血管靶向性载VEGF165基因纳米粒子在治疗肢体缺血的实验研究
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摘要
背景和目的
近年来,外周动脉疾病的发病率逐年升高,尤其是下肢缺血性疾病己经成为危害人类健康的严重疾病。目前针对下肢缺血性疾病主要手段是药物、外科手术、腔内治疗等。但是对于远端流出道严重狭窄尤其是合并糖尿病的患者往往没有手术机会,目前还缺乏令人满意的治疗方法。应用各种方法和手段促进新生血管生成,促进缺血肢体侧枝循环的建立,是有效可行的研究方向之一。
基因治疗是目前研究较多的治疗缺血性疾病的一项新技术。血管内皮生长因子(VEGF)在治疗缺血性疾病中的作用已经得到肯定,它能增加小血管的通透性、促进内皮细胞增殖和迁移,抑制内皮细胞凋亡,并能刺激内皮细胞产生纤溶酶原激活物,从而有力地促进血管新生。但是,目前对于VEGF基因治疗仍存在着许多局限性:(1)外源性VEGF虽能促进血管生成,但其靶向治疗能力差,因此要达到治疗下肢缺血目的所需的剂量较大,而增大剂量势必增加其副作用,甚至还有增加促肿瘤生成和加重糖尿病性视网膜病变的潜在风险。(2)病毒性基因载体转染效率高,但其促肿瘤生成、细胞毒性和免疫原性等安全性问题需要重视。后来发展的非病毒性载体,如阳离子聚合物,毒性小,安全性较高。其中壳聚糖作为一种天然阳离子聚合物的非病毒基因载体,显示了较好的前景。它具有细胞毒性低、生物相容性好、基因免疫性低等优点。其纳米粒子形式包载DNA,载药量较大,可以保护DNA在体内不被酶降解,还能起到缓慢释放DNA的效果。但其细胞转染效率较低,生物靶向性较差,因而也限制了它在基因治疗方向的应用。
因此,如何优化基因载体和增强局部治疗的靶向性是目前基因治疗需要解决的主要问题。
RGD肽是一类含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)的短肽,其作用是通过和整合素的特异性结合来实现的。整合素αvβ3是含有数个RGD位点的细胞受体,在血管生成过程中起到重要的作用。在成熟血管内皮细胞表面αvβ3整合素呈低水平表达,但在缺血状态下的新生血管内皮细胞中αvβ3的表达显著上升。利用外源性RGD对整合素αvβ3的靶向结合特性,可以携带促进新生血管生成的药物或基因进入体内缺血局部起治疗作用,从而治疗肢体缺血等疾病。
为提高壳聚糖纳米粒子非病毒基因载体的转染效率和生物靶向性,并将其应用于治疗缺血性疾病,本研究通过以共价键将RGD多肽结合在壳聚糖上,制备成纳米粒子基因载体,并包载VEGF165质粒,分别通过体外细胞实验和动物实验观察其靶向特异性和生物学效应,从而为基因靶向治疗缺血性疾病的临床应用提供实验依据。
内容和方法
1.RGD肽表面修饰壳聚糖载基因纳米粒子(CH-RGD)及FITC标记纳米粒子(FITC-CH-RGD)的制备,X射线光电子谱(XPS)进行材料定性,透射电镜测量纳米粒子的粒径,凝胶电泳阻滞实验确定最大质粒包封率的N/P值。
2.体外Hy926内皮细胞实验:
(1)利用激光共聚焦显微镜和流式细胞技术观察内皮细胞对FITC-CH-RGD和FITC-CH纳米粒子的结合和吞噬情况比较;
(2)包载pEGFP-C1质粒转染Hy926细胞,分为3组:CH-RGD-pEGFP组、CH-pEGFP组和Lipofetamine2000-pEGFP组,转染后48h荧光显微镜下观察转染效率;
(3)包载pIRES_2-VEGF_(165)质粒转染Hy926细胞,分为3组:CH-RGD-pVEGF_(165)组、CH-pVEGF_(165)组和Lipofetamine2000-pVEGF_(165)组,分别于转染后第1,3,7,14天取上清用ELISA法测定VEGF蛋白表达;
(4)倒置显微镜观察转染后各组细胞检测日期的形态及存活情况。
3.动物实验:
(1)建立小鼠股动脉结扎切断后肢缺血模型,根据实验需求分为CH-RGD-pVEGF_(165)治疗组、CH-pVEGF_(165)治疗组、pVEGF质粒治疗组和空白对照组。给药方式为患肢肌肉注射,于结扎股动脉后7天后给药。
(2)分别于给药后3天、7天、14天取材缺血侧腓肠肌组织和对侧腓肠肌组织;行HE染色、免疫组化染色(PCNA、VEGF)评价肌肉组织间的新生毛细血管生成、VEGF表达情况;Real Time-PCR检测VEGF mRNA含量及Western Blot检测VEGF表达含量。
结果
1.以共价键将RGD多肽结合在壳聚糖上,制成纳米粒子形式,X射线光电子谱证实产物为CH-RGD;透射电镜测得粒径范围为30～50nm;包载质粒DNA后,凝胶电泳阻滞实验显示最大质粒包封率时N/P值为2:1。
2.体外细胞实验:
将制得的FITC荧光标记RGD肽表面修饰的壳聚糖纳米粒子加入体外培养的Hv929细胞,通过流式细胞技术和激光共聚焦显微镜观察证实其在体外对内皮细胞具有很强的靶向结合特性,而且能促进内皮细胞的内吞作用。
包载EGFP质粒体外转染Hy926细胞实验中,CH-RGD-pEGFP组较CH-pVEGF_(165)组转染效率明显提高(35.7%vs 14.3%,p<0.001),较Lipofetamine2000-pEGFP组稍增高(35.7%vs 31.3%,p>0.05);
包载VEGF_(165)质粒体外转染Hy926细胞实验中,CH-RGD-VEGF_(165)转染组细胞上清液中VEGF浓度在转染后1、3、7、14天均较其他两组明显增高。(除第1天CH-RGD-VEGF_(165)转染组与Lipo-pVEGF_(165)组比较p>0.05外,其余p值均<0.01。)
CH-RGD-pVEGF_(165)组及CH-pVEGF_(165)组于转染后1、3、7、14天存活细胞总数均较Lipo-pVEGF_(165)组有显著性差异(p值均<0.01),而CH-RGD-pVEGF_(165)组和CH-pVEGF_(165)组之间无明显差异。表明CH-RGD在体外对内皮细胞无明显细胞毒性。
3.动物实验:
在后肢缺血模型小鼠中,HE染色和PCNA免疫组化染色显示CH-RGD-pVEGF_(165)治疗组较其余各组缺血肢体中新生毛细血管生成明显增多,而组织缺血表现较轻;每张HE染色切片在×100倍随机5个视野计数毛细血管的数目也明显增多(p值均<0.01);VEGF免疫组化显示毛细血管内皮和周围组织中VEGF表达显著增高;
Real-Time结果显示,CH-RGD-pVEGF_(165)治疗组较其余各组和健侧肌肉中VEGFmRNA的表达量明显增高。(p值均<0.01)
Western Blot结果显示:CH-RGD-pVEGF_(165)治疗组较其余各组和健侧肌肉中VEGF的表达显著增高。(p值均<0.01)
治疗后到14天观察,CH-RGD-pVEGF_(165)治疗组和CH-pVEGF_(165)治疗组未见患肢坏死、感染,组织水肿不明显。而pVEGF_(165)治疗组及空白对照组见足趾局灶性坏死,其中C组2只,占总数的33%;D组3只,占D组总数的50%。
结论
1.RGD肽表面修饰的CH纳米粒子在体外对内皮细胞具有很强的靶向结合特性,而且能促进内皮细胞的内吞作用。
2.RGD肽表面修饰的CH纳米粒子作为基因载体,其体外对内皮细胞的转染效率比壳聚糖纳米粒子明显提高,较Lipofetamine2000稍增高;包载VEGF_(165)质粒转染内皮细胞后,外分泌VEGF的浓度也显著增加。
3.RGD表面修饰的CH纳米粒子包载pVEGF_(165)治疗后肢缺血模型小鼠,毛细血管内皮和周围组织中VEGF表达显著增高,从而促进新生毛细血管生成,改善肌肉组织缺血。
4.CH-RGD纳米粒子作为基因载体在体外和动物体内具有较好的组织相容性,无明显细胞毒性。
Background and Objective
Gene therapy has been developed as a potential cure for the treatment of peripheral arterial diseases(PAD).However,viral vectors such as retrovirus and adenovirus have serious safety concerns such as potential oncogenicity,toxicity and immunogenicity. Non-viral strategies have been developed as an alternative for gene delivery,but also have their limitation in low transfection efficiency and poor target ability.
Chitosan(CH) is a polycationic biopolymer,and characterized as cheap, environmental-friendly production,good bacteriostatic effects and high biocompatibility.Studies showed it could serve as a non-virus gene vector.However, the efficiency and target ability still needed to improve.
Arg-Gly-Asp(RGD) peptides were found in extracellular matrix,and could promote attachment of cells as it could be recognized by the adhesion receptors on the cell membrane.Endothelia cells from tumor or neo-vasulariztion could specifically express some kind of RGD containing peptides,such asα_vβ_3,and supposed to enhance the affinity between cells to materials.
