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Cyplal 5' flanking DNA를 이용한 Cyplal전사 조절 인자의 연구

Title
Cyplal 5' flanking DNA를 이용한 Cyplal전사 조절 인자의 연구
Authors
김지은
Issue Date
1999
Department/Major
대학원 약학과
Publisher
이화여자대학교 대학원
Degree
Master
Abstract
Mouse Cyp1a1 5 flanking region이 Cyp1a1의 유전자 발현을 조절하는데 영향을 미치는 인자들을 luciferase reporter system을 사용하여 Hepa-I 세포에서 살펴보았다. Mouse Cyp1a1 5 flanking region을 luciferase gene 앞에 연결한 construct를 Hepa-I 세포에 transfection한 다음 저산소 상태(hypoxia)를 일으키는 cobalt chloride, desferrioxamine, picolinic acid와 일산화 질소에 관계된 화학 물질들인 sodiumnitroprusside, lipopolysaccharide, N^G-nitro-l-arginine, l-arginine을 전처치한 뒤, TCDD 혹은 3-MC를 처치하여 발현되는 luciferase의 활성을 측정하였다. TCDD 혹은 3-MC의 농도별, 시간별 처치에 따른 luciferase의 활성은 대조군에 비해 각각 1000-8000배, 100-400배 증가하였고, 이렇게 증가된 luciferase의 활성은 TCDD 혹은 3-MC의 투여 전에 처치한 hypoxia 유도 시약, nitric oxide 공급 시약, iNOS 유도 시약, flavonoids, dexamethasone에 의해 감소되었다. 그 감소된 정도는 TCDD 혹은 3-MC를 처치했을 때 유도되는 luciferase의 활성을 100%로 보았을 때, hypoxia 유도 화학 물질인 cobalt chloride, desferrioxamine, picolinic acid의 처치시 각각 5-11%, 2-4%, 17-25%로 감소되었고, nitric oxide 공급 시약인 sodiumnitroprusside를 처치했을 때 3-4%로, iNOS 유도 시약인 lipopolysaccharide의 처치시 3-7%로, 또 기질인 l-arginine의 처치시 4%로 감소되었다. 그리고, iNOS의 억제제인 N^G-nitro-l-arginine은 TCDD에 의해 유도되는 luciferase의 활성을 400-711%로 증가시켰으며 이 효과는 l-arginine에 의해 다시 11% 정도로 상쇄되었다. 한편, hypoxia와 nitric oxide간의 상호 교신을 알아보기 위하여 저 산소 상태를 일으키는 물질과 iNOS 억제제를 병용 처치했을 때 저 산소 상태에 의해 감소되었던 luciferase activity가 다시 증가됨을 관찰하였고 그 정도는 각 시약에 의해 감소되어 나타나는 luciferase의 활성을 100%로 보았을 때 cobalt chloride의 경우 500-800%로, desferrioxamine의 경우 720%로, 그리고 picolinic acid의 경우 316-360%로 증가하였다. 또한 flavonoid와 dexamethasone을 TCDD와 병용 처치했을 때도 luciferase의 활성이 감소됨을 관찰했는데 TCDD 혹은 3-MC 단독 처치시의 luciferase 활성을 100%로 보면 quercetin의 처치시 15, 17%로, morin의 경우 35, 27%로, methoxalen의 경우 42.5, 26%로, genistein의 경우 55%, 41%로, dexamethasone의 경우 27, 50%로 각각 감소하였다. 이들 결과로부터 저 산소 상태와 일산화 질소, 그리고 flavonoid 등의 인자들이 TCDD, 3-MC에 의해 유도되는 mouse Cyp1a1 유전자 발현을 저해함을 추측할 수 있었고 저 산소 상태와 일산화 질소의 신호 전달 체계에 상호 교신이 존재함과 이 교신이 Cyp1a1의 유전자 발현에 영향을 미치리라는 것을 추정할 수 있었다. ; In order to study the effect of transcription regulatory factors on the regulation of mouse Cyp1a1 expression, 5 flanking DNA of mouse cytochrome P450 1a1 was cloned into pGL3 basic vector encoding luciferase gene and pmCyp1a1-Luc was transfected into Hepa I cells and various chemicals were treated. Luciferase activity was induced 1000-8000 folds over that of control by 1nM TCDD (2,3,7,8-tetrachloro-p-dioxin) treatment and 100-400 folds over that of control by 1nM 3-MC treatment and these inductions were dose dependent. To find out the effect of hypoxic condition on the regulation of Cyp1a1 gene expression, pmCyp1a1-Luc construct was transfected into Hepa I cells and 100μM cobalt chloride, hypoxia mimicking reagent, 100μM desferrioxamine (DFX), iron-chelating agent, or 100μM picolinic acid (PA), another iron-chelating agent was administered before the treatment of 1nM TCDD or 1nM 3-MC. The luciferase activity induced by TCDD was decreased by all these reagents. These inhibitory effects of cobalt chloride, DFX, or PA on the luciferase activity were dose dependent and abolished by concomitant treatment with 1mM NG-nitro-l-arginine, inducible nitric oxide synthase (iNOS) inhibitor. To study the effect of nitric oxide, which is considered an inhibitor of Cyp1a1 in that it inhibited the transcription and enzyme activity of Cyp1a1, on the regulation of Cyp1a1 gene expression. pmCyp1a1-Luc construct was transfected into Hepa I cells and 100μM sodiumnitroprusside (SNP) which is a nitric oxide donor or 10ng/㎕ lipopolysaccharide (LPS), iNOS inducer, was administered before the treatment of 1nM TCDD or 3-MC. The luciferase activity induced by TCDD was decreased by these two reagents. These inhibitory effects of SNP or LPS on the luciferase activity were dose dependent and abolished by concomitant treatment with 1mM N^G-nitro-l-arginine. And when iNOS inhibitor, N^G-nitro-l-arginine was administered before the treatment with 1nM TCDD, the stimulatory effect of TCDD was enhanced and this effect was abolished by the addition of l-arginine to N^G-nitro-l-arginine pre treatment. Also, I studied the effect of flavonoids such as quercetin, morin, methoxalen, and genistein, and dexamethasone (DEX) by the above same method and the result showed that these flavonoids and dexamethasone also inhibited the luciferase activity induced by TCDD. These data strongly suggest that hypoxic condition downregulates Cyp1a1 gene expression by the mechanism that HIF-1α recruits ARNT (another protein induced by hypoxia) or through nitric oxide that is synthesized by iNOS with HRE (hypoxia response element) in its promoter and might be an inhibitory regulator on the Cyp1a1 gene expression in Hepa cells.
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일반대학원 > 생명·약학부 > Theses_Master
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