CAJ | 학술논문

目的:探讨葛根素对子宫内膜RL95-2细胞P450芳香化酶(aromatase P450,P450arom)表达的影响及其对RL95-2细胞P450arom基因表达的调控作用.方法:将梯度稀释的葛根素在不同时间点加入RL95-2细胞中,分别采用实时聚合酶链反应(real-timepolymerase chain reaction,RT-PCR)检测药物对RL95-2细胞P450arom-Mrna表达水平的影响.以人类基因组DNA为模板扩增人P450arom组织特异性启动子2(PⅡ)转录起始位点上游序列.PCR产物定向克隆到报告基因载体Pgl3-Basic中,构建人P450aromPⅡ系列报告基因质粒,并经限制性内切酶消化鉴定和基因测序证实.瞬时转染系列重组质粒至RL95-2细胞,加入葛根素刺激,分析相关转录因子,利用RT-PCR及Western blotting方法检测相关转录因子在葛根素作用下Mrna及蛋白表达的变化.结果:与溶剂对照组比较,低浓度葛根素会抑制P450aromMrna表达(P<0.01),抑制作用在6、12 h较显著(P<0.01).经酶切鉴定、基因测序、转录因子分析及启动子活性测定,成功构建了人P450aromPⅡ系列重组质粒(-1 017,-763,-543,-234～+8 bp).瞬时转染系列质粒至细胞中,于12 h后加入葛根素(10-7 mol/L)刺激,又12 h后检测荧光蛋白水平.结果表明,-763～-543 bp间的活性被抑制(P<0.05),通过转录因子分析找到了-410/-401 bp的AP-1(c-jun/c-fos).利用RT-PCR、Western blotting及荧光素酶活性检测发现葛根素(10-7mol/L)对AP-1(c-jun/c-fos)的抑制作用平行于对P450arom的作用.结论:低浓度葛根素对子宫内膜RL95-2细胞P450芳香化酶基因表达的抑制作用很可能是受上游调控转录因子AP-1(c-jun/c-fos)的影响.
목적:탐토갈근소대자궁내막RL95-2세포P450방향화매(aromatase P450,P450arom)표체적영향급기대RL95-2세포P450arom기인표체적조공작용.방법:장제도희석적갈근소재불동시간점가입RL95-2세포중,분별채용실시취합매련반응(real-timepolymerase chain reaction,RT-PCR)검측약물대RL95-2세포P450arom-Mrna표체수평적영향.이인류기인조DNA위모판확증인P450arom조직특이성계동자2(PⅡ)전록기시위점상유서렬.PCR산물정향극륭도보고기인재체Pgl3-Basic중,구건인P450aromPⅡ계렬보고기인질립,병경한제성내절매소화감정화기인측서증실.순시전염계렬중조질립지RL95-2세포,가입갈근소자격,분석상관전록인자,이용RT-PCR급Western blotting방법검측상관전록인자재갈근소작용하Mrna급단백표체적변화.결과:여용제대조조비교,저농도갈근소회억제P450aromMrna표체(P<0.01),억제작용재6、12 h교현저(P<0.01).경매절감정、기인측서、전록인자분석급계동자활성측정,성공구건료인P450aromPⅡ계렬중조질립(-1 017,-763,-543,-234～+8 bp).순시전염계렬질립지세포중,우12 h후가입갈근소(10-7 mol/L)자격,우12 h후검측형광단백수평.결과표명,-763～-543 bp간적활성피억제(P<0.05),통과전록인자분석조도료-410/-401 bp적AP-1(c-jun/c-fos).이용RT-PCR、Western blotting급형광소매활성검측발현갈근소(10-7mol/L)대AP-1(c-jun/c-fos)적억제작용평행우대P450arom적작용.결론:저농도갈근소대자궁내막RL95-2세포P450방향화매기인표체적억제작용흔가능시수상유조공전록인자AP-1(c-jun/c-fos)적영향.
Objective: To study the effects of puerarin on the aromatase P450 (P450arom) mRNA expression and the effects of low-dose puerarin on transcription factors of the P450arom gene (PⅡ) 5'-flanking region. Methods: The effects of puerarin on the P450arom mRNA expression were determined by real-time polymerase chain reaction (RT-PCR). The 5'-flanking region was amplified by PCR using human genomic cDNA as a template. By means of the restriction sites and sequence confirmation, the PCR product was cloned into reporter vector. Series of sequential deletion reporter constructs were transiently transfected into RL95-2 cells which were treated with or without puerarin. Luciferase activity was measured by DuaI-Luciferase Reporter Assay System and Luminoskan Ascent luminometer. Furthermore, by using web-based search program, the most possible cis-acting elements and transcription factors were evaluated.Results: The data demonstrated that low-dose puerarin treatment could decrease P450arom expression at mRNA level compared to dimethyl sulphoxide (DMSO) treatment (P<0.01 ), and puerarin (10<'-7> mol/L) had a time-course effect on P450arom mRNA expression, which reached the bottom at 12 h (P<0. 01). Cells transfected with the --763/+8 bp constructs showed decrease in relative luciferase activity after puerarin (10<'-7>mol/L) treatment compared to DMSO treatment (P<0.05), indicating an essential regulatory site between --763 bp and --543 bp responsible for the transcription suppression by puerarin. Furthermore, the most possible transcription factors, which turned out to be AP-1 (c-jun/c-fos) at --410/--401 bp were also evaluated. The activity of exogenous AP-1 was reduced after 12 hours of puerarin treatment OP<0.05). The inhibition of C-lUn mRNA also showed a time-course effect, which bottomed out at 12 h in parallel with that of P450arom(P<0.01). The protein level of c-jun was also down-regulated by puerarin (10<'-7> mol/L) treatment at 12h.Conclusion: The suppression of P450arom expression and activity may be associated with the down-regulation of transcription factor AP-1/c-jun. This partially explains the mechanisms whereby puerarin treats endometriosis.