本研究使用能夠穿透到真皮層的UVA，和真皮層含量最多的纖維母細胞，來探討玻尿酸是否可以有效地緩和光老化所造成的細胞外間質 (ECM)的降解以及恢復細胞的彈性模數。
實驗證實UVA照射真皮層纖維母細胞後，會使細胞受到傷害，而導致細胞生存率 (Cell viability) 降低，而在ECM方面，UVA照射會促進真皮層纖維母細胞表現IL-1β，而產生發炎反應，然後促進降解ECM的MMP-1和MMP-3的基因表現，除此之外，UVA能夠降低膠原蛋白和彈性蛋白的新生成。在膠原蛋白和彈性蛋白的新生成降低，MMP-1和MMP-3大量表現的情況下，使用原子力顯微鏡 (AFM) 來測試細胞的彈性模數變化，結果發現細胞彈性因為UVA照射時間增長而彈性變小。
纖維母細胞照射UVA後，導致光老化，而使用玻尿酸可以降低因為UVA和IL-1β所引起的MMP-1和MMP-3基因表現，因此達到降緩ECM降解的效果。在ECM新生成方面，玻尿酸或許可以回復少許的彈性蛋白以及膠原蛋白之新生成。除此之外，玻尿酸也可以有效恢復因為UVA照射所降低的細胞彈性。總和這些結果，玻尿酸只具有緩和UVA所誘導的纖維母細胞光老化之能力，卻無法將纖維母細胞回復到正常狀態，雖然如此，玻尿酸在治療光老化方面還是具有相當的潛力。

In this research, we used UVA irradiation, which can penetrate into dermis, and fibroblasts, the most abundant cells in dermis, to investigate the effect of UV light on dermis, such as inflammation, ECM degradation and elasticity losing. Moreover, this research is focused on “The influence of hyaluronic acid (HA) on UVA treated dermal fibroblasts”. We would like to find out whether HA can effectively relief ECM degradation, and restore the elasticity of UVA-damaged fibroblasts.
Prolonged exposure to UVA radiation can damage fibroblasts and led variation in cell morphology and reduction in cell viability. Besides, UVA radiation can induce IL-1β expression on fibroblasts and then promote MMP-1 and MMP-3 expression, which can accelerate ECM degradation. On the other hand, prolonged exposure to UVA radiation reduced collagen and elastin synthesis on fibroblasts. Due to the acceleration of ECM degradation and the reduction of ECM synthesis, AFM was used to analyze the elasticity reduction on UVA-damaged fibroblasts.
UVA irradiation causes photoaging on fibroblasts. UVA damaged fibroblasts with HA treatment can down-regulate the gene expression of MMP-1, MMP-3, and then slow down ECM degradation. On the other hand, HA maybe restored elastin and collagen synthesis in UV-damaged fibroblasts. Based on the slowdown of ECM degradation, UVA-damaged fibroblast elasticity can be effectively restored by HA treatment. In summary, HA can relief the photoaging conditions on fibroblasts, but may not be able to return fibroblasts to normal, healthy state. Although HA cannot fully recover UVA-damaged fibroblasts, HA is still potential for repairing photoaging skin.

1. Julie A. Pasco, Margaret J. Henry, Geoffrey C. Nicholson, Sharon L. Brennan, Mark A. Kotowicz. Behavioural and physical characteristics associated with vitamin D status in women.Bone 44 (2009) 1085–1091.

2. Jorg Reichrath. The challenge resulting from positive and negative effects of sunlight: How much solar UV exposure is appropriate to balance between risks of vitamin D deficiency and skin cancer. Progress in Biophysics and Molecular Biology 92 (2006) 9–16

3. Jennifer K. Wagner, Esteban J. Parra, Heather L. Celina Jovel, Mark D. Shriver. Skin Responses to Ultraviolet Radiation: Effects of Constitutive Pigmentation, Sex, and Ancestry. PIGMENT CELL RES 15 (2002) 385–390.

