肝细胞生长因子基因修饰的人骨髓间充质干细胞促进大鼠肢体血管新生

Human Mesenchymal Stem Cells Modified by Hepatocyte Growth Factor Gene Promote Angiogenesis of Local Limb Ischemia in Rats

目的评价肝细胞生长因子(Hepatocyte growth factor,HGF)修饰的人骨髓间充质干细胞(Human bone marrow mesenchymal stem cells,hMSC)体内促血管新生效应,探讨其作用机制。方法结扎Wistar大鼠右侧股动脉,建立局部肢体缺血模型。将hMSC、hMSC/Ad-HGF或PBS,通过肌肉注射接种于缺血区,21d后以对侧肢体为正常对照,进行激光多普勒灌注成像测定、HE染色和免疫组化检查,评价骨骼肌微血管密度。以MTT、Transwell及内皮细胞体外管状结构形成实验,评价hMSC/Ad-HGF对血管内皮细胞的体外增殖、迁移及成管状能力的影响。结果 21d后,hMSC/Ad-HGF和hMSC组血流灌注水平明显高于对照组(P<0.01),前者接近PBS组,显著高于hMSC组(P<0.05)。组织学分析表明,每高倍视野肌肉组织中,hMSC/Ad-HGF组微血管数量最高,PBS组和hMSC组次之,对照组最低(P<0.01)。hMSC/Ad-HGF培养上清可明显促进ECV304细胞的增殖,刺激作用高于表皮生长因子(20ng/mL,P<0.001);促进细胞跨膜迁移和管状样结构形成,强于表皮生长因子。结论 hMSC/Ad-HGF可促进血管内皮细胞的增殖、迁移及血管结构重建,从而引发血管新生效应。

Objective To evaluate the angiogenesis-promoting activities of human mesenchymal stem cells （hMSCs） modified by hepatocyte growth factor （HGF） in vivo and further investigate the underlying mechanisms. Methods Wistar rat limb isehemia model was developed by ligation of the right femoral artery, hMSC were infected by an adenoviral vector carrying human HGF cDNA. Aliquots of hMSC/Ad-HGF, hMSC or PBS were injected intramuscularly into the ischemie area of a rat limb ischemia model and 21 days later, laser Doppler peffusion imaging was performed to detect the blood flow of the limbs. Mierovessel density （MVD） was evaluated by HE staining and immunohistochemistry on skeletal muscle sections. Evaluation of proliferation, migration and forming tube-like structures on ECV304 cells impacted by hMSC/Ad-HGF was determined by MTF, Transwell migration and tubercular structure formation assays. Results In contrast to PBS, injection of hMSC/Ad-HGF and hMSC significantly improved the blood flow peffusion of the ischemic limbs. The relative means of the blood perfusion in hMSC/Ad-HGF-treated rats were comparable to those of the left legs and greater than those of hMSC- transplanted legs （P〈0.05）. The amounts of micro-vessels per high-fold field were highest in the hMSC/Ad-HGF-treated muscles, followed by those of PBS and hMSC, and all of them were greatly higher than those of normal controls （P〈0.01）. The supernatants of hMSC/Ad-HGF were shown to promote the proliferation of ECV304 cells and the stimulation activity was greater than that from recombinant human epithelial growth factor at a concentration of 20 ng/mL （P〈0.001）. In agreement, the eonditioned medium of hMSC/Ad-HGF exhibited a greater activity than epithelial growth factor to enhance the transmembrane migration and tubereular structure formation of ECV304 cells. Conclusion HGF gene-modified human mesenchymal stem cells promote the proliferation, migration and vascular formation of endothelial cells, thus activating the angiogenesis process.