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研究生:陳意舒
研究生(外文):Yi-Shu Chen
論文名稱:原位注射標靶性微脂粒應用於乳癌治療
論文名稱(外文):In situ injection of Targeting-Liposome therapy of Breast cancer
指導教授:駱俊良
指導教授(外文):Chun-Liang Lo
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學工程學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:77
中文關鍵詞:薑黃素咪喹莫特原位注射乳癌微脂粒
外文關鍵詞:CurcuminImiquimodin situ injectionBreast cancerliposome
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在本研究中合成具標靶性分子(Cholesterol-Glucosamine)與抗癌藥物薑黃素(Curcumin)與免疫藥物咪喹莫特(Imiquimod)製備成具標靶性微脂粒,平均粒徑大小為97.67 nm,粒徑分佈為0.21再與透明質酸 (Hyaluronic acid)混合成高分子水溶液,以原位注射的方式打入腫瘤,達到縮小腫瘤以及避免藉由淋巴轉移至身體他處以利手術治療時可以完全切除進而減少復發風險。
在實驗結果中首先確認了具標靶性微脂粒的穩定性,並且可以有效包覆姜黃素與咪喹莫特。而體外試驗中,發現薑黃素具有有效毒殺小鼠乳腺癌細胞株(4T1 cell line),另也證實具有標靶葡萄糖的微脂粒效果比不具有標靶葡萄糖的微脂粒更佳。最後體內試驗證實了具標靶性微脂粒混合多醣類溶液以原位注射給藥,可以有效的抑制腫瘤生長,且將藥物的副作用減輕。綜合上所述,本研究的原位注射標靶性微脂粒在對乳癌的治療中,具有有效的應用價值。
In this study, a synthesis of Cholesterol-Glucosamine and an anticancer drug Curcumin and an immunology drug Imiquimod were prepared to Targeting-liposome with an average particle size of 97.67 nm and a particle size polydispersity of 0.21.Then the Targeting- liposome solution is mixed with the hyaluronic acid to form a high molecular weight solution, which is injected into the tumor by in situ injection to reduce the tumor size and avoid lymphatic metastasis to the body for surgical treatment. It can be completely resected to reduce the risk of relapse.
In the experimental results. First, we confirmed that the stability of Targeting-liposome, and efficiently loaded curcumin and imiquimod. In vitro, curcumin was found to be effective in killing mouse breast cancer cell lines (4T1 cell line), and it was also confirmed that the liposome effect with target glucose was better than that of non-target glucose. Finally, in vivo experiments confirmed that the Targeting-liposome with the hyaluronic acid to form a high molecular weight solution, was administered by in situ injection, which can effectively inhibit tumor growth and reduce the side effects of the drug. In summary, the in situ injection of Targeting-liposome in this study has an effective application value in the treatment of breast cancer.
目錄
國立陽明大學碩士學位論文審定同意書 #
致謝 i
中文摘要 ii
ABSTRACT iii
目錄 iv
表目錄 ix
圖目錄 x
第一章 研究動機 1
第二章 文獻探討 3
2.1 乳癌 3
2.1.1 簡介 3
2.1.2 治療策略 4
2.1.3 手術治療 5
2.1.4 放射治療 6
2.1.5 賀爾蒙治療 8
2.1.6 化學治療 8
2.1.7 標靶治療 9
2.2 奈米藥物載體 10
2.2.1 藥物控制釋放 10
2.2.2 奈米藥物載體系統 11
2.3 微脂粒 13
2.3.1 微脂粒簡介 13
2.3.2 微脂粒組成 14
2.3.3 微脂粒製備方式 15
2.4 免疫治療 16
2.4.1 免疫治療發展 16
2.4.2 先天性免疫 18
2.4.3 後天性免疫 20
