期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

Tet-on系统诱导esp1基因表达对A_(549)细胞染色体的影响 被引量：1

Effect of conditional expression of esp1 gene by Tet-on system on ploidy in A_549
下载PDF
导出
摘要 目的观察肺腺癌Am细胞经Tet-on系统诱导表达espl基因后其染色体数目的改变。方法利用PTet—on基因表达调控系统，先后将调控质粒Ptet-on和反应质粒PTK-Hyg与PTRE-EGFP-espl以合适的比例用FuGENE6脂质体共转染入A-细胞，经G418和潮霉素筛选成活并带有绿色荧光的细胞为转染成功细胞，挑选8个单细胞克隆并扩增培养，加入强力霉素诱导espl表达。用RT-PCR检测不同浓度强力霉素诱导以及强力霉素诱导不同时间的espl的表达量，并采用传统秋水仙素方法以及流式细胞技术检测诱导不同时间段A549／Ptet-on／TK-Hyg／PTRE-EGFP-espl细胞克隆染色体数目的改变。结果100～200ng／ml浓度范围的强力霉素诱导espl表达量最高，诱导1h即可有espl基因的表达，至48h表达量到达高峰之后逐步减少。在诱导24h后逐步出现染色体数目减少。结论上调espl基因对肿瘤细胞异常染色体有一定的抑制作用，对肿瘤治疗有潜在的应用价值。 Objective To construct a conditional expression vector containing espl in order to observe the effect on the ploidy in A549 cells. Methods Tet-on regulating plasmid was transferred into A549 cells under the Dox induced Tet on regulating system and stable expression of Tet-on was established in An49 cells through G418 selection. The response plasmids of recombinant PTK Hyg and PTRE-EGFP-espl were transferred into positive A549/Tet-on cells,and stable expression of espl was established through hygromycin and green fluorescence selection. Eight single cellular clones were taken and cultivated in amplification. Positive A549/Tet on/TK-Hyg/IRE-EGFP-espl cells,whose expression were induced by Dox showed well dose-response in expression of espl with different concentration and time of Dox induction, were selected by reverse transcriptase-polymerasc chain reaction（RT-PCR）. The changes of chromosome in different A549/Tet-on/TK-Hyg/IRE-EGFP-espl cell clone which were induced different time were detected by the traditional colcbicines method and flow cytometry （FCM）. Results The best Dox concentration induced high cspl expression is from 100ng/ ml to 200ng/ ml. The espl could express after lb induce by Dox and the high expression occured after 48b induction,after that the expression of espl begin to descent. The chromosome of positive A549/Tet-on/TK-Hyg/IRE-EGFP-espl cells had no changes until were induced 24h by Dox. Conclusion The up-regulated expression of espl has certain inhibitory effect on abnormal division of tumor chromosome,so it has potential value in the treatment of cancer.
作者 刘丽莎 刘昕 程英
机构地区 第三军医大学基础医学部分子遗传学教研室
出处 《重庆医学》 CAS CSCD 2006年第20期1862-1864,共3页 Chongqing medicine
关键词 A549细胞 espl PTet-on 强力霉素 染色体 A549 cell espl PTet-on doxycycline chromosome
分类号 R346 [医药卫生—基础医学]
  • 相关文献

参考文献10

  • 1Mark L.Conditional control of gene expression in the mice[J].Nature Rev Genet,2001,8:743
  • 2Jeffrey RG,Jone R.Induce gene expression in the nervous system of transgenicmice[J].Annu Rev Neurosci,1998,21:377
  • 3Gossen M,Bujard H.Tight control of gene expression in mammalian cells by tetracycline responsive promoters[J].Proc Natl Acad Sci USA,1992,89(12):3347
  • 4Ciosk R,Zachariae W,Michaelis C,et al.An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast[J].Cell,1998,93:1067
  • 5Waizenegger I,Gimenez-Abian JF,Wernic D,et al.Regulation of human separase by securin binding and autocleavage[J].Curr Biol,2002,12:1368
  • 6Chestukhin A,Pfeffer C,Millig S,et al.Processing,localization,and requirement of human separase for normal anaphase progression[J].PNAS,2003,100(8):4574
  • 7Nasmyth K,Peter JM,Uhlmann F,et al.Splitting the chromosome cutting the tiesthat bind sister chromatids[J].Science,2000,288:1379
  • 8Uhlmann F.Secured cutting:controlling separase at the metaphase to anaphasetransition[J].EMBO Rep,2001,21(61):487
  • 9由莉越,刘昕.Separase上调对肿瘤细胞染色体分离作用的研究及条件表达载体的构建[J].第三军医大学学报,2005,27(6):497-499. 被引量：4
  • 10罗勇军,刘昕.实时荧光定量PCR标准品的制备及应用[J].重庆医学,2005,34(3):414-415. 被引量：37

