GDF15 derived from Senescent Fibroblasts Stimulates Melanogenesis in Human Melanocytes
- 주제(키워드) Skin pigmentation , Senescent fibroblasts , Melanocytes , GDF15 , b-catenin
- 발행기관 아주대학교
- 지도교수 강희영
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029327
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Background Senescent fibroblasts play a role in aging pigmentation. In our previous RNA sequencing data in senescent fibroblasts, the Growth differentiation factor 15 (GDF-15) gene was found to be significantly upregulated. Objective We investigated the role of senescent fibroblast-derived GDF15 in the regulation of human pigmentation. Methods Normal human melanocytes were co-cultured with fibroblasts infected with GDF15 lenti-virus or sh-GDF15 and melanogenesis was analyzed. The pigmentation were also assessed in the ex vivo skin culture. Results The melanin contents and tyrosinase activity were significantly increased in melanocytes co-cultured with fibroblasts infected with GDF15 lenti-virus. The mRNA and protein expression levels of melanogenesis-associated proteins, microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly up-regulated. The GDF15 stimulated b-catenin signaling in melanocytes. Consistently, the pigmentation were significantly reduced in melanocytes co-cultured with fibroblasts infected with sh-GDF15. The stimulatory action of GDF15 in pigmentation was further confirmed in ex vivo cultured skin. Conclusion Senescent fibroblast-derived GDF15 stimulates skin pigmentation and it may play a role in the aging pigmentation.
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ABSTRACT ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ⅰ
TABLE OF CONTENTS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ⅱ
INTRODUCTION ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1
Ⅱ. MATERIALS AND METHODS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 4
1. Cell culture ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 4
2. Enzyme-linked immunosorbent assay (ELISA) ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5
3. Senescence Associated b-Galactosidase (SA-b-Gal) Staining ‧‧‧‧‧‧‧‧ 5
4. In vitro model of senescent fibroblasts ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5
5. Ex vivo skin organ culture and pigmentation assay in cultured skin ‧ 6
6. Melanin content and tyrosinase activity assay ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 6
7. Lentivirus and adenovirus production ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 7
8. Semiquantitative real-Time PCR Analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 7
9. Western blot analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 8
10. Immunocytochemistry and immunohistochemical analysis ‧‧‧‧‧‧‧‧‧‧‧ 8
11. Statistical analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 9
Ⅲ. RESULTS ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 10
1. UV induced senescent fibroblasts stimulate skin pigmentation.‧‧‧‧‧‧ 10
2. GDF15 expression is increased in senescent fibroblasts and photo-aged hyperpigmented skin. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16
3. GDF15 derived from senescent fibroblasts stimulates melanogenesis in human melanocytes. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 23
4. GDF15 stimulates b-catenin signaling. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 35
Ⅳ. DISCUSSION ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 40
Ⅴ. REFERENCES ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 45
국문요약 ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 50
