Wnt信号通路关键基因β—catenin在RA诱导的P19胚胎性癌细胞神经分化 …

Ｗｎｔ信号参与了小鼠早期神经发育。我们以往的实验结果表明,Ｗｎｔ信号可引起Ｐ１９胚胎性癌细胞的神经分化。为进一步了解Ｗｎｔ信号在Ｐ１９神经分化过程中行使功能的时间,我们以Ｗｎｔ信号通路关键成员β－ｃａｔｅｎｉｎ是否定位在细胞核中作为考察Ｗｎｔ信号是否能传递到细胞核内调控下游基因活性的指标,分析了Ｗｎｔ信号在Ｐ     

神经钙粘着蛋白在P19神经元分化中的作用

利用ＲＴ－ＰＣＲ技术,我们检测Ｐ１９细胞体外神经元分化过程中神经钙粘着蛋白（Ｎ－ｃａｄｈｅｒｉｎ）的表达模式。结果显示,该基因在上述过程中存在上调和下调过程,与体内中枢神经系统发育过程的表达模式十分相近。在此基础上,我们将神经钙粘着蛋白基因ｃＤＮＡ全长转入Ｐ１９细胞,通过药物筛选,得到稳定表达钙粘着蛋白的细胞株。     

在肾癌细胞系GRC—1细胞周期不同阶段Wnt—5A基因的？…

Ｗｎｔ基因所编码的蛋白质与许多生长因子一样具有分泌型生长因子的结构特点,其家族成员Ｗｎｔ－５Ａ是许多恶性肿瘤的自分泌生长因子,在肾细胞癌中表达显著升高。为研究在细胞周期的不同阶段生长因子ＷＮＴ－５Ａ在转录水平的表达情况,我们采用胸腺嘧啶双阻断及高压笑气处理的方法,使肾细胞癌细胞系ＧＲＣ－１细胞同步化。用半定量反转录多聚酶链反应对处于细胞周期不同阶段的细胞ｃＤＮＡ进行扩增,Ｓ期与Ｇ１,Ｍ期Ｗｎｔ－５     

β—连环素在信息传导中的研究进展

β－连环素（β－ｃａｔ）是实质细胞粘附连接处ｃａｄｈｅｒｉｎ／ｃａｔ复合体的胞内组织部分。近年发现,β－ｃａｔ还参与Ｗｎｔ信息传导途径及酪氨酸蛋白激酶信息传导途径,参与细胞的生长、分化、死亡及癌症发生。本文对β－ｃａｔ的基因、蛋白质结构、βｃａｔ在Ｗｎｔ信息途径及酪氨酸蛋白激酶信息途径的作用作一综述。     

高分化及低分化鼻咽癌细胞株中Epstein－Barr病毒潜伏感染膜蛋白（LMP1）基因的原位杂交与克隆及序列分析

应用染色体原位杂交、ＰＣＲ扩增、克隆及核苷酸序列分析等方法,分析了ＣＮＥ１和ＣＮＥ３细胞株中的潜伏感染膜蛋白（ＬＭＰ１）基因。ＣＮＥ１是来自我国东北的高分化鼻咽癌细胞株,ＣＮＥ３是来自广西的低分化鼻咽癌细胞株。染色体原位杂交结果表明,ＣＮＥ１细胞中ＬＭＰ１基因存在于细胞核内,整合在第一号染色体上,ＣＮＥ３中ＬＭＰ１基因则随机存在于细胞核内及多条染色体上。用ＰＣＲ方法分别从ＣＮＥ１及ＣＮＥ３中扩增得到了ＬＭＰ１基因片段（外显子３）,核苷酸序列分析证明,来自ＣＮＥ１的ＬＭＰ１与来自Ｂ９５－８细胞的ＬＭＰ１核苷酸序列同源性极高,达９９．５％,而ＣＮＥ３的ＬＭＰ１基因与Ｂ９５－８的ＬＭＰ１基因同源性为９３％。     

