腺病毒-VEGF基因重组体促进预构皮瓣成活的实验研究

Improve survival of prefabricated flap with adenovirus vectors encoding for VEGF in rats

目的:应用大鼠预构皮瓣模型,探讨基因治疗技术产生的血管内皮生长因子促进预构皮瓣血管新生和皮瓣存活的可能性,为临床上寻找加速预构皮瓣成熟的新方法提供实验依据。方法:20只SD大鼠每只腹部两侧各构建一个预构皮瓣,共构建40个皮瓣,每只大鼠两侧皮瓣按随机原则进行不同的处理,分别归于实验组或对照组,每组各20个皮瓣。于大鼠腹部两侧各标记3cm×2cm矩形预构区,短边平行于腹股沟韧带,自尾侧短边中点向后纵向切开后肢皮肤,剥离出长约2cm的股动静血管束,远端结扎切断。在两侧预构区域的中轴线上,于真皮与肉膜层间各制作一皮下隧道,实验组的隧道壁皮下组织内注射携带有VEGF基因的腺病毒,同法向所有对照组的隧道壁软组织内注射等量生理盐水。将已剥离好的血管束向颅侧翻转置入相应皮下隧道内。所有已植入股血管的预购区域2周后均被制成以植入血管束为蒂的岛状皮瓣,从两组中各取一个皮瓣进行免疫组化染色,观察有无VEGF生成,其余岛状皮瓣均缝回原处。形成岛状皮瓣后第七天观察皮瓣存活及血管新生情况。结果:实验组与对照组的皮瓣平均存活率分别为(90.48±1.89)%、(69.75±2.36)%,其差异有统计学意义(P<0.01);血管放射显影图上,实验组植入血管周围见广泛白色显影,尤以血管两端明显,而对照组新生血管显影仅局限于植入血管周围;组织学切片显示实验组植入血管周围新生血管丰富,以毛细血管为主,并见肉芽成份,对照组新生血管相对较少,两组间新生小血管管腔大小则无明显差异;免疫组化检测显示仅实验组皮瓣中有VEGF表达。结论:腺病毒-VEGF基因重组体能通过促进预构皮瓣的血管新生,增加预构皮瓣的存活率。

Objective This study investigated the feasibility of local administration of adenovirus vectors encoding for VEGF to induce regional angiogenesis and improve the survival of prefabricated flap in rat prefabricated flap model, so as to provide experimental evidence for a new method with which we can accelerate maturation of prefabricated flap in clinics. Methods 40 prefabricated flaps were created on the left and right abdominal walls in 20 SD rats. Two prefabricated flaps in each rat were randomly allocated to experiment group or control group, and each group contains 20 flaps. In all animals, two 3cm×2cm rectangular skin flaps were designed obliquely on the abdominal wall so that the short side would lie parallel to inguinal ligament. Longitudinal incisions were made on both hind limbs starting from the midpoint of the short side down to the ankle. 2cm long femoral artery, vein were dissected as a bundle and ligated distally. A tunnel was created in the subcutaneous tissue between dermis and panniculus carnosus at the central axis of each planned skin flap. The subcutaneous tissue around the tunnel was injected with adenovirus vectors encoding for VEGF （Ad-VEGF） in experiment group, and with saline in equal amounts in control group. The prepared femoral vessel bundle was then turned over and passed through correspond tunnel. Two weeks later, abdominal island flap based solely on the implanted vessel was elevated. Select one flap from each group to observe the expression of VEGF. Other flaps were resutured into position. Flap viability and neovascularisation were evaluated on postoperative day 7 after the second surgical intervention. Results There was a significant increase in mean survival rate of prefabricated flaps in the Ad-VEGF group compared to the control group： Ad-VEGF, （90.48±1.89）%vs. saline, （69.75±2.36）（P〈0.01）. Microangiographic studies showed widespread neovascularisation around the pedicle-especially in the proximal and distal end-in experiment group, but new vessel formation is confined to the vicinity of the pedicle in control group;Histological examination done under high-power magnification （×100） revealed rich vascularity and mild inflammation surrounding the implanted vessel in experiment group, but small amount of new vessel was found in control group. The calibres of the new vessel from two groups were similar; Immunohistochemical stain showed that the VEGF was expressed in the survival tissue of the flap treated with Ad-VEGF, but it was not found in the control group. Conclusion Adenovirus-mediated VEGF gene therapy can increase the survival of prefabricated flaps through inducing regional angiogenesis.