非病毒載體大約可分為兩大類：微脂粒載體與高分子載體系統。先前許多的研究都致力於提昇非病毒載體之轉染效率，最常被使用的方式大多是利用化學反應對載體進行修飾。在本研究中，我們利用輔助劑以提昇非病毒載體之轉染效率，這個方式可以有效避免因化學反應所產生的載體純化與定性等問題。
我們使用不同雙醣分子與聚乙稀亞胺、或是不同的微脂粒載體系統混合，以探討其對於轉染與質體核酸傳遞效率之影響。利用質體核酸進入細胞後產生的綠色螢光蛋白質表現，與使用ethidium monoazide（EMA）螢光標記後的質體核酸於細胞內之螢光強度，分別表示為轉染效率與質體核酸傳遞效率。
研究發現，當聚乙稀亞胺/質體核酸複合物與海藻醣混合後，可以有效提昇轉染效率。然而當海藻醣以其他種類的雙醣分子替換後，便不具有提昇轉染效率的效果。利用海藻醣於轉染前5-120分鐘對動物細胞進行培養，轉染效率會降低30-50％，但是卻不會對質體核酸的傳遞效率造成影響。這表示轉染效率的降低並非由於胞飲作用的活性被抑制所致。在轉染完成後以海藻醣對動物細胞進行培養，結果發現對轉染效率並無影響。這表示海藻醣的存在並不影響細胞內蛋白質合成的機制。實驗也發現，海藻醣於轉染時存在會抑制質體核酸傳遞進入細胞的效率。此外，海藻醣於轉染時的存在時間多寡，也會影響轉染效率的提昇幅度。
對於微脂粒載體系統，實驗結果也發現纖維雙醣對實驗中所有的微脂粒載體都具有提昇轉染效率的作用，而麥芽醣則會抑制DOTAP/Cholesterol與LPD的質體核酸複合物之轉染效率。對質體核酸於動物細胞之傳遞效率的結果分析，發現大多數實驗所使用的雙醣分子，都能提昇DOTAP，DOTAP/Cholesterol與DOTAP/DOPE與質體核酸複合物的傳遞效率，但對於DC-Chol/DOPE與質體核酸複合物卻會產生抑制傳遞效率的效果。大體而言，轉染效率與傳遞效率間，呈現出相對的關係。
我們發現，使用海藻醣與纖維雙醣能有效提昇微脂粒載體的轉染效率。此外，當海藻醣與聚乙稀亞胺/質體核酸複合物共同存在時，才能提昇複合物之轉染效率。這或許是由於海藻醣會影響聚乙稀亞胺/質體核酸複合物在細胞中的傳遞過程與機制所引起。

Nonviral vectors mainly consist of two major classes: lipid-based and polymer-based gene delivery systems. Different strategies have been adopted to enhance the levels of transgene expression mediated by nonviral vectors. A most commonly used approach is to modify the carriers through chemical reactions. In this study, we using enhancers to improve the transfection efficiency of nonviral vectors can circumvent the needs of chemical modifications as well as subsequent purification and characterization of nonviral vectors.
In this study, different disaccharides were incorporated into the vectors prepared with DNA/polyethylenimine（PEI）, DOTAP/protamine/DNA (LPD) or DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide（EMA） covalently labeled plasmid, respectively.
We found that incorporating trehalose into the transfection reagents could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intra- cellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. The presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose.
For the lipid-based delivery system, cellobiose was found to be effective for all the lipid vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study.
In conclusion, we showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance transgene expression. Besides, only during the transfection process when DNA-PEI complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intra- cellular trafficking.

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