脂质体介导的VEGF质粒肝脏内注射对肝缺血再灌注损伤的保护作用

Protection of Intrahepatic Injection of Liposome-mediated VEGF Plasmid against Ischemia-reperfusion Liver Injury

目的探讨肝脏内注射脂质体包裹的血管内皮生长因子(VEGF)表达质粒对肝缺血再灌注损伤的影响及可能的作用机制。方法实验兔随机分为假手术组、肝缺血再灌注组、重组VEGF治疗组(缺血前20 min经门静脉肝脏内注射脂质体包裹的VEGF表达质粒),制作肝缺血再灌注损伤模型,分别于术毕、术后2、6、12、24 h共5个时间点检测肝功能及血浆超氧化物歧化酶(SOD)活性、黄嘌呤氧化酶(XO)活性。于术毕6 h取各组部分动物肝组织,以RT-PCR技术检测肝组织Fas mRNA水平,以流式细胞仪检测细胞凋亡率。于术毕24 h处死动物,取缺血部分肝组织,制备病理切片,在光镜及电镜下观察细胞结构的变化。结果肝缺血再灌注复流后6 h,血清谷丙转氨酶(ALT)升高至峰值,其后呈下降趋势,于术毕6、12、24 h,肝缺血再灌注组ALT值显著高于重组VEGF治疗组,差异均有显著性意义(均P<0.05)。肝缺血再灌注组SOD呈下降趋势,XO呈上升趋势,重组VEGF治疗组于术毕6 h以后SOD呈上升趋势,XO呈下降趋势,与肝缺血再灌注组比较差异均具有显著性意义(P<0.05或P<0.01)。重组VEGF治疗组的Fas mRNA水平及肝细胞凋亡率均显著低于肝缺血再灌注组(均P<0.01),光镜及电镜下观察,重组VEGF治疗组的肝细胞损伤亦较肝缺血再灌注组明显减轻。结论肝缺血前肝组织内预注射脂质体包裹的含有VEGF基因的质粒对肝细胞具有明显的保护作用,其作用机制是通过提高肝细胞的抗氧化能力,从而下调Fas mRNA的表达,抑制肝细胞的凋亡来实现的。

Objective To explore the effect of intrahepatic injection of liposome-mediated VEGF plasmid on ischemia-reperfusion liver injury and its mechanism.Methods Rabbits were randomly divided into normal group,ischemia-reperfusion group and recombinant VEGF therapy group（liposome-mediated transfer of VEGF plasmid into liver via portal vein 20 min before ischemia of liver）.The model of liver ischemia-reperfusion injury was established.Liver function and the activity of SOD,XO in blood were determined at the 0,2nd,6th,12th,and 24th h after operation.RT-PCR technique was applied to detect the expression level of Fas mRNA in liver tissues of every group,and flow cytometry was used to measure cell apoptosis rate at the 6th h after operation.At the 24th h after operation,all rabbits were killed and liver tissues of ischemia were taken to make pathological sections for observing the morphology and microstructure under the light microscopy and electron microscopy.Results The level of ALT in recombinant VEGF therapy group was markedly reduced as compared with ischemia-reperfusion group at the 6th,12th,and 24th h after operation（P〈0.05）.The activity of SOD in recombinant VEGF therapy group was significantly higher than in ischemia-reperfusion group at the 6th,12th,and 24th h after operation.The activity of XO in recombinant VEGF therapy group was significantly lower than that in ischemia-reperfusion group at the 6th,12th,and 24th h after operation（P〈0.05 or P〈0.01）.In addition,there was significant difference in the expression of Fas mRNA and cell apoptosis rate between recombinant VEGF therapy group and ischemia-reperfusion group（P〈0.01）.The injury of hepatocytes in recombinant VEGF therapy group was significantly alleviated as compared with that in ischemia-reperfusion group under the light microscopy and electron microscopy.Conclusion Liposome-mediated transfer of VEGF plasmid into liver before ischemia of liver can obviously protect hepatocytes by increasing anti-oxidative ability,decreasing the expression of Fas mRNA,and finally inhibiting hepatocyte apoptosis.