【作者】 王浩；

【导师】 王静云； 陈麒先；

【作者基本信息】 大连理工大学 ， 生物工程（专业学位）， 2021， 硕士

【摘要】 中心法则提出之后,人们认识到疾病发生的根源是基因发生有害缺陷或突变,基因治疗应运而生。但由于基因本身易被降解的缺陷,需要载体来负载基因进行治疗。病毒载体虽然具有极高的基因转染效率,但其免疫原性致命,且外源基因可能会永久整合到宿主细胞染色体中,存在潜在的诱癌风险,近年来,随着纳米医学技术的发展,非病毒载体由于便于功能化修饰在递送基因过程中取得理想的效果,成为目前研究的热点。本研究针对非病毒载体在递送基因过程中所面临的多级生理屏障,设计合成了一种具有多级响应性的基因递送系统。以聚赖氨酸(PLys)为核心,二硫键(S-S)交联结合基因,温敏中间层聚丙基丙烯酰胺(PNIPAM)保护基因,聚乙二醇(PEG)外壳延长血液循环时间,主动靶向(RGD)增加肿瘤富集,基质金属蛋白酶2(MMP-2)响应短肽(GPLGVRG)去PEG化增加内吞以及胞内运输效率。本研究为基因治疗提供了一种可行性策略,具体研究内容如下:1、阳离子载体的合成与表征通过酰胺反应、点击反应和开环聚合反应等合成方法合成了多个基因递送载体PEG-PLys-SH、PEG-GPLGVRG-PLys-SH、RGD-PEG-GPLGVRG-PLys-SH、PNIPAM-PLys-SH.2、载体/基因复合物的制备及理化性质通过琼脂糖凝胶电泳实验和肝素竞争实验验证了载体结合基因的能力及稳定性;动态光散射(DLS)观察复合物的粒径及电位,各载体/基因复合物粒径80-90 nm,电位3-5 m V,接近电中性;MTT细胞毒性实验证实了细胞存活率高,载体生物相容性好。3、载体及载体/基因复合物各级微环境响应性及基因转染通过琼脂糖凝胶电泳实验和肝素竞争实验验证了复合物胞内还原环境响应性;核酸酶保护、DLS、透射电子显微镜(TEM)、核磁共振氢谱(~1H NMR)验证温敏复合物温度响应性,温度由室温升高至生理温度,中间屏障由亲水变为疏水;激光共聚焦显微镜(CLSM)、流式细胞仪(FCM)验证主动靶向性,加入RGD细胞摄取效率增加约3倍;凝胶渗透色谱(GPC)、DLS、CLSM、FCM、乳酸脱氢酶(LDH)活性实验验证MMP响应去PEG化性能,去PEG化之后载体分子量有明显变化,复合物电位升高,细胞摄取能力增加约6倍,溶酶体逃逸速度加快;倒置荧光显微镜观察了复合物的基因转染能力,加入主动靶向RGD以及去PEG化策略都提升了复合物的基因转染能力。4、温敏载体/基因复合物联合索拉菲尼体内抑癌能力通过荷瘤小鼠体内抑癌实验,证明相较于单基因和药物治疗组,联合治疗组肿瘤体积增长最为缓慢,抑癌效果最为明显;苏木精-伊红(HE)染色结果表明所有治疗组均对小鼠主要器官没有明显的毒副作用;血管内皮生长因子(VEGF)免疫组化验证了治疗基因通过抑制VEGF表达达到抗肿瘤效果。更多还原

【Abstract】 After the central principle was put forward,it is recognized that the root cause of the disease is the harmful defect or mutation of the gene,and gene therapy came into being.However,due to the defect that the gene itself is easily degraded,a vector is needed to carry the gene for treatment.Although viral vectors have extremely high gene transfection efficiency,their immunogenicity is fatal,and foreign genes may be permanently integrated into the host cell chromosomes,and there is a potential risk of cancer induction.In recent years,with the development of nano medical technology,since non viral vectors are convenient for functional modification to achieve ideal results in the process of gene delivery,they have become a hot spot in current research.In this study,aiming at the multi-level physiological barriers faced by non-viral vectors in the process of gene delivery,a gene delivery system with multi-level responsiveness was designed and synthesized.Within the new gene delivery system,polylysine(PLys)is as the core,disulfide bond(S-S)cross-links and bind genes,temperature-sensitive intermediate layer polypropylacrylamide(PNIPAM)protects genes,polyethylene glycol(PEG)shell prolongs blood circulation time,active targeting(RGD)increases tumor enrichment,and matrix metalloproteinase 2(MMP-2)cleave short peptide(GPLGVRG)de PEGylation to increase endocytosis and intracellular transport efficiency.This study provides a feasible strategy for gene therapy,the research content is as follows:1.Synthesis and characterization of cationic carrierMultiple gene delivery vectors PEG-PLys-SH,PEG-GPLGVRG-PLys-SH,RGD-PEG-GPLGVRG-PLys-SH,PNIPAM-PLys-SH were synthesized by synthetic methods such as amide reaction,click reaction and ring-opening polymerization reaction.2.Preparation and physical and chemical properties of vector/gene complexThe agarose gel electrophoresis experiment and heparin competition experiment verified the ability and stability of the vector to bind genes;the particle size of each vector/gene complex was between 80-90 nm determined by dynamic light scattering(DLS),and the potential of the complexes was between 3-5 m V;MTT cytotoxicity experiments have confirmed that the cell survival rate is high,demonstrating that the carrier has good biocompatibility.3.Microenvironmental responsiveness of vectors and vector/gene complexes and gene transfection of vector/gene complexes at all levelsThe agarose gel electrophoresis experiment and heparin competition experiment verified the responsiveness of the vector/gene complexes to intracellular reduction environment;nuclease protection,DLS,transmission electron microscopy(TEM),proton nuclear magnetic resonance spectroscopy(~1H NMR)verified the temperature response of the temperature-sensitive complex.With the rise of temperature from room temperature to physiological temperature,the intermediate barrier changes from hydrophilic to hydrophobic;laser confocal microscopy(CLSM)and date from flow cytometry(FCM)demonstrated that the introduction of RGD,an active targeting group,-can increased the uptake efficiency of vector/gene complexes in cells by about 3 times;gel permeation chromatography(GPC),DLS,CLSM,FCM,lactate dehydrogenase(LDH)activity experiments displayed the performance of MMP in response to de PEGylation.After de PEGylation,the molecular weight of the carrier changes significantly,the potential of vector/gene complexes increases,the uptake capacity in cells was increased by about 6 times,and the lysosomal escape speed was accelerated;the gene transfection ability of the vector/gene complexes was observed by the inverted fluorescence microscope,and the introduction of active targeting RGD and de PEGylation strategies all improved the gene transfection ability of vector/gene complexes.4.Tumor suppression ability of temperature-sensitive vector/gene complex combined with sorafenib in vivoIn vivo tumor suppression experiments of tumor-bearing mice,it was proved that compared with the only gene or drug treatment groups,the tumor volume in the combined treatment group increased the most slowly and the tumor suppression effect was the most obvious.Hematoxylin-eosin(HE)staining showed that all the treatment groups had no obvious toxic and side effects on the main organs of mice.Immunohistochemistry of VEGF confirmed the antitumor effect of the therapeutic gene by inhibiting expression ofvascular endothelial growth factor(VEGF).更多还原