人支气管上皮细胞GCLC基因调控区ARE及E-box顺式调控元件调控功能的研究  

The study on regulatory function of the cis-regulating elements of ARE and E-box in the human GCLC gene

作　　者：邹国明[2] 李冰[1] 冉丕鑫[1] 

机构地区：[1]广州医学院第一附属医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：《中华生物医学工程杂志》2007年第3期133-138,共6页Chinese Journal of Biomedical Engineering

基　　金：广东省自然科学基金团队项目（编号05200239）

摘　　要：目的通过对人γ-谷氨酰半胱氨酸酶催化亚单位（GCLC）基因调控区的ARE及E-box顺式调控元件诱导缺失突变，研究它们在人支气管上皮细胞内对GCLC基因转录调控功能的影响。方法应用PCR方法克隆人GCLC基因调控区的大部分核苷酸序列，构建表达虫荧光索酶报告基因的野生型质粒PL45。采用PCR重叠延伸产生特异位点诱变的方法对ARE及E—box顺式调控元件进行缺失突变，构建相应的突变型质粒PLdel—ARE及PLdel—E—box。通过瞬时基因转染方法将上述质粒转染人支气管上皮细胞系16HBE，观察基因突变后的基因转录调控功能。结果质粒PL45的虫荧光素酶相对活性值为9949．75±1441．33、质粒PLdel—ARE的虫荧光素酶相对活性值为21258．75±1563．64、质粒PLdel-E—box的虫荧光素酶相对活性值为17082．00±89．51。与野生型质粒PL45相比，PLdel—ARE、PIAel-E-box突变型质粒表达的虫荧光酶相对活性值显著上升。结论ARE及E．box顺式调控元件具有负性调控作用。Objective To investigate the regulatory function of the cis- regulating elements of ARE and E-box of the human GCLC gene by gene depletion mutation. Methods The majority regulating region of human GCLC gene was cloned. The wild plasmid PL45 and the mutated plasmids PLdel-ARE and PLdel- E- box were constructed by PCR Gene regulating functions of these plasmids were investigated by transient transfection in the human bronchus epithelial cell line 16HBE. Results The relative activities of the luciferase of the PL45, PLdel-ARE, PLdel- E-box were 9949.75 ±1441.33,21258.75 ±1563. 64 and 17082. 00 ± 89. 51 respectively. Compared with PL45, the expression of PLdel-ARE and PLdel- E-box increased significantly. Conclusion The ARE and E-box cis-regulafing elements down-regulate the transcriptions of the GCLC gene.

关 键 词：基因转录调控 转录调控元件ARE 转录调控元件E—box 诱变 

分 类 号：R[医药卫生]