醣化去乙醯幾丁質與不同來源之幾丁聚醣之特性探討與基因轉染率之比較

Characterization of Sacchachitosan and Comparison of Gene Transfection Efficiency with Various Sources of Chitosan

劉慧如

指導教授 ： 許 明 照 ； 蘇 慶 華

臺北醫學大學/口腔學院/生物醫學材料研究所/碩士(2002年)

摘要

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。

關鍵字

醣化去乙醯幾丁質 ； 基因療法 ； 基因轉染率 ； 幾丁聚醣

並列摘要

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.

並列關鍵字

sacchachitosan ； gene therapy ； gene transfection efficiency ； chitosan ； GFP

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