Steinstraesser L, Hirsch T, Beller J, Mittler D, Sorkin M, Pazdierny G, Jacobsen F, Eriksson E, Steinau H U
Department for Plastic Surgery, Burn Center, BG University Hospital Bergmannsheil, Ruhr University Bochum, Germany.
J Gene Med. 2007 Nov;9(11):949-55. doi: 10.1002/jgm.1099.
Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds.
HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed.
Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression.
Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.
将基因转移至烧伤创面可能为传统且往往效果欠佳的局部及全身应用治疗药物以促进创面愈合提供一种替代方法。本研究的目的是评估并优化向烧伤创面进行瞬时非病毒基因递送的潜力。
使用纯质粒DNA（pDNA）或与脂质体2000、FuGENE6或DOTAP-胆固醇复合，将荧光素酶或β-半乳糖苷酶转基因转染至HaCaT细胞。通过生物发光和荧光测定表达情况。40只雄性Sprague-Dawley大鼠皮内注射裸pDNA、脂质体复合物或载体对照，注射部位为未烧伤皮肤、浅度、部分厚度或全层烫伤。在24小时、48小时或7天后处死动物，并评估转基因表达。
向HaCaT细胞的基因转移显示，DOTAP/胆固醇的总体表达最高（77.85纳克荧光素酶/毫克蛋白质），其次是脂质体2000（33.14纳克荧光素酶/毫克蛋白质）。源自pDNA的基因转移至浅度烧伤创面在烧伤组中表达最高（0.77纳克荧光素酶/毫克蛋白质）。然而，源自脂质体复合物的基因转移至浅度烧伤和未烧伤皮肤并未显示出更高的表达。
脂质体2000和DOTAP/胆固醇脂质体复合物显示出显著增强基因转移，而体外未检测到裸DNA的转染。与体外研究相反，裸DNA是在实验性烧伤创面成功实现基因递送的唯一试剂。这些发现突出了作为一种治疗方法的基因递送体外分析的预测性有限。
