局部应用血管内皮生长因子及骨髓间充质干细胞对超比例随意皮瓣成活率的影响

Topical application of vascular endothelial growth factor and bone marrow mesenchymal stem cell caused effects to the survival rate of random flap

目的研究血管内皮生长因子(VEGF)及人骨髓间充质干细胞(MSC)联合应用对超比例随意皮瓣成活率的影响。方法采用密度梯度离心法和贴壁筛选法分离、培养人MSC,鉴定其纯度及生物学特性。40只Wistar大鼠随机分成空白对照组、VEGF组、MSC组、MSC+VEGF联合应用组,每只大鼠背部设计2 cm×5 cm带蒂皮瓣,术后连续14 d观察皮瓣愈合情况。术后第14天处死各组动物,计算皮瓣成活百分率;切取距蒂部4 cm处皮瓣行组织学检查,观察远蒂端皮瓣的微血管数量。结果 1)经流式细胞术和成脂、成骨诱导方法鉴定,本研究分离并培养的MSC具有多向分化潜能。2)经临床观察,各组大鼠均未出现免疫排斥反应。空白对照组、VEGF组、MSC组、MSC+VEGF组动物的皮瓣成活百分率分别为55.4%±4.4%、70.7%±6.3%、65.1%±7.1%、93.4%±9.4%,3个实验组皮瓣成活百分率均高于空白对照组(P<0.05),MSC+VEGF组成活百分率最高,高于其他3组,差异均有统计学意义(P<0.05)。3)组织学检查可见:与其他3组相比,MSC+VEGF组的肉芽组织最为丰富,成纤维细胞多,新生毛细血管最多。结论1)采用贴壁筛选法和密度梯度离心法结合的方法可以得到纯化且具有多向分化潜能的MSC;2)人MSC具有无免疫反应性或免疫反应性低的特点,跨物种应用不会引起免疫排斥反应;3)VEGF和MSC联合应用具有协同作用,可在短期内重建缺血区域的血液循环,解决超比例皮瓣的远蒂端缺血问题,提高皮瓣成活率和创面组织的修复质量。

Objective Experimental study on the survival rate of random flap using vascular endothelial growth factor （VEGF） and bone marrow mesenchymal stem cell （MSC）. Methods MSC was isolated through the combined utilization of density gradient centrifugation and adherence screening methods. MSC was cultured and its purity and biological properties were identified. Forty Wistar rat models were established and divided into control group, VEGF group, MSC group and MSC＋VEGF group. Designed 2 cmx5 cm pedicle flap on each rat＇s back. The flaps were ob- served for 14 d after operation. On the 14th day, the rats were killed. The survival rates of random flap in each group were counted, and the histological sections were stained at the flap which was 4 cm far from the pedicle, and the number of microvascular was observed. Results 1）MSC was detected by flow cytometry, adipogenic and osteo- genic induction detection. The results showed that MSC could differentiate to fat and cartilage by induction and certified that the MSC had multi-differentiation potential. 2）Immune rejection did not appear on each animal. The survival rate of random flap in control group, VEGF group, MSC group and MSC＋VEGF group were 55.4%~4.4%, 70.7%~6.3%, 65.1%＋7.1%, and 93.4%~9.4%, respectively. The survival rate of each experimental group was higher than control group （P〈0.05）. The survival rate of MSC＋VEGF group was the highest （30〈0.05）. 3）The histological examination showed that the granulation tissue, fibroblast and neovascularity was most abundant in MSC＋VEGF group. Conclusion l）The purified and multi-differentiated MSC can be obtained by density gradient eentrifugation and ad-herence screening methods. 2）The human MSC has no immunogens or minimal immunogenicity, immunological rejection would not be caused when it is transmitted in cross-species. 3）VEGF and MSC have synergetic effect when they are used together, it can rebuild the blood circulation of the ischemic region in a short time, solve ischemia of the flap far from the pedicle, improve survival rate of the flap and enhance the repair quality of the wound.