傷口修補的過程可劃分成三個相互影響的過程：凝血發炎期、增生期與重組期，在此期間以角質細胞、纖維母細胞與血管內皮細胞為主的遷徙與增殖，掌握了傷口床周遭血管新生與傷口癒合的速度。然而糖尿病患者由於代謝功能異常進而破壞或延遲了這些癒合過程，使末肢傷口容易發展成稱為糖尿病潰瘍的慢性傷口。近年來硫化氫 (hydrogen sulfide, H2S) 氣體在生物體內作為信息傳遞物質的作用機制逐漸被解開，文獻指出硫化氫能夠促進血管內皮細胞的p38與ERK1/2蛋白磷酸化，提升細胞增殖、遷徙與管狀結構的形成。硫氫化鈉 (sodium hydrosulfide, NaHS) 是最常見且成本低廉的硫化氫前驅物之一，溶於水後會在短時間釋出大量硫化氫，但因硫化氫的半衰期短且瞬時濃度太高，無法合適且有效率的供細胞利用，因此設計具有硫化氫緩釋能力的藥物載體系統，已成為重要課題。本研究利用乳化法混合paraffin wax (PW) 與 1-tetradecanol (TD)，製備出搭載硫氫化鈉的蠟微米球，球體所提供的疏水性結構障礙能夠減緩水分子與硫氫化鈉接觸的時間，達到 48 小時緩慢釋放硫化氫的效果。在細胞實驗方面，藉由人類臍帶靜脈內皮細胞 (human umbilical vein endothelia cell, HUVEC) 的增殖、管狀形成實驗，以及人類表皮層角質細胞 (human epidermis keratinocyte) 的遷徙實驗，發現本硫化氫緩釋系統具有誘導血管新生與傷口癒合的潛力。在動物實驗方面，本研究針對第二型糖尿病 db/db小鼠背上的傷口進行治療，發現相對於直接投藥以及未治療之組別，投以硫化氫緩釋系統可顯著提升傷口癒合速度。此結果證實本系統能夠有效包覆硫氫化鈉，降低其在生物體中的解離速度，進而達成緩釋硫化氫的效果，對於慢性傷口之癒合有顯著幫助。

Impaired angiogenesis and induced refractory lesions of the chronic wounds are common complications observed in diabetes mellitus. Hydrogen sulfide (H2S) has been reported as a therapeutic gas transmitter, with the regulatory roles of cell migration and proliferation as well as capillary morphogenesis for endothelial cells. Sulfide salts such as sodium hydrosulfide (NaHS) have been used as a H2S precursor to study the biological effects of this gas compound. However, when encountered water, NaHS dissociates and generates a large amount of H2S over a very short time period. To overcome this concern, a hydrophobic microsphere system that is composed of paraffin wax (PW) and 1-tetradecanol (TD) and encapsulated with NaHS was developed. It was found that the NaHS-encapsulated microsphere system could sustain release of H2S for 48 h. As compared to that treated with free form NaHS, sustained release of H2S from the as-prepared microsphere system showed a significant promotion of human umbilical vein endothelia cell (HUVEC) proliferation and human epidermis keratinocyte migration, suggesting that sustained H2S release promotes the appearance of an angiogenesis phenotype. In the in vivo study, the wound closure rate in db/db diabetic mice was significantly faster in the group that was treated with the microsphere system than that treated with free form NaHS and the untreated control group. These experimental results demonstrate that the prepared microsphere system exhibited the features of bioavailability and protect the function of NaHS from rapid dissociation, suggesting that the sustained release of H2S is beneficial to chronic diabetic wounds in reference to the frequently treatment of free form NaHS.

摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VIII
第一章 緒論 1
1-1傷口癒合 (wound healing) 1
1-2糖尿病潰瘍 (diabetic ulcer) 3
1-3硫化氫與硫氫化鈉 (hydrogen sulfide and sodium hydrosulfide) 4
1-3-1硫化氫的發現與其促進血管新生的潛力 4
1-3-2硫化氫前驅物－硫氫化鈉 7
1-4相變化材料 (phase change materials, PCM) 9
1-5乳化法 (Emulsion method) 10
1-6研究目的與動機 11
1-7實驗架構圖 12
第二章 製程與實驗 13
2-1實驗藥品 13
2-2製備搭載硫氫化鈉之wax microspheres (NaHS-WMPs) 13
2-3物化性分析 (physicochemical characterization) 14
2-3-1場發射掃描式電子顯微鏡 (field emission scanning electron microscopy, FESEM) 14
2-4計算硫氫化鈉包覆含量 (calculation of loading content) 15
2-5硫化氫體外釋放實驗 15
2-6 NaHS-WMPs穩定性測試 (stability of NaHS-WMPs) 16
2-7細胞實驗 (in vitro study) 16
2-7-1細胞培養 16
2-7-2 HUVEC增殖實驗 17
2-7-3角質細胞遷徙實驗 18
2-7-4 Tube formation實驗 19
2-7-5 ELISA檢測HUVEC的p38蛋白之磷酸化 19
2-8 動物實驗 (in vivo study) 20
2-8-1 實驗設計 (study design) 20
2-8-2 建立type II糖尿病小鼠慢性傷口 21
2-8-3 追蹤傷口癒合面積 21
2-8-4 H&E染色與免疫染色 21
2-8-5 載體材料之生物可降解性測試 22
第三章 結果與討論 23
3-1以FESEM觀察NaHS-WMPs的表面型態 23
3-2硫氫化納包覆含量 (loading content of NaHS in NaHS-WMPs) 23
3-3硫化氫體外釋放曲線 (H2S releasing profile) 24
3-4 NaHS-WMPs穩定性測試 27
3-5細胞實驗 (in vitro study) 27
3-5-1 NaHS對促進HUVEC增殖的初步測試 28
3-5-2 NaHS對刺激角質細胞遷徙的初步測試 28
3-5-3 NaHS-WMPs 使用於HUVEC增殖實驗 29
3-5-4 NaHS-WMPs 使用於HUVEC管狀形成實驗 30
3-5-5 NaHS-WMPs 使用於HUVEC p38蛋白磷酸化實驗 32
3-5-6 NaHS-WMPs 使用於角質細胞遷徙實驗 33
3-6動物實驗 (in vivo study) 33
3-6-1傷口面積追蹤 33
3-6-2組織病理切片分析 35
3-6-3新生血管染色及血管密度定量 37
3-6-4載體材料之生物可降解性測試 38
第四章 結論 39
參考文獻 40

[1] Gurtner G. C., Werner S., Barrandon Y., Longaker M. T. Wound Repair and Regeneration. Nature. 2008; 453:314-21.
[2] Boateng J. S., Matthews K. H., Stevens H. N., Eccleston G. M. Wound Healing Dressings and Drug Delivery Systems: A Review. J Pharm Sci. 2008; 97:2892-923.
[3] Aonghus O’Loughlin, Timothy O’Brien. Topical Stem and Progenitor Cell Therapy for Diabetic Foot Ulcers, Stem Cells in Clinic and Research, Dr. Ali Gholamrezanezhad (Ed.), InTech, 2011; Chapter 23, pp581.
[4] Michael H. Shanik, Yuping Xu, Jan Škrha, Rachel Dankner, Yehiel Zick, Jesse Roth
Diabetes Care Feb 2008; 31, S262-S268.
[5] http://www.who.int/mediacentre/factsheets/fs312/en/
[6] Ceriello A. Diabetes mellitus: a hypercoagulable state. Coagulation activation in diabetes mellitus: the role of hyperglycaemia and therapeutic prospects. Diabetologia. 1993; 36:1119-25.
[7] Brem H., Tomic-Canic M. Cellular and Molecular Basis of Wound Healing in Diabetes. J. Clin. Invest. 2007; 117:1219–1222.
[8] Eming S. A., Krieg T., Davidson J. M. Inflammation in Wound Repair: Molecular and Cellular Mechanisms. J. Invest Dermatol. 2007; 127:514–25.
[9] Edwards R. et al. Bacteria and Wound Healing. Curr Opin Infect Dis.2004;17:91-6.