In this study,we tried to investigate the target ability and gene transfer-efficiency of chitosan nanoparticles modified with RGD as a new vector with plasmid DNA in vitro, and the therapy effect with plasmid vascular endothelial growth factor 165(pVEGF165) for angiogenic gene therapy in ischemic limbs of mice.
Methods
1.Nanoparticles of chitosan(CH) and chitosan linked with RGD(CH-RGD) were made by complex coacervation method,and were labeled with fluorescent isothiocyanate (FITC).With Hy926 cultured cell line system,the adhesion and endocytosis of cells to the compounds were assessed with flow cytometry and confocal laser microscopy, respectively.Using a report gene,pEGFP,CH-RGD-pEGFP nano-gene transfer system was compared to CH-pEGFP nano-gene transfer system and another polycationic polymer,lipofetamine2000,in the transfection efficiency with Hy926 cells in vitro.And then using a therapeutic gene,humen pVEGF_(165),the CH-RGD-pVEGF165 nano-gene transfer system,CH-pVEGF165 nano-gene transfer system and lipofetamine2000-pVEGF165 gene transfer system were transfected with Hy926 cells in vitro,then compared the VEGF expression in the supernatant detected with the
ABC-ELISA method and the number of living cells 1 day,3,7 and 14 days after transfection.
2.We made mice hindlimb ischemia models and divided them into four groups in random,CH-RGD-pVEGF165 nano-gene system,CH-pVEGF165 nano-gene system, pVEGF165 and equal dose sterilized distilled water were separately intramuscular injected into the ischemic limbs.The gastrocnemius of ischemic limbs were made to paraffin sections for HE stained,PCIMA and VEGF immunohistochemistry stained to observe the capillary density and VEGF expression in 7,14 and 28 days after treatment.Real-Time PCR method and western blot method were also used to observed the VEGF expression in the ischemic limbs after treatment.
Results
1.In vitro:
The adhesion and endocytosis via Chitosan-RGD-nanoparticles were much better than Chitosan-nanoparticles,expressing as more fluorescent positive cells 99.51%vs. 95.57%(p>0.05),and more intensity of fluorescent 132.47 vs.72.28(p<0.05). Report gene system showed CH-RGD-pEGFP had a significant high transfection efficiency compared with CH-pEGFP,35.7%vs.14.3%(p<0.001),and a little high transfection efficiency compared with lipofetamine2000-pEGFP,35.7%vs 31.3%, (p>0.05).The CH-RGD-pVEGF165 also had a significant high VEGF expression in the supernatant compared with CH-pVEGF165 and lipofetamine2000-pVEGF165 in 1 day,3,7 and 14 days after transfection.The cytotoxicitiy of CH-RGD nanoparticles were significant lower than Iipofetamine2000 by caculating the living cells(p<0.01).
2.In vivo:
The capillary density were significant higher in CH-RGD-pVEGF165 group than the other three groups(p<0.01),and the muscular cells were not severely ischemic changed.Both VEGF mRNA and VEGF expression were significant high in CH-RGD-pVEGF165 group than the other three groups(p<0.01),which were detected by Real-Time PCR method and western blot method separately.CH-RGD-pVEGF165 group had no limb necrosis.
Conclusion
1.Chitosan nanoparticles modified with RGD could improve the adhesion and endocytosis of cells in vitro,and increase gene transfection efficiency.
2.CH-RGD-pVEGF165 nano-gene system had the potential advances of target ability and therapeutic effect in the gene therapy for the treatment of limb ischemia.
3.CH-RGD nanoparticles showed low cytotoxicity and high biocompatibility as gene vector in vitro and in vivo.
近年来,外周动脉疾病的发病率逐年升高,尤其是下肢缺血性疾病己经成为危害人类健康的严重疾病。目前针对下肢缺血性疾病主要手段是药物、外科手术、腔内治疗等。但是对于远端流出道严重狭窄尤其是合并糖尿病的患者往往没有手术机会,目前还缺乏令人满意的治疗方法。应用各种方法和手段促进新生血管生成,促进缺血肢体侧枝循环的建立,是有效可行的研究方向之一。
基因治疗是目前研究较多的治疗缺血性疾病的一项新技术。血管内皮生长因子(VEGF)在治疗缺血性疾病中的作用已经得到肯定,它能增加小血管的通透性、促进内皮细胞增殖和迁移,抑制内皮细胞凋亡,并能刺激内皮细胞产生纤溶酶原激活物,从而有力地促进血管新生。但是,目前对于VEGF基因治疗仍存在着许多局限性:(1)外源性VEGF虽能促进血管生成,但其靶向治疗能力差,因此要达到治疗下肢缺血目的所需的剂量较大,而增大剂量势必增加其副作用,甚至还有增加促肿瘤生成和加重糖尿病性视网膜病变的潜在风险。(2)病毒性基因载体转染效率高,但其促肿瘤生成、细胞毒性和免疫原性等安全性问题需要重视。后来发展的非病毒性载体,如阳离子聚合物,毒性小,安全性较高。其中壳聚糖作为一种天然阳离子聚合物的非病毒基因载体,显示了较好的前景。它具有细胞毒性低、生物相容性好、基因免疫性低等优点。其纳米粒子形式包载DNA,载药量较大,可以保护DNA在体内不被酶降解,还能起到缓慢释放DNA的效果。但其细胞转染效率较低,生物靶向性较差,因而也限制了它在基因治疗方向的应用。
因此,如何优化基因载体和增强局部治疗的靶向性是目前基因治疗需要解决的主要问题。
RGD肽是一类含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)的短肽,其作用是通过和整合素的特异性结合来实现的。整合素αvβ3是含有数个RGD位点的细胞受体,在血管生成过程中起到重要的作用。在成熟血管内皮细胞表面αvβ3整合素呈低水平表达,但在缺血状态下的新生血管内皮细胞中αvβ3的表达显著上升。利用外源性RGD对整合素αvβ3的靶向结合特性,可以携带促进新生血管生成的药物或基因进入体内缺血局部起治疗作用,从而治疗肢体缺血等疾病。
为提高壳聚糖纳米粒子非病毒基因载体的转染效率和生物靶向性,并将其应用于治疗缺血性疾病,本研究通过以共价键将RGD多肽结合在壳聚糖上,制备成纳米粒子基因载体,并包载VEGF165质粒,分别通过体外细胞实验和动物实验观察其靶向特异性和生物学效应,从而为基因靶向治疗缺血性疾病的临床应用提供实验依据。
内容和方法
1.RGD肽表面修饰壳聚糖载基因纳米粒子(CH-RGD)及FITC标记纳米粒子(FITC-CH-RGD)的制备,X射线光电子谱(XPS)进行材料定性,透射电镜测量纳米粒子的粒径,凝胶电泳阻滞实验确定最大质粒包封率的N/P值。
2.体外Hy926内皮细胞实验:
(1)利用激光共聚焦显微镜和流式细胞技术观察内皮细胞对FITC-CH-RGD和FITC-CH纳米粒子的结合和吞噬情况比较;
(2)包载pEGFP-C1质粒转染Hy926细胞,分为3组:CH-RGD-pEGFP组、CH-pEGFP组和Lipofetamine2000-pEGFP组,转染后48h荧光显微镜下观察转染效率;
(3)包载pIRES_2-VEGF_(165)质粒转染Hy926细胞,分为3组:CH-RGD-pVEGF_(165)组、CH-pVEGF_(165)组和Lipofetamine2000-pVEGF_(165)组,分别于转染后第1,3,7,14天取上清用ELISA法测定VEGF蛋白表达;
(4)倒置显微镜观察转染后各组细胞检测日期的形态及存活情况。
3.动物实验:
(1)建立小鼠股动脉结扎切断后肢缺血模型,根据实验需求分为CH-RGD-pVEGF_(165)治疗组、CH-pVEGF_(165)治疗组、pVEGF质粒治疗组和空白对照组。给药方式为患肢肌肉注射,于结扎股动脉后7天后给药。
(2)分别于给药后3天、7天、14天取材缺血侧腓肠肌组织和对侧腓肠肌组织;行HE染色、免疫组化染色(PCNA、VEGF)评价肌肉组织间的新生毛细血管生成、VEGF表达情况;Real Time-PCR检测VEGF mRNA含量及Western Blot检测VEGF表达含量。
结果
1.以共价键将RGD多肽结合在壳聚糖上,制成纳米粒子形式,X射线光电子谱证实产物为CH-RGD;透射电镜测得粒径范围为30～50nm;包载质粒DNA后,凝胶电泳阻滞实验显示最大质粒包封率时N/P值为2:1。
2.体外细胞实验:
将制得的FITC荧光标记RGD肽表面修饰的壳聚糖纳米粒子加入体外培养的Hv929细胞,通过流式细胞技术和激光共聚焦显微镜观察证实其在体外对内皮细胞具有很强的靶向结合特性,而且能促进内皮细胞的内吞作用。
包载EGFP质粒体外转染Hy926细胞实验中,CH-RGD-pEGFP组较CH-pVEGF_(165)组转染效率明显提高(35.7%vs 14.3%,p<0.001),较Lipofetamine2000-pEGFP组稍增高(35.7%vs 31.3%,p>0.05);
包载VEGF_(165)质粒体外转染Hy926细胞实验中,CH-RGD-VEGF_(165)转染组细胞上清液中VEGF浓度在转染后1、3、7、14天均较其他两组明显增高。(除第1天CH-RGD-VEGF_(165)转染组与Lipo-pVEGF_(165)组比较p>0.05外,其余p值均<0.01。)
CH-RGD-pVEGF_(165)组及CH-pVEGF_(165)组于转染后1、3、7、14天存活细胞总数均较Lipo-pVEGF_(165)组有显著性差异(p值均<0.01),而CH-RGD-pVEGF_(165)组和CH-pVEGF_(165)组之间无明显差异。表明CH-RGD在体外对内皮细胞无明显细胞毒性。
3.动物实验:
在后肢缺血模型小鼠中,HE染色和PCNA免疫组化染色显示CH-RGD-pVEGF_(165)治疗组较其余各组缺血肢体中新生毛细血管生成明显增多,而组织缺血表现较轻;每张HE染色切片在×100倍随机5个视野计数毛细血管的数目也明显增多(p值均<0.01);VEGF免疫组化显示毛细血管内皮和周围组织中VEGF表达显著增高;
Real-Time结果显示,CH-RGD-pVEGF_(165)治疗组较其余各组和健侧肌肉中VEGFmRNA的表达量明显增高。(p值均<0.01)
Western Blot结果显示:CH-RGD-pVEGF_(165)治疗组较其余各组和健侧肌肉中VEGF的表达显著增高。(p值均<0.01)
治疗后到14天观察,CH-RGD-pVEGF_(165)治疗组和CH-pVEGF_(165)治疗组未见患肢坏死、感染,组织水肿不明显。而pVEGF_(165)治疗组及空白对照组见足趾局灶性坏死,其中C组2只,占总数的33%;D组3只,占D组总数的50%。
结论
1.RGD肽表面修饰的CH纳米粒子在体外对内皮细胞具有很强的靶向结合特性,而且能促进内皮细胞的内吞作用。
2.RGD肽表面修饰的CH纳米粒子作为基因载体,其体外对内皮细胞的转染效率比壳聚糖纳米粒子明显提高,较Lipofetamine2000稍增高;包载VEGF_(165)质粒转染内皮细胞后,外分泌VEGF的浓度也显著增加。
