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研究生:郭忠民
研究生(外文):Chung-Ming Kuo
論文名稱:葡萄糖對人類皮膚先天性免疫之作用:糖尿病傷口癒合不良病人之體外分子機轉研究
論文名稱(外文):Effects of glucose on innate immunity in skin: an in vitro study of molecular mechanisms on impaired wound healing in diabetic patients.
指導教授:吳慶軒吳慶軒引用關係
指導教授(外文):Ching-Shiuan Wu
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:81
中文關鍵詞:葡萄糖防禦素TLR先天性免疫細胞移動增生
外文關鍵詞:HBDTLRinnate immunityglucosemigrationproliferation
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糖尿病(Diabetic mellitus,DM) 病人是因為體內血糖調控失衡而造成,嚴重時引發許多組織器官慢性併發症。糖尿病病人當皮膚有傷口時,容易因為外來環境中病原體引起皮膚感染而造成皮膚傷口癒合不良,人類的皮膚是最常與致病原接觸的部位,而表皮為人體先天性免疫防禦機制之一,更是對抗微生物感染的第一道防線。角質細胞能夠藉由製造各種抗微生物蛋白,來保護人體免於病原體之感染。β防禦素 (β-defensin) 是角質細胞所產生之主要的抗微生物蛋白,具有廣效的抗微生物活性。Toll-like receptors (TLRs) 能夠辨識外來病原體,提供訊息傳遞抵抗入侵微生物,在人體先天性免疫的功能也扮演重要地位。過去的研究報告顯示糖尿病鼠β-defensins有減少之情形。本論文將藉著高濃度D-glucose之刺激來模擬糖尿病病人體內高血糖濃度之情況,並透過 Lipopolysaccharide (LPS) 與 phorbol myristate acetate (PMA) 的作用模擬傷口感染病原體的情形,來探討 β-defensin 及其上游之主要調控者 TLRs 之表現。結果發現高濃度的葡萄糖能抑制人類角質細胞的生長,而以LPS刺激高濃度葡萄糖作用之角質細胞無法刺激其增生,另一方面,PMA 刺激高濃度葡萄糖作用之角質細胞對其增生無明顯影響。在 HBDs 之表現結果方面發現,高濃度葡萄糖作用下角質細胞在 human β-defensin-2 (HBD-2) 與 HBD-3 的表現均下降,HBD-1 則不受影響。此外,角質細胞在 TLR-2 與 TLR-4之表現則不受葡萄糖濃度之影響。高濃度葡萄糖作用下再以LPS刺激角質細胞其HBD-2、HBD-3 與 TLR-4 之表現明顯低於正常濃度葡萄糖之作用。另一方面,高濃度葡萄糖作用下再以 PMA 刺激角質細胞其 HBD-2、HBD-3 與 TLR-4 之表現均明顯低於正常濃度葡萄糖之作用。在刮傷試驗 (in vitro scratch assay) 結果顯示,高濃度葡萄糖能夠抑制角質細胞的移動,而 LPS 刺激高濃度葡萄糖作用之角質細胞並無法促進角質細胞的移動。高濃度葡萄糖作用下以 PMA 刺激角質細胞其p38 MAPK的表現明顯低於正常濃度葡萄糖之作用。綜合上述研究結果得知,高濃度的葡萄糖的作用會抑制角質細胞表面辨識病原體接受器 TLR-4 之表現,進而造成 p38 MAPK 之表現減少,使得角質細胞在 HBD-2 及 HBD-3 之表現減低。本研究成果顯示高濃度葡萄糖能夠抑制皮膚先天性免疫之正常功能,而造成糖尿病病人傷口反覆性感染,進而使得傷口不易癒合。
目錄 1
中文摘要 3
英文摘要 5
壹、 緒論
一、前言 7
二、先天性(非專一)免疫 8
三、防禦素的特性與分類 8
四、Human β-defensin 的成員介紹 10
五、Toll-like receptor的角色與作用 11
六、Toll-like receptor與Human β-defensin的相關訊息傳遞途徑 12
七、研究目的 14
貳、 材料與實驗方法
一、人類角質細胞的分離與培養 15
二、細胞外基質蛋白(extracellular matrix, ECM)塗覆 16
三、使用不同濃度D-glucose作用人類角質細胞 16
四、計數角質細胞分析細胞的增生能力 17
五、LPS與PMA對角質細胞的刺激 17
六、alamarBlue Assay法檢測細胞增生能力 17
七、角質細胞移動能力試驗-刮傷試驗 (in vitro scratch assay) 18
八、反轉錄聚合酶連鎖反應 18
九、西方點墨法(Western blotting) 22
十、統計分析方法 24
參、 實驗結果
一、 高濃度葡萄糖對體外培養人類角質細胞增生之研究 26
二、 高濃度葡萄糖對人類角質細胞HBD-1、HBD-2、
HBD-3、HBD-4、TLR-2、TLR-4 mRNA表現的影響 27
三、 高濃度葡萄糖下LPS與PMA刺激人類角質細胞
產生HBD-1、HBD-2、HBD-3、HBD-4、TLR-2、
TLR-4 mRNA表現的影響 29
四、 LPS對高濃度葡萄糖作用之角質細胞移動之影響 32
五、 LPS與PMA對高濃度葡萄糖作用之角質細胞內訊息
分子的影響 33
肆、 討論 35
伍、 未來研究方向 41
陸、 圖表 42
柒、 附錄 70
捌、 參考文獻 72
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