【作者】 唐锋；

【导师】 管珩； 刘昌伟； 李拥军； 郑月宏；

【作者基本信息】 中国协和医科大学 ， 外科学， 2009， 博士

【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pIRES₂-VEGF₁₆₅质粒转染Hy926细胞,分为3组:CH-RGD-pVEGF₁₆₅组、CH-pVEGF₁₆₅组和Lipofetamine2000-pVEGF₁₆₅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-pVEGF₁₆₅治疗组、CH-pVEGF₁₆₅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-pVEGF₁₆₅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VEGF₁₆₅质粒体外转染Hy926细胞实验中,CH-RGD-VEGF₁₆₅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-VEGF₁₆₅转染组与Lipo-pVEGF₁₆₅组比较p>0.05外,其余p值均<0.01。)CH-RGD-pVEGF₁₆₅组及CH-pVEGF₁₆₅组于转染后1、3、7、14天存活细胞总数均较Lipo-pVEGF₁₆₅组有显著性差异（p值均<0.01）,而CH-RGD-pVEGF₁₆₅组和CH-pVEGF₁₆₅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-pVEGF₁₆₅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-pVEGF₁₆₅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-pVEGF₁₆₅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-pVEGF₁₆₅治疗组和CH-pVEGF₁₆₅治疗组未见患肢坏死、感染,组织水肿不明显。而pVEGF₁₆₅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VEGF₁₆₅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pVEGF₁₆₅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。更多还原

【Abstract】 Background and ObjectiveGene 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_vβ₃,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.Methods1.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 pVEGF₁₆₅,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.Results1.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.Conclusion1.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.更多还原