基因遞送系統之中選擇以chitosan作為轉染載體的優點在於：它是天然高分子，對於細胞毒性較低，而且因本身帶有多量的正電荷，能與欲轉染之DNA以靜電力(electrostatic force)自然的結合，在遞送至細胞的過程中，又能與細胞膜表面的靜電荷以天然的靜電引力牽滯住，使其能自然嵌合入細胞膜以胞飲作用(endocytosis)的形式進入細胞；在細胞質內遞送過程又不會因酸鹼值改變及酵素降解的影響，仍能將DNA順利帶入細胞核中，並且可以被複製及表現。
現階段chitosan在基因載體的材料使用，是將甲殼動物的幾丁質外殼(chitin)去乙醯化後所得之chitosan。在這次的論文當中要討論的是，取用來自真菌類(fungi)的子實體(SACCHACHITIN) ，在50 %氫氧化鈉溶液中，經不同溫度及不同時間的去乙醯化作用之後得到的chitosan (為與來自甲殼類之chitosan有所區別，將之名為sacchachitosan)，在作為基因遞送系統之載體方面，是否也和來自甲殼動物的市售商品具有同樣的轉染效果。
實驗結果顯示出：在產率方面，以130°C、2小時所得的產率最高(13.6％)。另外，不同的反應溫度及時間將會影響到sacchachitosan去乙醯化程度的不同，相對地其分子量大小也將受到影響。以電泳方式觀察sacchachitosan包覆DNA的效果，發現市售chitosan可包覆的比例(chitosan / DNA 1 : 4 )高於sacchachitosan( 1 : 1 )。在細胞體外轉染試驗中，不同反應條件所得之sacchachitosan對於HeLa細胞轉染能力的效力及表現雖然不若市售chitosan來得顯著，但經由螢光顯微鏡仍可以看出醣化去乙醯幾丁質具有基因轉染效果。

Several methods for nonviral gene therapy are being developed based on principles of advanced drug delivery, and development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired disease by the gene therapy. With regard to nonviral vectors, major attention is paid to cationic polymers which are able to condense large DNA into smaller structures as well as to mask the negative DNA charges, necessary for transfecting cells. Chitosan, a natural polysaccharide which is partially deacetylated glucosamine, is being widely used as a pharmaceutical excipient so far as to apply on gene delivery system. Commercial chitosan all are obtained from crustacean shells. In view of mimic main structure, a new biomaterial for gene delivery vector, sacchachitosan, which is purified from Ganoderma cell wall being abundant of chitin, was examined.
Following the purification processes, the most products were able to be manufactured under deacetylation condition in 50 % NaOH solution with heating at 130°C for 2h (yield about 13.6 %). Structural analysis by TLC revealed that these products mainly contained the same structural units of N-acetylglucosamine and glucosamine as commercial products. Observation from electrophoresis, sacchachitosan condensed in a less extent with DNA (< chitosan:DNA 1:1) than that with commercial chitosan (> chitosan:DNA 1:4). Co-culturing chitosan-DNA complexes with HeLa cells, autofluorescence performance through fluorescent microscopy was observed indicating transfection was achieved. According to the FASCan measurement, the chitosan-DNA complexes with different brands of commercial chitosan showed apparent transfection efficiency, whereas sacchachitosan-DNA complexes showed less significant but still detectable expression of transfection.

