臺灣博碩士論文加值系統

皮膚是與外界的第一道屏障，具有保護、調節、感受及代謝的功能，而膚質的特性能反映出角質層的健康，且影響影響微生物分布與種類，並存在著互相調節的關係。過去研究也發現皮膚表面的共生細菌、真菌、病毒皆與疾病進展有關。肌膚的保水能力下降會容易導致過敏，如異位性皮膚炎的狀況為表皮水分散失增加所造成以及與特定菌影響相關。而過去的研究顯示皮膚不同性質存在著不同的微生物比例。本篇以菌相研究針對12位無皮膚疾病之台灣人，年齡介於23歲至43歲間，探討臉部兩個區域膚質（額頭與臉頰）與皮膚菌相之間的關係，並檢測三種膚質數據，即皮膚含水量、皮脂率和水份散失率，評估膚質與皮膚菌相和腸道菌相的相關性。研究結果發現額頭的保濕度與臉頰TEWL高低和微生物菌相組成有關。此外，除了臉頰保濕度和額頭皮脂率與其genus含量相關聯性不大外，共28個菌屬分別和額頭與臉頰的皮膚特性具顯著相關性。其中，臉頰的皮脂率有七個菌屬為Proteobacteria菌門，臉頰水分散失共六個菌屬和Actinobacteria與 Bacteroidetes 菌門相關。臉部的菌相比例也與其他研究比較不同國家與地理區域的差異，研究結果將有助於後續皮膚生理臨床研究，最終達到利用菌相來改善皮膚疾病和維持臉部肌膚的健康。

The skin serves as a physical and immunological barrier protecting the human body from invasion by various foreign organisms. In human skin, there are interactions between endogenous innate and adaptive immunity functions and the exogenous environment. Loss of skin hydration leads to skin allergies, such as atopic dermatitis. Patients with atopic dermatitis have symptoms including increases in transepidermal water loss (TEWL) and defects in the barrier function of the stratum corneum, which is also found in other skin diseases such as psoriasis and eczema. Previous studies show that skin microbiomes are highly correlated with the disease progression of skin disorders and with skin health. In this study, we detected facial and cheek bacteria communities and three skin biophysical parameters: hydration, sebum, and TEWL, from 12 healthy Taiwan volunteers, aged 23 to 43 years old. Our purpose was to evaluate the association between the skin microbiota and skin characteristics. The results showed that levels of hydration on the forehead and TEWL on the cheek were associated with the composition of the microbiota. Except for the hydration level on the cheek and sebum levels on the forehead, most of the skin’s biophysical parameters were associated with the genus content. A total of 26 genera were significantly correlated with skin characteristics on the forehead and cheek. Sebum levels on the cheek were correlated with seven genera of Proteobacteria. A total of six genera on the cheek belonging to Actinobacteria and Bacteroidetes phyla were correlated with TEWL level. We believe these findings will contribute to subsequent clinical research, and provide potential clinical applications using microbiota to treat skin diseases and maintain facial skin health.

