真皮成纤维细胞中 VEGF 的过度表达加速血管生成和伤口愈合功能：体外和体内研究,Scientific Reports

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真皮成纤维细胞中 VEGF 的过度表达加速血管生成和伤口愈合功能：体外和体内研究
Scientific Reports ( IF 3.8 ) Pub Date : 2022-11-02 , DOI: 10.1038/s41598-022-23304-8
Forough Shams ₁ , Hamideh Moravvej ₂ , Simzar Hosseinzadeh _{3,

4} , Ebrahim Mostafavi _{5,

6} , Hadi Bayat _{4,

7} , Bahram Kazemi ₁ , Mojgan Bandehpour ₁ , Elnaz Rostami ₈ , Azam Rahimpour _{3,

4} , Hamidreza Moosavian ₉

Affiliation

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Medical Nanotechnology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA.
Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Animal Sciences and Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

成纤维细胞是结缔组织的主要细胞，在伤口愈合的增殖和成熟阶段具有关键作用。这些细胞可以分泌各种细胞因子、生长因子和胶原蛋白。血管内皮生长因子 (VEGF) 是成纤维细胞迁移过程中的一个独特因子，通过诱导伤口愈合级联组分，如血管生成、胶原沉积和上皮化。本研究旨在创建 VEGF ₁₆₅过表达成纤维细胞以评估伤口愈合中的血管生成功能。在体外，一种新型重组表达载体 pcDNA3.1(-)-VEGF 被制备并转染到成纤维细胞中。在用潮霉素选择成纤维细胞后，通过定量和定性方法研究重组细胞的 VEGF 表达。研究了聚氨酯-醋酸纤维素 (PU-CA) 支架的机械、物理和生存特性。最后，在体内，在第 0、2、5、12 和 15 天评估了四组血管生成潜力，包括对照组、PU-CA、含有成纤维细胞的 PU-CA 和表达 VEGF 的细胞。收集伤口活组织检查和愈合过程在不同的日子进行组织病理学评估。qRT-PCR 显示与成纤维细胞相比，基因操作细胞中的 VEGF 过表达（七倍）。重组 VEGF 表达也通过蛋白质印迹法得到证实。在手术后的第二天，经过处理的成纤维细胞比其他组表现出更多的血管生成，抗 CD31 抗体也证实了这一点。与其他组相比，基因操作 Hu02 和 Hu02 组的伤口闭合面积百分比在第 5 天显示伤口面积显着减少。这些发现表明，过表达成纤维细胞中的VEGF ₁₆₅在愈合过程的早期导致血管生成增强和肉芽组织形成，这可以在伤口愈合受损的患者中显示其治疗潜力，并为基因治疗提供功能支持。

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Overexpression of VEGF in dermal fibroblast cells accelerates the angiogenesis and wound healing function: in vitro and in vivo studies

Fibroblasts are the main cells of connective tissue and have pivotal roles in the proliferative and maturation phases of wound healing. These cells can secrete various cytokines, growth factors, and collagen. Vascular endothelial growth factor (VEGF) is a unique factor in the migration process of fibroblast cells through induces wound healing cascade components such as angiogenesis, collagen deposition, and epithelialization. This study aimed to create VEGF₁₆₅ overexpressing fibroblast cells to evaluate angiogenesis function in wound healing. In vitro, a novel recombinant expression vector, pcDNA3.1(-)-VEGF, was produced and transfected into the fibroblast cells. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying and qualifying methods. Mechanical, physical, and survival properties of polyurethane-cellulose acetate (PU-CA) scaffold were investigated. Finally, in vivo, the angiogenic potential was evaluated in four groups containing control, PU-CA, PU-CA with fibroblast cells, and VEGF-expressing cells on days 0, 2, 5, 12 and 15. Wound biopsies were harvested and the healing process was histopathologically evaluated on different days. qRT-PCR showed VEGF overexpression (sevenfold) in genetically-manipulated cells compared to fibroblast cells. Recombinant VEGF expression was also confirmed by western blotting. Manipulated fibroblast cells represented more angiogenesis than other groups on the second day after surgery, which was also confirmed by the antiCD31 antibody. The percentage of wound closure area on day 5 in genetically-manipulated Hu02 and Hu02 groups showed a significant reduction of wound area compared to other groups. These findings indicate that overexpression of VEGF₁₆₅ in fibroblast cells results in enhanced angiogenesis and formation of granulated tissue in the early stage of the healing process, which can show its therapeutic potential in patients with impaired wound healing and also provide functional support for gene therapy.

更新日期：2022-11-03

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