质粒pUDKH基因治疗大鼠肢体动脉闭塞病的实验研究

Plasmid pUDKH gene therapy of rat acute hindlimb ischemia:an experimental study

目的 研究携带人肝细胞生长因子 (HGF)基因的质粒pUDKH对大鼠肢体动脉闭塞病的治疗效果及最低有效剂量。方法 手术结扎大鼠左侧后肢股动脉 ,造成急性缺血性血管病模型。并随机分为 5组 ,每组 10只 ,在手术后即刻将 pUDKH以 5 0 μg、10 0 μg、2 0 0 μg、4 0 0 μg、0 μg剂量一次多点注射于结扎部位肌肉内 ,并于注射后第 10天 ,评价各组血管新生及肌肉组织的情况。另 12只大鼠手术后即刻局部多点注射 2 0 0 μgpUDKH( 6只 )或pUDK( 6只 ) ,注射后不同时间点 (第 3,5 ,10 ,14 ,2 1天 )检测骨骼肌组织中HGF的表达。结果 于转移pUDKH质粒后第 3、5、10、14天肌肉标本与对照组比较均可见较强的HGF的表达。第 10天观察到pUDKH组 (除 5 0 μg组外 ) ,在肌肉组织间或软组织中有丰富的小血管形成。半定量评价各组血管再生程度 ,pUDKH 10 0 μg组 ( 0 96± 0 0 7)、2 0 0 μg组 ( 1 97± 0 91)、4 0 0 μg组 ( 2 2 6± 1 0 0 )与对照组 ( 0 2 7± 0 0 4 )之间差异有显著意义 (P <0 0 5 )。结论 人肝细胞生长因子基因治疗大鼠肢体动脉闭塞病是有效的 ,且以 10 0 μg组为最低有效剂量。

Objective To investigate the effect of plasmid pUDKH carrying human hepatocyte growth factor(HGF) gene on rat acute limb ischemia and to find the lowest effective dosage Methods Ligation of femoral artery of one hindlimb in Wistar rats was performed The rats were randomly divided into 5 groups of 10 rats: pUDKH 50 μg, 100 μg, 200 μg, and 400 μg groups and a vacant vector pUDK group (control group) The plasmids were injected once directly into the ischemic limb muscle (5 sites around ligation position) immediately after ligation At day 10, the muscles were removed and stained with H E to assess histologically the blood vessel formation HGF expression was detected in the muscle tissue of another 12 rats on days 3,5,10,14,21,30 after injection of pUDKH or pUDK, 200μg each per animal Results HGF expression was detected clearly in muscle tissue on days 3,5, 10, 14 in pUDKH groups In HGF transfected animals(except for 50 μg group) neoformation of blood vessels in muscles and soft tissues was found, while it was not seen in controls By semi quantitation of the degree of vessel formation, significant differences between pUDKH groups (0 96±0 07, 1 97±0 91, 2 26±1 00 for 100 μg, 200 μg, 400 μg, respectively) and the control group (0 27±0 04) ( P <0 05) were noted In addition, injection of pUDKH could alleviate the severity of degeneration of muscular tissue due to ischemia Conclusion Human HGF gene therapy is effective in treating rat acute limb ischemia with the lowest effective dose of 100 μg