摘要 .i
ABSTRACT ii
誌 謝. iii
Table of Contents iv
List of Tables vi
List of Figures vii
Chapter 1 Introduction 1
1.1 Background 1
1.1.1 Skin and Skin Microbiota 1
1.1.2 Microorganisms in Humans ..4
1.1.3 The Next-generation Sequencing Technology..5
1.1.4 Human Microbiome Project ..6
1.1.5 Hydration, Sebum, and TEWL in Human Facial Skin ..8
1.1.6 Gut Flora ..9
1.1.7 Gut Microbiota Influence Skin . . 10
1.2 Motivation 10
1.3 Research Goals 12
1.4 Significance of This Research 12
Chapter 2 Related Works . 14
Chapter 3 Materials and Methods . . 16
3.1 Study Design and Sample Collection 16
3.2 DNA Extraction . 17
3.3 Library Construction and Sequencing 18
3.4 Quality Control of 16S rRNA Sequencing Data and Taxonomic Assignment 19
3.5 Bacterial Community Analysis 19
3.6 Statistical analyses . . 20
Chapter 4 Results 21
4.1 Hydration, TEWL and Sebum Levels from Cheek and Forehead Regions.. .. 21
4.2 Sequencing Data and Statistics 22
4.3 Bacterial Diversity and Communities Comparisons of Forehead Hydration Levels . 234.4 Bacterial Diversity and Communities Comparisons of the TEWL Level of the Cheek . 26
4.5 The relationship between skin biophysical parameters and abundance of bacteria ... 28
4.6 Bacterial Diversity and Communities Comparisons in Stools 30
4.7 Comparisons of Bacterial Diversity and Communities among Stool, Forehead, and Cheek . 32
Chapter 5 Discussion and Conclusion 34
5.1 Discussion 34
5.2 Conclusion .. 37
References ... 39
Appendix I - Supplementary Figures . 44

References
1. Grice, E.A., et al., Topographical and temporal diversity of the human skin microbiome. Science, 2009. 324(5931): p. 1190-2.
2. Grice, E.A. and J.A. Segre, The skin microbiome. Nat Rev Microbiol, 2011. 9(4): p. 244-53.
3. Costello, E.K., et al., Bacterial community variation in human body habitats across space and time. Science, 2009. 326(5960): p. 1694-7.
4. Fahlen, A., et al., Comparison of bacterial microbiota in skin biopsies from normal and psoriatic skin. Arch Dermatol Res, 2012. 304(1): p. 15-22.
5. Zeeuwen, P.L., et al., Microbiome dynamics of human epidermis following skin barrier disruption. Genome Biol, 2012. 13(11): p. R101.
6. Roth, R.R. and W.D. James, Microbiology of the skin: resident flora, ecology, infection. J Am Acad Dermatol, 1989. 20(3): p. 367-90.
7. Chiller, K., B.A. Selkin, and G.J. Murakawa, Skin microflora and bacterial infections of the skin. J Investig Dermatol Symp Proc, 2001. 6(3): p. 170-4.
8. Oh, J., et al., The altered landscape of the human skin microbiome in patients with primary immunodeficiencies. Genome Res, 2013. 23(12): p. 2103-14.
9. Capone, K.A., et al., Diversity of the human skin microbiome early in life. J Invest Dermatol, 2011. 131(10): p. 2026-32.
10. Belkaid, Y. and J.A. Segre, Dialogue between skin microbiota and immunity. Science, 2014. 346(6212): p. 954-9.
11. Cogen, A.L., V. Nizet, and R.L. Gallo, Skin microbiota: a source of disease or defence? Br J Dermatol, 2008. 158(3): p. 442-55.
12. Cogen, A.L., et al., Staphylococcus epidermidis antimicrobial delta-toxin (phenol-soluble modulin-gamma) cooperates with host antimicrobial peptides to kill group A Streptococcus. PLoS One, 2010. 5(1): p. e8557.
13. Gallo, R.L. and T. Nakatsuji, Microbial symbiosis with the innate immune defense system of the skin. J Invest Dermatol, 2011. 131(10): p. 1974-80.
14. Statnikov, A., et al., Microbiomic signatures of psoriasis: feasibility and methodology comparison. Sci Rep, 2013. 3: p. 2620.
15. Gao, Z., et al., Substantial alterations of the cutaneous bacterial biota in psoriatic lesions. PLoS One, 2008. 3(7): p. e2719.
16. Kong, H.H., et al., Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis. Genome Res, 2012. 22(5): p. 850-9.
17. Alekseyenko, A.V., et al., Community differentiation of the cutaneous microbiota in psoriasis. Microbiome, 2013. 1(1): p. 31.
18. Fitz-Gibbon, S., et al., Propionibacterium acnes strain populations in the human skin microbiome associated with acne. J Invest Dermatol, 2013. 133(9): p. 2152-60.
19. Oh, J., et al., Biogeography and individuality shape function in the human skin metagenome. Nature, 2014. 514(7520): p. 59-64.