[10] Yu T., Robotham J. L., Yoon Y. Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. PNAS. 2006; 103:2653-8.
[11] Uemura S., Matsushita H., Li W., Glassford A. J., Asagami T., Lee K. H., Harrison D. G., Tsao P. S. Diabetes mellitus enhances vascular matrix metalloproteinase activity: role of oxidative stress. Circ Res. 2001; 88:1291-8.
[12] Lan C. C., Wu C. S., Huang S. M., Wu I. H., Chen G. S. High-glucose environment enhanced oxidative stress and increased interleukin-8 secretion from keratinocytes: new insights into impaired diabetic wound healing. Diabetes. 2013; 62 (7):2530-8.
[13] Potter C. F., Kuo N. T., Farver C. F., McMahon J. T., Chang C.H., Agani F. H., Haxhiu M. A., Martin R. J. Effects of Hyperoxia on Nitric Oxide Synthase Expression, Nitric Oxide Activity, and Lung Injury in Rat Pups. Pediatr. Res. 1999; 45:8–13.
[14] Saap. L.J., Falanga. V. Debridement Performance Index and Its Correlation with Complete Closure of Diabetic Foot Ulcers. Wound Repair Regen. 2002; 10:354–359
[15] Wang R. Physiological Implications of Hydrogen Sulfide: a Whiff Exploration That Blossomed. Physiol Rev. 2012; 92:791-896.
[16] Savage J. C., Gould D. H. Determination of Sulfides in Brain Tissue and Rumen Fluid by Ion-Interaction Reversedphase High-Performance Liquid Chromatography. Journal of Chromatography, 1990; 526:540-545.
[17] Goodwin L. R., Francom D., Dieken F. P., Taylor J. D., Warenycia M.W., Reiffenstein R. J., Dowling G. Determination of Sulfide in the Brain Tissue by Gas Dialysis/Ion Chromatography: Postmortem Studies and Two Case reports. Journal of Analytical Toxicology, 1989; 13:105-109.
[18] Barr L. A., Calvert J. W. Discoveries of Hydrogen Sulfide as a Novel Cardiovascular Therapeutic. Circ J. 2014;78:2111-8.
[19] Wallace J. L. Hydrogen Sulfide-Releasing Anti-Inflammatory Drugs. Trends Pharmacol. Sci. 2007; 28:501-505.
[20] Wen Y. D., Wang H., Kho S. H., Rinkiko S., Sheng X., Shen H. M., Zhu Y. Z.. Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress. PLoS One. 2013; 8:e53147.
[21] Yang G., Wu L., Jiang B., Yang W., Qi J., Cao K., Meng Q., Mustafa A. K., Mu W., Zhang S., Snyder S. H., Wang R. H2S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine Gamma-Lyase. Science. 2008; 322:587-590.
[22] Coletta C., Papapetropoulos A., Erdelyi K., Olah G., Módis K., Panopoulos P., Asimakopoulou A., Gerö D., Sharina I., Martin E., SzaboHydrogen C. Sulfide and Nitric Oxide are Mutually Dependent in the Regulation of Angiogenesis and Endothelium-Dependent Vasorelaxation. PNAS. 2008; 109:9161-9166.
[23] Papapetropoulos A., Pyriochou A., Altaany Z., Yang G., Marazioti A., Zhou Z., Jeschke M.G., Branski L. K., Herndond D. N., Wang R., Szabo C. Hydrogen Sulfide is an Endogenous Stimulator of Angiogenesis. PNAS. 2009; 106:21972-21977.
[24] Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK. Tissue engineering: creation of long-lasting blood vessels. Nature. 2004; 428 (6979):138-9.
[25] Melero-Martin JM, De Obaldia ME, Kang SY, Khan ZA, Yuan L, Oettgen P, Bischoff J. Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells. Circ Res. 2008; 103:194-202.
[26] Li L., Manuel S-T., Tan Ch.-H., Whitemand M., Moore P. K. GYY4137, A Novel Hydrogen Sulfide-Releasing Molecule, Protect against Endotoxic Shock in the Rat. Free Radical Biology & Medicine. 2009; 47:103-113.
