皮膚鱗狀上皮細胞癌(cutaneous squamous cell carcinoma)是一種惡性且易轉移的皮膚腫瘤，在皮膚癌的發生率中排名第二位，近十年來發生率有明顯上升的趨勢。臨床上治療方法包括外科手術、放射治療、化學治療，以及尚於研究階段的治療方式有免疫療法以及基因療法。
WWOX基因位於人類染色體16q23.3-24.1的位置，可產生含雙色胺酸功能區氧化還原酶(ww domain-containing oxidoreductase)。近年來的研究顯示相較於正常細胞，WOX1 蛋白質在腫瘤細胞的表現量較低、甚或是完全不表現，可能是造成腫瘤生成的因素之一。TP53基因研究迄今已三十餘年，當外來或內生性壓力產生時，在細胞反應調節上p53蛋白扮演重要角色。TP53基因主要功能在於調節轉錄作用，調節細胞週期靜止及細胞凋亡。在皮膚癌的發生過程中，會因UV光長期暴露而使DNA受損產生顯著性的TP53基因突變。
基因傳遞(gene delivery)的技術在發展基因療法(gene therapy)上佔了很重要的部分。基因傳遞的路徑很多，若要採用非侵入性的方式則需要一個有效的載體以達到基因傳遞的目的。PEI為帶正電荷的聚合物載體，各種研究證實能成功的將基因傳送入各種不同的細胞，而各細胞適合之轉染條件有所不同，因此用於不同細胞之轉染條件仍須確認。
本研究之目的在探討PEI作為皮膚基因輸送載體之可行性。藉由WWOX基因與TP53基因作為目標抗癌基因，將於體外皮膚細胞實驗觀察其轉染效率與細胞凋亡效應，並評估複合物投與腫瘤組織後之抗癌表現。
Gel retardation 試驗研究結果顯示，PEI/DNA複合物比例大於4:1即可形成有效包覆之複合物。藉由Flow cytometry研究轉染效率，其結果顯示對於SCC-15及HaCaT兩細胞株，在相同的PEI濃度下，轉染效率皆與DNA量成正比，而與DNA種類無關；PEI載體的轉染效率皆低於Lipofectamine載體。細胞凋亡反應方面，在SCC-15細胞中由WWOX基因引起的凋亡反應，不因DNA含量增加而上升，而p53基因有劑量依存性，而DNA種類並未影響細胞凋亡反應。對於HaCaT而言，WWOX基因與p53基因引起的凋亡反應皆與DNA含量相關，並與DNA種類無關；而PEI載體在HaCaT產生之細胞凋亡反應皆高於Lipofectamine，對於SCC-15則與Lipofectamine所產生之細胞凋亡反應略同。基於上述研究結果，選擇DNA含量為2μg/mL(N/P ratio 8:1)評估其蛋白質表現。結果顯示，給與含有WWOX-pEGFPC1基因的複合物相較於對照組皆會使WWOX蛋白質表現明顯增加；對於HaCaT，使用PEI作為載體於WWOX蛋白質表現量與Lipofectamine之間無差異；對於SCC-15，使用PEI於WWOX蛋白質表現量除了在WWOX-pEGFPC1 2μg/mL較Lipofectamine高，其餘組別兩者之間並無差異。給與含有p53-DsRedN1基因的複合物相較於對照組皆會使p53(DsRed)蛋白質表現明顯增加，使用PEI時除了在WWOX-pEGFPC1 1μg/mL合併p53-DsRedN11μg/Ml SCC-15表現較高外，其餘組別兩者之間並無差異。動物腫瘤生長抑制結果則顯示相較於對照組，PEI/WWOX-pEGFPC1、PEI/WWOX-pEGFPC1+ PEI/p53-pDsRedN1、Lipo/WWOX-pEGFPC1及PEI/p53-pDsRedN1皆能明顯抑制腫瘤生長，其中又以PEI/WWOX-pEGFPC1最有抑制效果。
本研究利用WWOX基因及p53基因作為抗癌基因研究，由動物體內試驗研究結果發現WWOX基因及p53基因對於鱗狀上皮細胞癌確實有相當程度的抑制腫瘤效果。利用PEI作為基因傳遞載體，於細胞轉染效率、細胞凋亡效應、蛋白質表現皆與Lipofectamine載體無差異，由於PEI具易取得且經濟之優點，因此PEI與WWOX基因及p53基因的複合物對於未來臨床抗癌具有相當潛力，
亦可做為未來相關皮膚基因傳遞研究之參考。