4. Arun D. Singh, Ian G. Rennie, Stefan Seregard, Michael Giblin, John McKenzie. Sunlight Exposure and Pathogenesis of Uveal Melanoma. SURVEY OF OPHTHALMOLOGY VOLUME 49 (2004).

5. M. Ichihashi, M. Ueda, A. Budiyanto, T. Bito, M. Oka, M. Fukunaga, K. Tsuru, T. Horikawa. UV-induced skin damage. Toxicology 189 (2003) 21-39

6. Yasuhiro Matsumura and Honnavara N. Ananthaswamy. Toxic effects of ultraviolet radiation on the skin. Toxicology and Applied Pharmacology 195 (2004) 298– 308

7. C.S. Zerefos. Long-term ozone and UV variations at Thessaloniki, Greece. Physics and Chemistry of the Earth 27 (2002) 455–460

8. Hyun-kyung Choi, Dong-hyun Kim, Jin Wook Kim, Sulaiman Ngadiran, Mohamad Roji Sarmidi, Chang Seo. Labisia pumila extract protects skin cells from photoaging caused by UVB irradiation. Journal of Bioscience and Bioengineering VOL. 109 No. 3, (2010) 291–296,

9. Linan Wang, KanakoSaito, MasaakiToda, TomohideHori, Mie Torii, NingMa, NaoyukiKatayama, HiroshiShiku, Kagemasa Kuribayashi, TakumaKato. UV irradiation after immunization induces type 1 regulatory T cells that suppress Th2-type immune responses via secretion of IL-10. Immunobiology 215 (2010) 124–132

10. Young-Kwon Seo, Su-Hyun Jung, Kye-Yong Song, Jung-Keug, Chang-Seo. Anti-photoaging effect of fermented rice bran extract on UV-induced normal skin fibroblasts. Eur Food Res Technol 23 (2010) 1163–1169

11. Misato Hashizume, Masahiko Mihara. High molecular weight hyaluronic acid inhibits IL-6-induced MMP production from human chondrocytes by up-regulating the ERK inhibitor, MKP-1. Biochemical and Biophysical Research Communications 403 (2010) 184–189

12. C.-T. Wang, Y.-T. Lin, B.-L. Chiang, Y.-H. Lin, S.-M. Hou. High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes from patients with early osteoarthritis. OsteoArthritis and Cartilage 14 (2006) 1237-1247

13. Beele. H. Artificial skin: past, present and future. The International Journal of Artificial Organs 25 (2002) 163-173

14. Haake A. R., Holbrook. K The structure and development of skin. In Freedberg, et al,ed. Fitzpatrick’s Dermatology in General Medicine 5th edn (1999)

15. Wickett R. R., Visscher M. O. Structure and function of the epidermal barrier. Am J Infect Control 34 (2006) 98-110

16. Different Parts of the Skin. http://skincancer.dermis.net/content/e01geninfo/e7/index_eng.html

17. Nocka K., Majumder S., Chabot. B. Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice-evidence for an impaired c-kit kinase in mutant mice. Gene Dev 3 (1989) 816-26

18. Masaki, H., T. Atsumi and H. Sakurai. Detection of hydrogen peroxide and hydroxyl radicals in murine skin fibroblasts under UVB irradiation. Biochemical and Biophysical Research Communications 206 (1995) 474-479
19. Tsukahara K., Takema Y., Moriwaki S., Tsuji N., Suzuki Y., Fujimura T., Imokawa G. Selective Inhibition of Skin Fibroblast Elastase Elicits a Concentration-Dependent Prevention of Ultraviolet B-Induced Wrinkle Formation. The Journal of investigative dermatology. 117(3) (2001) 671-677

20. Naderi-Hachtroudi L., Peters T., Brenneisen P., Meewes C., Hommel C., Razi-Wolf Z., Lars A. Schneider L. A., Schu‥ller J., Wlaschek M., Scharffetter-Kochanek K.. Induction of Manganese Superoxide Dismutase in Human Dermal Fibroblasts. Arch Dermatol. 138 (2002) 1473-1479