2.5 材料性質與應用 21
2.5.1 葡萄糖-Glucose(Glu) 21
2.5.2 薑黃素-Curcumin(CUR) 23
2.5.3 咪喹莫特-Imiquimod(IMQ) 24
第三章 材料與方法 26
3.1 實驗藥品 26
3.2 實驗儀器與裝置 29
3.3 膽固醇官能基修飾 31
3.3.1 Cholesterol-COOH改質 31
3.3.2 Cholesterol-NHS ester合成 32
3.3.3 Cholesterol-Glucosamine合成 33
3.4 材料性質分析 34
3.4.1 核磁共振氫譜分析 34
3.4.2 傅立葉轉換紅外線光譜分析 34
3.5 具標靶性微脂粒製備 35
3.6 具標靶性微脂粒之基本分析 36
3.6.1 粒徑大小、粒子分佈及表面電位分析 36
3.6.2 有效包覆率與載藥率分析 36
3.6.3 具標靶性微脂粒穩定性測試 37
3.6.4 穿透式電子顯微鏡型態分析 37
3.6.5 具標靶性微脂粒藥物釋放分析 38
3.7 具標靶性微脂粒體外試驗 39
3.7.1 細胞種類 39
3.7.2 細胞培養液及磷酸鹽緩衝溶液配置 39
3.7.3 細胞繼代 40
3.7.4 細胞計數 40
3.7.5 具標靶性微脂粒細胞毒性測試 41
3.7.6 具標靶性微脂粒細胞吞噬測試 41
3.8 具標靶性微脂粒體內試驗 43
3.8.1 乳癌動物模型建立 43
3.8.2 具標靶性微脂粒混合透明質抑制腫瘤效果 43
3.8.3 具標靶性微脂粒混合透明質酸抑制腫瘤效果組織切片 44
3.8.4 小鼠血液分析 44
第四章 結果與討論 45
4.1 膽固醇官能基改質鑑定 45
4.1.1 Cholesterol-COOH改質鑑定 45
4.1.2 Cholesterol-NHS ester合成鑑定 47
4.1.3 Cholesterol-Glucosamine合成鑑定 49
4.2 具標靶性微脂粒基本性質分析 51
4.2.1 粒徑大小、粒子分佈及表面電位分析 51
4.2.2 有效包覆率及載藥率分析 53
4.2.3 穩定性測試 55
4.2.4 穿透式電子顯微鏡型態分析 57
4.2.5 藥物釋放分析 58
4.3 具標靶性微脂粒體外試驗 59
4.3.1 具標靶性微脂粒細胞毒性測試 59
4.3.2 具標靶性微脂粒細胞吞噬測試 61
4.4 具標靶性微脂粒體內試驗 63
4.4.1 具標靶性微脂粒混合透明質酸抑制腫瘤之效果 63
4.4.2 具標靶性微脂粒混合透明質酸抑制腫瘤效果組織切片 65
4.4.3 小鼠血液分析 67
第五章 結論 69
第六章 參考文獻 70

表目錄
表2-1、乳癌賀爾蒙治療表 8
表2-2、乳癌化學治療表 9
表3-1、各組微脂粒的組成成分比 35
表4-1、各組微脂粒的基本性質 52
表4-2、各組微脂粒的藥物含量 54

圖目錄
圖1-1、實驗概念圖 2
圖2-1、乳癌分期示意圖 4
圖2-2、乳癌的治療策略圖 5
圖2-3、乳癌手術治療示意圖 6
圖2-4、乳癌放射治療示意圖 7
圖2-5、乳癌標靶治療示意圖 10
圖2-6、藥物釋放示意圖 11
圖2-7、奈米藥物載體圖 12
圖2-8、乳癌奈米藥物傳輸示意圖 13
圖2-9、微脂粒示意圖 15
圖2-10、微脂粒製備示意圖 16
圖2-11、腫瘤微環境免疫機制示意圖 ………………...……………17
圖2-12、先天性免疫綜觀示意圖 ………………...…………………19
圖2-13、乳癌T細胞免疫療法示意圖 21
圖2-14、Warburg Effect示意圖 22
圖2-15、Curcumin作用機制示意圖 23
圖2-16、Imiquimod作用機制示意圖 24
圖2-17、巨噬細胞分化示意圖 25
圖3-1、Cholesterol-COOH合成 ………………...………………..31
圖3-2、Cholesterol-NHS ester合成 ………………...…………….32
圖3-3、Cholesterol-Glucosamine合成 ………………...…….……33
圖4-1、Cholesterol-COOH 1H NMR鑑定圖 46
圖4-2、Cholesterol-COOH FT-IR光譜圖 46
圖4-3、Cholesterol-NHS ester 1H NMR鑑定圖 48
圖4-4、Cholesterol-NHS ester FT-IR光譜圖 48
圖4-5、Cholesterol-Glucosamine 1H NMR鑑定圖 50
圖4-6、Cholesterol-Glucosamine FT-IR光譜圖 50
圖4-7、微脂粒在37℃下穩定度測試(A) size (B) PDI 56
圖4-8、微脂粒在4℃下穩定度測試(A) size (B) PDI 56
圖4-9、GI、GIC的TEM影像 57
圖4-10、GI、GIC藥物釋放曲線 58
圖4-11、微脂粒與抗癌藥物之細胞毒性測試。(A) L929, 24 hr;(B) L929, 48 hr 60
圖4-12、微脂粒與抗癌藥物之細胞毒性測試。(A) 4T1, 24 hr;(B) 4T1, 48 hr 60
圖4-13、微脂粒與抗癌藥物之細胞毒性測試。(A) RAW, 24 hr;(B) RAW, 48 hr 60
圖4-14、具標靶性微脂粒4T1 CELL LINE吞噬測試 (A) 流式細胞分析儀結果圖 (B)量化分析圖 62
圖4-15、具標靶性微脂粒RAW CELL LINE吞噬測試 (A) 流式細胞分析儀結果圖 (B)量化分析圖 62
圖4-16、治療後小鼠腫瘤大小 64
圖4-17、治療後小鼠體重變化 64
圖4-18、治療後小鼠取下的腫瘤大小比較 64
圖4-19、腫瘤組織切片結構分布 66
圖4-20、治療後小鼠血液指數 68
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