人参皂苷Rb1对NEP基因启动子活性的影响  

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

作　　者：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] 

机构地区：[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

出　　处：《山东大学学报（医学版）》2013年第10期42-48,53,共8页Journal of Shandong University:Health Sciences

基　　金：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)

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2.4 kb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2.4 kb 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^-857 bp(RegionⅠ)及-100^-82 bp(RegionⅣ)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^-534 bp(RegionⅡ)及-223^-179 bp(RegionⅢ)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),RegionⅣ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RegionⅣ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2.4 kb DNA片段有较强的启动子活性,Rb1能明显增加其活性。NEP基因2.4 kb DNA片段有2个正调控区(RegionⅠ和RegionⅣ)和2个负调控区(RegionⅡ和RegionⅢ),Rb1通过RegionⅣ发挥正调控作用。Objective To construct a series of neprilysin （NEP） gene promoter deletion mutant-luciferase reporter plas- mids and explore sites in the NEP promoter region that are related to the regulation of the NEP promoter activity by gin- senoside Rbl. Methods The NEP promoter-luciferase reporter gene plasmid pGL3-nep2.4 was constructed by using a 2.4 kb DNA fragment upstream of the NEP gene and the luciferase reporter vector pGL3-basic. A series of 5＇ NEP pro- moter deletion mutant luciferase reporter plasmids derived from pGL3-nep2.4 were obtained via Erase-a-Base System. Human neuroblastoma SH-SY5Y ceils transfected with the mutants were treated with Rbl, and the NEP promoter activi- ties in them were determined by dual-luciferase reporter assay. Results The recombinant pGL3-nep2.4 plasmid contai- ning the NEP promoter was successfully constructed. Duai-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 Rbl, 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 - 82 bp （ 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 -534 bp （ Region II ） or -223 to -179 bp （ Region III ） region upstream of the NEP gene was deleted, respectively. After Rbl 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 Reg

关 键 词：人参皂苷RB1 神经内肽酶 启动子 

分 类 号：R741.02[医药卫生—神经病学与精神病学]