臺灣博碩士論文加值系統

肝細胞癌（HCC）是肝臟中最常見的原發型惡性腫瘤，並且位居癌症死因第三名。肝動脈化療栓塞廣泛應用於無法進行手術切除的肝細胞癌（HCC），因此本研究旨在設計具新穎性的化療栓塞試劑。該栓塞劑是將結蘭膠微球包覆透明質酸/聚乙烯亞胺-阿黴素的奈米顆粒（HH / PH-DOX）。組氨酸分別與透明質酸、聚乙烯亞胺共軛形成HH、PH產物，1 H NMR指出在8.064 ppm（-N = CH-）和6.979 ppm（-N-CH = C-）有新的尖端值出現，表透明質酸的“methenyl group”與組氨酸的咪唑環有起反應，兩者有成功共軛成功。而在待測物PH的1 H NMR結果，發現了7.714 ppm（-N = CH-）和6.959 ppm（-N-CH = C-）新的尖端值出現，表明了有成功地獲得PH。從Zeta電位的變化，我們發現阿黴素可以成功地被承載於HH / PH-DOX奈米顆粒。透過W / O乳化法製備出包覆HH/PH-DOX的結蘭膠微粒，平均粒徑300±100微米。從細胞攝取試驗結果得知，於24小時共同培養後，HH/PH-DOX可以藉由內吞作用進至HepG2細胞裡頭。綜合以上所述之結果，本論文認為包覆HH/PH-DOX的結蘭膠微粒具有成為栓塞劑的潛能性。

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and the third cause of cancer death. Transcatheter arterial chemoembolization has most widely been applied to hepatocellular carcinoma (HCC) for patients who are not eligible for surgery. The study aims to design noval chemoembolization agent for hepatoma therapy. The chemoembolization agent is based on gellan gum microsphere which contain hyaluronan/polyethylenimine-doxorubicin nanoparticles (HH/PH-DOX). Hyaluronan conjugated with histidine (HH) and Polyethylenimine conjugated with histidine (PH) were synthesized, 1H NMR spectrum showed that the peaks of HH were at 8.064 ppm (-N=CH-) and 6.979 ppm (-N-CH=C-) which were ascribed to the methenyl group located in imidazole group of Histidine. The newly appeared peaks at 7.714 ppm (-N=CH-) and 6.959 ppm (-N-CH=C-) revealed that PH was successfully obtained. From the change of zeta potential, we found the doxorubicin can encapsulate into HH/PH matrix successfully. The gellan gum microsphere contain HH/PH-DOX were fabricated using a W/O emulsion method, and the diameter of these microspheres was 300± 100 um. The cellular uptake test, results showed that HH/PH-DOX could be into HepG2 cells by endocytosis after co-incubation for 24h. From these datas, the gellan gum microsphere with HH/PH-DOX nano particles have the potential as the chemoembolization agent in the future.

中文摘要…………………………………………….......................................i
英文摘要……………………………………………......................................ii
目錄……………………………………………….........................................iii
圖目錄……………………………………………….....................................vi
表目錄…………………………………………….......................................viii
第一章、緒論…………………………………………………………………1
1-1肝癌 ……………………………………………………………………...1
1-2肝癌動脈栓塞療法……………………………………………………….3
1-3結蘭膠（Gellan gum）簡介………………………………………………7
1-4聚乙烯亞胺（Polyethyleneimine;PEI）簡介……………………………9
1-5透明質酸（Hyaluronic acid; HA）簡介…………………………………12
1-6鹽酸多柔比星（Doxorubicin．HCl）簡介………………………………14
1-7藥物動力學簡介…………………………………………………….......16
1-8研究動機………………………………………………………………...17
第二章、實驗材料與方法
2-1 實驗藥品……………………………………………………………….18
2-2 實驗儀器與裝置……………………………………………………….19
2-3 Hyaluronic acid / polyethylenimine-doxorubicin nanoparticles (HH/PH-DOX)合成…………………………………………………..20
2-3.1 Hyaluronic acid-Histidine（HH）合成……………………………….21
2-3.2 Polyethylenimine-Histidine（PH）合成………………………………21
2-3.3 Hyaluronic acid / polyethylenimine-doxorubicin nanoparticles (HH/PH-DOX)合成…………………………………………………22
2-4 HepG2 細胞進行HH/PH-DOX攝取…………………………………..22
2-5純Gellan gum 微粒製備……………………………………………….23
2-6 Gellan gum微粒包覆HH/PH-DOX奈米顆粒製備……………………24
2-7 Gellan gum微粒包覆HH/PH-DOX進行體外藥物釋放模擬…………24
2-8 動物模型的栓塞建立………………………………………………….25
第三章、結果與討論……………………………………………………….26
3-1核磁共振光譜儀（NMR）………………………………………………26
3-1.1 HH NMR……………………………………………………………...26
3-1.2 PH NMR………………………………………………………………26
3-2介面電位………………………………………………………………...26
3-3 HH/PH-DOX粒徑分析…………………………………………………27
3-4螢光顯微鏡下HepG2攝取HH/PH-DOX的情形……………………...28
3-5純gellan gum微粒結果呈現……………………………………………28
3-5.1巨觀圖和掃描式電子顯微鏡下的外觀型態及表面結構……………28
3-5.2粒徑分析圖……………………………………………………………29
3-6 Gellan gum微粒包覆HH/PH-DOX奈米顆粒的結果呈現……………30
3-6.1掃描電子顯微鏡下的外觀型態和表面結構…………………………30
3-6.2粒徑分析…………................................................................................31
3-7 Gellan gum微粒包覆HH/PH-DOX體外藥物釋放模擬………………32
3-7.1繪製Doxorubicin的標準曲線………………………………………..32
3-7.2體外藥物釋放曲線圖（pH7.4）………………………………………..32
3-8動物模型栓塞情況分析………………………………………………...33
第四章、結論………………………………………………………………..34
參考文獻……………………………………………………………………35
附錄…………………………………………………………………………42

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