基因療法(Gene therapy)是將帶有治療效果的一段基因利用細胞轉染(transfection)的方式送入特定的標的細胞或器官中取代發生病變或缺失的基因並促使其表現以達到治療目的的方法。但裸露且本身帶負電的基因易受核酸酶的分解及細胞膜表面的負電性所排斥，無法有效地進入細胞中達到應有的治療效果。因此，便需要載體(Vector)的保護及幫助，以增加細胞轉染的效率(transfection efficiency)。病毒性載體(viral vector)雖然有不錯的細胞轉染效果，但容易引起不良反應，甚而致死。有鑑於此，發展非病毒性載體(non-viral vector)便成了許多研究者的目標。最近的研究指出，幾丁聚醣(chitosan)具有成為有效的非病毒性載體的發展潛力。
　　幾丁聚醣是幾丁質經由去乙醯化(deacetylation)而得，具有相當良好的生物相容性及生物可分解性，且幾乎無細胞毒性。此外，幾丁聚醣為帶有正電的聚合物，能夠和帶負電的DNA以一定的比例藉由電性作用而緊密結合成為帶正電的複合物，保護去氧核醣核酸不受核酸酶的分解，並因複合物帶有正電易於和帶負電的細胞膜表面結合，使基因能順利進入細胞中，而有效促進細胞轉染的效率。
　　文獻指出幾丁聚醣之轉染效率隨著細胞株種類、幾丁聚醣分子量大小、去乙醯化程度(deacetylation)、細胞培養環境而有所不同。為進一步研究，本研究選擇基底細胞瘤(Basal cell carcinoma，BCC)，株化角質細胞(Human keratinocyte，HaCaT)，肝癌細胞(human hepatoma cells， Huh7)及胃癌細胞(human gastric cells，AGS)，並利用reporter gene, pCMV-β,探討不同分子量的幾丁聚醣water soluble chitosan、chitosan 100、chitosan 500，及low molecular weight chitosan在這四株細胞中細胞轉染的效果，並探討不同的轉染條件對幾丁聚醣在轉染效果上的影響以及幾丁聚醣之細胞毒性。同時亦利用pEGFP-C1和幾丁聚醣形成複合物進行轉染，觀察蛋白質表現之分布情形。
　　研究結果顯示，除了water soluble chitosan外，其餘三種chitosan均能夠和質體形成複合物。在轉染試驗中，chitosan 100在BCC細胞株之轉染效率最高，HaCaT，及AGS次之，Huh7則最低。另外，轉染效率在皮膚細胞株（BCC、HaCaT）較其他細胞株來的高。另一方面，轉染效率及最佳轉染效率之重量比是根據分子量及細胞種類而有所不同，為cell-dependent。細胞毒性試驗之結果顯示，三種不同的幾丁聚醣在四個不同的細胞株上均無細胞毒性。螢光顯微鏡之影像亦可以觀察到chitosan之轉染情形及蛋白質分布狀態。
由本研究可知，幾丁聚醣為安全無毒，和基因結合後能促進基因之轉染，是一個具有發展潛力的基因載體。由於幾丁聚醣之轉染效率具有細胞特異性，本研究之結果可供作相關細胞或組織之轉染參考。

　　Gene therapy refers to the transfection of DNA encoding a therapeutic gene into the targeted cells or organs with expression of the transgene. Recently, gene therapy has received attention due to its potential application in the replacement of dysfunctional gene and treatment of acquired diseases. The electronegativity of the naked DNA tends to inhibit itself from entering most negatively-charged cell membrane. Moreover, the unprotected DNA is rapidly degraded by nuclease. The most important issue in gene therapy is to develop a safe and effective gene transfection system. Several methods have been developed to enhance the transfer of DNA into cells for gene therapy. Virus vectors are very effective, but they have fatal drawbacks such as immune response and oncogenic effects when used in vivo. Non-viral vectors, generally consisting of cationic liposomes and linear cationic polymers, have several advantages over viral vectors. However, non-viral gene delivery has its limitations such as toxicity and low transfection efficiency. Chitosan is a cheap, biocompatible, biodegradable, and non-toxic polymer that forms polyelectrolyte complex with DNA.

　　In this study, three different chitosans, chitosan 100, chitosan 500, and low molecular weight chitosan, were characterized and evaluated as a gene delivery vector in four different human cell lines. The objectives were to investigate the formation of plasmid/chitosan complex, and to evaluate the transfection efficiency of the complexes which chitosans formed with plasmid, pCMV-β, in human cell lines, including basal cell carcinoma (BCC), human immortalized keratinocytes (HaCaT), human hepatoma cells (Huh7), and human gastric carcinoma cells (AGS). The cytotoxicity of three different chitosans was determined by MTT assay, and localization of reporter gene, pEGFP-C1, expression in the transfected cells was examined using microscopes.

　　The reaults showed that all chitosans formed complex with plasmids in appropriate weight ratios except water soluble chitosan. At a plasmid/chitosan weight ratio between 1:1~1:10, the most effective transfection efficiency was obtained at weight ratio of 1:5 in BCC cells, and the transfection efficiency of chitosans in Huh7 was lower than other cells. The transfection efficiency of chitosans in skin cell lines (BCC and HaCaT) is higher than other cell lines (Huh7 and AGS). The influence of molecular weight of chitosans in transfection efficiency appeared to be cell-dependent. In addition, DNA/chitosans complex showed negligible cytotoxicity for four different cell lines. Microscopic examination revealed that the gene expression products were distributed in the entire cell including cytosol and nuclei.

　　In conclusion, this study demonstrated that plasmid DNA formed complexes with chitosan 100, chitosan 500, and low molecular weight chitosan, and was effectively transfected into four different human cells lines. Therefore, the non-cytotoxic chitosans have the potential for safe and efficient non-viral gene delivery.

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