Hojman Pernille, Gissel Hanne, Andre Franck M, Cournil-Henrionnet Christelle, Eriksen Jens, Gehl Julie, Mir Lluis M
Department of Oncology, 54B1 Copenhagen University Hospital Herlev, Herlev, Denmark.
Hum Gene Ther. 2008 Nov;19(11):1249-60. doi: 10.1089/hum.2008.059.
Gene transfer by electroporation is gaining momentum now that high-level, long-term expression of transgenes is being obtained. Several different pulse regimens are efficient, yet little information is available about the physiological muscular response to gene electrotransfer. This paper provides a comprehensive evaluation of the physiological and molecular effects on host tissue after DNA electrotransfer. We have tested several pulse regimens with special emphasis on the pulse combination of a short (100 microsec) high-voltage (HV) pulse followed by a long low-voltage (LV) pulse used for DNA electrotransfer, comparing it with 8 HV pulses designed to ensure extensive permeabilization of the muscle membrane. Using both mouse and rat skeletal muscle tissue, we investigated cell permeabilization by the 51Cr-labeled EDTA assay, lactate dehydrogenase release, Na+ and Ca2+ influx, K+ efflux, ATP release, and water content, as well as muscle function both in vivo and ex vivo, Hsp70 induction, and histology. In all these assays, the HVLV pulse combination gave rise to minimal disturbance of cell function, in all cases significantly different from results when using 8 HV pulses. The evaluated parameters were normalized after 1 week. The addition of DNA caused significantly more transmembrane exchange, and this may be due to entrance of the DNA through the membrane. In conclusion, this study comprehensively documents the immediate effects of DNA electrotransfer and shows that only slight cell disturbances occur with the HVLV pulses used for gene transfer. This is highly important, as minimal perturbation of cell physiology is essential for efficient transgene expression.
随着转基因高水平、长期表达的实现，通过电穿孔进行基因转移正日益受到关注。几种不同的脉冲方案都很有效，但关于基因电转移后肌肉生理反应的信息却很少。本文全面评估了DNA电转移后对宿主组织的生理和分子影响。我们测试了几种脉冲方案，特别强调了用于DNA电转移的短（100微秒）高压（HV）脉冲后接长低压（LV）脉冲的脉冲组合，并将其与旨在确保肌肉膜广泛通透化的8个HV脉冲进行比较。使用小鼠和大鼠骨骼肌组织，我们通过51Cr标记的EDTA测定、乳酸脱氢酶释放、Na+和Ca2+内流、K+外流、ATP释放和水分含量来研究细胞通透化，以及体内和体外的肌肉功能、Hsp70诱导和组织学。在所有这些测定中，HVLV脉冲组合对细胞功能的干扰最小，在所有情况下都与使用8个HV脉冲时的结果有显著差异。评估参数在1周后恢复正常。DNA的加入导致了明显更多的跨膜交换，这可能是由于DNA通过膜进入细胞所致。总之，本研究全面记录了DNA电转移的即时效应，并表明用于基因转移的HVLV脉冲只会引起轻微的细胞干扰。这非常重要，因为细胞生理的最小扰动对于有效的转基因表达至关重要。
