转基因骨髓间充质干细胞移植与组织工程皮肤的血管化

背景：组织工程化皮肤移植后常因缺乏足够的血管化而导致低灌注和缺血损伤。利用转基因技术，可将血管生成调控因子基因导入目的细胞，使其表达某些调控因子，进而利于血管生成。 目的：观察转基因骨髓间充质干细胞移植促进组织工程皮肤血管化基因治疗的可行性。 设计、时间及地点：随机对照动物实验，于2008-03/11在江西省南昌大学第一附属医院烧伤研究所完成。 材料：人骨髓间充质干细胞由试验者取自临床骨髓穿刺检查骨髓正常的患者。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。16只新西兰大白兔用于皮肤缺损模型的制作，分为基因转染骨髓间充质干细胞组、骨髓间充质干细胞组、真皮支架材料组进行移植。 方法: 采用密度梯度离心结合贴壁法分离培养人骨髓间充质干细胞，以pShuttle-CMV/VEGF165质粒转染骨髓间充质干细胞。于兔背部两侧制备2 cm×2 cm 的正方形全层皮肤缺损3个，并分别以人血管内皮细胞生长因子165转染骨髓间充质干细胞、骨髓间充质干细胞、不含细胞的真皮支架材料植入全层皮肤缺损创面。 主要观察指标：观察局部组织微血管密度变化及移植物成活率。 结果：术后7 d，3组创口周围皮肤均无明显红肿及炎症反应,移植物与创面接触紧密，基因转染骨髓间充质干细胞组、骨髓间充质干细胞组可见部分真皮泛红, 真皮支架材料组不明显，术后3周创面基本愈合，基因转染骨髓间充质干细胞组创面的毛细血管密度较骨髓间充质干细胞组、真皮支架材料组明显增高(P < 0.01)，14 d时3组中基因转染骨髓间充质干细胞组血管密度仍最高，但3组数据无统计学差异。术后二三周基因转染骨髓间充质干细胞组移植物成活率最高(P < 0.01)。 结论：血管内皮细胞生长因子转染骨髓间充质干细胞移植可改善和促进局部血管再生，促使新的毛细血管从创面长入移植的组织工程皮肤，从而促进组织工程皮肤的早期血管化及创面愈合。

Transplantation of transgeneic human bone mesenchymal stem cells and the neovascularization of tissue-engineered skin

BACKGROUND: Tissue-engineered skins usually lead to hypoperfusion-ischemia injury. Transgenic technology can import angiogenesis regulatory factor gene into target cells, to express certain regulatory factor protein, which contributing to neovascularization. OBJECTIVE: To investigate the feasibility of transgeneic human bone mesenchymal stem cells (BMSCs) for accelerating neovascularization of tissue-engineered skin. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Laboratory of Burns, the First Affiliated Hospital of Nanchang University between March and November 2008. MATERIALS: BMSCs were harvested from bone marrow of patients without the severe diseases in clinical examination. The E.coli containing plasmid pShuttle-CMV/VEGF165 was kindly provided by doctor Gaoyong, who worked in Wuhan Xiehe Hospital. Following skin defect models preparation, 16 New Zealand rabbits were divided into transgeneic BMSCs, BMSCs and dermal scaffold groups. METHODS: Human MSCs were isolated with density gradient centrifugation and adherent method, and then transfected with pShuttle-CMV/VEGF165 plasmid. Rabbits were prepared three full thickness skin defects with each 2 cm×2 cm on the two sides of the back. Transgeneic BMSCs, BMSCs and dermal scaffold were implanted, respectively. MAIN OUTCOME MEASURES: Changes of local microvessel density and graft survival rate. RESULTS: At 7 days after operation, there was no obvious red swelling or inflammatory reaction in wound, the graft was contacted tightly with wound surface. Part of dermis showed red in the transgeneic BMSCs, and BMSCs group, the wound surface basic healed at the 3 weeks after operation. The capillary density of wound tissue of transgeneic BMSCs group was significantly higher than those of MSCs and dermal scaffold groups (P < 0.01). At 14 days after operation, the capillary density of wound tissue of the transgeneic BMSCs group was highest in those three groups. However, there was no significant difference among the three groups. At the second week and third week after operation, there was significant difference in the survival rate between the three groups. The graft survival rate reached the peak at 2 or 3 weeks after operation in the transgeneic BMSCs group. CONCLUSION: BMSCs transfected with gene VEGF can improve local angiogenesis, accelerate the new capillary grows from the wound into the transplanted tissue-engineered skin, thereby, facilitate early revascularization of tissue-engineered skin and wound healing.