呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

中国生物工程杂志

研究报告

呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

李升锦周向东

重庆医科大学附属第二医院呼吸科重庆医科大学附属第二医院呼吸科

Analysis of cis-acting elements on respiratory mucin MUC5AC expression and regulation in pulmonary A549 cell line

全文: PDF HTML

摘要： 目的：探讨呼吸道黏蛋白（mucin,MUC）5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法：应用DNA重组技术，构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术，在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白（specificity protein）-1和核因子(nuclear factor, NF)-κB结合位点单独突变体，并测定NE刺激的转染细胞荧光素酶相对活性。结果：成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加，而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体（pGL3E-MUC5AC-NF-кB-MU）荧光素酶相对光强度增加，而NE不能诱导Sp-l结合位点单独突变体（pGL3E-MUC5AC-SP-1-MU）荧光素酶表达增加。结论：MUC5AC 5'上游序列中-324～-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件，位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用，该位点可能作为靶向性基因治疗的关键调控元件。

Abstract: Objective：To study the mechanisms of neutrophil elastase (NE) induced expression of respiratory mucin MUC5AC. Methods：Using gene recombination techniques,four luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter were constructed.Site-directed mutagenesis technique was used to establish mutants of Sp-l and NF-кB site in MUC5AC gene promoter; above plasmids and intracontrol plasmid pRL-TK were co-transfected into human pulmonary A549 cells with eukaryotic gene transfection techniques; the relative luciferase activities were detected in the transfected A549 cells. Results：Series of luciferase reporter gene containing different sequences of human MUC5AC pmmotor were constructed successfully.NE could increase the expression of luciferase reporter gene plasmid containing -1300bp,-689bp and-324bp version of MUC5AC promoter in the transfected A549 cells (P<0.05)，but could not increase the expression of luciferase reporter gene plasmid containing -64bp version of MUC5AC promoter .NE could increase the expression of luciferase reporter gene plasmid containing mutated NF-кB version (P<0.05 vs contro1)of MUC5AC promoter in the transfected A549 cells，The induction by NE decreased markedly when the Sp-l elements in MUC5AC promoter have been mutated. Conclusions：There is an up-regulative element of gene transcription in the region of -324 to-64bp in MUC5AC gene upstream,Sp-l site of promotor mediates NE-induced MUC5AC expression in human A549 cell

收稿日期: 2006-11-21 出版日期: 2007-02-25

通讯作者: 周向东

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	周向东
	李升锦

引用本文:

李升锦,周向东. 呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析[J]. 中国生物工程杂志, .

. Analysis of cis-acting elements on respiratory mucin MUC5AC expression and regulation in pulmonary A549 cell line. China Biotechnology, .

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I2/29

No related articles found!

Viewed

Full text

Abstract

Cited

Shared

Discussed