Basner-Tschakarjan E, Mirmohammadsadegh A, Baer A, Hengge U R
Department of Dermatology, Heinrich Heine-University Düsseldorf, Düsseldorf, Germany.
Gene Ther. 2004 May;11(9):765-74. doi: 10.1038/sj.gt.3302221.
The skin is an interesting organ for human gene therapy due to accessibility, immunologic potential and synthesis capabilities. In this study, we attempted to visualize and measure the uptake of naked FITC-labeled plasmid by FACS analysis detecting up to 15% internalization in a dose- and time-dependent manner. Cycloheximide treatment inhibited the uptake by >90%, suggesting a protein-mediated uptake. The inhibition of different internalization pathways demonstrated that blocking macropinocytosis (by amiloride and N,N-dimethylamylorid) reduced DNA uptake by >85%, while the inhibition of clathrin-coated pits (by chlorpromazine) and caveolae (by nystatin/filipin III) did not limit the uptake. Colocalization studies using confocal laser microscopy revealed a time-dependent accumulation of plasmid DNA in endosomes and lysosomes. When a green fluorescent protein (GFP) expression vector was used, specific GFP-RNA became detectable by reverse transcriptase-PCR, whereas measurable amounts of protein could not be identified in FACS experiments. To detect the potential DNA receptors on the keratinocyte surface, membrane proteins were extracted and subjected to South-Western blotting using digoxigenin-labeled calf thymus and lambda-phage DNA. Two DNA-binding proteins, ezrin and moesin, known as plasma membrane-actin linkers, were identified by one- and two-dimensional-South-Western blots and matrix-assisted laser desorption and ionization-mass spectrometry. Ezrin and moesin are functionally associated with a number of transmembrane receptors such as the EGF, CD44 or ICAM-1 receptor. Taken together, naked plasmid DNA seems to enter human keratinocytes through different pathways, mainly by macropinocytosis. Two DNA-binding proteins were identified that seemed to be involved in binding/trafficking of internalized DNA.
由于皮肤易于接触、具有免疫潜力和合成能力，因此它是人类基因治疗中一个有趣的器官。在本研究中，我们试图通过流式细胞术分析来可视化和测量裸荧光素异硫氰酸酯（FITC）标记质粒的摄取情况，检测到高达15%的内化呈剂量和时间依赖性。放线菌酮处理抑制摄取>90%，提示为蛋白质介导的摄取。对不同内化途径的抑制表明，阻断巨胞饮作用（通过阿米洛利和N，N-二甲基阿米洛利）可使DNA摄取减少>85%，而抑制网格蛋白包被小窝（通过氯丙嗪）和小窝（通过制霉菌素/菲律宾菌素III）并不限制摄取。使用共聚焦激光显微镜的共定位研究揭示了质粒DNA在内体和溶酶体中的时间依赖性积累。当使用绿色荧光蛋白（GFP）表达载体时，通过逆转录聚合酶链反应（RT-PCR）可检测到特异性的GFP-RNA，而在流式细胞术实验中未鉴定出可测量量的蛋白质。为了检测角质形成细胞表面潜在的DNA受体，提取膜蛋白，并用地高辛标记的小牛胸腺和λ噬菌体DNA进行South-Western印迹分析。通过一维和二维South-Western印迹以及基质辅助激光解吸电离质谱法鉴定出两种DNA结合蛋白，即埃兹蛋白和膜突蛋白，它们是质膜-肌动蛋白连接蛋白。埃兹蛋白和膜突蛋白在功能上与许多跨膜受体相关，如表皮生长因子（EGF）、CD44或细胞间黏附分子-1（ICAM-1）受体。综上所述，裸质粒DNA似乎通过不同途径进入人角质形成细胞，主要是通过巨胞饮作用。鉴定出两种似乎参与内化DNA结合/运输的DNA结合蛋白。
