【作者】 欧阳德群；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 近年来,随着基因转移技术的日趋成熟,肝癌的基因治疗成为生物科学和临床医学的研究热点之一。基因治疗的载体,以及载体相关的免疫反应、细胞毒性与安全性问题是限制基因治疗发展和临床应用的瓶颈。目前基因治疗的研究和临床应用中常用的病毒载体和非病毒载体都存在无法克服的局限性。纳米粒基因转运载体是近年发展起来的一种新型的非病毒基因转运载体。它是将DNA、RNA等基因治疗分子包裹在纳米颗粒之中或吸附在其表面,同时也在颗粒表面耦联特异性的靶向分子,如特异性配体、单克隆抗体等,通过靶向分子与细胞表面特异性受体结合,在细胞摄取作用下进入胞内,实现安全有效的靶向性基因治疗。纳米粒材料的选择是成功进行纳米基因转运和治疗的关键。所选择的材料必须具有生物适应性和可降解型的,或者易于从体内排泄,而不产生有害的降解产物,且无免疫原性的物质,不会引起机体的免疫排斥反应。高分子生物降解材料制备的纳米颗粒具有稳定、无毒、无抗原性、生物相容性好、对所转运基因的表达有控释作用及对基因有保护作用等优点,是良好的纳米基因转运载体材料。研究证实,纳米粒与DNA的连接多通过静电吸引作用来完成。DNA的磷酸骨架所带的负电荷只能与表面带正电性的载体结合。因此,需要利用生物活性分子对纳米颗粒的表面进行改性,使其表面携带阳离子物质,以起到防止纳米颗粒团聚,并有利于结合DNA分子的作用。本研究中结果显示,以氨基硅烷化Fe₃O₄纳米颗粒作为基因转运载体所形成的氨基硅烷化Fe₃O₄纳米粒/DNA复合物,能保护所携带的DNA免受核酸酶的降解和超声剪切作用。同时,本研究发现,在较低浓度下Fe₃O₄纳米颗粒与氨基硅烷化Fe₃O₄纳米颗粒对肝癌（HepG2）细胞无明显的细胞毒性,只在高浓度下才会表现出一定的细胞毒性作用。在体外基因转运中,该纳米颗粒可有效转运pEGFP-C1报道基因表达质粒进入HepG2细胞,其转运效率为39%,略高于相同条件下脂质体的转染效率,但无统计学差异（*P＞0.05,N=10）。在外加磁场的条件下氨基硅烷化Fe₃O₄磁性纳米粒的转染效率明显增强。在此基础之上,本研究以氨基硅烷化Fe₃O₄磁性纳米粒作为基因转运载体,以重组人真核细胞表达质粒pcDNA3.1（+）-p53作为治疗基因,对HepG2细胞的荷瘤小鼠模型进行以氨基硅烷化Fe₃O₄磁性纳米粒作为基因转运载体在体内转运重组人真核细胞表达质粒pcDNA3.1（+）-p53的转染,利用RT-PCR、Western blot等方法检测HepG2细胞中转染入的外源野生型p53的表达情况,结果发现,在HepG2细胞中成功导入了外源野生型p53基因,并能表达p53蛋白。本实验结合局部热疗研究基因治疗与局部热疗的协同效应及对荷瘤小鼠的肿瘤抑制情况,设计了对照组、局部热疗组、氨基硅烷化Fe₃O₄纳米粒作转导pcDNA3.1（+）-p53基因治疗组和联合治疗组4个实验组,结果显示,热疗和野生型p53基因治疗都能抑制荷肝癌裸鼠转移瘤的生长。热疗和野生型p53基因联合应用可达到较好的协同作用,比单一的热疗或基因治疗能更好的抑制肝癌转移瘤的生长。联合治疗组、基因治疗组、热疗治疗组的抑瘤率分别为84.07%,59.43%,47.19%。更多还原

【Abstract】 Recently,the methods of gene delivery are gradually developing and have made great progresses.So the gene therapy of HCC has been as a hot spot on the biology and clinical medicine field.The gene delivery vectors and vectors associated immune response, cell toxicity and safety are the bottleneck of research and clinic application of gene therapy.The traditional gene delivery vectors including viral vectors and non-viral vectors have some disadvantages that are hard to overcome.Nanoparticle（NP） as a new non-viral gene delivery vector is recently developed.Gene therapeutical molecules including DNA,RNA,etc,which are encapsulated into or loaded on the surface of nanoparticles.While specific monoclonal antibodies of the target cell are linked on the surface of nanoparticle.The nanoparticles/DNA complexes can be taken into target cells by receptor-mediated endocytosis resulting in an effective target gene therapy.Selecting proper materials is the key point for nanoparticle-mediated gene delivery and gene therapy.The nanoparticle materials must be very good to biodegradability or easy to be discharged,and to have non-immunogenic.The nanoparticles made by biodegradable polymer have some advantages,such as stability,non-toxicity,good biocompatibility and the loaded DNA delivery can be controlled and protected from degradation.It is proved that the binding between nanoparticles and DNA was accomplished by electrostatic attraction.The negatively charge of phosphate backbone of DNA could be binded to the positively charged of vector.Therefore,modification of the nanoparticles surface properties with biomolecules makes the nanoparticles surface carring possitve charge which can protect the condensotion of NPs,it is avaliable to the carried gene delivery.In this study,we used functional Fe₃O₄ nanoparticles modified by APTES as a gene delivery vector,and found that functional Fe₃O₄ nanoparticles -DNA complexes could protect the DNA from degradation of DNaseI and clip of ultrasound.While we found that Fe₃O₄ nanoparticles had no obvious cell toxicity to HepG2 at lower concentration,but showed cell toxicity at high concentration.The pEGEP-C1 expression plasmid could be transfected effectively into HepG2 cells by APTES-modified Fe₃O₄ nanoparticles in vitro.The transfection rate was 39%,which was slightly higher than that of liposome（P＞0.05）,but the transfecation rate could be increased by addition of extra-magnetic field.Using functional Fe₃O₄ NPs as gene delivery vector and eucryotic expression plasmid pEGEP-C1 as therapeutical gene were performed in this study,and the expression of wt-p53 transfected in HepG2 cells was measured by RT-PCR and Western blot.The results showed that transfection of exogenous wild-type p53 gene to HepG2 cells was successful and could be expression of p53 protein.In this experiment,the nude rats were randomly diveded into 4 groups:control group,local thermotherapy group,amino-functional of Fe₃O₄ nanoparticles for transduction pcDNA3.1（+）-p53 gene（functional gene） therapy group,and combination of thermotherapy and group functional gene therapy.The results showed that thermotherapy and functional gene therapy can inhibit the growth of metastatic tumors in nude mice.However the effect of combined therapy was better than that of the thermotherapy or functional gene therapy alone.The inhibiting rate of the tumor growth in combined therapy group,functional gene therapy group and thermotherapy group was 84.07%,59.43%and 47.19%, respectively.更多还原