γ-谷氨酰半胱氨酸合成酶基因调控的初步研究  

Preliminary study of γ-GCS gene regulation

作　　者：程璘令 李冰[2] 涂洪斌[2] 刘启才[2] 冉丕鑫[1] 

机构地区：[1]广州医学院广州呼吸疾病研究所,510120 [2]广州医学院实验医学研究中心

出　　处：《现代临床医学生物工程学杂志》2006年第4期328-332,共5页Journal of Modern Clinical Medical Bioengineering

基　　金：广东省自然科学基金(06301136)

摘　　要：目的研究抗氧化基因———大鼠γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因的功能区及其调控表达。方法克隆1.76 kb大鼠γ-GCS基因的重链亚单位———GCLC基因的上游调控序列,并构建含荧光素酶基因的报道载体GCLC-Luc(GCLC/pGL-3)。利用嵌顿缺失方法构建GCLC基因的缺失体,并将其表达载体转染大鼠肺泡上皮细胞,在细胞中分析该基因的调控区域。通过该方法初步发现GCLC基因的上游调控序列的-745^-705 bp属负性调控区域。利用EMSA和supersh ift证实大鼠肺泡上皮细胞GCLC基因负调控区域的E-box元件能与转录因子USF1/USF2特异性的结合。将USF的真核表达载体和逆转录病毒表达载体分别导入肺泡上皮细胞,观察GCLC基因的转录活性和GCLC蛋白的表达情况。结果GCLC基因的-403^-111 bp和-705^-613 bp区段属正调控区域;-745^-705 bp属负调控区域。EMS和supersh ift证实上游刺激因子(USF)能与该负调控区域的E-box元件结合抑制GCLC基因的转录和表达。结论发现GCLC基因其中2个正调控区域(-403^-111 bp与-705^-613 bp)和1个负调控区域(-745^-705 bp),其中负调控区域-745^-705 bp上的E-box元件通过与USF结合介导GCLC基因的负性表达调控。Objective To analyze the characteristic of regulatory region of rat γ-GCS catalytic subunit gene （GCLC gene）. Methods A 1. 76 kb 5＇-flanking region of the rat GCLC was cloned and constructed into pGL-3 enhancer vector which included Luciferase reporter gene, Exonuclease Ⅲ was used to cut the 5＇-flanking region of rat GCLC gene unidirectionally into deletion mutants of different length, and all mutants were used to transfect rat alveolar epithelium cells, then the regulatory region of the gene was determined by luciferase activity assay, GCLC gene - 745 - - 705 bp region was found to be a negative regulatory region, EMSA and antibody supershift assay were used to verify that E-box element of the negative regulatory region can be specifically bound by its corresponding transcriptional factors USF1/USF2 in GCLC gene of rat alveolar epithelium cells （ CCL-149 cells）. The role of E-box element of this region was identified by co-transfection of USF1/USF2 expression vectors （or retroviral vectors expressing USF1/USF2） with GCLC promoter-luciferase reporter constructs in the cells. Results Two DNA regions - 403 - - 111 bp and -705- -613 bp of GCLC gene were involved in positive gene expression regulation. A newly identified region - 745 - - 705 bp of GCLC gene was involved in negative gene expression regulation, The interaction between E-box and USF suppressed the expression of GCLC gene. Conclusions Two positive regulatory regions and one negative regulatory region of GCLC gene are found, The E-box element within the negative regulatory region mediates the negative expression regulation of GCLC gene by the binding with transcriptional factors USFI/USF2,

关 键 词：慢性阻塞性肺病 Γ-谷氨酰半胱氨酸合成酶 氧化应激 E-box元件 大鼠 

分 类 号：R346[医药卫生—基础医学]