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研究生:張台香
研究生(外文):Tai-Hsiang Chang
論文名稱:利用動物模式探討高壓氧氣治療對於腫瘤細胞之影響
論文名稱(外文):The effect of hyperbaric oxygen therapy to tummor cell in vivo
指導教授:陳紹原
指導教授(外文):Shao-Yuan Chen
學位類別:碩士
校院名稱:國防醫學院
系所名稱:海底醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:74
中文關鍵詞:高壓氧氣治療
外文關鍵詞:hyperbaric oxygen therapytumor hypoxia
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高壓氧氣治療 (hyperbaric oxygen therapy, HBOT) 是指在大於1絕對大氣壓 (atmosphere absolute, ATA) 的治療艙內給予患者間歇性吸入100%的氧氣。臨床上常用來治療一氧化碳中毒、問題傷口、減壓症等。由於高壓氧氣治療對腫瘤細胞的影響並不明確,加上對於高濃度的氧氣有促進細胞生長作用的疑慮,使得高壓氧氣治療一直被列為癌症治療的相對禁忌症。而目前的研究報告也指出腫瘤細胞在快速生長的過程中,壞死區域周圍會產生缺氧的情形,另外,缺氧也會造成基因不穩定及突變,進一步增加腫瘤細胞抵抗走向細胞凋亡的能力。除此之外,缺氧亦會誘發缺氧誘發因子 (Hypoxia-Inducible Factor-1, HIF-1)進而造成VEGF (Vascular Endothelial Growth Factor)、Met (tyrosine kinase receptor) 等因子過度表現,使得腫瘤細胞持續增生甚至轉移。本實驗的目的在建立腫瘤缺氧動物模式,探討給予高壓氧氣治療是否可以改善腫瘤缺氧表現進而抑制腫瘤生長甚至轉移。
實驗分為兩部份進行,第一部分首先觀察腫瘤細胞在生長過程中體積變化與缺氧表現,並確立腫瘤缺氧模式,利用異種移植腫瘤細胞至SCID mice,待腫瘤細胞生長至第30、45天時,分別犧牲老鼠,利用免疫組織化學染色法觀察腫瘤細胞內部缺氧表現與血管分佈情形,同時觀察腫瘤體積變化。第二部份則待腫瘤缺氧模式確立後,即腫瘤細胞生長至第45天,體積達500 mm3,即可隨機分組進行實驗。高壓氧氣組 (n=6) 每天給予2.5絕對大氣壓的高壓氧氣90分鐘,共10次,高濃度氧氣組 (n=6) 每天給予1絕對大氣壓的高壓氧氣90分鐘,共10次,控制組 (n=6) 則不給予高壓氧氣處置。每週2次測量腫瘤體積,分別於治療結束第0、14、28天,犧牲老鼠,利用免疫組織化學染色法觀察腫瘤細胞內部缺氧表現與血管分佈情形,並使用西方墨點法觀察Met、VEGF等蛋白質表現。
實驗結果顯示給予高壓氧氣與高濃度氧氣處理後,高壓氧氣組的腫瘤體積在治療結束第0、14、28天皆小於控制組,且達統計上意義 (P<0.05);而免疫組織染色觀察高壓氧氣組與控制組相比缺氧範圍減少;非缺氧區域,血管分佈明顯增加。因此,利用高壓氧氣治療實體腫瘤,可以改善腫瘤內部缺氧情形,並限制腫瘤體積生長,同時增加血管表現,然而就免疫組織染色觀察Met、VEGF表現則三組間並無明顯差別。
Hyperbaric Oxygen Therapy (HBOT) is the administration of 100% oxygen at atmospheric pressures greater than that at sea level. Hyperbaric oxygen therapy is commonly utilized to treat clinical diseases, such as carbon monoxide poisoning, decompression disease, arterial gas embolism, osteomyelitis, sever anemia, wound healing, and so on. However, malignant disease has been recognized as a contraindication to HBO2 since it might promote residual tumor growth. On the other hand, many studies have described the rapidly growing progression of tumor incurring hypoxia that would be close to the necrotic areas in tumors. Under certain hypoxic condition, tumor cell could become unstable and make gene mutation. Most genes induced by hypoxia are regulated by the hypoxia-induced factor -1 (HIF-1), a protein playing a very important role in tumor development. Vascular endothelial growth factor (VEGF) is also regulated by HIF-1 under transcription lever. Meanwhile, hypoxia activates transcription of the met protooncogene, resulting to higher levels of Met. The overexpression of HIF, VEGF and Met may be able to keep tumor cells proliferation and even metastasis.
