摘要
基因电转染到皮肤在医疗应用的发展中包括DNA疫苗、癌症治疗、伤口愈合或局部皮肤疾病的治疗都具有特别的益处。然而,由于控制DNA电转染到人体组织内的机制了解不够,使得目前这类的临床应用很有限。当今大多数的研究都是在啮齿类动物模型中进行的,但啮齿类皮肤与人类皮肤在细胞组成和结构方面都有很大的差异。我们用组织工程学的方法来研究在人体组织中基因转染的机制。根据自组装方法培养原代人类皮肤成纤维细胞,生成三维重建的人类真皮组织。在这项研究中,我们显示了利用毫秒级的电脉冲对重建的皮肤组织细胞进行有效的电通透,而不影响到它们的活性。一个报告基因成功地电转移到人体组织,检测其基因表达48小时。有趣的是,转染后的细胞单独位于组织的上表面,与质粒DNA溶液紧密接触。此外,我们报告的证据表明电转染的成功取决于胶原蛋白丰富的组织质粒内的移动性,而不是细胞增殖的状态。总之,组织工程学除了提供可靠的动物实验的替代方法之外,还是人体组织中的基因转染机制的体外研究中行之有效的生物工具。
关键词: 传递,质粒DNA,电穿孔,细胞外基质,基因治疗,皮肤,组织工程学
Current Gene Therapy
Title:Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue
Volume: 16 Issue: 2
Author(s): Moinecha Madi, Marie-Pierre Rols, Laure Gibot
Affiliation:
关键词: 传递,质粒DNA,电穿孔,细胞外基质,基因治疗,皮肤,组织工程学
摘要: Gene electrotransfer into the skin is of particular interest for the development of medical applications including DNA vaccination, cancer treatment, wound healing or treatment of local skin disorders. However, such clinical applications are currently limited due to poor understanding of the mechanisms governing DNA electrotransfer within human tissue. Nowadays, most studies are carried out in rodent models but rodent skin varies from human skin in terms of cell composition and architecture. We used a tissue-engineering approach to study gene electrotransfer mechanisms in a human tissue context. Primary human dermal fibroblasts were cultured according to the self-assembly method to produce 3D reconstructed human dermal tissue. In this study, we showed that cells of the reconstructed cutaneous tissue were efficiently electropermeabilized by applying millisecond electric pulses, without affecting their viability. A reporter gene was successfully electrotransferred into this human tissue and gene expression was detected for up to 48h. Interestingly, the transfected cells were solely located on the upper surface of the tissue, where they were in close contact with plasmid DNA solution. Furthermore, we report evidences that electrotransfection success depends on plasmid mobility within tissue- rich in collagens, but not on cell proliferation status. In conclusion, in addition to proposing a reliable alternative to animal experiments, tissue engineering produces valid biological tool for the in vitro study of gene electrotransfer mechanisms in human tissue.
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Moinecha Madi, Marie-Pierre Rols, Laure Gibot , Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue, Current Gene Therapy 2016; 16 (2) . https://dx.doi.org/10.2174/1566523216666160331125052
DOI https://dx.doi.org/10.2174/1566523216666160331125052 |
Print ISSN 1566-5232 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5631 |
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