纳米血管内皮生长因子在治疗下肢缺血促进血管再生成中的作用

The effect of vascular endothelia growth gactor encapsulated in nanoparticles on chronic limb ischemia

目的 利用家兔后肢缺血模型 ,观察纳米材料聚乳酸聚乙醇酸共聚物 (poly dl lactic co glycolicacid ,PLGA) ,包载血管内皮生长因子 (VEGF16 5 )基因 ,经局部肌肉注射后 ,外源基因在局部缺血组织中的转染及强度 ,以及缺血部位的血管新生状况。 方法 制备VEGF16 5 真核表达质粒 ,制备包载VEGF16 5 基因的纳米粒子 ,并检测其理化性质和体外释放曲线。建立家兔后肢缺血模型 2 4只 ,其中4只为对照组 ,只行股动脉及其分支结扎切断术 ;2只为空白纳米粒子组 ,局部缺血肌肉注射空白纳米粒子 ;10只应用裸质粒VEGF16 5 转染 ,8只采用纳米技术包载VEGF16 5 转染。直接缺血部位肌肉内多点注射 ,进行局部定位基因转染。术后 14d行血管造影 ,了解缺血部位侧枝形成情况。处死兔子 ,取股二头肌 ,内收大肌 ,做病理切片 ,免疫组化染色 ,观察VEGF16 5 的表达 ,测定毛细血管密度。应用逆转录 聚合酶链反应了解VEGF16 5 在骨骼肌中的表达 ,并对不同的转染技术进行半定量分析。 结果 转基因治疗 14d后 ,转染VEGF基因组血管造影可见明显新生血管和侧枝循环形成 ,免疫组化染色可见VEGF16 5 蛋白表达水平增高 ,缺血肌肉血管数增多 ,纳米VEGF16 5 治疗组与裸质粒VEGF16 5 治疗组的毛细血管密度明显高于对照组 ,有显著性差异

Objective To experimentally investigate direct intramus cular gene transfer of nanoparticles encoding vascular endothelial growth factor for the treatment of peripheral artery disease. Methods The human VEGF 165 cDNA was cloned into the eukaryotic expression vector PIRES2 under the control of cytomegalovirus promoter/enhancer. The recombinant gene was transferred into a rabbit model of chronic hindlimb ischemia by naked plasmid and nanoparticle respectively. Ischemia was induced in the hindlimb of New Zealand White rabbits by ligation of the distal external iliac artery and complete excision of the femoral artery and all its branches. At day 7 postoperation animals received VEGF 165 plasmid (10 intramuscular) or nanoparticle-VEGF 165 (8 intramuscular). With RT-PCR, immunohistochemistry analysis,and angiography,the expression and biological effects of VEGF 165 gene in experimental animals were investigated. Results Two weeks after initiation of therapy, angiography showed that the transfer of VEGF 165 gene stimulated the formation of focal neovessels and established collateral circulation.The adductor muscle of ischemic limbs was histologically examined at day 14.Capillary density was increased among VEGF 165 -tranfected rabbits,especially Nano-VEGF 165 -treated animals(Naked VEGF 165 plasmid=50 18 per mm 2,Nano-VEGF 165 =81 22 per mm 2,Control=29 54 per mm 2,P<0 05).RT-PCR showed that the transcription and expression of VEGF 165 gene in experimental group were significantly higher than those of control groups. Conclusions Intramuscular administration of VEGF 165 induces collateral artery augmentation in the rabbit model of chronic limb ischemia. Nanoparticle can act as a vector to transfect specific gene and it will benefit gene transfer.