3.RGD表面修饰的CH纳米粒子包载pVEGF_(165)治疗后肢缺血模型小鼠,毛细血管内皮和周围组织中VEGF表达显著增高,从而促进新生毛细血管生成,改善肌肉组织缺血。
4.CH-RGD纳米粒子作为基因载体在体外和动物体内具有较好的组织相容性,无明显细胞毒性。
Background and Objective
Gene therapy has been developed as a potential cure for the treatment of peripheral arterial diseases(PAD).However,viral vectors such as retrovirus and adenovirus have serious safety concerns such as potential oncogenicity,toxicity and immunogenicity. Non-viral strategies have been developed as an alternative for gene delivery,but also have their limitation in low transfection efficiency and poor target ability.
Chitosan(CH) is a polycationic biopolymer,and characterized as cheap, environmental-friendly production,good bacteriostatic effects and high biocompatibility.Studies showed it could serve as a non-virus gene vector.However, the efficiency and target ability still needed to improve.
Arg-Gly-Asp(RGD) peptides were found in extracellular matrix,and could promote attachment of cells as it could be recognized by the adhesion receptors on the cell membrane.Endothelia cells from tumor or neo-vasulariztion could specifically express some kind of RGD containing peptides,such asα_vβ_3,and supposed to enhance the affinity between cells to materials.
In this study,we tried to investigate the target ability and gene transfer-efficiency of chitosan nanoparticles modified with RGD as a new vector with plasmid DNA in vitro, and the therapy effect with plasmid vascular endothelial growth factor 165(pVEGF165) for angiogenic gene therapy in ischemic limbs of mice.
Methods
1.Nanoparticles of chitosan(CH) and chitosan linked with RGD(CH-RGD) were made by complex coacervation method,and were labeled with fluorescent isothiocyanate (FITC).With Hy926 cultured cell line system,the adhesion and endocytosis of cells to the compounds were assessed with flow cytometry and confocal laser microscopy, respectively.Using a report gene,pEGFP,CH-RGD-pEGFP nano-gene transfer system was compared to CH-pEGFP nano-gene transfer system and another polycationic polymer,lipofetamine2000,in the transfection efficiency with Hy926 cells in vitro.And then using a therapeutic gene,humen pVEGF_(165),the CH-RGD-pVEGF165 nano-gene transfer system,CH-pVEGF165 nano-gene transfer system and lipofetamine2000-pVEGF165 gene transfer system were transfected with Hy926 cells in vitro,then compared the VEGF expression in the supernatant detected with the
ABC-ELISA method and the number of living cells 1 day,3,7 and 14 days after transfection.
2.We made mice hindlimb ischemia models and divided them into four groups in random,CH-RGD-pVEGF165 nano-gene system,CH-pVEGF165 nano-gene system, pVEGF165 and equal dose sterilized distilled water were separately intramuscular injected into the ischemic limbs.The gastrocnemius of ischemic limbs were made to paraffin sections for HE stained,PCIMA and VEGF immunohistochemistry stained to observe the capillary density and VEGF expression in 7,14 and 28 days after treatment.Real-Time PCR method and western blot method were also used to observed the VEGF expression in the ischemic limbs after treatment.
Results
1.In vitro:
The adhesion and endocytosis via Chitosan-RGD-nanoparticles were much better than Chitosan-nanoparticles,expressing as more fluorescent positive cells 99.51%vs. 95.57%(p>0.05),and more intensity of fluorescent 132.47 vs.72.28(p<0.05). Report gene system showed CH-RGD-pEGFP had a significant high transfection efficiency compared with CH-pEGFP,35.7%vs.14.3%(p<0.001),and a little high transfection efficiency compared with lipofetamine2000-pEGFP,35.7%vs 31.3%, (p>0.05).The CH-RGD-pVEGF165 also had a significant high VEGF expression in the supernatant compared with CH-pVEGF165 and lipofetamine2000-pVEGF165 in 1 day,3,7 and 14 days after transfection.The cytotoxicitiy of CH-RGD nanoparticles were significant lower than Iipofetamine2000 by caculating the living cells(p<0.01).
2.In vivo:
The capillary density were significant higher in CH-RGD-pVEGF165 group than the other three groups(p<0.01),and the muscular cells were not severely ischemic changed.Both VEGF mRNA and VEGF expression were significant high in CH-RGD-pVEGF165 group than the other three groups(p<0.01),which were detected by Real-Time PCR method and western blot method separately.CH-RGD-pVEGF165 group had no limb necrosis.
Conclusion
1.Chitosan nanoparticles modified with RGD could improve the adhesion and endocytosis of cells in vitro,and increase gene transfection efficiency.
2.CH-RGD-pVEGF165 nano-gene system had the potential advances of target ability and therapeutic effect in the gene therapy for the treatment of limb ischemia.
3.CH-RGD nanoparticles showed low cytotoxicity and high biocompatibility as gene vector in vitro and in vivo.
引文
1 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease.[J]Hypertension 2004,44:203-209.
2 Kim HJ,Jang SY,Park JI,et al.Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease.IJjExp Mol Med 2004,36:336-344.
3 Huang M,Khor E,Lim LY.Uptake and cytotoxicity of chitosan molecules and nanoparticles:effects of molecular weight and degree of deacetylation[J].PharmRes,2004,21(2):344-353.
4 P Erbacher,S Zou,T Bettinger et al.Chitosan-based vector/DNA complexes for gene delivery:biophysical characteristics and transfection ability[J].Pharra Res.1998,15:1332～1339.
5 Brooks PC,Clark RA,Cheresh DA.Requirement of vascular integrin alpha v beta 3 for angiogenesis[J].Science.1994 Apr 22;264(5158):569-71.
6 Haubner R,Bruchertseifer F,Bockl M,et al.Synthesis and bio-logical evaluation of a 99mTc-labelled cyclic RGD peptide for imaging the a vβ 3 expression [J].J Nucl Med,2004,43(1):26-32.
7 Kyung-Han Lee,Kyoung-Ho Jung,Sung-Hee Song.Radiolabeled RGD Uptake and alpha v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs[J].J Nucl Med 2005;46:472～478.
8 Eric T.Choi,M.Faisal Khan,et al.beta 3-Integrin Mediates Smooth Muscle Cell Accumulation in Neointima After Carotid Ligation in Mice [J].2004;109:1564-1569.
9 Jiasheng Zhang,Svetlana Krassilnikova,Amir A.et al.α v β 3-Targeted detection of arteriopathy in transplanted human coronary arteries:an autoradiographic study[J].FASEB J.2005 Nov;19(13):1857-9.
10 Kyung-Han Lee,Kyoung-Ho Jung,Sung-Hee Song.Radiolabeled RGD Uptake and alpha v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs.J Nucl Med 2005;46:472～478.
11 Tigli RS,GtimUsderelioglu M.Evaluation of RGD-or EGF-immobilized chitosan scaffolds for chondrogenic activity.Int J Biol Macromol.2008 Aug 15;43(2):121-8.Epub 2008 Apr 11.
12 Mao HQ,Roy K,Troung-Le VL,et al.Chitosan-DNA nanoparticles as gene carriers:synthesis,characterization and transfection efficiency[J].J Controlled Release,2001,70(3):399- 421.
