【摘要】 背景:研究证实血管内皮生长因子能够诱导新生血管形成,人羊膜间充质干细胞也可通过多种机制改善皮瓣移植的缺血再灌注损伤。因此,用血管内皮生长因子基因修饰人羊膜间充质干细胞为促进皮瓣成活提供了可能。目的:探索局部应用血管内皮生长因子基因修饰人羊膜间充质干细胞对超长比例随意皮瓣成活的影响。方法:将30只成年SD大鼠随机分为Ⅰ、Ⅱ、Ⅲ组,每组10只,在其背部左右分别构建2 cm×8 cm超长缺血随意皮瓣模型,皮瓣蒂部位于髂棘水平,分别为Ⅰ-left,Ⅰ-right,Ⅱ-left,Ⅱ-right,Ⅲ-left,Ⅲ-right。皮瓣掀起后即刻,Ⅰ-left、Ⅱ-left注射0.5m LLG-DMEM培养基划分为对照组,Ⅰ-right、Ⅲ-left注射0.5m L细胞浓度为1×10⁹ L^-1的人羊膜间充质干细胞悬液划分为实验组1,Ⅱ-right、Ⅲ-right注射0.5 mL细胞浓度为1×109L-1转染血管内皮生长因子基因的人羊膜间充质干细胞悬液划分为实验组2,每个皮瓣共注射5点,每点注射0.1m L。移植后即刻、24,48h以及4,7d,激光多普勒血流监测仪监测皮瓣中部和蒂部的血流灌注值,移植后7d观察各组皮瓣的成活率,取成活皮瓣组织通过组织学观察各组皮瓣毛细血管密度,荧光显微镜观察人羊膜间充质干细胞在皮瓣内的分布及存活状况,Westernblot检测皮瓣组织中血管内皮生长因子蛋白水平。结果与结论:①实验组2皮瓣成活率、血流灌注值、皮瓣毛细血管密度明显高于实验组1及对照组,实验组1皮瓣成活率、血流灌注值、皮瓣毛细血管密度明显高于对照组,差异有显著性意义（P <0.05）;②在荧光显微镜下观察发现,实验组1、实验组2皮瓣组织内有CM-Dil标记的人羊膜间充质干细胞;③各组皮瓣组织血管内皮生长因子蛋白表达均呈阳性,实验组2皮瓣组织血管内皮生长因子蛋白表达明显高于实验组1及对照组,实验组1皮瓣组织血管内皮生长因子蛋白表达明显高于对照组,差异有显著性意义（P <0.05）;④结果证实,在超长随意皮瓣中、远部位应用人羊膜间充质干细胞可明显改善皮瓣成活率;人羊膜间充质干细胞经血管内皮生长因子基因转染后能够分泌更多的血管内皮生长因子蛋白,进而促进超长随意皮瓣血管新生,提高皮瓣成活率。更多还原

【Abstract】 BACKGROUND: Studies have confirmed that vascular endothelial growth factor can induce angiogenesis, and human amnion-derived mesenchymal stem cells improve the repair of ischemia-reperfusion injury using flap transplantation via various mechanisms. Therefore, the modification of human amnion-derived mesenchymal stem cells using vascular endothelial growth factor gene promotes the survival of skin flaps.OBJECTIVE: To investigate the effect of human amnion-derived mesenchymal stem cells modified by vascular endothelial growth factor gene on the survival of ultra-long random skin flaps.METHODS: Thirty healthy adult Sprague-Dawley rats were randomly divided into group I, group II and group III, 10 rats in each group. Two ultra-long ischemic random skin flap models were constructed at the left and right back of each rat, and the skin flap pedicle was located at iliac spine level. These skin flaps received the following treatment immediately after removal: The flaps were divided into group I-left, I-right;II-left, II-right; III-left and III-right. 0.5 mL of LG-DMEM was injected in the skin flaps in I-left and II-left groups(control group); 0.5 mL of 1×10~9/L human amnion-derived mesenchymal stem cells was injected into the skin flaps in I-right and III-left groups(experimental group 1); 0.5 mL of1×10~9/L vascular endothelial growth factor-transfected human amnion-derived mesenchymal stem cells was injected into the flaps in II-right and III-right groups(experimental group 2). There were five injection sites for each flap, 0.1 mL per each site. The blood perfusion in the middle and pedicle of each skin flap was monitored using laser Doppler flow meter immediately, 1, 2, 4 and 7 days after transplantation. The survival rate of skin flaps in all groups was measured at 7 days after transplantation. The capillary density in survived skip flaps was histologically observed. The distribution and survival rate of CM-Dil labeled human amnion-derived mesenchymal stem cells were observed by fluorescent microscope. The vascular endothelial growth factor protein level was detected by western blot.RESULTS AND CONCLUSION:(1) The survival rate of skin flaps, blood perfusion, and capillary density showed significantly difference among groups(P < 0.05) and ranked as follows: experimental group 2 > experimental group 1 > control group.(2) Under fluorescent microscope, CM-Dil labeled human amnion-derived mesenchymal stem cells were visible in the flaps in the experimental groups 1 and 2.(3)Vascular endothelial growth factor positively expressed in all the flaps, while the protein expression of vascular endothelial growth factor was ranked as follows: experimental group 2 > experimental group 1 > control group and there were significant differences among groups(P <0.05). To conclude, the application of human amnion-derived mesenchymal stem cells in the middle and distal parts of ultra-long random skin flap can remarkably improve the survival rate of skin flaps, and moreover transfection with vascular endothelial growth factor can increase the secretion of vascular endothelial growth factor, further improving the effects of human amnion-derived mesenchymal stem cells promoting angiogenesis and survival rate of ultra-long random skip flaps.更多还原