20. Human Microbiome Project, C., Structure, function and diversity of the healthy human microbiome. Nature, 2012. 486(7402): p. 207-14.
21. Belkaid, Y. and S. Tamoutounour, The influence of skin microorganisms on cutaneous immunity. Nat Rev Immunol, 2016. 16(6): p. 353-66.
22. Murillo, N. and D. Raoult, Skin microbiota: overview and role in the skin diseases acne vulgaris and rosacea. Future Microbiol, 2013. 8(2): p. 209-22.
23. Roth, R.R. and W.D. James, Microbial ecology of the skin. Annu Rev Microbiol, 1988. 42: p. 441-64.
24. Somerville, D.A., The normal flora of the skin in different age groups. Br J Dermatol, 1969. 81(4): p. 248-58.
25. Leeming, J.P., K.T. Holland, and W.J. Cunliffe, The microbial ecology of pilosebaceous units isolated from human skin. J Gen Microbiol, 1984. 130(4): p. 803-7.
26. Gardner, S.E., et al., The neuropathic diabetic foot ulcer microbiome is associated with clinical factors. Diabetes, 2013. 62(3): p. 923-30.
27. Caporaso, J.G., et al., QIIME allows analysis of high-throughput community sequencing data. Nat Methods, 2010. 7(5): p. 335-6.
28. Kong, H.H. and J.A. Segre, Skin microbiome: looking back to move forward. J Invest Dermatol, 2012. 132(3 Pt 2): p. 933-9.
29. Aziz, Q., et al., Gut microbiota and gastrointestinal health: current concepts and future directions. Neurogastroenterol Motil, 2013. 25(1): p. 4-15.
30. Franzosa, E.A., et al., Relating the metatranscriptome and metagenome of the human gut. Proc Natl Acad Sci U S A, 2014. 111(22): p. E2329-38.
31. Zupancic, M.L., et al., Analysis of the gut microbiota in the old order Amish and its relation to the metabolic syndrome. PLoS One, 2012. 7(8): p. e43052.
32. Loman, N.J., et al., Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol, 2012. 30(5): p. 434-9.
33. Koren, O., et al., A guide to enterotypes across the human body: meta-analysis of microbial community structures in human microbiome datasets. PLoS Comput Biol, 2013. 9(1): p. e1002863.
34. Human Microbiome Project, C., A framework for human microbiome research. Nature, 2012. 486(7402): p. 215-21.
35. Integrative, H.M.P.R.N.C., The Integrative Human Microbiome Project: dynamic analysis of microbiome-host omics profiles during periods of human health and disease. Cell Host Microbe, 2014. 16(3): p. 276-89.
36. Voegeli, R., et al., A novel continuous colour mapping approach for visualization of facial skin hydration and transepidermal water loss for four ethnic groups. Int J Cosmet Sci, 2015. 37(6): p. 595-605.
37. Muegge, B.D., et al., Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans. Science, 2011. 332(6032): p. 970-4.
38. LeBlanc, J.G., et al., Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Curr Opin Biotechnol, 2013. 24(2): p. 160-8.
39. Carmody, R.N. and P.J. Turnbaugh, Host-microbial interactions in the metabolism of therapeutic and diet-derived xenobiotics. J Clin Invest, 2014. 124(10): p. 4173-81.
40. Sommer, F. and F. Backhed, The gut microbiota--masters of host development and physiology. Nat Rev Microbiol, 2013. 11(4): p. 227-38.
41. Bengmark, S., Gut microbiota, immune development and function. Pharmacol Res, 2013. 69(1): p. 87-113.
42. Goldsmith, J.R. and R.B. Sartor, The role of diet on intestinal microbiota metabolism: downstream impacts on host immune function and health, and therapeutic implications. J Gastroenterol, 2014. 49(5): p. 785-98.