13 张海玲,朱敦皖,董霞.壳聚糖载基因纳米粒子荧光标记的优化研究[J].生物医学工程与临床,2008,(1)12.
14 R J Mumper,J J Wang,J M Claspell et al.Proc.Int Symp.Control [J].Release Bioact.Mater,1995,122:178-179.
15 Mizeje W,Gerald J.Role of integrins in cancer:survey of ex-pression patterns[J].Pro Soc Exp Biol Med,1999,222(2):124.
16 Unser M,Aldroubi A,Eden M.The L2 Polynomial Spline Pyramid[J].IEEE Trans Pattern Analysis and machine intelligence,1993,15(4):364 - 379.
17 Unser M.Spline:A Perfect Fit for Signal and Image Processing[J].IEEE Signal Processing,1999,16(6):22 - 38.
18 Kai Temming,Raymond M.Schiffelers,et al.RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature[J].Drug Resistance Updates 8(2005) 381-402.
19 Patrick M.Winter.Anne M.et al.Endothelial alpha(v)beta3integrin-targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis[J].Arterioscler Thromb Vasc Biol.2006Sep;26(9):2103-9.
20 PierschbacherM D,Ruoslahti E.Cell Attachment Activity of Fibronectin Can Be Dup licated by Small Synthetic Fragments of theMolecule[J].Nature,1984,309:30- 33.
21 CHEN Yi-xin,XU Xue- ming,HONG Shui-gen,et al.RGD-Tachyp lesin Inhibits Tumor Gr owth[J].Cancer Res,2001,61:2434- 2438.
22 Buckley C D,Pilling D,Henriquez N V,et al.RGD Peptides Induce Apoptosis by Direct Cas pase Activation[J].Nature,1999,397(6719):534-539.
23 Lebaron RG,Athanasiou KA.Extracellular matrix cell adhesion peptides:functional applications in orthopedic materials[J].Tissue Eng,2000,6:85 - 103.
24 Zamir E,Geiger B.Molecular complexity and dynamics of cell-matrix adhesions[J].J Cell Sci,2001,114:3583 - 3590.
25 Geiger B,Bershadsky A.Assembly and mechanosensory function of focal contacts[J].Curr Opin Cell Biol,2001,13:584 - 592.
26 Petit V,Thiery JP.Focal adhesions:structure and dynamics[J].Biol Cell,2000,92:477 - 494.
27 Van der Flier A,Sonnenberg A.Function and interactions of integrins [J].Cell Tissue Res,2001,305:285 - 298.
28 Leavesley DI,Schwartz MA,Rosenfeld M et al.Integrin β 1 and β 3mediated endothelial cell migration is triggered through distinct signaling mechanisms.J Cell Biol 121:163?170.
29 Polrorak Z,Cohen T,Sivan R,et al.VEGF145,a secreted VEGF isform that binds to extracellular matrix[J].J Biol Chem,1997,272:7151.
30 Senger DR.Molecular framework for angiogenesis:a complex web of interactions between extravasated plasma proteins and endothelial cell proteins induced by angiogenic cytokines[J].Am J Pathol 1996,149:17.
31 Spyridopoulos I,Brogi E,Kearney M,et al.Vascular endothelial growth factor inhibits endothelial cell apoptosis induced by tumor necrosis factor- α:balance between growth and death signals[J].J Mol Cell Cardil 1997,29:1321-1330.
32 Stromblad S,Becher JC,Yebra M,et al.Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin α v β 3 during angiogenesis[J].J Clin Invest 1996,98:426?433.
33 Takeo Abumiya,Jacinta Lucero,Ji Hoe Heo,et al.Activated Microvessels Express Vascular Endothelial Growth Factor and Integrin V3 During Focal Cerebral Ischemia[J].Journal of Cerebral Blood Flow & Metabolism,1999,19,1038-1050.
34 Jones MC,Caswell PT,Moran-Jones K,et al.VEGFR1(Flt1) Regulates Rab4Recycling to Control Fibronectin Polymerization and Endothelial Vessel Branching[J].Traffic 2009 Mar 17.[Epub ahead of print].
35 He Y,Jiang Y,Wang J,et al.Prevalence of peripheral arterial disease and its association with smoking in a population-based study in Beijing,China[J].J Vasc Surg.Aug 2006;44(2):333-338.
36 管珩,刘志明,李光伟等.50岁以上糖尿病人群周围动脉闭塞性疾病相关因素分析[J],中华医学杂志2007;87(1):23-27.
37 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
38 Hitlock PR,Hackett NR,Leopold PL,et al.Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs[J].Mol Ther 2004,9:67-75.
39 Ohara N,Koyama H,Miyata T,et al.Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia[J].Gene Therapy 2001,8:837-845.
40 Ishii S,Koyama H,Miyata T,et al.Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia[J].J Vase Surg 2004,39:629-638.
41 KondohK,Koyama H,Miyata T,et al.Conduct ion performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor[J].Cardiovasc Res 2004,61:132-142.
42 Vincenti V,Cassano C,Roechi M,et al.Assignment of the vascular endothelial growth factor gene to human chromosome 6p21,3[J].Circulation,1996,93:1493.
43 Messina LM,Brevetti LS,Chang DS,et al.Therapeutic angiogenesis for critical limb ischaemia:invited commentary[J].J Control Release,2002,78:285.
44 Mustonen T,Alitalo K.Endothelial receptor tyrosine kinases involved in angiogenesis[J].J Cell Biol,1995,129:895-898.
45 Watanabe Y,Lee SW,Detmar M,et al.Vascular permeability factor/wascular endothelial growth factor(VPF/VEGF)delays and induces escape from senescence in human dermal microvascular endothelial cells [J].Oncogene,1997,14:2025-2032.
46 Conffinhal T,Silver M,Keaey M,et al.Impaired collateral vessel deveopment associated with reduced expression of vascular endothelial growth factor in ApoE mice[J].Circulation,1999,99:3188.
47 Schultz A,Lavie L,Hochebery I,et al.Iner individual heterogenety in the hypoxic regulation of VEGF significance for the development of the coronary artery collateral circulation[J].Circulation,1999,100:547.
48 Rajagopalan S,Mohler ER,3rd,Lederman RJ,et al.Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease:a phase Ⅱ randomized,double-blind,controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication[J].Circulation,2003,108:1933-1938.
49 Henry TD,Annex BH,McKendall GR,et aL.The VIVA trial:Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis[J].Circulation,2003,107:1359-1365.
50 Jeon,S.W.Kang,H.W.Lim,D.et al,Synergistic effect of sustained delivery of basic fibroblast growth factor and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs[J].Biomaterials 27 (2006),pp.1617-1625.
51 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
52 Sharif F,Daly K,Crowley J,et al.Current status of catheter-and stent-based gene therapy[J].Cardiovasc Res 2004,64:208-216.
1 He Y,Jiang Y,Wang J,et al.Prevalence of peripheral arterial disease and its association with smoking in a population-based study in Beijing,China[J].J Vasc Surg.Aug 2006;44(2):333-338.
2 管珩,刘志明,李光伟等,50岁以上糖尿病人群周围动脉闭塞性疾病相关因素分析[J],中华医学杂志2007:87(1):23-27.
3 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
4 Fishbein I,Alferiev IS,Nyanguile 0,et al.Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents[J].Proc Natl Acad Sci USA 2006,103:159-164.
5 Rosengart TK,Lee LY,Patel SR,et al.Angiogenesis gene therapy:phase Ⅰ assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease[J].Circulation 1999,100:468-474.
6 Rome JJ,Ahayani V,Flugelman MY,et al.Anatomic barriers influence the distribution of in vivo gene transfer into the arterial wall,Modeling with microscopic tracer particles and verification with a recombinant adenoviral vector[J].Arteriascler Thromb,1994,14:148-161.
7 彭应旭,廖工铁。骨髓靶向给药制剂研究进展[J]。中国药学杂志,2000,35(2):73-75.
8 Alexiou C.Magnetic mitoxantrone nanoparticle detection by histology,X-ray and MRI after magnetic tumor targeting.Journal of Magnetism and Magnetic Materials[J],2001,225:187-193.
9 王斌。靶向抗肿瘤药物载体系统研究近况[J]。广东药学院学报,1998,14(4):311-314.
10 Goodwin S,Peterson C,Hoh C,et al.Targeting and retention of magnetic targeted carriers(MTCs) enhancing intra-arterial chemotherapy[J].Journal of Magnetism and Magnetic Materials,1999,194:132-139.
11 Lobenberg R,Araujo L,Kreuter J.Body distribution of azidothymidine bound to nanoparticles after oral administration[J].Eur J pharm Biopharm,1997,44:127-132.
12 Lobenberg,R.Body distribution of azidothymidine bound to hexyl-cyanoacrylate nanoparticles after i.v.injection to rats[J].Journal of Controlled Reseased,1998,50:21-30.
13 陆彬。 药物新剂型与新技术[M]。北京:人民卫生出版社,1998.
14 Oliveira M C D.Delivery of antisense oligonucleotides by means of pH-sensitive liposomes[J].Journal of Controlled Release,1997,48:179-184.
15 Torchilin VP.Drug targeting.Eur J Pharm Sci,2000,11 Suppl 2:81-91.
16 Davis S S.Biomedical application of nanotechnology-implications for drug targeting and gene therapy[J].Tibtech,1997,15:217-224.
