皮膚鱗狀上皮細胞癌(cutaneous squamous cell carcinoma)是一種惡性且易於轉移的皮膚腫瘤，在皮膚癌的發生率中排名第二位，近十年來甚至有明顯上升的趨勢。皮膚鱗狀上皮細胞癌是發生於表皮上基底層(suprabasal epidermis)的鱗狀細胞，常見的發生原因有紫外線的照射、游離輻射線的暴露、人類乳頭狀瘤病毒(HPV)感染、長期接觸化學致癌物質、器官移植接受者、基因變異及傷口久不癒合等。臨床上已用於治療皮膚鱗狀上皮細胞癌的方法有外科手術、放射治療及化學治療，尚於研究階段的治療方式有免疫療法及基因療法。
WWOX基因是一種腫瘤抑制基因，位於人類染色體16q23.3-24.1的位置，可轉譯出WWOX/FOR/WOX1家族蛋白。過去研究發現許多癌細胞本身的WWOX基因表現偏低，包括皮膚鱗狀上皮細胞癌，並藉由許多實驗證明WWOX蛋白質具有腫瘤抑制的效果；更進一步的研究將WWOX基因藉由病毒性載體送入肺癌、乳癌、攝護腺癌和胰臟癌等癌細胞，能成功觀察到抑制腫瘤生長的效果。因此，將WWOX基因送入皮膚鱗狀上皮細胞癌，對於皮膚癌的治療而言非常值得研究。
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。
本研究藉由PEI特性分析以及細胞實驗尋找適合皮膚鱗狀上皮細胞癌的PEI轉染條件，觀察WWOX基因送入皮膚鱗狀上皮細胞癌所產生的細胞凋亡反應，從中挑選最適合的條件進行in vivo試驗。此外，藉由皮膚局部給藥的方式將WWOX基因傳送進入皮膚，並以組織切片觀察WWOX基因表現之位置，並推測複合物進入皮膚之路徑。
研究結果顯示，N/P ratio大於4:1時PEI能與WWOX基因形成有效複合物，且複合物的表面電荷隨著N/P ratio增加而增加，最後趨近於+60mV。PEI載體之細胞毒性及產生之細胞凋亡比例，隨著PEI濃度增加而增加；PEI/WWOX複合物之轉染效率隨著N/P ratio增加而增加，而產生之細胞凋亡比例則在N/P ratio 10:1時達到最大值。計算由WWOX基因引起之細胞凋亡比例，N/P ratio 10:1之複合物最大，N/P ratio 8:1之複合物次高。綜合上述結果，考慮轉染效率、細胞凋亡比例及載體細胞毒性等因素，篩選出N/P ratio 8:1之複合物做為in vivo皮膚局部給藥之條件。
PEI/WWOX複合物(N/P ratio 8:1)能成功地將WWOX基因送入正常裸鼠皮膚之毛囊，並在毛囊周圍組織表現細胞凋亡反應。而在SCC-15 tumor xenograft之裸鼠皮膚上雖能觀察到表層皮膚之綠色螢光表現，但複合物無法到達較深層之腫瘤組織，亦無法觀察到對映之細胞凋亡反應。腫瘤皮膚局部給藥之研究，需考慮tumor xenograft之皮膚表面型態學變化，設計適當之給藥頻次及觀察時間點，此部分有待更多之研究。本研究可做為相關皮膚基因傳遞研究之參考。

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. However, the etiology is largely unknown. Cutaneous SCC is malignant and tends to spread or metastasize. It is believed to originate from the squamous cells located at the suprabasal epidermis. Several methods for treatment of squamous cell carcinoma are acceptable, including surgery, radiotherapy, chemotherapy, immunotherapy and gene therapy. Factors that influence the choice of treatment include the site, grade, and stage of the primary tumor; patient age; and general medical condition.
Human WWOX gene, encoding the WWOX/FOR/WOX1 family proteins, is mapped to a fragile site on chromosome 16q23.2. Loss of heterozygosity of WWOX gene has been shown in numerous types of cancers, including squamous cell carcinoma. WOX1 protein is considered as a candidate tumor suppressor protein. In several studies, the restoration of WWOX gene in lung cancer cells, breast cancer cells, prostate cancer cells and pancreatic cancer cells prevents their growth both in vitro and in vivo.
The technology of gene delivery plays an important role in the development of gene therapy. Several methods have been developed to transfer DNA into cells for gene therapy. One of the non-invasive gene delivery method is the use of gene vectors or carriers. Polyethylenimine (PEI) is a cationic polymer-based gene carrier, many researches prove that PEI can transfer genes into different kind of cells under different transfection condition.
In this study, PEI 25kDa was used as a gene vector. The PEI/WWOX complexes of different N/P ratio were characterized by zeta potential measurement and gel retardation assay. Cytotoxicity, transfection efficiency and percentage of cell apoptosis were analyzed in SCC cells, and to determine optimal transfection conditions of PEI/WWOX complex for in vivo experiments. After topical administration of PEI/WWOX complex for 24 hours to the skin of nude mice, the skin tissue was cryosectioned to examine the localization of gene expression.
The results demonstrated that the WWOX gene can bind with PEI when N/P ratios are greater than 4:1, and zeta potential was increased with the increase of N/P ratio (with a final value of +60mV). Cytotoxicity and apoptotic effect of PEI also increased with PEI concentration. The transfection efficiency of PEI/WWOX complex increased with N/P ratio, while apoptotic effect achieved a maximal value at N/P ratio of 10:1. Based on these results, PEI/WWOX complex of N/P ratio 8:1 was selected for topical administration in vivo.
In in vivo experiments, PEI successfully delivered WWOX gene into hair follicle of normal skin, and apoptosis was induced around hair follicle. However, WWOX gene was expressed only on surface of the SCC-15 tumor xenografted skin.
Further studies are required to improve delivery of PEI/WWOX complex to tumor xenografted skin.

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