臺灣博碩士論文加值系統

不論在醫學美容或臨床上，抑制皮膚黑色素(melanin)生成皆是極為重要的一項需求，雖然目前已知藥物中已有部分應用於抑制黑色素生成之研究，但仍然無法達到最佳效果。幾丁聚醣(chitosan)為一種具有良好生物相容性及抗菌性的天然材料，此項研究係探討幾丁聚醣對皮膚美白之效果，更針對三個階段（黑色素生成、黑色素小體(melanosome)運送及角質細胞對黑色素小體進行胞吞作用）分別作探討。欲釐清幾丁聚醣之特性對抑制黑色素生成所造成的影響，我們也將不同分子量及不同去乙醯度(degree of deacetylation, D.D.)之幾丁聚醣與B16F10黑色素瘤細胞(melanoma cells)進行培養進而探討在基礎條件或有加黑素細胞激素（alpha melanocyte-stimulation hormone, αMSH）刺激條件下對黑色素生成量進行分析。此外，我們也建立了一個共培養系統研究人類初代黑色素細胞(melanocytes)與人類角質細胞株(HaCaT)欲研究於幾丁聚醣培養環境下之相互作用。研究結果指出不同分子量之幾丁聚醣對於抑制黑色素生成之效果相同，而增加其去乙醯度則會使黑色素生成量顯著減少。並且，幾丁聚醣於前述研究美白效果之三個階段（黑色素生成、黑色素小體運送及角質細胞對黑色素小體進行胞吞作用）皆有顯著抑制的結果。根據結果，由於幾丁聚醣對於皮膚美白有全面性效用，因此我們認為這項研究將可提供一種方法利用幾丁聚醣達到有效抑制黑色素生成之目的。

Decreasing skin pigmentation is an unmet need encountered in various medical or cosmetic conditions. Although some pharmaceutical agents are available for inhibiting melanogenesis, their effects are not ideal. Chitosan, a natural compound extracted from shell fish sources, is a well-known biocompatible and anti-bacterial material. The present research aimed to investigate the skin whitening effects of chitosan on three related processes—melanin synthesis, melanosomes release and melanosomes uptake; and evaluate the possible mechanisms involved. To clarify how the characteristic of chitosan inhibited the melanin synthesis, different molecular weight and different degree of deacetylation (D.D.) of chitosan were added into the culture medium with B16F10 melanoma cells in the basal or αMSH-stimulated condition. Also, our co-culture system is able to study the melanosomes release and uptake between human melanocytes and human keratinocytes HaCaT cell line under the presence of chitosan. Our results have revealed that molecular weight of chitosan would not affect melanogenesis while increasing D.D. of chitosan would enhance the depigmentation effect. Moreover, chitosan can significantly inhibit melanin synthesis and melanosomes release on pigment cells; and lessen melanosomes uptake on keratinocytes. Given the inhibition effect of chitosan on these three processes, we suggest that chitosan is a potential candidate as the treatment of hyperpigmentation disorders.

中文摘要 i
ABSTRACT ii
CONTENTS iii
LIST OF FIGURE vi
Chapter 1 Introduction 1
1.1 Cells 1
1.1.1 Melanocytes 1
1.1.2 Keratinocytes 3
1.2 Melanogenesis 4
1.2.1 Biosynthesis Pathway 4
1.2.2 Regulation by UVB, Endocrine, Paracrine, and Autocrine Factors 5
1.3 Hyperpigmentation Disorders 7
1.4 Current Clinical Treatment of Hyperpigmentation 8
1.4.1 Regulation of Melanogenic Enzymes 8
1.4.2 Inhibition of Melanosome Transfer 10
1.4.3 Dispersion of Melanin and Acceleration of Skin Turnover 10
1.4.4 Other Treatment of Pigmented Lesions 10
1.5 Notable Depigmenting and Lightening Agents 11
1.5.1 Chemical Agents 11
1.5.2 Biological Agents 14
1.6 Establishment of a Melanocytes-Keratinocytes Co-culture System 14
1.7 Chitosan 15
1.8 Motive and Aims 17
1.9 Research Framework 19
Chapter 2 Materials and Methods 20
2.1 Materials 20
2.1.1. Chemicals and Reagents 20
2.1.2. Cell Culture 21
2.2 Experimental Equipments 21
2.3 Solution Formula 21
2.3.1. DMEM-HG Culture Medium 21
2.3.2. Modified Melanocyte Medium (MMM) 22
2.3.3. Treatment-containing Culture Medium 22
2.4 Methods 23
2.4.1. Cell Culture 23
2.4.1.1. Melanoma B16F10 23
2.4.1.2. Human Melanocytes 23
2.4.1.3. Human Keratinocytes Cell line HaCaT 23
2.4.1.4. Establishment of a Melanocytes-HaCaT Co-culture System 24
2.4.2. Preparation of various modified chitosan 25
2.4.2.1. FITC-labeled chitosan 25
2.4.2.2. Different Degree of Deacetylated Chitosan 26
2.4.3. Isolation of Melanosomes Secreted by Melanocytes 27
2.4.4. Cell Viability Test 28
2.4.5. Determination of Melanin Content 28
2.4.5.1. Melanoma B16F10 and Human Melanocytes 28
2.4.6. Determination of Tyrosinase activity 29
2.4.7. Observation of FITC-chitosan in culture medium 30
2.4.8. Examination of Melanosomes Uptake in HaCaT 30
2.4.9. Western Blot analysis 31
2.4.10. Statistical Analysis 31
Chapter 3 Results & Discussions 32
PART I. Chitosan effect on Melanogenesis 32
3.1. A Dose Dependent Effect of Exogenous Chitosan On Depigmentation 32
3.1.1. Cell Viability 32
3.1.2. Inhibition on Melanin Content 32
3.2. The Effects of Chitosan Monomers on Depigmentation 33
3.2.1. Cell Viability 33
3.2.2. Inhibition on Melanin Content 34
3.3. The Effects of Chitosan with Different Molecular Weight on Depigmentation 35
3.3.1. Cell Viability 35
3.3.2. Inhibition on Melanin Content 35
3.4. The Effects of Chitosan with Different Degree of Deacetylation on Depigmentation 35
3.4.1. The Characterization of Chitosan for the Degree of Deacetylation (D.D.) 35
3.4.2. Cell Viability 36
3.4.3. Inhibition on Melanin Content 37
3.5. Observation of Melanoma Cells Cultured with FITC-chitosan 38
3.6. Investigating the Role of Chitosan in Molecular Mechanisms of Melanogenesis Inhibition 39
3.6.1. Inhibitory Effect on the Melanogenic-related Protein Expression 39
3.6.2. Inhibition on Tyrosinase Activity 40
PART II. Chitosan Effect on Melanosomes Release and Uptake 42
3.7. The Effect of Chitosan on Melanosomes Release from Melanocytes 42
3.8. The Effect of Chitosan on Melanosomes Uptake on Keratinocytes Phagocytosis 43
3.8.1. Observation in a Keratinocytes Mono-culture System 43
3.8.2. Observation in a Melanocytes-HaCaT Co-culture System 44
3.8.3. The Effects of Chitosan on PAR-2 Expression 45
3.9. The Overall Effects of Chitosan on Depigmentation, Melanosomes Release and Uptake in a Melanocytes-Keratinocytes Co-culture System 46
Chapter 4 Conclusion 47
Chapter 5 References 49
Chapter 6 Figures 54

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