17 Balram Bhargava,Krishna Reddy,Ganesan Karthikeyan,et al.A Novel Paclitaxel-Eluting Porous Carbon-Carbon Nanoparticle Coated,Nonpolymeric Cobalt-Chromium Stent:Evaluation in a Porcine Model[J].Catheterization and Cardiovascular Interventions 2006,67:698-702.
18 Whitlow PL,Bass TA,Kipperman RM,et al.Results of the study to determine rotablator and transluminal angioplasty strategy[J].Am J Cardiol,2001,87(6):699-705.
19 Min Huang,Samadhi N,Vitharana,et al.Polyelectrolyte Complexes Stabilize and Controllably Release Vascular Endothelial Growth Factor[J].Biomacromolecules,2007,8(5),1607-1614.
20 Guzman L,Labhasetwar V,Song C,et al.Local intraluminal infusion of biodegradable polymeric nanoparticles.A novel approach for prolonged drug delivery after balloon angioplasty[J].Circulation,1996,94:1441-1448.
21 Labhasetwar V,Song CX,Levy RJ,et al.Nanoparticle drug delivery system for restenosis[J].Advanced Drug Delivery Reviews,1997,24:63-85.
22 Moon HT,Lee YK,Han JK,et al.A novel formulation for controlled release of heparin-DOCA conjugate dispersed as nanoparticles in polyurethane film[J].Biomaterials,2001,22(3):281-289.
23 Tillmann Cyrus,Huiying Zhang,John S.Allen,Intramural Delivery of Rapamycin With avP 3-Targeted Paramagnetic Nanoparticles Inhibits Stenosis After Balloon Injury[J].Arterioscler Thromb Vase Biol 2008:28:820-826.
24 李拥军,管珩,徐意瑶等.纳米粒子载体在防治血管损伤后再狭窄中的应用[J].基础医学与临床,2002,22(2).
25 李拥军,管珩,刘昌伟等.反义单核细胞趋化蛋白-1基因移植静脉内膜增殖的实验研究[J].中华外科杂志,2001,39,9.
26 徐意瑶,李拥军,管珩等.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[J].中华外科杂志,2004,42(1).
27 Sun-Woong Kang,Hee-Won Lim,Sang-Woo Seo,et al.Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs[J].Biomaterials 2008,29(8):1109-1117.
28 Hongchi Jiang,Tiemin zhang,Xueying Sun,et al.Vascular Endothelial Growth Factor Gene Deliver by Magnetic DNA Nanospheres Ameliorates Limb Ischemia in Rabbits[J].Journal of Surgical Research 2005,126,48-54.
29 Kyung-Han Lee,Kyoung-Ho Jung,Sung-Hee Song.Radiolabeled RGD Uptake and alpha v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs[J].J Nucl Med 2005;46:472-478.
30 唐锋,管珩,李拥军等.RGD肽连接壳聚糖载基因纳米粒子的体外试验研究[J].国际生物医学工程杂志,2009,39.
1 He Y,Jiang Y,Wang J,et al.Prevalence of peripheral arterial disease and its association with smoking in a population-based study in Beijing,China[J].J Vasc Surg.Aug 2006;44(2):333-338.
2 管珩,刘志明李光伟等,50岁以上糖尿病人群周围动脉闭塞性疾病相关因素分析[J],中华医学杂志2007:87(1):23-27.
3 Baker AH.Designing gene delivery vectors for cardiovascular gene therapy[J].Prog Biophys Mol Biol 2004,84:279-299.
4 Burns JC,Friedraann T,Driever W et al.Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors:concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells[J].Proc Natl Acad Sci USA 90:1993,8033-8037.
5 Chen S,Kapturczak M,Loiler SA,et al.Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus sero-type 1 and 5 vectors[J].Hum Gene Ther 2005,16:235-247.
6 Conklin LD,McAninch RE,Schulz D,et al.HIV-based vectors and angiogenesis following rabbit hindlimb ischemia[J].J Surg Res 2005,123:55-66.
7 Denby L,Nicklin SA,Baker AH.Adeno-associated virus(AAV)-7 and -8poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation[J].Gene Therapy 2005,12:1534-1538.
8 Mao HQ,Roy K,Troung- Le VL,et al.Chitosan- DNA nanoparticles as gene carriers:synthesis,characterization and transfection efficiency[J].J Controlled Release,2001,70(3):399-421.
9 Putnam D.Polymers for gene delivery across length scales[J].Nat Mater 2006,5:439-451.
10 Hedman M,Hartikainen J,Syvanne M,et al.Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia:phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT)[J].Circulation 2003,107:2677-2683.
11 Sharif F,Daly K,Crowley J,et al.Current status of catheter-and stent-based gene therapy[J].Cardiovasc Res 2004,64:208-216.
12 Wang K,Kessler PD,Zhou Z,et al.Local adenoviral-mediated inducible nitric oxide synthase gene transfer inhibits neointimal formation in the porcine coronary stented model[J].Mol Ther 2003,7:597-603.
13 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
14 Koren B,Weisz A,Fischer L,Gluzman Z,et al.Efficient transduction and seeding of human endothelial cells onto metallic stents using bicistronic pseudo-typed retroviral vectors encoding vascular endothelial growth factor[J].Cardiovasc Revasc Med 2006,7:173-178.
15 Fishbein I,Alferiev IS,Nyanguile 0,et al.Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents[J].Proc Natl Acad Sci USA 2006,103:159-164.
16 Sharif F,Hynes SO,McMahon J,et al.Gene-eluting stents:comparison of adenoviral and adeno associated viral gene delivery to the blood vessel wall in vivo [J].Hum Gene Ther 2006,17:741-750.
17 Klugherz BD,Song C,DeFelice S,et al.Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus [J].Hum Gene Ther 2002,13:443-454.
18 Rome JJ,Shayani V,Newman KD,et al.Adenoviral vector-mediated gene transfer into sheep arteries using a double-balloon catheter[J].Hum Gene Ther 1994,5:1249-1258.
19 FolkmanJ,ShingY.Angiogenesis [J].J Biol Chem 1992,267:10931-10934.
20 Whitlock PR,Hackett NR,Leopold PL,et al.Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs[J].Mol Ther 2004,9:67-75.
21 Ohara N,Koyama H,Miyata T,et al.Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia[J].Gene Therapy 2001,8:837-845.
22 Ishii S,Koyama H,Miyata T,et al.Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia[J].J Vase Surg 2004,39:629-638.
23 Kondoh K,Koyama H,Miyata T,et al.Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor[J].Cardiovasc Res 2004,61:132-142.
24 Kusumanto YH,vanWeel V,Mulder NH,et al.Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia:a double-blind randomized trial[J].Hum Gene Ther 2006,17:683-691.
25 Rajagopalan S,Shah M,Luciano A,et al.Adenovirus-mediated gene transfer of VEGF(121)improves lower-extremity endothelial function and flow reserved].Circulation 2001,104:753-755.
26 Kim HJ,Jang SY,Park JI,et al.Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery diseased].Exp Mol Med 2004,36:336-344.
27 Isner JM,Walsh K,Symes J,et al.Arterial gene transfer for therapeutic angiogenesis in patients with peripheral artery disease[J].Hum Gene Ther 1996,7:959-988.
28 Baumgartner I,Pieczek A,Manor 0,et al.Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemiad]-Circulation 1998,97:1114-1123.
29 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial diseased]-Hypertension 2004,44:203-209.
30 Rajagopalan S,Mohler Ⅲ ER,Lederman RJ,et al.Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease:a phase Ⅱ randomized,double-blind,controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication[J].Circulation 2003,108: 1933-1938.
31 Kusumanto YH,vanWeel V,Mulder NH,et al.Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia:a double-blind randomized trial[J].Hum Gene Ther 2006,17:683-691.
32 Epstein AL.HSV-1-based amplicon vectors:design and applications [J].Gene Therapy 2005,12 (Suppl 1):S154-S158.
33 Baker AH.Gene therapy for bypass graft failure and restenosis[J].Pathophysiol Haemost Thromb 2002,32:389-391.
34 Akowuah EF,Gray C,Lawrie A,et al.Ultrasound-mediated delivery of TIMP-3 plasmidDNA into saphenous vein leads to increased lumen size in a porcine inteposition graft model [J].Gene Therapy 2005,12:1154—1157.
35 West NE,Qian H,Guzik TJ,et al.Nitric oxide synthase (nNOS)gene transfer modifies venous bypass graft remodeling:effects on vascular smooth muscle cell differentiation and superoxide production[J].Circulation 2001,104:1526-1532.
36 Wan S,George SJ,Nicklin SA,et al.Over-expression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts[J].Mol Ther 2004,9:689-698.
37 George SJ,Angelini GD,Capogrossi MC,et al.Wild-type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis [J].Gene Therapy 2001,8:668-676.
38 Wan S,George SJ,Nicklin SA,Yim AP,Baker AHet al.Over-expression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts[J].Mol Ther 2004,9:689-698.
39 West NE,Qian H,Guzik TJ,et al.Nitric oxide synthase (nNOS)gene transfer modifies venous bypass graft remodeling:effects on vascular smooth muscle cell differentiation and superoxide production [J].Circulation 2001,104:1526-1532.
40 Wolff RA,Ryomoto M,Stark VE,et al.Antisense to transforming growth factor-betal messenger RNA reduces vein graft intimal hyperplasia and monocyte chemotactic protein 1[J].J Vase Surg 2005,41:498-508.
