意义(药物流产组蛋白表达量分别为13869±541、19251±1503、139492±918明显低于负压吸宫组22017±235、30543±729、37237±710)。结论:米非司酮可以通过抑制早孕绒毛中Oct4、Sox2、Nanog表达,发挥抗早孕作用。     

Dppa2 基因启动子区Oct4 结合位点突变对其活性的影响

目的：探讨Dppa2 基因5'' 端启动子区Oct4 结合位点突变对Dppa2基因启动子活性的影响。方法：PCR 扩增包括Oct4 结合 位点的Dppa2 基因5'' 端转录起始点上游-2439～+293 bp 的启动子序列,片段长度为2732 bp。将该片段连接到pGL3-Basic 载体, 构建野生型pGL3-2439表达载体。采用定点突变法,将-1959～-1957 位碱基的GCA突变成TAG,构建Oct4 结合位点突变型 pGL3-mo2439 表达载体。用上述两种表达载体、PGL3-basic 载体和Oct4 表达载体分别瞬时转染HEK 293 细胞。细胞培养48 h 后,利用双荧光素酶报告系统测定各组细胞表达的荧光素酶的相对活性。结果：经琼脂糖凝胶电泳及测序鉴定,证实野生型 (pGL3-2439)和突变型(pGL3-mo2439)载体构建成功。荧光素酶活性测定结果显示,转染Dapp2 基因启动子野生型pGL3-2439 表 达载体的细胞组荧光素酶的相对活性为16.307,突变型pGL3-mo2439 表达载体的细胞组荧光素酶的相对活性为10.634。Oct4 结 合位点突变后,Dppa2 基因启动子区转录活性较野生型降低了35 %。结论：Dppa2基因5''端启动子区-1959～-1957 位的Oct4 结 合位点突变可能导致Dppa2 基因启动子活性下降     

Over-expression of Oct4 in human esophageal squamous cell carcinoma

The Octamer 4 gene (Oct4) is a master pluripotency controller that has been detected in several types of tumors. Here, we examine the expression of Oct4 in human esophageal squamous cell carcinoma (ESCC). We found that punctate Oct4 protein was expressed in most (93.7%) ESCC samples but it was not observed in esophageal mucosa. Some ESCC cells had the capacity to form tumorospheres; those with an Oct4⁺-rich cell phenotype had increased proliferation and Oct4 mRNA levels compared to those of differentiated cells in culture or xenograft tumors. The over-expression of Oct4 in ESCCs suggests that it is a potential target for ESCC therapy. Oct4 could be a useful tumor marker in an immunohistochemical panel designed to differentiate between ESCC and esophageal mucosa. Expression of Oct4 in tumorospheres might indicate the presence of a population of ECSCs and its expression in xenograft tumors suggests that Oct4 is also associated with tumor metastasis.     

Sox regulates transcription of the sea urchin arylsulfatase gene

A 50 bp region from -194 bp to -144 bp of the arylsulfatase gene (HpArs) of the sea urchin, Hemicentrotus pulcherrimus, is related to the temporally regulated expression of this gene. This region contains a Sox (Sry-related HMG box)-binding site, and the introduction of sequence mutations to this site significantly reduced the activity of the HpArs promoter, even in the presence of the C15 enhancer, which consists of HpOtx and CAAT motifs. A protein that binds to the Sox-binding site in the 50 bp region of the HpArs gene was detected in nuclear extracts of mesenchyme blastulae and a protein synthesized in vitro using SoxB1 cDNA of another sea urchin, Strongylocentrotus purpuratus, also bound to this Sox site. These results suggest that HpSox, which is maternally expressed and remains abundant by the pluteus stage, is clearly implicated in regulation of the HpArs gene. The presence of a negatively acting cis element in this 50 bp region has also been detected.     

同源域蛋白Nanog与胚胎干细胞的自我更新研究进展

胚胎干细胞作为一种具有自我更新能力的细胞,可以在体外无限对称性分裂,同时保持未分化状态,具有向各种类型细胞分化的潜能.基于这一特性,胚胎干细胞（embryonic stem cell,ES细胞）有着极其广阔的应用前景。维持ES细胞自我更新的机制至今尚未阐明,推测ES细胞的自我更新机制是一个包括细胞外刺激、细胞内多种因子共同参与的复杂的网络调节系统。近年来发现同源域蛋白Nanog在这个网络调节系统中处于中心地位,对ES细胞自我更新的维持起着关键作用。本文就近年来关于Nanog在ES细胞自我更新维持中的作用,以及它与其他信号通路之间的对话,阐明ES细胞自我更新的维持机制。