核转染：一种皮肤基因转移的新方法？
Nucleofection: a new method for cutaneous gene transfer?
作者信息
Jacobsen Frank, Mertens-Rill Janine, Beller Juergen, Hirsch Tobias, Daigeler Adrien, Langer Stefan, Lehnhardt Marcus, Steinau Hans-Ulrich, Steinstraesser Lars
机构信息
Department of Plastic Surgery, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bochum 44789, Germany.
出版信息
J Biomed Biotechnol. 2006;2006(5):26060. doi: 10.1155/JBB/2006/26060.
BACKGROUND
Transfection efficacy after nonviral gene transfer in primary epithelial cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with the established transfection reagent FuGENE6.
METHODS
Primary human keratinocytes (HKC), primary human fibroblasts (HFB), and a human keratinocyte cell line (HaCaT) were transfected with reporter gene construct by FuGENE6 or Amaxa Nucleofector device. At corresponding time points, beta-galactosidase expression, cell proliferation (MTT-Test), transduction efficiency (X-gal staining), cell morphology, and cytotoxicity (CASY) were determined.
RESULTS
Transgene expression after nucleofection was significantly higher in HKC and HFB and detected earlier (3 h vs. 24 h) than in FuGENE6. After lipofection 80%-90% of the cells remained proliferative without any influence on cell morphology. In contrast, nucleofection led to a decrease in keratinocyte cell size, with only 20%-42% proliferative cells.
CONCLUSION
Related to the method-dependent increase of cytotoxicity, transgene expression after nucleofection was earlier and higher than after lipofection.
背景
原代表皮细胞中非病毒基因转染后的转染效率有限。本研究的目的是比较最近可用的核转染方法与已确立的转染试剂FuGENE6的转染效率。
方法
用FuGENE6或Amaxa核转染仪将报告基因构建体转染原代人角质形成细胞（HKC）、原代人成纤维细胞（HFB）和人角质形成细胞系（HaCaT）。在相应时间点，测定β-半乳糖苷酶表达、细胞增殖（MTT试验）、转导效率（X-gal染色）、细胞形态和细胞毒性（CASY）。
结果
核转染后HKC和HFB中的转基因表达明显更高，且比FuGENE6转染更早检测到（3小时对24小时）。脂质体转染后80%-90%的细胞仍能增殖，对细胞形态无任何影响。相比之下，核转染导致角质形成细胞大小减小，只有20%-42%的增殖细胞。
结论
与方法依赖性细胞毒性增加相关，核转染后的转基因表达比脂质体转染更早且更高。
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