關鍵詞：鱗狀上皮細胞癌，基因傳遞，polyethylenimine(PEI)，WWOX基因，
p53基因

Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin cancer. The incidence of this type of skin cancer has increased greatly for the past decade. The treatment for treatment of squamous cell carcinoma include surgery, radiotherapy, chemotherapy, immunotherapy and gene therapy.
Human WWOX gene, encoding the WWOX/FOR/WOX1 family proteins, is mapped to a fragile site on chromosome 16q23.2. WOX1 protein is considered as a candidate tumor suppressor protein. TP53 gene is located on the short arm of chromosome 17p13.1. It is well-known that p53 protein is important for cellular response to a wide variety of stressful stimulation. During the development of skin cancer, loon-term ultraviolet radiation (UVR) exposure can alter gene activity in keratinocytes, resulting notably p53 mutantion.
The technology of gene delivery plays an important role in the evelopment of gene therapy. Several methods have been developed to transfer DNA into cells for gene therapy. One of the non-invasive gene delivery methods is the use of gene vectors or carriers. Polyethylenimine (PEI) is a cationic polymer-based gene carrier,. Many researches demonstrated that PEI can transfer genes into different kind of cells under different transfection conditions.
The aim of this study was to examine the feasibility of using PEI as gene carrier to deliver WWOX gene TP53 gene to squamous cell carcinoma. The PEI/DNA complexes of different N/P ratios were characterized by gel retardation assay. Transfection efficiency, degree of cell apoptosis and protein expression were analyzed in SCC cells and HaCaT cells, in order to determine optimal transfection conditions for PEI/DNA complexes to be used in in vivo experiments.
Results of gel retardation assay showed that WWOX gene formed complexes with PEI when N/P ratios were greater than 4:1. It was demonstrated that transfection efficiency were related to the amount of DNA, but not to DNA types in both cell lines, and transfecton efficiency of PEI was lower than that of Lipofectamine. In SCC-15 cells, degree of apoptosis induced by WWOX gene were not related with DNA types. However, degree of apoptosis induced by p53 gene were dose-dependent. On the other hand, degree of apoptosis induced by both WWOX gene and p53 gene in HaCaT cell was associated with DNA content but not with DNA types. Degree of apoptosis induced by PEI/DNA complex was more prominent than lipo/DNA complex in HaCaT cells, while they were comparable in SCC-15 cells.
Based on the above data, vector/DNA 2μg/mL (N/P ratio 8:1) was selected for the following evaluation. The results displayed that WWOX protein expression was significantly increased in comparison with control groups in both cell lines. For HaCaT cell, protein expression level was comparable between PEI and Lipo as vectors, while in SCC-15 Cell, protein expression level by PEI-mediated gene delivery was greater than Lipo. Direct injection of vector/DNA complexes into tumor-bearing mice demonstrated that PEI/WWOX pEGFPC1, PEI/WWOX-pEGFPC1+ PEI/p53-pDsRedN1, Lipo/WWOX-pEGFPC1 and PEI/p53-pDsRedN1 complexes significantly inhibited tumor growth in comparison to the control. Among these groups, the effect from PEI/WWOX-pEGFPC1 complex was the most prominent.
In summary, the current study demonstrated that both WWOX gene and p53 gene can be effectively delivered to squamous cell carcinoma by PEI and Lipofectamine and effectively reduced tumor size in vivo. The effect of PEI was comparable to Lipofectamine in terms of transfection efficiency, apoptotic effect and protein expression, with the advantages of easy accessibility and cost-effectiveness. It is concluded PEI with WWOX gene and p53 gene complexes is a potential therapeutics for squamous cell carcinoma.

Keywords：Squamous cell carcinoma (SCC), WWOX gene, p53 gene, gene delivery, polyethylenimine(PEI)

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