43. Chen, J., X. He, and J. Huang, Diet effects in gut microbiome and obesity. J Food Sci, 2014. 79(4): p. R442-51.
44. Erejuwa, O.O., S.A. Sulaiman, and M.S. Ab Wahab, Modulation of gut microbiota in the management of metabolic disorders: the prospects and challenges. Int J Mol Sci, 2014. 15(3): p. 4158-88.
45. Hold, G.L., et al., Role of the gut microbiota in inflammatory bowel disease pathogenesis: what have we learnt in the past 10 years? World J Gastroenterol, 2014. 20(5): p. 1192-210.
46. Arumugam, M., et al., Enterotypes of the human gut microbiome. Nature, 2011. 473(7346): p. 174-80.
47. Wu, G.D., et al., Linking long-term dietary patterns with gut microbial enterotypes. Science, 2011. 334(6052): p. 105-8.
48. Roberfroid, M., et al., Prebiotic effects: metabolic and health benefits. Br J Nutr, 2010. 104 Suppl 2: p. S1-63.
49. Blottiere, H.M., et al., Human intestinal metagenomics: state of the art and future. Curr Opin Microbiol, 2013. 16(3): p. 232-9.
50. Clemente, J.C., et al., The impact of the gut microbiota on human health: an integrative view. Cell, 2012. 148(6): p. 1258-70.
51. Bowe, W.P. and A.C. Logan, Acne vulgaris, probiotics and the gut-brain-skin axis - back to the future? Gut Pathog, 2011. 3(1): p. 1.
52. Volkova, L.A., I.L. Khalif, and I.N. Kabanova, [Impact of the impaired intestinal microflora on the course of acne vulgaris]. Klin Med (Mosk), 2001. 79(6): p. 39-41.
53. Miyazaki, K., et al., Bifidobacterium fermented milk and galacto-oligosaccharides lead to improved skin health by decreasing phenols production by gut microbiota. Benef Microbes, 2014. 5(2): p. 121-8.
54. Mizukoshi, K. and H. Akamatsu, The investigation of the skin characteristics of males focusing on gender differences, skin perception, and skin care habits. Skin Res Technol, 2013. 19(2): p. 91-9.
55. Enomoto, T., et al., Effects of bifidobacterial supplementation to pregnant women and infants in the prevention of allergy development in infants and on fecal microbiota. Allergol Int, 2014. 63(4): p. 575-85.
56. Oh, J., et al., Shifts in human skin and nares microbiota of healthy children and adults. Genome Med, 2012. 4(10): p. 77.
57. Lax, S., et al., Longitudinal analysis of microbial interaction between humans and the indoor environment. Science, 2014. 345(6200): p. 1048-52.
58. Leung, M.H., D. Wilkins, and P.K. Lee, Insights into the pan-microbiome: skin microbial communities of Chinese individuals differ from other racial groups. Sci Rep, 2015. 5: p. 11845.
59. Mukherjee, S., et al., Sebum and Hydration Levels in Specific Regions of Human Face Significantly Predict the Nature and Diversity of Facial Skin Microbiome. Sci Rep, 2016. 6: p. 36062.
60. Clemente, J.C., et al., The microbiome of uncontacted Amerindians. Sci Adv, 2015. 1(3).
61. Klindworth, A., et al., Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res, 2013. 41(1): p. e1.
62. Caporaso, J.G., et al., Moving pictures of the human microbiome. Genome Biol, 2011. 12(5): p. R50.
63. Oh, J., et al., Temporal Stability of the Human Skin Microbiome. Cell, 2016. 165(4): p. 854-66.
64. Sekirov, I., et al., Gut microbiota in health and disease. Physiol Rev, 2010. 90(3): p. 859-904.
65. Sanford, J.A. and R.L. Gallo, Functions of the skin microbiota in health and disease. Semin Immunol, 2013. 25(5): p. 370-7.
66. Egert, M., R. Simmering, and C.U. Riedel, The Association of the Skin Microbiota With Health, Immunity, and Disease. Clin Pharmacol Ther, 2017.
67. Maguire, M. and G. Maguire, The role of microbiota, and probiotics and prebiotics in skin health. Arch Dermatol Res, 2017.