金属硫蛋白 - 潜伏膜蛋白融合基因的体外转录调控

ＥＢ病毒（ＥＢＶ）是一种与地区性伯基特氏淋巴瘤、鼻咽癌、何杰金氏病等多种人体肿瘤有关的疱疹病毒．已往的研究表明,潜伏膜蛋白（ＬＭＰ）基因是ＥＢＶ最可能的致瘤基因．为制备ＬＭＰ基因转基因小鼠,探讨ＬＭＰ的体内致瘤作用,首先构建了含鼠金属硫蛋白－１（ＭＴ－１）基因调控区和ＬＭＰ基因编码区的ｐＢＲ３２２－ＭＴ－ＬＭＰ质粒,并用电击法将该质粒与ｐＫＪ１－Ｎｅｏ质粒共转染人胃癌细胞株ＭＧＣ,对ＭＴ－ＬＭＰ基因在转染细胞中的整合、转录情况及重金属镉和镍对该融合基因的转录调控进行了研究．结果表明：（１）两质粒共转染效率为８６．７％;（２）ＰＣＲ和Ｓｏｕｔｈｅｒｎ杂交分析显示,完整的ＭＴ－ＬＭＰ基因已整合入转染的ＭＧＣ细胞基因组,且在不同的转染细胞克隆中,ＭＴ－ＬＭＰ基因整合的方式及拷贝数不同,拷贝数从１到１９不等;（３）ＲＴ－ＰＣＲ和Ｎｏｒｔｈｅｒｎ杂交分析证实,ＭＴ－ＬＭＰ基因不仅在转染的ＭＧＣ中能够转录,而且在１０μｍｏｌ／Ｌ镉诱导下,ＭＴ－ＬＭＰ基因转录增强,平均增高约１．４倍．结果说明,在ＭＴ－１基因调控区指导下,ＬＭＰ基因不但有ｍＲＮＡ水平的表达,而且其表达受重金属镉的调控,上述结果为制备ＭＴ－ＬＭＰ转基因小鼠     

人鼻咽癌细胞基因组中瘤基因Ha－ras－1的Dnase Ⅰ超敏感位点的初步研究

ＤＮａｓｅⅠ超敏感位点的研究能够发现潜在的调控基因转录活化的位点,比较正常人外周血有核细胞,淋巴瘤细胞株Ｐ３ＨＲ１和人鼻咽癌低分化磷癌细胞株ＨＯｎＥ１和ＨＮＥ２中Ｈａ－ｒａｓ－１瘤基因的ＤＮａｓｅⅠ超敏感位点发现,只有ＨＯＮＥ１和ＨＮＥ２细胞基因组中存在一个ＤＮａｓｅⅠ超敏感位点,位于第一个外显子上游０．３７ｋｂ处,上述结果提示正常白细胞和Ｐ３ＨＲ１细胞中Ｈａ－ｒａｓ－１基因处于失活状态,而在鼻咽癌细胞基因组中则处于活化状态,它的活化可能与０．３７ｋｂ处的ＤＮＡ序列有密切的关系。     

缺氧诱导因子—1

氧是在细胞内呼吸氧化还原反应中电子的最终接受体。缺氧可以引起急性或长期的生理或病理生理反应,在这些过程中缺氧诱导了许多基因的转录。缺氧诱导因子－１（ＨＩＦ－１）是可与红细胞生成素基因（ＥＰＯ）３＇增强子特异结合的由缺氧诱导的ＤＮＡ结合蛋白,可激活Ｈｅｐ ３Ｂ细胞ＥＰＯ的基因转录。并且由于它可在很多细胞被缺氧诱导,包括生成ＥＰＯ和不生成ＥＰＯ细胞,以及许多缺氧诱导的基因都具有ＨＩＦ－１结合位点等特点     

