血管内皮生长因子165基因修饰人骨髓间充质干细胞复合兔脱细胞真皮基质构建活性组织工程皮肤

Construction of tissue-engineered skin with vascular endothelial growth factor 165 gene-modified human bone marrow mesenchymal stem cells and rabbit acellular dermal matrix

背景:利用组织工程原理构建的人工皮肤替代物用于皮肤移植可有效解决供皮不足这一难题,但组织工程皮肤很大程度上受制于移植物血管网缺乏造成的细胞供养障碍而导致失效。目的:以血管内皮生长因子165基因修饰的人骨髓间充质干细胞复合兔脱细胞真皮基质,拟构建活性组织工程皮肤。设计、时间和地点:以基因工程为基础的组织构建实验,于2008-03/06在南昌大学第一附属医院烧伤研究所完成。材料:普通级新西兰大白兔5只用于提取脱细胞真皮基质原料,由南昌大学医学院动物科学部提供,人骨髓间充质干细胞取自临床骨髓穿刺检查正常的患者,pShuttle-CMV/VEGF165质粒转化大肠杆菌菌种由武汉协和医院郜勇博士惠赠。方法:无菌条件下切取兔耳全层皮肤,将2cm×2cm皮片置于0.25%胰酶-EDTA消化液中去掉表皮,再置入曲拉通X-100溶液使真皮细胞完全脱净,磷酸盐缓冲液反复清洗,即为脱细胞真皮基质,4℃保存备用。采用密度梯度离心与贴壁筛选法体外分离培养人骨髓间充质干细胞,取传至第3代细胞,按5×105/孔密度接种,待细胞达80%融合时进行脂质体介导的pShuttle-CMV/VEGF165质粒转染,接种于制备好的脱细胞真皮基质上体外联合培养。主要观察指标:骨髓间充质干细胞与脱细胞真皮基质的形态观察,组织工程皮肤结构观察。结果:体外培养的骨髓间充质干细胞大部分呈梭形,基因转染后细胞形态及活性无明显影响;脱细胞真皮基质呈瓷白色,柔韧有弹性。血管内皮生长因子165基因修饰的骨髓间充质干细胞与脱细胞真皮基质共培养2d后,所构建的组织工程皮肤呈淡红色,质软,接种细胞生长状态正常,苏木精-伊红染色及扫描电镜观察可见在脱细胞真皮基质的间隙中有大量长梭形细胞附着生长。结论:血管内皮生长因子165基因转染的人骨髓间充质干细胞在兔脱细胞真皮基质中生长良好,可在体外联合构建活性组织工程皮肤。

BACKGROUND： Tissue-engineered skins for skin transplantation can effectively solve the problem of supply deficiency. However tissue-engineered skin are limited by cell supply disorder due to lack of a vascular plexus. OBJECTIVE： To construct tissue-engineered skin with vascular endothelial growth factor （VEGF） 165 gene-modified human bone marrow mesenchymal stem cells （BMSCs） as well as rabbit acellular dermal matrix. DESIGN, TIME AND SETTING： A gene-engineered based tissue construction experiment was performed at the Institute of Burns of First Affiliated Hospital of Nanchang University between March and June 2008. MATERIALS： Acellular dermal matrix was harvested from 5 mature New Zealand rabbits, which were supplied by Department of Animal Science. Medical College, Nanchang University. BMSCs were harvested from normal patients in clinical examination of bone marrow aspiration, pShuttle-CMV/VEGF165 plasmid transformation bacillus coli strain was gifted by Dr. Hao from Wuhan Union Hospital, China. METHODS： Full-thickness skin was sterilely obtained from rabbit ears. 2 cm×2 cm free skin graft was placed in 0.25% pancreatin-EDTA to remove epidermis, in Qulatong X-100 solution to completely take off hypodermal cells, repeatedly rinsed in phosphate buffer saline. Thus, acellular dermal matrix was collected, and kept at 4 ℃ for usage. BMSCs were separated from normal patients by combining density gradient centrifugation with adhering method. At the third passage, BMSCs were incubated at a density of 5×10^5 per well. The vector pShuttle-CMV/VEGF165 was transfected into BMSCs by liposome mediated at the time of 80% confluence, which were seeded onto the surface of acellular dermal matrix. MAIN OUTCOME MEASURES： Morphology of BMSCs and acellular dermal matrix; tissue-engineered skin structure. RESULTS： The. cells cultured in vitro assumed spindle shape mostly; VEGF transfection had no significantly inhibitory effect on BMSCs. Acellular dermal matrix presented as white, pliable and elastic sheet. Two days after VEGF-modified BMSCs seeded to acellular dermal matrix, the soft tissue-engineered skin appeared rose pink. The cells with spindle shape adhered to petri dish, and grew well. By hematoxylin-eosin staining and scanning electron microscopy, there were plenty of long-spindle shape cells cohered into the interspace of acellular dermal matrix. CONCLUSION： VEGF 165 gene transfected human BMSCs grew well on acellular dermal matrix, and active tissue-engineered skin can be constructed in vitro.