二级参考文献10

  • 1Ciosk R, Zachariae W, Michaelis C, et al. An ESp1/PDs1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast[J]. Cell, 1998, 93(6): 1067-1076.
  • 2Jensen S, Segal M, Clarke D J, et al. A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1[J]. J Cell Biol, 2001, 152(1): 27-40.
  • 3Uhlmann F, Wernic M, Poupart E, et al. Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast[J]. Cell, 2000, 103(3): 375-386.
  • 4Waizenegger I, Gimenez Abian J F, Wernic D, et al. Regulation of Human Separase by Securin Binding and Autocleavage[J]. Curr Biol, 2002, 12(16): 1368-1378.
  • 5Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[J]. Proc Natl Acad Sci U S A, 1992, 89(12): 5547-5551.
  • 6Gossen M, Freundlieb S, Bender G, et al. Transcriptional activation by tetracyclines in mammalian cells[J]. Science, 1995, 268(5218): 1766-1769.
  • 7Gupta S, Schoer R A, Egan J E, et al. Inducible,reversible, and stable RNA interference in mammalian cells[J]. Proc Natl Acad Sci U S A, 2004, 101(7): 1927-1932.
  • 8Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference[J]. J Virol, 2003, 77(16): 8957-8961.
  • 9张鋆.荧光实时定量PCR技术初探[J].生命科学趋势,2003,12(1):1-28.
  • 10萨姆布鲁克J 弗里奇EF 曼尼阿蒂斯T.分子克隆实验指南[M].北京：科学出版社,1999..

共引文献39

同被引文献10

  • 1刘丽莎,刘昕.esp1基因上调表达对A549细胞凋亡影响的研究[J].第三军医大学学报,2006,28(8):828-830. 被引量：1
  • 2刘丽莎,刘昕,刘永康.A549细胞转染esp1基因后生物学行为的变化[J].郑州大学学报（医学版）,2006,41(5):884-886. 被引量：1
  • 3Uhlmann F. The mechanism of sister chromatid cohesion[ J]. Exp Cell Res, 2004, 296( 1 ) :80 - 85.
  • 4Oueralt E, Uhlmann F. More than a separase [ J ]. Nat Cell Biol, 2005, 7 (10) : 930 -932.
  • 5Waizenegger I, Gimenez-Abian J F, Wernic D, et al. Regulation of human separase by securin binding and autocleavage [ J ]. Curr Biol, 2002, 12(16): 1368-1378.
  • 6Kumada K, Yao R, Kawaguchi T, et al. The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase[ J]. J Cell Biol,2006,172(6) :835 - 846.
  • 7Nagao K, Yanagida M. Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase[J]. Genes Cells, 2006, 11(3) : 247 -260,
  • 8Sullivan M, Hornig N C, Porstmann T, et al. Studies on substrate recognition by the budding yeast separase [ J ]. J Biol Chem, 2004, 279 (2) : 1191 -1196.
  • 9Shi Q, King R W. Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines [J]. Nature, 2005, 437 (7061) : 1038 - 1042.
  • 10Potempa J, Sroka A, Imamura T, et al. Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of muhidomain protein complexes [J]. Curt Protein Pept Sci, 2003, 4(6) : 397 -407.

引证文献1

重庆医学
2006年 第20期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部