[27] Li L., Whiteman M., Guan Y.Y., Neo K.L., Cheng Y., Lee S.W., Zhao Y., Baskar R., Tan C. H., Moore P.K. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation. 2008; 117:2351-60.
[28] Li L., Moore P. K. Putative Biological Roles of Hydrogen Sulfide in Health and Disease: a Breath of Not So Fresh Air? Trends Pharmacol Sci. 2008; 29:84-90.
[29] Cai W. J., Wang M. J., Moore P. K., Jin H. M., Yao T., Zhu Y. C. The novel proangiogenic effect of hydrogen sulfide is dependent on Akt phosphorylation. Cardiovasc Res. 2007; 76:29-40.
[30] Dhaese I., Van C. I., Lefebvre R. A. Mechanisms of action of hydrogen sulfide in relaxation of mouse distal colonic smooth muscle. Eur J Pharmacol. 2010; 628:179-86.
[31] Benavides G. A., Squadrito G. L., Mills R. W., Patel H. D., Isbell T. S., Patel R. P., Darley-Usmar V. M., Doeller J. E., Kraus D. W. Hydrogen sulfide mediates the vasoactivity of garlic. PNAS. 2007; 104:17977–17982.
[32] Lee Z. W., Zhou J., Chen C. S., Zhao Y., Tan C. H., Li L., Moore P. K., Deng L. W. The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo. PLoS One. 2011;6:e21077.
[33] Wallace J. L., Dicay M., McKnight W., Martin G. R. Hydrogen sulfide enhances ulcer healing in rats. FASEB J. 2007; 21:4070-6.
[34] Ali H., Opere C., Singh S. In vitro-Controlled Release Delivery System for Hydrogen Sulfide Donor. AAPS PharmSciTech. 2014; 15:910-9.
[35] Dr. Dong Choon Hyun, Nathanael S. Levinson,Prof. Unyong Jeong, Younan Xia Emerging Applications of Phase‐Change Materials (PCMs): Teaching an Old Dog New Tricks. Angew. Chem. Int. Ed. 2014; 53:3780-3795.
[36] L.F. Cabeza, A. Castell, C. Barreneche, A. de Gracia, A.I. Fernández. Materials used as PCM in thermal energy storage in buildings: a review, Renewable and Sustainable Energy Reviews 2011; 15:1675–1695.
[37] http://www.pcmproducts.net
[38] http://www.pcmproducts.net
[39] http://www.rubitherm.com
[40] Choi S-W, Zhang Y, Xia Y. A Temperature-Sensitive Drug Release System Based on Phase-Change Materials. Angew. Chem. Int. Ed. 2010;49 (43):7904-7908.
[41] Hughes M. N., Centelles M. N., Moore K. P. Making and working with hydrogen sulfide The chemistry and generation of hydrogen sulfide in vitro and its measurement in vivo: A review. Free Radic Biol Med. 2009; 47:1346-53.
[42] Geventman, L. H.: 1999, ‘Solubility of Selected Gases in Water’, in Lide, D. R. (ed.), CRC Handbook of Chemistry and Physics, 1999–2000, 80th edn., CRC Press, Boca Raton, Florida, pp. 8–86–8–90.
[43] Staton C. A., Stribblinh SM, Tazzyman S, et al. Current methods for assaying angiogenesis in vitro and in vivo. Int J Exp Pathol, 2004; 85:233-248.
[44] Yang C. T., Zhao Y., Xian M., Li J. H., Dong Q., Bai H. B., Xu J. D., Zhang M. F. A novel controllable hydrogen sulfide-releasing molecule protects human skin keratinocytes against methylglyoxal-induced injury and dysfunction. Cell Physiol Biochem. 2014; 34 (4):1304-17.
[45] Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003; 9:685-93.
[46] Koch, Distler. Vasculopathy and disordered angiogenesis in selected rheumatic diseases: rheumatoid arthritis and systemic sclerosis. Arthritis Research & Therapy. 2007; 9 (Suppl 2):S3.
[47] http://www.mohw.gov.tw/cht/DOS/Statistic.aspx