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山东大学学报(医学版) ›› 2013, Vol. 51 ›› Issue (10): 42-48.

• 基础医学 • 上一篇    下一篇

人参皂苷Rb1对NEP基因启动子活性的影响

张晓金1，孙高英1，杨玲玲2，郭元芳1，郝秀玉1，郝建荣1,毕文祥1   

  1. 1.山东大学医学院生物化学与分子生物学研究所， 济南 250012；
    2.北京市红十字血液中心， 北京 100088
  • 收稿日期:2013-01-23 出版日期:2013-10-10 发布日期:2013-10-10
  • 通讯作者: 毕文祥， E-mail：biwenxiang@sdu.edu.cn
  • 基金资助:

    国家自然科学基金青年科学基金(8000146)；山东省中青年科学家科研奖励基金(BS2010YY035)

Effects of ginsenoside Rb1 on the activity of the NEP gene promoter

ZHANG Xiao-jin1, SUN Gao-ying1, YANG Ling-ling2, GUO Yuan-fang1, HAO Xiu-yu1, HAO Jian-rong1, BI Wen-xiang1   

  1. 1. Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, China;
    2. Beijing Red Cross Blood Center, Beijing 100088, China
  • Received:2013-01-23 Online:2013-10-10 Published:2013-10-10

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摘要：

目的  构建NEP启动子缺失体-荧光素酶报告基因质粒，探查神经内肽酶(NEP)基因启动子中与人参皂苷Rb1影响NEP启动子活性有关的位点。方法   用NEP基因5′上游启动区2.4kb片段和荧光素酶报告基因载体pGL3-basic构建NEP启动子-荧光素酶报告基因质粒pGL3-nep2.4；用Erase-a-Base System对pGL3-nep2.4质粒2.4kb DNA插入片段5′端进行缺失，构建NEP启动子缺失体-荧光素酶报告基因质粒；用Rb1处理NEP启动子缺失体转染的人神经母细胞瘤SH-SY5Y细胞，检测双荧光素酶活性，观察Rb1对NEP启动子活性的影响。结果  成功构建了含NEP启动子DNA序列的重组质粒pGL3-nep2.4。荧光素酶活性检测显示，转染pGL3-nep2.4质粒的SH-SY5Y细胞荧光素酶活性是转染pGL3-basic的13.1倍(P<0.01)。Rb1处理可使转染pGL3-nep2.4质粒的SH-SY5Y细胞荧光素酶活性增加，其是对照组的2.9倍(P<0.01)；细胞荧光素酶活性检测亦显示，NEP基因上游启动区-894~-857bp(Region Ⅰ)及-100~-82bp(Region Ⅳ)片段缺失后，启动子活性分别是缺失前的29%(P<0.01)和25%(P<0.01)，-559~-534bp(Region Ⅱ)及-223~-179bp(Region Ⅲ)片段缺失后，启动子活性分别是缺失前的5.12倍(P<0.01)及1.81倍(P<0.01)。Rb1处理后，RegionⅠ、Ⅱ和Ⅲ缺失体质粒转染细胞荧光素酶活性均与对照组无统计学差异(P>0.05)，Region Ⅳ缺失质粒pGL3-226转染细胞荧光素酶活性也与对照组无明显差异(P>0.05)，而含Region Ⅳ质粒pGL3-244转染细胞荧光素酶活性则是对照组的1.61倍(P<0.01)。结论  NEP基因5′上游2.4kb DNA片段有较强的启动子活性，Rb1能明显增加其活性。NEP基因2.4kb DNA片段有2个正调控区(RegionⅠ和Region Ⅳ)和2个负调控区(RegionⅡ和Region Ⅲ)，Rb1通过Region Ⅳ发挥正调控作用。

关键词: 人参皂苷Rb1；神经内肽酶；启动子

Abstract:

Objective  To construct a series of neprilysin (NEP) gene promoter deletion mutant-luciferase reporter plasmids and explore sites in the NEP promoter region that are related to the regulation of the NEP promoter activity by ginsenoside Rb1. Methods  The NEP promoter-luciferase reporter gene plasmid pGL3-nep2.4 was constructed by using a 2.4kb DNA fragment upstream of the NEP gene and the luciferase reporter vector pGL3-basic. A series of 5′ NEP promoter deletion mutant luciferase reporter plasmids derived from pGL3-nep2.4 were obtained via Erase-a-Base System. Human neuroblastoma SH-SY5Y cells transfected with the mutants were treated with Rb1, and the NEP promoter activities in them were determined by dual-luciferase reporter assay. Results  The recombinant pGL3-nep2.4 plasmid containing the NEP promoter was successfully constructed. Dual-luciferase reporter assay showed that the luciferse activity in SH-SY5Y cells transfected with pGL3-nep2.4 was 13.1 times of that in the cells transfected with pGL3-basic(P<0.01). After SH-SY5Y cells transfected with pGL3-nep2.4 were treated with Rb1, the luciferase activity in them was 2.9 times of that in the control cells (P<0.01). Dual-luciferase reporter assay also showed that the NEP promoter activity decreased to 29% (P<0.01) or 25%  (P<0.01) of that in the corresponding control group after the -894  to -857 bp (Region Ⅰ) or -100  to -82bp (Region Ⅳ) region upstream of the NEP gene was deleted, whereas it increased to 5.12 (P<0.01) or 1.81 times (P<0.01) of that in the corresponding control group after the -559  to -534bp (Region Ⅱ) or -223  to -179bp (Region Ⅲ) region upstream of the NEP gene was deleted, respectively. After Rb1 treatment, the luciferase activity in SH-SY5Y cells transfected with the mutant deleting Regions Ⅰ, Ⅱ or Ⅲ was no different from that in the control cells (P>0.05), and in SH-SY5Y cells transfected with the mutant pGL3-244 containing Region Ⅳ, it was 1.61 times of that in the control cells (P<0.01) while in SH-SY5Y cells transfected with the mutant pGL3-226 containing no Region Ⅳ, it was different from that in the control cells (P>0.05). Conclusion  The 2.4kb DNA fragment upstream of the NEP gene has the strong promoter activity that could be increased by Rb1. Two positive (Region Ⅰ and Ⅳ) and two negative (Region Ⅱ and Ⅲ) regulatory regions are identified in the NEP promoter, and the increase of the NEP promoter activity caused by Rb1 is related to Region IV.

Key words: Ginsenoside Rb1; Neprilysin; Promoter

中图分类号: 

  • R741.02
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