表面活性物质相关蛋白表达的调控研究进展

特异性的肺表面活性物质相关蛋白（ＳＰ）包括亲水性的ＳＰ－Ａ、ＳＰ－Ｄ和疏水性的ＳＰ－Ｂ、ＳＰ－Ｃ．它们的表达与合成受众多生理、病理因素影响。本文综述了该领域的研究进展。１．ＳＰ表达的组织细胞特异性调控：只有肺内某些细胞（肺泡Ⅱ型细胞、Ｃｌａｒａ细胞等）能合成分泌ＳＰ,这可能是由ＳＰ基因中特定序列决定的,如ＳＰ－Ｂ基因的细胞特异性表达的调控成分。２．ＳＰ表达的发育期调控：ＳＰ基因属发育控制基因家族。在人类妊娠前３ｍｏｎ胎肺中,ＳＰ基本不表达：妊娠１５－１８ｗｋ时,气管、支气管上皮细胞即可见ＳＰ－Ｂ、ＳＰ－ＣｍＲＮＡｓ和表达蛋白,它们可能比ＳＰ－Ａ出现早：妊娠１９－２０ｗｋ时可见ＳＰ－ＡｍＲＮＡ和表达蛋白,胎肺组织在体外无激素条件下培养可很快诱导ＳＰ表达,在妊娠后３ｍｏｎ内,各ＳＰｍＲＮＡｓ及其表达蛋白水平与磷脂水平平行升高,也与ＳＰ降低表面张力的特性逐渐增强相关,羊水中可检出这些蛋白,板层体的出现与ＳＰ－Ｂ的表达密切相关,而比ＳＰ－Ａ的表达早,胎肺发育过程中ＳＰｍＲＮＡｓ增加至少部分是由于其基因转录率升高,可能同时也与翻译增加有关。３．糖皮质激素对ＳＰ表达的调控：糖皮质激素对ＳＰ－Ａ表达的调控极复杂,且与剂量     

大鼠肝纤维化过程中组织金属蛋白酶抑制因子I基因的原位表达

为了解组织金属蛋白酶抑制因子１（ＴＩＭＰ－１）基因在实验性肝纤维化形成中的作用,我们应用地高辛原位杂交技术对大鼠肝组织在四氯化碳（ＣＣｌ４）诱发肝纤维化形成过程中ＴＩＭＰ－１ｍＲ－ＮＡ的表达进行了研究。结果表明,ＣＣｌ４肝损伤早期（４周）肝组织中间质细胞（血管及窦内皮细胞及贮脂细胞）中显示ＴＩＭＰ－１的过度表达;ＣＣｌ４肝纤维化早期（８周）及肝纤维化晚期（１２周）,肝组织中间质细胞的ＴＩＭＰ－１表达持续维持在高水平。结果提示,肝间质细胞（内皮细胞及贮脂细胞）是肝内ＴＩＭＰ－１的主要来源细胞;ＴＩＭＰ－１的异常表达是大鼠肝纤维化过程中较早出现的分子变化,与肝纤维化发生有关;纤维化晚期持续高表达的ＴＩＭＰ－１通过抑制胶原酶而在肝纤维化持续进展中起重要作用。     

A role of N-cadherin in neuronal differentiation of embryonic carcinoma P19 cells.

N-cadherin is one of the important molecules for cell to cell interaction in the development of the central nervous system (CNS). In this report, we have shown that N-cadherin mRNA and protein were increased rapidly in retinoic acid (RA)-induced neuronal differentiation of embryonic carcinoma P19 cells. To explore possible roles for N-cadherin during this process, N-cadherin-overexpressing P19 cell lines were established. These transfected cells could differentiate into neurofilament-expressing neurons in the absence of RA. RT-PCR revealed that the expression patterns of development-related genes, such as Oct-3/4, nestin, Notch-1, and Mash-1 were similar between the transfected P19 cells and the RA-induced wild-type P19 cells during their neuronal differentiation. On the contrary, the Wnt-1 gene was up-regulated in the N-cadherin-overexpressing P19 cells, but could not be detected in the wild-type P19 cells. These results suggest N-cadherin may play a role in neuronal differentiation of P19 cells, possibly through the Wnt-1 signaling pathway.     

Structural characterization of Escherichia coli sialic acid synthase

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.     

DIXDC1 Promotes Retinoic Acid-Induced Neuronal Differentiation and Inhibits Gliogenesis in P19 Cells

Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. XT Jing and HT Wu contributed equally to this work.     

Differential expression of the mouse and human Thy-1 gene in embryonal carcinoma cells.

Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.