Adenoviral gene delivery to primary human cutaneous cells and burn wounds
Adenoviral gene delivery to primary human cutaneous cells and burn wounds
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DOI：
10.2119/2006-00031.hirsch
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发表时间：
2006-09-01
影响因子：
5.7
通讯作者：
Steinstraesser, Lars
中科院分区：
文献类型：
--
作者：
Hirsch, Tobias;von Peter, Sebastian;Steinstraesser, Lars
腺病毒将治疗基因转移到表皮和真皮细胞是治疗皮肤病和促进伤口愈合的一种有趣的方法。本研究的目的是评估腺病毒基因转染后皮肤和烧伤创面的体外和体内转染效果，用不同浓度的腺病毒构建物（eGFP）转染原代角化细胞（HKC）、成纤维细胞（HFB）和HaCaT细胞。转染效率和细胞毒性测定至30天。用流式细胞仪和荧光仪定量表达。用台锥蓝排斥法测定45只雄性Sprague Dawley大鼠的细胞毒性，将2 × 10(8) pfu的Ad5-CMV-Lac:Z或载体对照注射到浅浅部分厚度的烧伤或未烧伤皮肤中。动物分别于治疗后48小时、7天和14天实施安乐死。转染后48 h转染率最高：HKC为79%，HFB为70%，HaCaT为48%。30 d各组均可检测到eGFP的表达（P < 0.05）。10天后观察腺病毒载体对HFB和30天后观察HaCaT的细胞毒作用。烧伤皮肤报告基因在体内的表达明显高于未烧伤皮肤（P = 0.004）。基因表达量在第2 ~ 7天下降，第14天无显著表达。本研究表明腺病毒介导的表皮原代细胞和细胞系基因转移是可行的。上皮细胞的体外基因转移有望用于接受自体角质细胞片的严重烧伤患者。使用腺病毒载体在烧伤创面中瞬间传递皮肤基因，在创面组织中产生显著浓度至少一周。基于这些发现，我们假设瞬时皮肤腺病毒基因递送伤口愈合促进因子具有临床应用潜力。
The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery, Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method, 45 male Sprague Dawley rats received 2 x 10(8) pfu of Ad5-CMV-Lac:Z or carrier control intradermally into either superficial partial thickness scold burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays, The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0.004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least I week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application.