使用不同組成的正電性微脂粒，探討體外視網膜色素上皮細胞之轉殖效果

Transfection Effects of In Vitro Retinal Pigment Epithelial Cells by Different Components of Cationic Liposome

陳志明(Chi-Ming Chan)

指導教授 ： 劉得任

臺北醫學大學/口腔學院/生物醫學材料研究所/碩士(2005年)

摘要

本研究的目的在於使用不同組成的正電性微脂粒，探討體外視網膜色素上皮細胞之轉殖效果。我們利用綠螢光蛋白質基因，去檢測視網膜色素上皮細胞之蛋白質螢光表現。本實驗並使用了不同種類的正電性脂質、微脂粒中不同莫爾比的正電性脂質及其助手脂質(膽固醇及DOPE)，不同組成的小微泡單層微脂粒(SUV)及多層微脂粒(MLV)去比較其基因轉殖效果。我們並嘗試使用幾丁聚醣作為基因載體。 結果顯示本實驗所使用的正電性微脂粒，雖有不同的轉殖效果，但均能成功的轉殖至視網膜色素上皮細胞。而中性的微脂粒，其轉殖效果郤不佳。本實驗顯示DOTAP 是最佳的正電性脂質，而微脂粒中較高莫爾比的正電性脂質及DOPE，較低莫爾比膽固醇及多層微脂粒組成，能改善其轉殖效果。而利用幾丁聚醣作為基因載體，其轉殖效果不佳。當添加正電性脂質及DOPE至幾丁聚醣，其轉殖效果仍然不佳。我們發現1:4莫爾比之DOTAP:DOPE及2:1重量比之正電性微脂粒:DNA，是最佳的正電性微脂粒組成。 在視網膜色素上皮細胞之基因轉殖，找出最適當的正電性微脂粒組成，對其正常及帶病細胞之基因表現，在分子生物之研究，是相當重要的。

關鍵字

正電性微脂粒 ； 視網膜色素上皮細胞 ； 幾丁聚醣

並列摘要

The purpose of this study was to investigate the transfection effects of in vitro retinal pigment epithelial (ARPE) cells by different components of cationic liposome. In order to show the transfection efficacy of green fluorescent protein (GFP) gene to the ARPE cells, we use different kinds of cationic lipids, different molar ratio of the cationic lipid components and helper lipids [cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE)] in the liposomes. Moreover, we compared the transfection efficacy of small unilamellar vesicles (SUV) and multilamellar vesicles (MLV). Furthermore, we tried to use chitosan as the gene carriers. The results show that all cationic liposomes used in this study successfully transfected ARPE cells with different efficiencies. But the transfection efficacy for the neutral liposome was poor. N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) showed the best cationic lipid for the gene transfection. Higher molar ratio of positively charged lipid component and DOPE, lower molar ratio of cholesterol and formulation of MLV improved the transfection efficacy. For chitosan mediated gene transfection, the efficacy was not satisfactory. When adding the cationic lipid and DOPE to the chitosan, the transfection efficacies were still poor. In our study, we showed the optimal cationic liposome formulations of DOTAP:DOPE (molar ratio) in 1:4 and cationic liposome (CL):DNA in 2:1 (w/w) with increased transfection efficiency to the ARPE in vitro. Optimization of gene delivery to ARPE cell line cultures by different components of cationic liposomes is particularly important when considering molecular biological studies on gene expression in normal and diseased cells.

並列關鍵字

Cationic liposomes ； Retinal pigment epithelial cells ； Chitosan

參考文獻

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