21. Fisher G. J., Wang Z. Q., Datta S. C., Varani J., Kang S., and Voorhees J. J., Pathophysiology of Premature Skin Aging Induced by Ultraviolet Light. N Engl J Med, 337 (13) (1997) 1419-1429

22. Manuskiatti W., and Maibach H. I., Hyaluronic acid and skin: wound healing and aging. International Journal of Dermatology, 35 (8) (1996) 539-544

23. B. Joddar, S. Ibrahim, A. Ramamurthi. Impact of delivery mode of hyaluronan oligomers on elastogenic responses of adult vascular smooth muscle cells. Biomaterials 28 (2007) 3918–3927

24. Chandrasekhar R. Kothapalli , Anand Ramamurthi. Induced elastin regeneration by chronically activated smooth muscle cells for targeted aneurysm repair. Acta Biomaterialia 6 (2010) 170–178

25. Dai G., Freudenberger T., Zipper P., Melchior A., Grether-Beck S., Rabausch B., Groot J. D.,Twarock S., Hanenberg H., Homey B., Krutmann J., Reifenberger J., and Fischer. J. W.Chronic Ultraviolet B Irradiation Causes Loss of Hyaluronic Acid from Mouse Dermis Because of Down-Regulation of Hyaluronic Acid Synthases. The American Journal of Pathology, 171 (5) (2007) 1451-1461

26. H. Birkedal-Hansen, W. G. I. Moore, M. K. Bodden, L J. Windsor, B. Birkedal-Hansen, A. DeCarlo, J. A. Engler. Matrix Metalloproteinases: A Review. Critical Reviews in Oral Biology and Medicine, 4(2) (1993) 197-250

27. F. Bruge, E. Venditti, L. Tiano, G.P. Littarru, E. Damiani. Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies. Journal of Biotechnology 156 (2011) 153– 162

28. Kaoru Suganuma, Hiroaki Nakajima, Mamitaro Ohtsuki, Genji Imokawa. Astaxanthin attenuates the UVA-induced up-regulation of matrixmetalloproteinase-1 and skin fibroblast elastase in human dermal fibroblasts. Journal of Dermatological Science 58 (2010) 136–142

29. Laure Rittie, Gary J. Fisher. UV-light-induced signal cascades and skin aging. Ageing Research Reviews 1 (2002) 705–720

30. Meinhard Wlaschek, Iliana Tantcheva-Poor, Lale Naderi, Wenjian Ma, Lars Alexander Schneider, Ziba Razi-Wolf, Jutta Schuller, Karin Scharffetter-Kochanek. Solar UV irradiation and dermal photoaging. Journal of Photochemistry and Photobiology B: Biology 63 (2001) 41–51

31. J. Brinckmann, Y. A il, H.H. Wolff, P.K. M ller. Collagen synthesis in (sun-)aged human skin and in fibroblasts derived from sun-exposed and sun-protected body sites. Journal of Photochemistry and Photobiology B: Biology 27 (1995) 33-38

32. Ji-Young Bae, Jung-Suk Choi, Yean-Jung Choi, Seung-Yong Shin, Sang-Wook Kang, Seoung Jun Han, Young-Hee Kang. (-)Epigallocatechin gallate hampers collagen destruction and collagenase activation in ultraviolet-B-irradiated human dermal fibroblasts: Involvement of mitogen-activated protein kinase. Food and Chemical Toxicology 46 (2008) 1298–1307

33. Barbara Iovine, Massimiliano Nino, Carlo Irace, Maria Assunta Bevilacqua, Giuseppe Monfrecola. Ultraviolet B and A irradiation induces fibromodulin expression in human fibroblasts in vitro. Biochimie 91 (2009) 364-372

34. Naoko Kondo, Hitoshi Takeda, Takahide Kaneko, Takayuki Aizu, Ryuta Moritsugu, Atsushi Kon, Nakano Hajime, Katsumi Hanada. A method for rapid evaluation of photoaging by measuring fluorescence intensity of green fluorescent protein due to elastin promoter Activity. Journal of Dermatological Science Supplement 2 (2006) S31-S37