41 Akowuah EF,Gray C,Lawrie A,et al.Ultrasound-mediated delivery of TIMP-3 plasmidDNA into saphenous vein leads to increased lumen size in a porcine inteposition graft model [J].Gene Therapy 2005,12:1154-1157.
42 Grube E,PRoject of Ex-vivo Vein graft ENgineering via Transfection (PREVENT)Ⅱ trial[J].Presented at the annual Amercian HeartAssociation Scientific Sessions,November 11-14,2001,Anahe im,Cali forn i a,2001.
43 Mann MJ,Whittemore AD,Donaldson MC,et al.Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy:the PREVENT single-centre,randomised,controlled trial[J].Lancet 1999,354:1493-1498.
44 Alexander JH,Ferguson Jr TB,Joseph DM,et al.The PRoject of Ex-vivo Vein graft ENgineering via Transfection IV (PREVENT IV)trial:study rationale,design,and baseline patient characteristics[J].Am Heart J 2005,150:643-649.
45 ConteMS,Lorenz TJ,BandykDF,et al.Design and rationale of the PREVENT Ⅲ clinical trial:edifoligide for the prevention of infrainguinal vein graft failure[J].Vase Endovascular Surg 2005,39:15-23.
46 Burt HM,Hunter WL.Drug-eluting stents:a multidisciplinary success story[J].Adv Drug Deliv Rev 2006,58:350-357.
47 Joner M,Finn AV,Farb A,et al.Pathology of drug-eluting stents in humans:delayed healing and late thrombotic risk[J].J Am Coll Cardiol 2006,48:193-202.
48 Laitinen M,Hartikainen J,Hiltunen MO,et al.Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty[J].Hum Gene Ther 2000,11:263-270.
49 Hedman M,Hartikainen J,Syvanne M,et al.Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia:phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT)[J],Circulation 2003,107:2677-2683.
50 Johnson TW,Wu YX,Herdeg C,et al.Stent-based delivery of tissue inhibitor of metallo-proteinase-3 adenovirus inhibits neointimal formation in porcine coronary arteries[J].Arterioscler Thromb Vase Biol 2005,25:754-759.
51 Fishbein I,Alferiev IS,Nyanguile 0,et al.Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents[J].Proc Natl Acad Sci USA 2006,103:159-164.
52 Rosengart TK,Lee LY,Patel SR,et al.Six-month assessment of a phase I trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA[J].Ann Surg 1999,230:466-470;discussion 470-462.
53 Rosengart TK,Lee LY,Patel SR,et al.Angiogenesis gene therapy:phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease [J].Circulation 1999,100:468-474.
54 Simari RD,O'Brien T.Clinical trials in vascular gene therapy:the missing piece of the puzzle [J].Arch Pathol Lab Med 2002,126:317-319.
55 Yla-Herttuala S,Markkanen JE,Rissanen TT.Gene therapy for ischemic cardiovascular diseases:some lessons learned from the first clinical trials[J].Trends Cardiovasc Med 2004,14:295-300.
2 Kim HJ,Jang SY,Park JI,et al.Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease.IJjExp Mol Med 2004,36:336-344.
3 Huang M,Khor E,Lim LY.Uptake and cytotoxicity of chitosan molecules and nanoparticles:effects of molecular weight and degree of deacetylation[J].PharmRes,2004,21(2):344-353.
4 P Erbacher,S Zou,T Bettinger et al.Chitosan-based vector/DNA complexes for gene delivery:biophysical characteristics and transfection ability[J].Pharra Res.1998,15:1332～1339.
5 Brooks PC,Clark RA,Cheresh DA.Requirement of vascular integrin alpha v beta 3 for angiogenesis[J].Science.1994 Apr 22;264(5158):569-71.
6 Haubner R,Bruchertseifer F,Bockl M,et al.Synthesis and bio-logical evaluation of a 99mTc-labelled cyclic RGD peptide for imaging the a vβ 3 expression [J].J Nucl Med,2004,43(1):26-32.
7 Kyung-Han Lee,Kyoung-Ho Jung,Sung-Hee Song.Radiolabeled RGD Uptake and alpha v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs[J].J Nucl Med 2005;46:472～478.
8 Eric T.Choi,M.Faisal Khan,et al.beta 3-Integrin Mediates Smooth Muscle Cell Accumulation in Neointima After Carotid Ligation in Mice [J].2004;109:1564-1569.
9 Jiasheng Zhang,Svetlana Krassilnikova,Amir A.et al.α v β 3-Targeted detection of arteriopathy in transplanted human coronary arteries:an autoradiographic study[J].FASEB J.2005 Nov;19(13):1857-9.
10 Kyung-Han Lee,Kyoung-Ho Jung,Sung-Hee Song.Radiolabeled RGD Uptake and alpha v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs.J Nucl Med 2005;46:472～478.
11 Tigli RS,GtimUsderelioglu M.Evaluation of RGD-or EGF-immobilized chitosan scaffolds for chondrogenic activity.Int J Biol Macromol.2008 Aug 15;43(2):121-8.Epub 2008 Apr 11.
12 Mao HQ,Roy K,Troung-Le VL,et al.Chitosan-DNA nanoparticles as gene carriers:synthesis,characterization and transfection efficiency[J].J Controlled Release,2001,70(3):399- 421.
13 张海玲,朱敦皖,董霞.壳聚糖载基因纳米粒子荧光标记的优化研究[J].生物医学工程与临床,2008,(1)12.
14 R J Mumper,J J Wang,J M Claspell et al.Proc.Int Symp.Control [J].Release Bioact.Mater,1995,122:178-179.
15 Mizeje W,Gerald J.Role of integrins in cancer:survey of ex-pression patterns[J].Pro Soc Exp Biol Med,1999,222(2):124.
16 Unser M,Aldroubi A,Eden M.The L2 Polynomial Spline Pyramid[J].IEEE Trans Pattern Analysis and machine intelligence,1993,15(4):364 - 379.
17 Unser M.Spline:A Perfect Fit for Signal and Image Processing[J].IEEE Signal Processing,1999,16(6):22 - 38.
18 Kai Temming,Raymond M.Schiffelers,et al.RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature[J].Drug Resistance Updates 8(2005) 381-402.
19 Patrick M.Winter.Anne M.et al.Endothelial alpha(v)beta3integrin-targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis[J].Arterioscler Thromb Vasc Biol.2006Sep;26(9):2103-9.
20 PierschbacherM D,Ruoslahti E.Cell Attachment Activity of Fibronectin Can Be Dup licated by Small Synthetic Fragments of theMolecule[J].Nature,1984,309:30- 33.
21 CHEN Yi-xin,XU Xue- ming,HONG Shui-gen,et al.RGD-Tachyp lesin Inhibits Tumor Gr owth[J].Cancer Res,2001,61:2434- 2438.
22 Buckley C D,Pilling D,Henriquez N V,et al.RGD Peptides Induce Apoptosis by Direct Cas pase Activation[J].Nature,1999,397(6719):534-539.
23 Lebaron RG,Athanasiou KA.Extracellular matrix cell adhesion peptides:functional applications in orthopedic materials[J].Tissue Eng,2000,6:85 - 103.
24 Zamir E,Geiger B.Molecular complexity and dynamics of cell-matrix adhesions[J].J Cell Sci,2001,114:3583 - 3590.
25 Geiger B,Bershadsky A.Assembly and mechanosensory function of focal contacts[J].Curr Opin Cell Biol,2001,13:584 - 592.
26 Petit V,Thiery JP.Focal adhesions:structure and dynamics[J].Biol Cell,2000,92:477 - 494.
27 Van der Flier A,Sonnenberg A.Function and interactions of integrins [J].Cell Tissue Res,2001,305:285 - 298.
28 Leavesley DI,Schwartz MA,Rosenfeld M et al.Integrin β 1 and β 3mediated endothelial cell migration is triggered through distinct signaling mechanisms.J Cell Biol 121:163?170.
29 Polrorak Z,Cohen T,Sivan R,et al.VEGF145,a secreted VEGF isform that binds to extracellular matrix[J].J Biol Chem,1997,272:7151.
30 Senger DR.Molecular framework for angiogenesis:a complex web of interactions between extravasated plasma proteins and endothelial cell proteins induced by angiogenic cytokines[J].Am J Pathol 1996,149:17.
31 Spyridopoulos I,Brogi E,Kearney M,et al.Vascular endothelial growth factor inhibits endothelial cell apoptosis induced by tumor necrosis factor- α:balance between growth and death signals[J].J Mol Cell Cardil 1997,29:1321-1330.
32 Stromblad S,Becher JC,Yebra M,et al.Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin α v β 3 during angiogenesis[J].J Clin Invest 1996,98:426?433.
33 Takeo Abumiya,Jacinta Lucero,Ji Hoe Heo,et al.Activated Microvessels Express Vascular Endothelial Growth Factor and Integrin V3 During Focal Cerebral Ischemia[J].Journal of Cerebral Blood Flow & Metabolism,1999,19,1038-1050.
34 Jones MC,Caswell PT,Moran-Jones K,et al.VEGFR1(Flt1) Regulates Rab4Recycling to Control Fibronectin Polymerization and Endothelial Vessel Branching[J].Traffic 2009 Mar 17.[Epub ahead of print].
