肝细胞生长因子基因治疗促进大鼠缺血后肢血管新生的研究  

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat hindlimb of ischemia

作　　者：孙晋津[1] 陈博[1] 郭亮 陈剑秋[1] 

机构地区：[1]天津医科大学第二医院血管外科,300211 [2]军事交通学院门诊部

出　　处：《中华老年医学杂志》2012年第9期805-809,共5页Chinese Journal of Geriatrics

基　　金：基金项目：天津市自然科学基金资助项目（08JCYBJC27600）;天津市高等学校科技发展基金项目（20040226）

摘　　要：目的构建肝细胞生长因子（HGF）真核细胞表达质粒pEGFP—HGF—C1，并观察该质粒对大鼠急性缺血肢体血管新生的作用。方法构建含有人HGFcDNA全长的真核细胞表达质粒pEGFPHGF—C1；脂质体法向大鼠原代骨骼肌细胞转染质粒并测定转染效率；酶联免疫吸附法（EusA）测定细胞上清液中HGF蛋白表达浓度。建立大鼠急性缺血肢体模型；裸质粒pEGFP—HGF-C1200ug／500ul行缺血肢体肌肉注射，术后第3、6、9天取标本行HGF免疫组织化学和蛋白印迹检测；术后第24天取标本行毛细血管密度测定，评价血管新生情况。结果成功构建含人HGFcDNA的真核细胞表达质粒pEGFP—HGF—C1，且可有效转染原代培养的大鼠骨骼肌细胞（0．8％），并表达HGF，第4天达到高峰为（5402．0±227．9）ng／L；蛋白印迹和免疫组织化学法均显示于术后第3、6、9天HGF表达量高于空载和空白对照组，差异有统计学意义（P〈O．05）；术后第24天治疗组毛细血管密度为10．81±2．35，与空载组6．11±0．90和空白组5．45±0．90比较，差异有统计学意义（P〈0．01）。结论构建的质粒pEGFP—HGF—C1转染大鼠肌肉组织后表达的HGF促进了缺血部位的血管新生。Objective To investigate the effect of plasmid pEGFP hepatocyte growth factor （HGF）-C1 on rat acute ischemia of hindlimb. Methods The eukaryotic expressed plasmid pEGFP- HGF-C1 carrying human HGF eDNA was constructed. The transfection efficiency and the expression level of HGF were evaluated by transfecting pEGFP-HGF-C1 into primary rat skeletal muscle cells. Ligation-induced ischemia of femoral artery of one hindlimb in Wistar rats was performed. The plasmids （200 ug/500 ul ） were injected once directly into the ischemic limb muscle （5 sites around ligation position） immediately after ligation. HGF expression was detected in the muscle tissue of rats on days 3,6 and 9 by immunochemical stain and Western blot. At day 24, the muscles were removed and stained with CD31 to assess histologically the capillary formation. Results The primary rat skeletal muscle cells could be transfected efficiently with pEGFP-HGF-C1 using LipofeetamineTM 2000 （0.8%）, and secreted HGF which peak concentration was （5402.0± 227.9 ） ng/L at 4 d. HGF expression was detected clearly in muscle tissue on days 3, 6, 9 in pEGFP-HGF-C1 groups. Western blot semi-quantitied analysis showed the levels of HGF expression were higher than control groups on days 3, 6, 9（P〈0.05）. A significant increase in capillary density was found in rats transfected with human HGF（10.81±2.35） as compared with sham group （6. 11±0.90） and control group （5.45± 0.90） at 24 d（P〈0.01）. Conclusions Intramuscular injection of naked human HGF plasmid may induce therapeutic angiogenesis in rat ischemic hindlimb models, as potential therapy for peripheral arterial disease.

关 键 词：肝细胞生长因子 质粒 缺血 新生血管化 生理性 

分 类 号：R363[医药卫生—病理学]