The purpose of this study was to establish tumor-hypoxia animal model, and to examine the effect of HBO2 therapy on tumor hypoxia, tumor growth and metastasis. First, we tested hypoxia and monitored the tumor growth separately on the 30th and 45th days after A549 tumor cell were implanted into SCID mice. Next, the tumor implanted mice were treated with NBO and HBO treatment 5 times a week at 2.5 atm absolute for 90 minutes (n=6), then sacrificed on 0, 14, 28 days . Immunohistochemical analysis was used to assist the examination on tumor hypoxia, vasculature expression of VEGF and Met. In the experiment, we have showed the significant differences (p<0.05) in the tumor volume, hypoxia and vasculature between the two growth (Control and HBO2). We concluded that hyperbaric oxygen treatment could decrease tumor hypoxic condition, change the density of tumor blood vessels and limit the growth of tumor. However, the detailed mechanism is still unclear further investigation.
目錄…………………………………………………………………………….I
表次…………………………………………………………………………...Ⅲ
圖次………………………………………………………………………….. IV
中文摘要……………………………………………………………………...Ⅴ
英文摘要……………………………………………………………………...Ⅶ
第一章 緒論
第一節、高壓氧氣治療…………………………………………………….1
第二節、高壓氧氣治療對腫瘤細胞之影響………………………………...5
第三節、腫瘤缺氧理論…………………………………………………….7
第四節、實驗動機…………………………………………………………11
第二章 實驗目的…………….………………………………………………12
第三章 實驗材料與實驗方法
第一節、試劑及抗體…………….…………………………………………13
第二節、緩衝液及SDS-PAGE之備製…………………………………….14
第三節、細胞株的培養……………………………………………………17
第四節、實驗動物…………………………………………………………18
第五節、腫瘤動物模式之建立……………………………………………19
第六節、高壓艙……………………………………………………………19
第七節、實驗分組…………………………………………………………20
壹、控制組
貳、高壓氧氣組
叁、高濃度氧氣組
第八節、實驗參數
壹、腫瘤體積…………………………………………………………...20
貳、腫瘤內部缺氧表現…………………………………………………21
叁、免疫組織化學染色 (Immunohistochemistry)……………………..21
肆、西方點墨法 (Western blot analysis)
一、細胞蛋白質萃取…………………………………………………23
二、蛋白質濃度之定量分析…………………………………………23
三、蛋白質樣本之處理………………………………………………24
四、西方點墨法………………………………………………………24
第九節、數據分析………………………………………………………….25
第四章、實驗結果
第一節、觀察A549細胞株進行異種移植的生長曲線與生長型態…….26
第二節、腫瘤細胞生長至第45天的內部缺氧表現比30天更明顯……26
第三節、經高壓氧氣處理後,限制腫瘤體積生長………………………27
第四節、高壓氧氣治療改善腫瘤缺氧,增加腫瘤內的血管表現...............28
第五節、高壓氧氣治療改善腫瘤缺氧,對VEGF及Met表現的影響...29
第五章 結論………………………………………………………………….31
第六章 討論
第一節、高壓氧氣治療對於腫瘤缺氧及血管生成的影響………………32
第二節、高壓氧氣治療對腫瘤細胞之影響………………………………36
第三節、未來方向…………………………………………………………37
第七章 參考資料…………………………………………………………….72
附錄一………………………………………………………………………...81
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