臺灣博碩士論文加值系統

自從光生物刺激理論被提出後，光照治療成為醫學上常使用的治療方式，特別是在傷口癒合的治療。近年來，發光二極體(Light emitting diode, LEDs)逐漸取代雷射成為新一代光照治療所使用的光源。本實驗利用先前所獲得的光照刺激最佳參數，持續探討以紅光LEDs刺激不同培養方式之人類皮膚纖維母細胞(human dermal fibroblast, HDF)及人類淋巴瘤細胞(U-937)的影響。光源波長為630 nm，功率15 mW/cm2，細胞所接受之能量為4 J/cm2。以細胞計數方式分析細胞數之變化，發現光照刺激可促進HDF之增生；亦可透過刺激U-937細胞，間接促進HDF之增生。以Sirius Red- Fast green染色法分析HDF膠原蛋白之生合成，發現光照刺激可促進HDF膠原蛋白之分泌；亦可透過刺激U-937細胞，間接促進HDF膠原蛋白之生合成。最後，使用DCFDA螢光染劑，測量HDF活性氧分子(Reactive oxygen species, ROS)產生的變化，實驗結果發現光照刺激並未顯著提升ROS之產生。

After the theory of photomodulation was proposed, light therapy has been widely used in clinical treatments, particularly in wound healing. Lately, light emitting diodes (LEDs) have gradually replaced lasers and became new generation of light sources in the light therapy. Here we describe the effects of red-light irradiation on different ways of culturing human dermal fibroblasts (HDFs) and human lymphoma cells (U-937) by using the optimal stimulating parameters obtained previously. The LEDs used had a wavelength of 630 nm. Its irradiance was 15 mW/cm2 and the radiant exposure was 4 J/cm2. The change in cell numbers was analyzed by direct cell counting. Significant enhancement of HDFs proliferation was found after light irradiation. The growth of HDFs was also promoted by incubating HDFs in the culture supernatant of illuminated U-937 cells. The secretion of total collagen by HDFs was estimated by using Sirius red- Fast green stain. The increase of the collagen secretion by HDFs was observed after irradiation. The secretion of collagen was also enhanced by incubating HDFs in the supernatant of light-stimulated U-937 cells. By using DCFDA analysis for the generation of reactive oxygen species (ROS) by HDFs, no significant difference was observed after illumination.

中文摘要 i
Abstract ii
誌謝 iii
目錄 iv
表目錄 viii
圖目錄 ix
第1章 緒論 1
1.1. 前言 1
1.2. 文獻回顧 2
1.3. 研究目的 12
第2章 低能量光照刺激細胞之理論依據 14
第3章 實驗材料與方法 18
3.1. 實驗儀器 18
3.2. 實驗藥品及來源 20
3.3. 藥品配置 23
3.3.1. DMEM培養基配置 23
3.3.2. RPMI 1640培養基配置 23
3.3.3. PBS配置 24
3.3.4. EDTA溶液配置 25
3.3.5. PMA配置 26
3.3.6. Picric acid配置 26
3.3.7. Fast green配置 26
3.3.8. Fast green / Sirius red配置 27
3.3.9. DCFDA配置 27
3.4. 細胞 28
3.4.1. 細胞來源 28
3.4.2. 細胞繼代培養 31
3.4.3. 細胞計數 32
3.4.4. 細胞冷凍保存 34
3.4.5. 細胞解凍 34
3.5. 分析方式 36
3.5.1. 膠原蛋白分析方式 36
3.5.2. ROS分析方式 37
3.5.3. 統計分析方式 37
3.6. 實驗架構 38
3.6.1. 光照刺激單獨培養之人類皮膚纖維母細胞 38
3.6.2. 光照刺激已分化之U-937上清液對人類皮膚纖維母細胞之影響 40
3.6.3. 光照刺激共培養已分化之U-937與人類皮膚纖維母細胞 42
3.6.4. 光照刺激對人類皮膚纖維母細胞產生ROS之影響 44
第4章 實驗結果 46
4.1. 光照刺激單獨培養之人類皮膚纖維母細胞 46
4.1.1. 細胞數之影響 46
4.1.2. 膠原蛋白之生合成 49
4.2. 光照刺激已分化之U-937上清液對人類皮膚纖維母細胞之影響 54
4.2.1. 細胞數之影響 54
4.2.2. 膠原蛋白之生合成 58
4.3. 光照刺激共培養已分化之U-937與人類皮膚纖維母細胞 64
4.3.1. 膠原蛋白之生合成 64
4.4. 光照刺激對人類皮膚纖維母細胞產生ROS之影響 69
第5章 討論 71
5.1. 光照刺激對於細胞數之影響 71
5.2. 光照刺激對於細胞膠原蛋白生合成之影響 72
5.3. 光照刺激細胞產生ROS之影響 73
5.4. phenol red對細胞接受光照刺激之影響 74
第6章 結論 76
第7章 未來展望 77
參考文獻 79

