Stable expression of human interlukin┐2gene in transfected keratinocyt
http://www.100md.com 《第四军医大学学报》 2001年第24期
Stableexpressionofhumaninterlukin┐2geneintransfectedkeratinocytes,INTRODUCTION,MATERIALSANDMETHODS,Cellcultureandtransfection,RESULTS,DISCUSSION,REFERKeywords:keratinocytes;gene therapy;human interlukin-2CLC
Abstract:AIM To investigate the stable expression and se-cretion of human interlukin-2(IL-2)gene in transfected ker-atinocytes and assess the possibility of keratinocytes as a tar-get cell for gene therapy.METHODS Primary epidermal keratinocytes were isolated，cultured in serum-free medium，transfected by Lipofectamine and selected with antibiotic G418.The expression and activity of IL-2were assessed with techniques of DNA dot blot，RNA dot blot，hybridization in situ，immunohistochemistry，Western blot and MTT colori-metric assay.RESULTS Positive signal was found in trans-fected keratinocytes by DNA dot blot，RNA dot blot，hy-bridization in situ and immunohistochemistry.Western blot analysis showed a specific band exhibiting a molecular weight of15000which was in accordance with that of IL-2.The ac-tivity of IL-2in the culture supernatants was27.7U·mL-1 in2nd-passage cells and15U·mL-1 in4th-passage cells.CONCLUSION Keratinocytes have the potential availablity for gene stable expression and secreting active transgene products;This observations support the feasibility of ker-atinocytes as a target cell for gene transfection ......
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