摘要
目的:了解甘草甜素对基质金属蛋白酶组织抑制因子-1基因启动子活性的调节作用,探讨甘草甜素的抗纤维化机制.方法:应用聚合酶链反应(PCR)扩增基质金属蛋白酶组织抑制因子-1基因启动子,命名为TIMP-1P,以T-A克隆法,将TIMP-1P基因片段连入载体pGEM-Teasy.将获得的质粒pGEMTeasy-TIMP-1P,与报告质粒pCAT3-basic分别用NheI和XhoI双酶切后构建TIMP-1启动子报告基因表达载体pCAT3-TIMP-1P,以重组表达质粒pCAT3-TIMP-1P瞬时转染HepG2细胞,4h后以0.1mmol甘草甜素刺激,48h后收获细胞.同时以转染pCAT3-basic的HepG2细胞为阴性对照,每组设复孔对照,用酶联免疫吸附法(ELISA)检测标本在415nm波长的吸光度值,其数值反映细胞中氯霉素乙酰转移酶(CAT)的表达水平.结果:构建的报告基因表达载体pCAT3-TIMP-1P经过序列分析和酶切鉴定正确.瞬时转染HepG2细胞后,pCAT3-basic的吸光度值为0.004±0.002,pCAT3-TIMP-1P为2.329±0.685,经方差分析两者差异有统计学意义(F=26.075,P<0.05),pCAT3-TIMP-1在HepG2细胞中能够启动CAT的表达.转染HepG2细胞的pCAT3-TIMP-1P经0.1mmol甘草甜素刺激后,其吸光度值为0.268±0.009,同期未经甘草甜素刺激的pCAT3-TIMP-1P吸光度值为0.490±0.153,方差分析两者差异有统计学意义(F=35.775,P<0.05).结论:甘草甜素可下调TIMP-1启动子的转录活性,抑制TIMP-1的基因表达.
AIM： To investigate the regulatory effect of glycyrrhizin on the tissue inhibitor of metalloproteinases-1（TIMP-1） expression and to explore its anti-fibrosis mechanism. METHODS： The TIMP-1 promoter was amplified by polymerase chain reaction（PCR）, and the product was named TIMP-1P. The TIMP-1P was cloned into pGEM- Teasy vector to obtain pGEM-Teasy-TIMP-1 P, and then the product and pCAT3-basic vector were digested by NhelⅠ and XhoⅠ to construct pCAT3-TIMP-1P. Then pCAT3-TIMP-1P was transfected into HepG2 cells and the cells were treated with 0.1 mmol glycyrrhizin for 48 h. The HepG2 cells transfected with pCAT3-basic were used as negative controls. The expression level of chloramphenicol acetyltransferase（CAT） in HepG2 cells was detected by enzyme-linked immunoassay （ELISA）. RESULTS： The expressive vector pCAT3-TIMP-1P was constructed and confirmed by restriction enzyme digestion and sequencing. The optical density（OD） of the cells transfected with pCAT3-TIMP-1P was signifi- cantly higher than that with pCAT3-basic（2.329 ± 0.685 vs 0.004 ± 0.002, F = 26.075, P 〈0.05）. After treatment with glycyrrhizin, the expression of CAT in the HepG2 cells transfected with pCAT3-TIMP-1P was notably decreased as compared with that in the same cells without glycyrrhizin treatment（OD： 0.268 ± 0.009 vs 0.490 ± 0.153, F= 35.775, P 〈0.05）. CONCLUSION： Glycyrrhizin can down-regulate the activity of TIMP-1 gene promoter as well as inhibit the expression of TIMP-1.
出处
《世界华人消化杂志》
CAS
北大核心
2005年第18期2183-2187,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金攻关项目
No.C03011402
No.C30070689
军队九五科技攻关项目
No.98D063
军队回国留学人员启动基金项目
No.98H038
军队十五科技攻关青年基金项目
No.01Q138
军队十五科技攻关面上项目
No.01MB135~~