35 He Y,Jiang Y,Wang J,et al.Prevalence of peripheral arterial disease and its association with smoking in a population-based study in Beijing,China[J].J Vasc Surg.Aug 2006;44(2):333-338.
36 管珩,刘志明,李光伟等.50岁以上糖尿病人群周围动脉闭塞性疾病相关因素分析[J],中华医学杂志2007;87(1):23-27.
37 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
38 Hitlock PR,Hackett NR,Leopold PL,et al.Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs[J].Mol Ther 2004,9:67-75.
39 Ohara N,Koyama H,Miyata T,et al.Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia[J].Gene Therapy 2001,8:837-845.
40 Ishii S,Koyama H,Miyata T,et al.Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia[J].J Vase Surg 2004,39:629-638.
41 KondohK,Koyama H,Miyata T,et al.Conduct ion performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor[J].Cardiovasc Res 2004,61:132-142.
42 Vincenti V,Cassano C,Roechi M,et al.Assignment of the vascular endothelial growth factor gene to human chromosome 6p21,3[J].Circulation,1996,93:1493.
43 Messina LM,Brevetti LS,Chang DS,et al.Therapeutic angiogenesis for critical limb ischaemia:invited commentary[J].J Control Release,2002,78:285.
44 Mustonen T,Alitalo K.Endothelial receptor tyrosine kinases involved in angiogenesis[J].J Cell Biol,1995,129:895-898.
45 Watanabe Y,Lee SW,Detmar M,et al.Vascular permeability factor/wascular endothelial growth factor(VPF/VEGF)delays and induces escape from senescence in human dermal microvascular endothelial cells [J].Oncogene,1997,14:2025-2032.
46 Conffinhal T,Silver M,Keaey M,et al.Impaired collateral vessel deveopment associated with reduced expression of vascular endothelial growth factor in ApoE mice[J].Circulation,1999,99:3188.
47 Schultz A,Lavie L,Hochebery I,et al.Iner individual heterogenety in the hypoxic regulation of VEGF significance for the development of the coronary artery collateral circulation[J].Circulation,1999,100:547.
48 Rajagopalan S,Mohler ER,3rd,Lederman RJ,et al.Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease:a phase Ⅱ randomized,double-blind,controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication[J].Circulation,2003,108:1933-1938.
49 Henry TD,Annex BH,McKendall GR,et aL.The VIVA trial:Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis[J].Circulation,2003,107:1359-1365.
50 Jeon,S.W.Kang,H.W.Lim,D.et al,Synergistic effect of sustained delivery of basic fibroblast growth factor and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs[J].Biomaterials 27 (2006),pp.1617-1625.
51 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
52 Sharif F,Daly K,Crowley J,et al.Current status of catheter-and stent-based gene therapy[J].Cardiovasc Res 2004,64:208-216.
1 He Y,Jiang Y,Wang J,et al.Prevalence of peripheral arterial disease and its association with smoking in a population-based study in Beijing,China[J].J Vasc Surg.Aug 2006;44(2):333-338.
2 管珩,刘志明,李光伟等,50岁以上糖尿病人群周围动脉闭塞性疾病相关因素分析[J],中华医学杂志2007:87(1):23-27.
3 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
4 Fishbein I,Alferiev IS,Nyanguile 0,et al.Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents[J].Proc Natl Acad Sci USA 2006,103:159-164.
5 Rosengart TK,Lee LY,Patel SR,et al.Angiogenesis gene therapy:phase Ⅰ assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease[J].Circulation 1999,100:468-474.
6 Rome JJ,Ahayani V,Flugelman MY,et al.Anatomic barriers influence the distribution of in vivo gene transfer into the arterial wall,Modeling with microscopic tracer particles and verification with a recombinant adenoviral vector[J].Arteriascler Thromb,1994,14:148-161.
7 彭应旭,廖工铁。骨髓靶向给药制剂研究进展[J]。中国药学杂志,2000,35(2):73-75.
8 Alexiou C.Magnetic mitoxantrone nanoparticle detection by histology,X-ray and MRI after magnetic tumor targeting.Journal of Magnetism and Magnetic Materials[J],2001,225:187-193.
9 王斌。靶向抗肿瘤药物载体系统研究近况[J]。广东药学院学报,1998,14(4):311-314.
10 Goodwin S,Peterson C,Hoh C,et al.Targeting and retention of magnetic targeted carriers(MTCs) enhancing intra-arterial chemotherapy[J].Journal of Magnetism and Magnetic Materials,1999,194:132-139.
11 Lobenberg R,Araujo L,Kreuter J.Body distribution of azidothymidine bound to nanoparticles after oral administration[J].Eur J pharm Biopharm,1997,44:127-132.
12 Lobenberg,R.Body distribution of azidothymidine bound to hexyl-cyanoacrylate nanoparticles after i.v.injection to rats[J].Journal of Controlled Reseased,1998,50:21-30.
13 陆彬。 药物新剂型与新技术[M]。北京:人民卫生出版社,1998.
14 Oliveira M C D.Delivery of antisense oligonucleotides by means of pH-sensitive liposomes[J].Journal of Controlled Release,1997,48:179-184.
15 Torchilin VP.Drug targeting.Eur J Pharm Sci,2000,11 Suppl 2:81-91.
16 Davis S S.Biomedical application of nanotechnology-implications for drug targeting and gene therapy[J].Tibtech,1997,15:217-224.
17 Balram Bhargava,Krishna Reddy,Ganesan Karthikeyan,et al.A Novel Paclitaxel-Eluting Porous Carbon-Carbon Nanoparticle Coated,Nonpolymeric Cobalt-Chromium Stent:Evaluation in a Porcine Model[J].Catheterization and Cardiovascular Interventions 2006,67:698-702.
18 Whitlow PL,Bass TA,Kipperman RM,et al.Results of the study to determine rotablator and transluminal angioplasty strategy[J].Am J Cardiol,2001,87(6):699-705.
19 Min Huang,Samadhi N,Vitharana,et al.Polyelectrolyte Complexes Stabilize and Controllably Release Vascular Endothelial Growth Factor[J].Biomacromolecules,2007,8(5),1607-1614.
20 Guzman L,Labhasetwar V,Song C,et al.Local intraluminal infusion of biodegradable polymeric nanoparticles.A novel approach for prolonged drug delivery after balloon angioplasty[J].Circulation,1996,94:1441-1448.
21 Labhasetwar V,Song CX,Levy RJ,et al.Nanoparticle drug delivery system for restenosis[J].Advanced Drug Delivery Reviews,1997,24:63-85.
22 Moon HT,Lee YK,Han JK,et al.A novel formulation for controlled release of heparin-DOCA conjugate dispersed as nanoparticles in polyurethane film[J].Biomaterials,2001,22(3):281-289.
23 Tillmann Cyrus,Huiying Zhang,John S.Allen,Intramural Delivery of Rapamycin With avP 3-Targeted Paramagnetic Nanoparticles Inhibits Stenosis After Balloon Injury[J].Arterioscler Thromb Vase Biol 2008:28:820-826.
24 李拥军,管珩,徐意瑶等.纳米粒子载体在防治血管损伤后再狭窄中的应用[J].基础医学与临床,2002,22(2).
25 李拥军,管珩,刘昌伟等.反义单核细胞趋化蛋白-1基因移植静脉内膜增殖的实验研究[J].中华外科杂志,2001,39,9.
26 徐意瑶,李拥军,管珩等.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[J].中华外科杂志,2004,42(1).
27 Sun-Woong Kang,Hee-Won Lim,Sang-Woo Seo,et al.Nanosphere-mediated delivery of vascular endothelial growth factor gene for therapeutic angiogenesis in mouse ischemic limbs[J].Biomaterials 2008,29(8):1109-1117.
28 Hongchi Jiang,Tiemin zhang,Xueying Sun,et al.Vascular Endothelial Growth Factor Gene Deliver by Magnetic DNA Nanospheres Ameliorates Limb Ischemia in Rabbits[J].Journal of Surgical Research 2005,126,48-54.
29 Kyung-Han Lee,Kyoung-Ho Jung,Sung-Hee Song.Radiolabeled RGD Uptake and alpha v Integrin Expression Is Enhanced in Ischemic Murine Hindlimbs[J].J Nucl Med 2005;46:472-478.
30 唐锋,管珩,李拥军等.RGD肽连接壳聚糖载基因纳米粒子的体外试验研究[J].国际生物医学工程杂志,2009,39.
1 He Y,Jiang Y,Wang J,et al.Prevalence of peripheral arterial disease and its association with smoking in a population-based study in Beijing,China[J].J Vasc Surg.Aug 2006;44(2):333-338.
2 管珩,刘志明李光伟等,50岁以上糖尿病人群周围动脉闭塞性疾病相关因素分析[J],中华医学杂志2007:87(1):23-27.
3 Baker AH.Designing gene delivery vectors for cardiovascular gene therapy[J].Prog Biophys Mol Biol 2004,84:279-299.
4 Burns JC,Friedraann T,Driever W et al.Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors:concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells[J].Proc Natl Acad Sci USA 90:1993,8033-8037.
5 Chen S,Kapturczak M,Loiler SA,et al.Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus sero-type 1 and 5 vectors[J].Hum Gene Ther 2005,16:235-247.
