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研究生:陳家琪
論文名稱:利用微脂體遞送基因進入肝細胞之最佳化劑型探討
論文名稱(外文):optimizing of liposomal formulation for gene delivery to liver cells
指導教授:張富雄
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:微脂體基因遞送
外文關鍵詞:polyethylenimineprotamine sulfatepoly-L-lysine
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中文摘要
基因治療的重要關鍵在於是否有一個有效的遞送系統能將具有功能性的基因送至細胞中表現,目前所使用的遞送系統,主要分為兩大類:病毒性載體與非病毒性載體。病毒性載體其遞送效率佳,但是對宿主所造成的毒性以及免疫反應的緣故,有安全上的顧慮,因此致力於非病毒性載體的研究。非病毒性載體具有使用簡單、容易大量製備、不易引起宿主免疫反應的特點,但是其遞送效率差,是目前所面臨到的最大瓶頸。目前已有數種非病毒性載體的遞送系統,其中以微脂體作為載體的遞送效率最好,又以正電荷微脂體最具有潛力,並且已廣泛應用在體外實驗中。但是微脂體的遞送效率仍是有限,而在文獻中有報導指出,利用陽離子聚合物可將DNA纏繞成一緻密結構,再與微脂體所形成之複合體其粒徑比DNA/liposome複合體小,利於細胞的內吞作用,並且會保護DNA避免受到酵素分解,增加DNA到核內表現的機會,提升遞送效率。
本論文主要研究利用不同的陽離子聚合物與正電荷微脂體形成不同劑型的複合體,來提高正電荷微脂體遞送基因至人類肝癌細胞的效率。在體外細胞轉染實驗中,利用陽離子脂質與輔助性脂質以適當比例製成正電荷微脂體,再添加魚精蛋白硫酸胺、聚-L-離胺酸、PEI等陽離子聚合物,以螢光基因(luciferase、EGFP)為報導基因來分析細胞轉染效率。在HepG2細胞中,與DNA/liposome複合體比較,DNA/protamine sulfate/liposome複合體轉染效率提高1.3倍,DNA/poly-L-lysine/liposome複合體提高1.5倍,DNA/PEI/liposome(LED)複合體則提高8.5倍;而在HepG2-HBV細胞中,DNA/protamine sulfate/liposome複合體提高1.4倍,DNA/poly-L-lysine複合體提高1.6倍,LED複合體則提高7倍。再者,以流式細胞儀分析EGFP此報導基因細胞轉染的效率,並用螢光顯微鏡觀察EGFP的表現,可以得知LED複合體遞送基因能力最高。因此利用此最佳劑型的微脂體來遞送反義去氧寡核醣核酸,經由西方墨點法觀察蛋白質改變情形。由實驗結果得知,所遞送的反義去氧寡核醣核酸能夠有效的抑制基因的表現,即使經過藥物作用將基因活化,其基因仍持續被抑制。由此可以知道所發展的遞送系統,不論是遞送質體DNA或是小片段DNA分子,都能有效率的將基因遞送至細胞中表現。
本實驗是利用陽離子聚合物所形成不同劑型的微脂體,增進正電荷微脂體基因遞送的效率,以得到最佳基因遞送系統,能夠有效的遞送完好的基因到細胞中表現或是遞送反義去氧寡核醣核酸抑制基因的表現,達到基因治療的目的。此發展對非病毒性載體有很大的貢獻,期許能夠進一步應用在動物活體的研究。
Abstract
A major aim of gene therapy is the efficient and specific delivery of therapeutic gene into the desired target tissues. At the present time, major gene delivery systems employ either viral or non-viral vectors. Viral vectors can mediate efficient gene transfer. However, there are drawbacks related to the viral elements such as immune and toxic reactions, and the potential for recombination with wild-type virus and insertional mutagenesis. Non-viral vectors, despite their relatively low efficiency, are less immunogenic than viral vectors and ease of manipulation, making them attractive gene delivery systems. Recent reports suggest that cationic liposome are potent non-viral vectors and have been widely used for gene delivery in vitro. In attempt to improve biological activity of the lipoplexes, condensing DNA with polycations prior to its mixture with cationic liposomes was shown to be advantageous. The addition of polycations, such as protamine sulfate、poly-L-lysine and PEI, were showen to condense DNA, to reduce the particle size of the complexes, to render DNA resistant to nuclease activity and to enhance transfection efficiency.
The aim of this study is to develop an effective non-viral gene transfer system by the combination of cationic liposomes and polycations, and increase gene transfer efficiency in human hepatocellular carcinoma cell lines. We used cationic lipid, DOTAP, in the presence of cholesterol as helper lipid. The polycations/cationic liposomes complexes (polyplexes) were used to be vectors and the pCMVLuc, pEGFP as reproter genes. In HepG2 cells, compare with DNA/liposomes complexes (lipoplexes), the protamine sulfate polyplex increased the transfection activity about 1.3-fold, the poly-L-lysine polyplex increased about 1.5-fold, and the LED (DNA/PEI/liposome) polyplex increased up to 8.5-fold. And in the HepG2-HBV cells, the protamine sulfate polyplex increased the transfection activity about 1.4-fold, the poly-L-lysine polyplex increased about 1.6-fold, and the LED polyplex increased up to 7-fold. In addition, the different liposomal formulations were futher characterized by the FAScan analysis and fluorescent microscopy. These data indicated that the LED polyplex is a better formulation in vitro, so we used this liposomal formulation to delivery antisense oligodeoxynucledtides and observed the protein expression by Western bolt analysis. It suggests that the gene expression was inhibited by antisense oligodeoxynucledtides and reduced the protein expression level. The inhibition effect was shown even in treating with anticancer drugs to activate gene expression.
The experimental results demonstrated that the polycations can enhance transfection efficiency, and we will use this delivery system to deliver wild type tumor suppressor gene into a cancer or to deliver antisense molecules to down-regulate the gene expression, achieved the successful gene therapy. The efficiency of this non-viral delivery system in vivo remains to be studied.
目錄
中文摘要……………………………………………………………………1
英文摘要……………………………………………………………………3
縮寫…………………………………………………………………………5
第一章 緒論………………………………………………………………6
1.1 基因治療……………………………………………………….6
1.2 微脂體…………………………………………………………14
1.3 陽離子脂質……………………………………………………24
1.4 魚精蛋白………………………………………………………28
1.5 報導基因………………………………………………………28
1.6 流式細胞儀……………………………………………………29
1.7 唐黴素…………………………………………………………30
1.8 研究動機與目的………………………………………………32
第二章 實驗材料……………………………………………………….33
第三章 實驗方法……………………………………………………….34
3.1 DNA結合能力分析………………………………………………....34
3.2 基因遞送之試驗…………………………………………………...34
3.3 不同劑型正電荷微脂體基因載體之製備………………………...38
3.4 反義去氧寡核醣核酸……………………………………………...41
第四章 實驗結果……………………………………………………….44
4.1 DNA/polycations/cationic liposome複合體對細胞轉染的影響44
4.2 反義去氧寡核醣核酸之遞送對於基因表現的影響……………...48
第五章 討論………………………………………………………………51
第六章 參考文獻…………………………………………………………57
圖表與說明……………………………………………………………….67
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