内皮型一氧化氮合酶基因疗法防治小血管吻合口血栓形成的实验研究

Preventive effection of gene therapy with eNOS gene on the formation of thrombi of vascular anastomotic site

目的探讨内皮型一氧化氮合酶(endothelial nitricoxide synthase,eNOS)基因局部转基因表达对小血管吻合口血栓形成的影响。方法大鼠30只,随机分为实验组和对照组,切断大鼠双侧股动脉,行原位端端吻合,并向吻合口血管腔内注入真核表达载体pcDNA3.0-eNOS质粒与脂质体Lipofectin 2000的混合溶液(实验组)或真核表达载体pcD-NA3.0质粒与脂质体Lipofectin 2000的混合溶液(对照组),使之在腔内滞留30min。术后d5在两组中随机各取5只大鼠,切取以吻合口为中心,长1.5cm股动脉,将其分成3等份,每份0.5cm,分别行RT-PCR、Western blotting和组织化学法检测以了解eNOS表达情况。其余大鼠于术后14d处死,切取以吻合口为中心0.5cm长血管段行病理形态学检测。结果试验组RT-PCR在591bp处出现明显条带;Western blotting检测试验组eNOS含量分别为0.517±0.140,明显高于对照组0.248±0.062.;组织化学法检测试验组一氧化氮(nitric oxide,NO)含量(7.6±0.3)μmol/L,明显高于对照组(2.4±0.2)μmol/L。实验组吻合口血栓出现率明显低于对照组(P<0.01)。结论脂质体介导的携带eNOS基因的真核表达载体可以在小血管吻合口中成功表达,并且能有效地防止小血管吻合口血栓的形成。

Objective To identify the effects of in vivo local expression of recombine eNOS gene on the thrombosis formation in vascular anastomotic site. Methods 30 rats were divided into treatment group （ n = 15 ） and control group （ n = 15 ）. End to end anastomoses were performed in rat femoral arteries. Lipofectin 2000 and recombinant pcDNA3.0-eNOS （ treatment group） or Lipofectin 2000 and pcDNA3.0 （ control group） were injected into anastomotic vascular lumen for 30 minutes. 5 rats of each group were executed 5 days after the operation to obtain 1.5 cm length vascular tissue. The vascular tissue was divided to 3 portiones equally. The expression of eNOS gene in vascular anastomotic site was detected by RT-PCR and Western blotting assay. The NO activity was measured by chemiluminescent assay. Other rats were executed 2 weeks after the operation to obtain 0. 5 cm length vascular tissue for pathological study. Results When the isolated RNA was performed with RT-PCR, 591 bp band appeared in the treatment group, but band could not be found in the control group. The eNOS and NO in treatment group were significantly higher than those in control group. Thrombosis rate fewer in the treatment group than that in the control group （P 〈 0.01 ）. Conclusion When pcDNA3.0-eNOS is transferred into anastomotic vascular wall, the foreign gene can produce eNOS at anastomotic sites and effectively prevent the formation of thrombosis.