35. Eric F. Bernstein, Yue Qiu Chen, Katsuto Tamai,Kennech J. Shepley, Kennech S. Resnik, Hui Zheng, Rocky Tuan, Alain Mauviel, Jouni Uitto. Enhance elastin and fibrillin gene expression in chronically photodamage skin. The Journal of investigative Dermatology (1994) 182-186

36. Alexander JP, Acott TS. Involvement of protein kinase C in TNFα regulation of trabecular matrix metalloproteinases and TIMPs. Invest Ophthalmol Vis Sci 42 (2001) 2831-2838.

37. Wlaschek M, Heinen G, Poswig A, Schwarz A, Krieg T, Scharffetter Kochanek K. UVA-induced autocrine stimulation of fibroblast-derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol 59 (1994) 550-556.

38. Rutter JL, Benbow U, Coon CI, Brinckerhoff CE. Cell-type specific regulation of human interstitial collagenase-1 gene expression by interleukin-1b (IL-1b) in human fibroblasts and BC-8701 breast cancer cells. J Cell Biochem 66 (1997) 322-336.

39. Ayumi Honda, Riichiro Abe, Teruhiko Makino, Osamu Norisugi, Yasuyuki Fujita, Hirokazu Watanabe, Jun Nishihira, Yoichiro Iwakura, Sho-ichi Yamagishi, Hiroshi Shimizu, Tadamichi Shimizu. Interleukin-1β and macrophage migration inhibitory factor (MIF) in dermal fibroblasts mediate UVA-induced matrix metalloproteinase-1 Expression. Journal of Dermatological Science 49 (2008) 63-72

40. Akiko Sasaki, Ken Sasaki, Yrjő T. Konttinen, Seppo Santavirta, Masatoshi Takahara, Hiroshi Takei, Toshihiko ogino, Michiaki Takagi. Hyaluronate inhibits the interleukin-1β-induced expression of matrix metalloproteinase (MMP)-1 and MMP-3 in human synovial cell. Tohoku J Exp. Med. 204 (2004) 99-107

41. 陳哲雄, 林俊勳, 林文瑞, 吳靖宙, 張憲彰.原子力顯微鏡成像原理與中文簡易操作書. 成功大學醫學工程所生醫感測實驗室.

42. 吳靖宙, 張憲彰. 掃描式探針顯微鏡於生物樣本的量測與應用.

43. Binning G. C.F. Quate, C. Gerber. Atomic force microscope and method for imaging surfaces with atomic resolution. Physical Review Letters 56 (1986) 930-933

44. Mitsui K., M Hara, A.Ikai. Mechanical unfolding of α2-marcoglobulin molecules with atomic force microscopy. FEBS Letters 385 (1996) 29-33

45. Dimitriadis, E.K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R.S.,. Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82 (2002) 2798–2810.

46. Xiaofang Cai, Xiaobo Xing, Jiye Cai, Qian Chen, Shixian Wu, Feicheng Huang. Connection between biomechanics and cytoskeleton structure of lymphocyte and Jurkat cells: An AFM study. Micron 41 (2010) 257–262

47. Liang, X.,Mao, G., Simon Ng, K.Y. Probing small unilamellar EggPC vesicles on mica surface by atomic force microscopy. Colloids Surf. B: Biointerfaces 34 (2004) 41-51.

48. Q.S. Li, G.Y.H. Lee, C.N. Ong, C.T. Lim. AFM indentation study of breast cancer cells. Biochemical and Biophysical Research Communications 374 (2008) 609–613.

49. C.-H. Hsieh, Y.-H. Lin, S. Lin. Tsai-Wu, C. H. Herbert Wu, C.-C. Jiang. Surface ultrastructure and mechanical property of human chondrocyte revealed by atomic force microscopy. Osteoarthritis and Cartilage 16 (2008) 480-488.