表目錄
表 1 1 與傷口癒合相關之細胞激素 4
表 3 1 PBS配置所需之藥品 25
表 3 2人類皮膚纖維母細胞 29
表 3 3人類淋巴瘤細胞 30


圖目錄
圖 1 1傷口癒合程序圖 4
圖 2 1電子傳遞鏈能量之轉移 16
圖 2 2光照刺激細胞粒腺體產生的初級反應 16
圖 2 3光照刺激細胞粒腺體產生的次級反應 17
圖 3 1顯微鏡下人類皮膚纖維母細胞之型態 28
圖 3 2顯微鏡下人類淋巴瘤細胞之細胞型態 31
圖 3 3血球計數盤九宮格 33
圖 3 4人類皮膚纖維母細胞單獨培養實驗流程 39
圖 3 5已分化之U-937上清液與人類皮膚纖維母細胞間接培養實驗流程 41
圖 3 6已分化之U-937與人類皮膚纖維母細胞共培養實驗流程 43
圖 3 7人類皮膚纖維母細胞ROS assay流程 45
圖 4 1光照刺激對單獨培養人類皮膚纖維母細胞之增殖影響 47
圖 4 2光照刺激對單獨培養人類皮膚纖維母細胞之collagen分泌量影響 52
圖 4 3光照刺激已分化之U-937上清液對人類皮膚纖維母細胞之增殖影響 56
圖 4 4光照刺激已分化之U-937上清液對人類皮膚纖維母細胞collagen分泌量影響 62
圖 4 5光照刺激對已分化之U-937與人類皮膚纖維母細胞共培養之collagen分泌影響 67
圖 4 6光照刺激對人類皮膚纖維母細胞產生ROS之影響 70
圖 7 1皮膚傷口癒合的時程 78
圖 7 2傷口癒合的發炎時期各種細胞分泌生長因子與細胞作用 78