6 Conklin LD,McAninch RE,Schulz D,et al.HIV-based vectors and angiogenesis following rabbit hindlimb ischemia[J].J Surg Res 2005,123:55-66.
7 Denby L,Nicklin SA,Baker AH.Adeno-associated virus(AAV)-7 and -8poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation[J].Gene Therapy 2005,12:1534-1538.
8 Mao HQ,Roy K,Troung- Le VL,et al.Chitosan- DNA nanoparticles as gene carriers:synthesis,characterization and transfection efficiency[J].J Controlled Release,2001,70(3):399-421.
9 Putnam D.Polymers for gene delivery across length scales[J].Nat Mater 2006,5:439-451.
10 Hedman M,Hartikainen J,Syvanne M,et al.Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia:phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT)[J].Circulation 2003,107:2677-2683.
11 Sharif F,Daly K,Crowley J,et al.Current status of catheter-and stent-based gene therapy[J].Cardiovasc Res 2004,64:208-216.
12 Wang K,Kessler PD,Zhou Z,et al.Local adenoviral-mediated inducible nitric oxide synthase gene transfer inhibits neointimal formation in the porcine coronary stented model[J].Mol Ther 2003,7:597-603.
13 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease[J].Hypertension 2004,44:203-209.
14 Koren B,Weisz A,Fischer L,Gluzman Z,et al.Efficient transduction and seeding of human endothelial cells onto metallic stents using bicistronic pseudo-typed retroviral vectors encoding vascular endothelial growth factor[J].Cardiovasc Revasc Med 2006,7:173-178.
15 Fishbein I,Alferiev IS,Nyanguile 0,et al.Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents[J].Proc Natl Acad Sci USA 2006,103:159-164.
16 Sharif F,Hynes SO,McMahon J,et al.Gene-eluting stents:comparison of adenoviral and adeno associated viral gene delivery to the blood vessel wall in vivo [J].Hum Gene Ther 2006,17:741-750.
17 Klugherz BD,Song C,DeFelice S,et al.Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus [J].Hum Gene Ther 2002,13:443-454.
18 Rome JJ,Shayani V,Newman KD,et al.Adenoviral vector-mediated gene transfer into sheep arteries using a double-balloon catheter[J].Hum Gene Ther 1994,5:1249-1258.
19 FolkmanJ,ShingY.Angiogenesis [J].J Biol Chem 1992,267:10931-10934.
20 Whitlock PR,Hackett NR,Leopold PL,et al.Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs[J].Mol Ther 2004,9:67-75.
21 Ohara N,Koyama H,Miyata T,et al.Adenovirus-mediated ex vivo gene transfer of basic fibroblast growth factor promotes collateral development in a rabbit model of hind limb ischemia[J].Gene Therapy 2001,8:837-845.
22 Ishii S,Koyama H,Miyata T,et al.Appropriate control of ex vivo gene therapy delivering basic fibroblast growth factor promotes successful and safe development of collateral vessels in rabbit model of hind limb ischemia[J].J Vase Surg 2004,39:629-638.
23 Kondoh K,Koyama H,Miyata T,et al.Conduction performance of collateral vessels induced by vascular endothelial growth factor or basic fibroblast growth factor[J].Cardiovasc Res 2004,61:132-142.
24 Kusumanto YH,vanWeel V,Mulder NH,et al.Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia:a double-blind randomized trial[J].Hum Gene Ther 2006,17:683-691.
25 Rajagopalan S,Shah M,Luciano A,et al.Adenovirus-mediated gene transfer of VEGF(121)improves lower-extremity endothelial function and flow reserved].Circulation 2001,104:753-755.
26 Kim HJ,Jang SY,Park JI,et al.Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery diseased].Exp Mol Med 2004,36:336-344.
27 Isner JM,Walsh K,Symes J,et al.Arterial gene transfer for therapeutic angiogenesis in patients with peripheral artery disease[J].Hum Gene Ther 1996,7:959-988.
28 Baumgartner I,Pieczek A,Manor 0,et al.Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemiad]-Circulation 1998,97:1114-1123.
29 Morishita R,Aoki M,Hashiya N,et al.Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial diseased]-Hypertension 2004,44:203-209.
30 Rajagopalan S,Mohler Ⅲ ER,Lederman RJ,et al.Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease:a phase Ⅱ randomized,double-blind,controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication[J].Circulation 2003,108: 1933-1938.
31 Kusumanto YH,vanWeel V,Mulder NH,et al.Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia:a double-blind randomized trial[J].Hum Gene Ther 2006,17:683-691.
32 Epstein AL.HSV-1-based amplicon vectors:design and applications [J].Gene Therapy 2005,12 (Suppl 1):S154-S158.
33 Baker AH.Gene therapy for bypass graft failure and restenosis[J].Pathophysiol Haemost Thromb 2002,32:389-391.
34 Akowuah EF,Gray C,Lawrie A,et al.Ultrasound-mediated delivery of TIMP-3 plasmidDNA into saphenous vein leads to increased lumen size in a porcine inteposition graft model [J].Gene Therapy 2005,12:1154—1157.
35 West NE,Qian H,Guzik TJ,et al.Nitric oxide synthase (nNOS)gene transfer modifies venous bypass graft remodeling:effects on vascular smooth muscle cell differentiation and superoxide production[J].Circulation 2001,104:1526-1532.
36 Wan S,George SJ,Nicklin SA,et al.Over-expression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts[J].Mol Ther 2004,9:689-698.
37 George SJ,Angelini GD,Capogrossi MC,et al.Wild-type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis [J].Gene Therapy 2001,8:668-676.
38 Wan S,George SJ,Nicklin SA,Yim AP,Baker AHet al.Over-expression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts[J].Mol Ther 2004,9:689-698.
39 West NE,Qian H,Guzik TJ,et al.Nitric oxide synthase (nNOS)gene transfer modifies venous bypass graft remodeling:effects on vascular smooth muscle cell differentiation and superoxide production [J].Circulation 2001,104:1526-1532.
40 Wolff RA,Ryomoto M,Stark VE,et al.Antisense to transforming growth factor-betal messenger RNA reduces vein graft intimal hyperplasia and monocyte chemotactic protein 1[J].J Vase Surg 2005,41:498-508.
41 Akowuah EF,Gray C,Lawrie A,et al.Ultrasound-mediated delivery of TIMP-3 plasmidDNA into saphenous vein leads to increased lumen size in a porcine inteposition graft model [J].Gene Therapy 2005,12:1154-1157.
42 Grube E,PRoject of Ex-vivo Vein graft ENgineering via Transfection (PREVENT)Ⅱ trial[J].Presented at the annual Amercian HeartAssociation Scientific Sessions,November 11-14,2001,Anahe im,Cali forn i a,2001.
43 Mann MJ,Whittemore AD,Donaldson MC,et al.Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy:the PREVENT single-centre,randomised,controlled trial[J].Lancet 1999,354:1493-1498.
44 Alexander JH,Ferguson Jr TB,Joseph DM,et al.The PRoject of Ex-vivo Vein graft ENgineering via Transfection IV (PREVENT IV)trial:study rationale,design,and baseline patient characteristics[J].Am Heart J 2005,150:643-649.
45 ConteMS,Lorenz TJ,BandykDF,et al.Design and rationale of the PREVENT Ⅲ clinical trial:edifoligide for the prevention of infrainguinal vein graft failure[J].Vase Endovascular Surg 2005,39:15-23.
46 Burt HM,Hunter WL.Drug-eluting stents:a multidisciplinary success story[J].Adv Drug Deliv Rev 2006,58:350-357.
47 Joner M,Finn AV,Farb A,et al.Pathology of drug-eluting stents in humans:delayed healing and late thrombotic risk[J].J Am Coll Cardiol 2006,48:193-202.
48 Laitinen M,Hartikainen J,Hiltunen MO,et al.Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty[J].Hum Gene Ther 2000,11:263-270.
49 Hedman M,Hartikainen J,Syvanne M,et al.Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia:phase Ⅱ results of the Kuopio Angiogenesis Trial (KAT)[J],Circulation 2003,107:2677-2683.
50 Johnson TW,Wu YX,Herdeg C,et al.Stent-based delivery of tissue inhibitor of metallo-proteinase-3 adenovirus inhibits neointimal formation in porcine coronary arteries[J].Arterioscler Thromb Vase Biol 2005,25:754-759.
51 Fishbein I,Alferiev IS,Nyanguile 0,et al.Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents[J].Proc Natl Acad Sci USA 2006,103:159-164.
52 Rosengart TK,Lee LY,Patel SR,et al.Six-month assessment of a phase I trial of angiogenic gene therapy for the treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA[J].Ann Surg 1999,230:466-470;discussion 470-462.
53 Rosengart TK,Lee LY,Patel SR,et al.Angiogenesis gene therapy:phase I assessment of direct intramyocardial administration of an adenovirus vector expressing VEGF121 cDNA to individuals with clinically significant severe coronary artery disease [J].Circulation 1999,100:468-474.
54 Simari RD,O'Brien T.Clinical trials in vascular gene therapy:the missing piece of the puzzle [J].Arch Pathol Lab Med 2002,126:317-319.
55 Yla-Herttuala S,Markkanen JE,Rissanen TT.Gene therapy for ischemic cardiovascular diseases:some lessons learned from the first clinical trials[J].Trends Cardiovasc Med 2004,14:295-300.