【摘要】 目的:探讨雌激素对载脂蛋白(Apo)AⅠ基因启动子不同区域转录调节活性的影响。方法:利用含荧光素酶报告基因的表达载体pGL2构建携带ApoAⅠ基因启动子不同区域片段的重组质粒和同时携带ApoCⅢ/AⅣ基因片段的重组质粒。阳离子脂质体法将重组质粒与pRL-null内参质粒共转染HepG2细胞,加入10μmol/L雌激素刺激24h检测细胞荧光素酶报告基因的荧光强度,以反映ApoAⅠ的转录水平。结果:pGL2/-256AⅠ,pGL2/-2500AⅠ,pGL2/-256AⅠCⅢAⅣ及pGL2/-2500AⅠCⅢAⅣ转染后雌激素组荧光素酶相对活性明显高于对照组(P<0.05);而pGL2/-41AⅠ及pGL2/-41AⅠCⅢAⅣ质粒转染后雌激素组与对照组荧光素酶活性差异无统计学意义(P>0.05)。结论:ApoAⅠ启动子-256~-41区域可能包含与雌激素作用相关的反应元件,受雌激素刺激后转录活性增强。更多还原

【Abstract】 Objective: To analyze the modulating mechanism of estrogen on different fractions of apolipoprotein (apo) AⅠgene promoter. Methods: The recombinants carrying different length DNA fragments of Apo AⅠ gene promoter with or without a 7 kb region ApoCⅢ/AⅣ intergenic region in basic pGL2 vector that containing lueiferase reporter gene were constructed. The final recombinants were transfected with pRL-null vector as an internal control to HepG2 cells by the cationic lipid method. The cells were treated with estradiol at 10 μmol/L concentration for 24 h, and then cells were collected and analysed. The activities of these luciferase enzymes were measured with Dual-GloTM Luciferase Assay System. Results: The recombinant expressions of luciferase enzymes including pGL2/-256AⅠ, pGL2/-2500AⅠ, pGL2/-256AⅠCⅢAⅣ and pGL2/-2500AⅠCⅢAⅣ were significantly increased in estradiol induced experiments, exception of the recombinant plasmids containing only the basal promoter (-41/+397 bp) with or without region of Apo CⅢ/AⅣ compared with those of control group(P < 0.05). Conclusion: The region of -256 bp to-41 bp in Apo AⅠpromoter may be the smallest reactive element for estrogen, which can increase transcription activity after estrogen stimulation.更多还原