目錄
附圖目錄………………………………………………………..І
附表目錄……………………………………………………...ІV
中文摘要………………………………………………………V
英文摘要…………………………………………………….VІІ
壹、 緒論
第一節 基因療法…………………………………….....1
1.1 病毒載體……………………………………….....2
1.2 非病毒載體……………………………………….3
第二節 基因遞送載體與細胞之交互作用…………….6
2.1 與血中物質間的交互作用……………………….6
2.2 免疫系統的辨識………………………………….7
2.3 血管壁的通透性………………………………….7
2.4 標的細胞的再吸收……………………………….8
2.5 內質體的釋放…………………………………….9
2.6 在細胞質中的安定性與細胞核的輸入………….9
第三節 chitin與chitosan的介紹……………………..10
3.1 chitin………………………………………......10
3.2 chitosan………………………………………..10
研究動機與目的……………………………………………...16
貳、實驗材料與方法
第一節 靈芝甲殼素之製備………………………..….17
第二節 chitosan/DNA複合物之製備………………....23
第三節 以chitosan/DNA複合物作為基因遞送系統之體外試驗(in vitro)………………………………...29
參、結果與討論
4.1 醣化去乙醯幾丁質之產率…………………….36
4.2 TLC結果……………………………………….36
4.3 Chitosan/DNA複合物電泳膠片結果…………40
4.4 Chitosan/DNA複合物以體外細胞試驗之基因轉染表現………………………………………….43
肆、結論………………………………………………………50
伍、附圖………………………………………………………52
陸、附表………………………………………………………78
柒、參考文獻…………………………………………………81
圖目錄
圖一、Retrovirus之結構圖……………………………………..…52
圖二、Adenovirus之結構………………………………………...52
圖三、Adeno-associated virus 之結構………………………..…..53
圖四、微脂粒之構造………………………………………..….…53
圖五、polyethyleneimine (PEI)之化學結構式………………..….54
圖六、chitosan之化學結構式………………………………..…...54
圖七、pEGFP-C1結構圖…………………………………….…...55
圖八、FACScan與FACSCalibur之液流系統簡圖……………...55
圖九、FACScan與FACSCalibur之光學系統簡圖……………...56
圖十、流式細胞儀之原理圖………………………………….…..56
圖十一、光電訊號之轉換處理，FSC ……………………………57
圖十二、光電訊號之轉換處理，SSC，FL1，FL2，FL3……….57
圖十三、靈芝子實體以不同時間、不同溫度進行去乙醯作用所得醣化去乙醯幾丁質(sacchachitosan)之產率…………....58
圖十四、薄層液相色層分析(Thin-Layer Chromatography)
--- 醣類分析……………………………………………59
圖十五、薄層液相色層分析(Thin-Layer Chromatography)
-- 醣胺類分析………………………………………… .60
圖十六、市售chitosan / DNA ( 2 μg / 2 μg ) 電泳圖…………. 61
圖十七、市售chitosan / DNA ( 2 μg / 4 μg ) 電泳圖………… .62
圖十八、市售chitosan / DNA ( 2 μg / 6 μg ) 電泳圖…………..63
圖十九、市售chitosan / DNA ( 2 μg / 8 μg ) 電泳圖…………..64
圖二十、市售chitosan / DNA ( 2 μg / 10 μg ) 電泳圖…………65
圖二十一、醣化去乙醯幾丁質(901) / DNA 各種比例之電泳圖..................................................................................66
圖二十二、各種醣化去乙醯幾丁質/ DNA 各種比例之電泳圖……………………………………………………..67
圖二十三、各種醣化去乙醯幾丁質/ DNA 各種比例之電泳圖……………………………………………………..68
圖二十四、HeLa cell (attached) 未經轉染之顯微鏡觀察……….69
圖二十五、HeLa cell (suspened) 未經轉染之顯微鏡觀察………70
圖二十六、HeLa cell (suspened) 未經轉染之螢光顯微鏡觀察…70
圖二十七、HeLa cell (attached) 經市售chitosan/DNA (1/1) 複合物轉染後之顯微鏡觀察……………………………..71
圖二十八、HeLa cell (attached)經市售chitosan/DNA (1/1) 複合物轉染後之螢光顯微鏡觀察…………………………..71
圖二十九、HeLa cell經sacchachitosan / DNA (1/1) 複合物轉染72小時後顯微鏡觀察…………………………….…….72
圖三十、HeLa cell經sacchachitosan / DNA (1/1) 複合物轉染72小時後以螢光顯微鏡觀察…………………………..72
圖三十一、HeLa cell經市售chitosan/DNA (1/1) 複合物轉染72小時後顯微鏡觀察……………………………….….73
圖三十二、HeLa cell經市售chitosan/DNA (1/1) 複合物轉染72小時後以螢光顯微鏡觀察……………………….....73
圖三十三、HeLa cell經sacchachitosan/DNA (1/1) 複合物轉染後繼代培養(P1)之顯微鏡觀察………………………..74
圖三十四、HeLa cell經sacchachitosan/DNA (1/1) 複合物轉染後繼代培養(P1)之螢光顯微鏡觀察…………………...74
圖三十五、HeLa cell經市售chitosan/DNA (1/1) 複合物轉染後繼代培養(P1)之顯微鏡觀察…………………………...75
圖三十六、HeLa cell經市售chitosan/DNA (1/1) 複合物轉染後繼代培養(P1)之螢光顯微鏡觀察……………………...75
圖三十七、FL2對細胞顆粒性分佈之點狀圖…………………...76
圖三十八、FL1之曲線分佈圖…………………………………...76
圖三十九、顆粒性變化對FL2之點狀圖………………………..77
表目錄
表一、以不同時間、溫度進行去乙醯化作用所得之醣化去乙醯幾丁質(Sacchachitosan)之產率……………………………….78
表二、市售chitosan流式細胞儀測得之FL1數值………………..79
表三、醣化去乙醯幾丁質流式細胞儀測得之FL1數值………….80

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