〔1〕劉清棋（2002）”傷口癒合市場分析”
〔2〕Witte M.B. and Barbul A.（1997）” General principles of wound healing. “ Surg Clin North Am. 77︰509-528.
〔3〕Takehara K.（2000）“ Growth regulation of skin fibroblasts.” J. Dermatol. Sci. 24︰70 – 77.
〔4〕Spörri B., Bickel M., Limat A., Ernst R. Waelti E.R., Hunziker T. and Wiesmann U.N.（1996）” Juxtacrine stimulation of cytokine production in cocultures of human dermal fibroblasts and T cells.” Cytokine 8︰631-635.
〔5〕Chen V., Croft D., Purkis P., and Kramer I.M.（1998）” Co-culture of synovial fibroblasts and differentiated U937 cells is sufficient for high interleukin-6 but not interleukin-1beta or tumour necrosis factor-alpha release.” Br. J. Rheumatol. 37︰148–156.
〔6〕Lee H.G. and Eun H.C.（1999）” Differences between fibroblasts cultured from oral mucosa and normal skin: implication to wound healing.” J. Dermatol. Sci. 21︰176-182.
〔7〕Yamamoto T., Hartmann K., Eckes B. and Krieg T.（2000）” Mast cells enhance contraction of three-dimensional collagen lattices by fibroblasts by cell-cell interaction: role of stem cell factor/c-kit.” Immunology. 99︰435-439.
〔8〕Skold C.M., Liu X.D., Umino T., Zhu Y.K., Ertl R.F., Romberger D.J. and Rennard S.I.（2000）” Blood monocytes attenuate lung fibroblast contraction of three-dimensional collagen gels in coculture.” Am J Physiol Lung Cell Mol Physiol. 279︰667–674.
〔9〕http://www.nas.org.uk/nas/jsp/polopoly.jsp?d=364&a=2619#logo
〔10〕邱國力（2000）”雷射在皮膚科的應用”
〔11〕Posten W., Wrone D.A., Dover J.S., Arndt K.A., Silapunt S. and Alam M.（2000）” Low-level laser therapy for wound healing mechanism and efficacy.” Dermatol. Surg. 31︰334-340.
〔12〕Young S., Bolton P., Dyson M., Harvey W. and Diamantopoulos C.（1989）” Macrophage responsiveness to light therapy.” Lasers Surg Med. 9︰497-505.
〔13〕Lubart R., Wollman Y., Friedmann H., Rochkind S. and Laulicht I..（1992）” Effects of visible and near-infrared lasers on cell cultures.” J. Photochem. Photobiol.B. 12︰305-310.
〔14〕Karu T., Pyatibrat L. and Kalendo G.（1995）” Irradiation with He-Ne laser increases ATP level in cells cultivated in vitro.” J Photochem Photobiol B. 27︰219-223.
〔15〕Yu H.S., Chang K.L., Yu C.L., Chen J.W. and Chen G.S.（1996）” Low-energy helium-neon laser irradiation stimulates interleukin-1 alpha and interleukin-8 release from cultured human keratinocytes.” J. Invest Dermatol. 107︰593-596.
〔16〕Stadler I., Evans R., Kolb B., Naim J.O., Narayan V., Buehner N. and Lanzafame R.J.（2000）” In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes.” Lasers Surg Med. 27︰255-261.
〔17〕Pereira A.N., Eduardo Cde P., Matson E. and Marques M.M.（2002）” Effect of low-power laser irradiation on cell growth and procollagen synthesis of cultured fibroblasts.” Lasers Surg Med. 31︰263-267.
〔18〕Moore P, Ridgway T.D., Higbee R.G., Howard E.W. and Lucroy M.D.（2005）” Effect of wavelength on low-intensity laser irradiation-stimulated cell proliferation in vitro.” Lasers Surg Med. 36︰8-12.
〔19〕Karu T.I.（1987）”Photobiological Fundamentals of low power laser therapy” IEEE J. Quantum Electron. 10︰23.
〔20〕許馨仁（2005）” 發光二極體光照刺激人類淋巴瘤細胞株影響之研究” 中原大學醫學工程系碩士學位論文.
〔21〕http://www.romana.com.tw/biolight.htm
〔22〕慧聰醫療器械商務網（2003）”創傷癒合(wound healing) ”
〔23〕http://www.dls.ym.edu.tw/lesson/arch.htm
〔24〕Karu T.I.（2003）” Low-power laser therapy.” Biomed Photonics Handbook. 48︰1-20.
〔25〕LeBel C.P. Ischiropoulos H. and Bondy S.C.（1990）” Evaluation of the Probe 2’7’-Dichlorofluorescin as an Indicator of Reactive Oxygen Species Formation and Oxidative Stress.” Chem. Res. Toxicol. 5︰227-231.
〔26〕http://www.rndsystems.com/mini_review_detail_objectname_MR02_CytokineWoundHealing.aspx
〔27〕van Breugel H.H. and Bar P.R.（1992）” Power density and exposure time of He-Ne laser irradiation are more important than total energy dose in photo-biomodulation of human fibroblasts in vitro.” Lasers Surg Med. 12︰528-537.
〔28〕Chen Y.J., Jeng J.H., Jane Yao C.C., Chen MH, Hou L.T. and Lan W.H.（2005）” Long-term effect of pulsed Nd-YAG laser irradiation on cultured human periodontal fibroblasts.” Lasers Surg Med. 36︰225-233.
〔29〕李文婷 ”人工皮膚的技術進展與產業概況”
〔30〕Adam J., Singer M.D., Richard A.F. and Clark M.D.（1999）” Mechanisms of Disease cutaneous wound healing.” N. Engl. J. Med. 341︰738-346.
〔31〕Alexandratou E., Yova D., Handris P., Kletsas D., Loukas S.（2002）” Human fibroblast alterations induced by low power laser irradiation at the single cell level using confocal microscopy.” Photochem Photobiol Sci. 1︰547-552.
〔32〕Bae G.. U., Seo D. W., Kwon H. K., Lee H. Y., Hong S., Lee Z. W., Ha K. S., Lee H. W. and Han J. W.（1999）”Hydrogen Peroxide activates p70S6k signaling pathway.”J. Biol. Chem. 274︰32596–32602.
〔33〕Vanden Hoek T. L., Becker L. B., Shao Z., Li C. and Schumacker P. T.（1998）”Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes”J. Biol. Chem. 273︰18092–18098.
〔34〕Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS.（1986）”Phenol red in tissue culture media is a weak estrogen: Implications concerning the study of estrogenresponsive cells in culture.”Proc Natl Acad Sci USA 83︰2496–2500.
〔35〕Ernst M, Schmid C, Froesch E.R.（1989）”Phenol red mimics biological actions of estradiol: enhancement of osteoblast proliferation in vitro and of type I collagen gene expression in bone and uterus of rats in vivo.”J Steroid Biochem. 33（5）︰907-914.
〔36〕Wesierska-Gadek J, Schreiner T, Gueorguieva M, Ranftler C.（2006）” Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells.” J Cell Biochem. 98（6）︰1367-1379