멜라닌 세포에서 tetrahydroquinoline 유도체의 MITF 발현 억제

다국어 초록 (Multilingual Abstract)

Alpha-melanocyte stimulating hormone (α-MSH) is the most important melanocyte-stimulating hormone in stimulating melanogenesis. α-MSH binds to melanocortin 1 receptor (MC1R), which induces the activation of adenylyl cyclase, followed by cAMP production. cAMP leads to phosphorylation of CREB transcription factors, which in turn stimulate microphthalmia-associated transcription factor (MITF) promoter activation. MITF directly binds to the promoter regions of tyrosinase. In turn, MITF induces expression of tyrosinase for skin pigmentation. The present study aimed to investigate the antimelanogenic effect of 1-propionyl-N-(2-hydroxyphenyl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (EL-B-02) on α-MSH-activated B16F0 melanoma cells. EL-B-02 did not display any significant cytotoxicity up to 100 μM. EL-B-02 efficiently reduced melanin productions induced by α-MSH, dibutyryl-cyclicAMP (db-cAMP) or histamine. Moreover, to investigate the in vivo effect of EL-B-02, i carried out topical treatment with EL-B-02 in UV-irradiated dorsal skins of guinea pig. EL-B-02 suppressed skin pigmentation as compared with the vehicle group. In addition EL-B-02 decreased UV-B induced tyrosinase/MITF expression in guinea pig skin tissue. Furthermore, EL-B-02 decreased α-MSH-induced mRNA and protein level of tyrosinase, also decreased α-MSH-induced transcriptional activity of tyrosinase, but EL-B-02 did not directly affect catalytic activity of cell-free tyrosinase. EL-B-02 decreased α-MSH-induced mRNA and protein level of MITF, also decreased transcriptional activity of MITF. EL-B-02 did not affect CREB and β-catenin phosphorylation but EL-B-02 decreased α-MSH-induced protein level of PAX3. Based on the findings, EL-B-02 has inhibitory effects for cellular melanogenesis by decreasing the expression of tyrosinase/MITF, resulting in the suppression of melanin synthesis. However, it is unknown how PAX3 is regulated in melanocytes. So it requires additional experiments. Furthermore, EL-B-02 showed significant depigmenting effects in guinea pig skin. Thus, here i suggest that EL-B-02 may have potential effects for skin whitening.

목차 (Table of Contents)

• I. 서론 8
• II. 실험 재료 및 방법 12
• 1. 실험 재료 12
• (1) 시약 12
• (2) 배지 및 완충 용액 13
• (3) 기기 14
• 2. 실험 방법 15
• (1) 시료 용액의 제조법 15
• (2) 세포 배양법 15
• (3) 세포 생존율 측정법 16
• (4) Melanin 측정법 17
• (5) Tyrosianse 활성 측정법 19
• (6) 단백질 발현 측정법 20
• (7) 역전사 중합효소 연쇄반응 21
• (8) 전사 활성 측정법 21
• (9) 핵 단백 추출법 21
• (10) Fontana-Masson 염색법 22
• (11) UV radiation 22
• (12) 통계처리 24
• III. 결과 25
• 1. 동물모델에서의 멜라닌 저해에 대한 효과 25
• 2. 동물조직에서의 Tyrosinase와 MITF의 생합성에 대한 효과 27
• (1) Tyrosinase와 MITF의 단백질 발현에 대한 효과 33
• (2) Tyrosinase와 MITF의 mRNA 발현에 대한 효과 33
• 3. 멜라닌 생성에 미치는 효과 29
• 4. 세포 생존율 미치는 효과 29
• 5. Tyrosinase activity에 대한 효과 31
• 6. Tyrosinase의 생합성에 대한 효과 33
• (1) Tyrosinase의 단백질 발현에 대한 효과 33
• (2) Tyrosinase의 mRNA 발현에 대한 효과 33
• 7. MITF의 생합성에 대한 효과 35
• (1) MITF의 단백질 발현에 대한 효과 35
• (2) MITF의 mRNA 발현에 대한 효과 35
• 8. Transcription factor에 대한 효과 37
• 9. Inhibitor들에 따른 멜라닌 생성에 미치는 효과 39
• 10. Inhibitor에 따른 MITF의 생합성에 대한 효과 42
• (1) Inhibitor에 따른 MITF의 단백질 발현에 대한 효과 42
• (2) Inhibitor에 따른 MITF의 mRNA 발현에 대한 효과 42
• IV. 고찰 51
• V. 결론 56
• 참고문헌 57