血管内皮生长因子基因治疗联合外科延迟法改善大鼠腹壁超范围轴型皮瓣成活的研究

目的探讨皮下注射血管内皮生长因子（vascular endothelial growth factor,VEGF）基因、外科延迟两种方法及其联合应用对大鼠腹壁超范围轴型皮瓣成活的影响。方法取雄性Wistar大鼠48只,体重400～450g,制作大鼠以腹壁浅动脉为血管蒂的8cm×8cm超范围轴型皮瓣模型。随机分为六组,每组8只,分别为空白对照组（A组）、术时基因治疗组（B组）、术前基因治疗组（C组）、单纯延迟组（D组）、延迟同时基因治疗组（E组）和延迟后基因治疗组（F组）。术后7d,计算各组的皮瓣成活率;取皮瓣组织标本行HE染色,检测平均微血管密度及内径;取皮瓣组织标本行VEGF免疫组织化学染色检测VEGF165的表达。结果各实验组皮瓣成活率均显著高于A组（P〈0.05）;实验组中,E组皮瓣成活率显著高于其他各组（P〈0.05）,余各实验组间差异无统计学意义（P〉0.05）。微血管密度：B、C、E、F组显著高于A、D组,差异有统计学意义（P〈0.05）;B、C、E、F组间以及A、D组间比较差异无统计学意义（P〉0.05）。微血管内径：D组显著大于E、F组,差异有统计学意义（P〈0.05）;而D组和E、F组均显著大于A、B、C组,差异有统计学意义（P〈0.05）。免疫组织化学染色示A组及D组有少量VEGF165沉积,染色深度明显浅于其他各组。B、C组和E、F组可见毛细血管内皮细胞胞浆内有棕褐色VEGF165抗原抗体复合物的沉积,染色较深,部分沉积物呈带状围绕血管腔。各组实验动物角膜层均未见新生血管。结论皮下注射pcDNA4-VEGF165和外科延迟均能有效改善大鼠皮瓣的成活,但二者作用机制不同,而延迟的同时皮下注射VEGF基因能进一步提高大鼠皮瓣成活率。

SURVIVAL OF RAT OVER-AREA ABDOMINAL AXIAL SKIN FLAP AFTER APPLICATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR GENE AND SURGICAL DELAY

Objective To investigate the effect of the vascular endothelial growth factor (VEGF) gene therapy, the surgical delay, and the combination of the two therapeutic approaches on the survival of the rat over-area abdominal axial skin flap. Methods In 48 male Wistar rats (weight, 400-450 g), a model of the abdominal axial skin flap supplied by the superficial epigastric blood vessel was created. The rats were randomly divided into 6 groups: Group A (the blank group), Group B (the gene-therapy-during-operation group), Group C (the gene-therapy-before-operation group), Group D (the merely-surgical-delay group), Group E (the gene-therapy-during-surgical-delay group), and Group F (the gene-therapy-after-surgical-delay group). Seven days after operation, the survival rate of the skin flap was measured; the specimens were harvested from the skin flap for a histological investigation of the microvessels and for an immunohistochemical staining to observe the expression of VEGF165. Results The average survival rate of the skin flap was significantly greater in each of the treated groups than in Group A (P<0.05); the rate was the greater in Group E (P<0.05), but with no statistically significant difference between the other treated groups (P>0.05). The average number of the microvessels was significantly greater in Groups B, C, E and F than in Groups A and D (P<0.05), but with no statistically significant difference between Groups B, C, E and F and between Groups A and D (P>0.05). The lumen diameter of the microvessels was significantly greater in Group D than in Groups E and F (P<0.05), and the diameter was significantly greater in Groups D, E and F than in the other groups (P<0.05). More deposition of VEGF DNA detected by the immunohistochemical staining was in Groups B, C, E and F than in Groups A and D. There was no newly-formed blood vessel in the rat cornea in the treated groups. Conclusion Both the administration of pcDNA4-VEGF165 and the surgical delay can improve the survival of the rat abdominal axial skin flap, but the mechanism of the effect is different in explanation. The combination of the two therapeutic approaches can achieve a better effect.