Jacobsen F, Mittler D, Hirsch T, Gerhards A, Lehnhardt M, Voss B, Steinau H U, Steinstraesser L
Plastic Surgery Research Department for Plastic Surgery, Burn Center, Handsurgery, Sarcoma Reference Center, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bochum, Germany.
Gene Ther. 2005 Oct;12(20):1494-502. doi: 10.1038/sj.gt.3302568.
Host defense peptides (HDP) are naturally occurring effector molecules of the innate immune system, which might be an alternative to currently used antibiotics. The objective of this study was to investigate the efficiency of transient cutaneous adenoviral transfection with human cathelicidin hCAP-18/LL-37 in infected burn wounds. Specific transgene expression was analyzed in vitro on mRNA and protein level using real-time PCR and Western-blot. Male Sprague-Dawley rats (n=40) received a second degree scald burn on both flanks (5% BSA), which were inoculated with 10(8) colony-forming units (CFU) Pseudomonas aeruginosa. Two days later, rats were randomized into the following groups: (1) adenoviral delivery of LL-37 (Ad5-hCAP-18, n=10), (2) synthetic host defense peptide LL-37 (1 mg; n=10), (3) carrier control (PBS, n=10) and (4) empty-virus control (Ad5-LacZ, n=10). Agents were injected intradermally and subcutaneously into both flanks. After either 2 or 7 days, skin samples were harvested and homogenized. CFU per gram tissue were determined. The hCAP-18/LL-37 expression was confirmed by real-time PCR and localized using in situ hybridization. In vitro transfection of cutaneous cells delivered a specific response on mRNA production. Western blot analysis revealed protein expression of hCAP-18/LL-37 in conditioned medium and cell pellet. The host defense peptide LL-37 was detectable after cleavage of the inactive pro-form hCAP-18/LL-37 with human elastase. Ad5-hCAP-18 showed a significant bacterial inhibition of approximately 10 000 fold compared to the control group (P<0.001) and 1000-fold (P<0.001) compared to the synthetic HDP LL-37 7 post-transfection. No inhibition was observed for the carrier or empty-virus control. Real-time PCR and in situ hybridization confirmed expression of hCAP-18/LL-37. In conclusion, transient cutaneous adenoviral delivery of the host defense peptide hCAP-18/LL-37 is significantly more effective than administration of synthetic host defense peptides and might be a potential adjunct for wound treatment in the near future.
宿主防御肽（HDP）是天然存在的固有免疫系统效应分子，可能是目前所用抗生素的替代品。本研究的目的是调查人cathelicidin hCAP-18/LL-37经皮腺病毒瞬时转染对感染烧伤创面的疗效。使用实时PCR和蛋白质印迹法在mRNA和蛋白质水平对体外特异性转基因表达进行分析。40只雄性Sprague-Dawley大鼠双侧胁腹接受二度烫伤（5%体表面积），并接种10⁸集落形成单位（CFU）铜绿假单胞菌。两天后，将大鼠随机分为以下几组：（1）腺病毒递送LL-37（Ad5-hCAP-18，n = 10），（2）合成宿主防御肽LL-37（1 mg；n = 10），（3）载体对照（PBS，n = 10）和（4）空病毒对照（Ad5-LacZ，n = 10）。将药物皮内和皮下注射到双侧胁腹。在2天或7天后，采集皮肤样本并匀浆。测定每克组织的CFU。通过实时PCR确认hCAP-18/LL-37表达，并使用原位杂交进行定位。皮肤细胞的体外转染对mRNA产生有特异性反应。蛋白质印迹分析显示在条件培养基和细胞沉淀中有hCAP-18/LL-37的蛋白质表达。用人类弹性蛋白酶切割无活性的前体形式hCAP-18/LL-37后可检测到宿主防御肽LL-37。与对照组相比，Ad5-hCAP-18在转染后7天显示出约10000倍的显著细菌抑制作用（P<0.001），与合成HDP LL-37相比有1000倍的抑制作用（P<0.001）。载体或空病毒对照未观察到抑制作用。实时PCR和原位杂交证实了hCAP-18/LL-37的表达。总之，宿主防御肽hCAP-18/LL-37经皮腺病毒瞬时递送比合成宿主防御肽给药显著更有效，可能在不久的将来成为伤口治疗的潜在辅助手段。
