【作者】 钟慧；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 疾病基因组学， 2007， 博士

【摘要】 【纳米载体的研究现状】对疾病的预防和治疗来讲，基因治疗是一种有效和经济的方法。高效的基因转移和表达是基因治疗的关键技术。目前常用的基因载体包括病毒载体和非病毒载体，且临床试验中75％以上利用病毒为载体来转运目的基因。虽然病毒载体具有较高的转染效率，但同时也存在着很多难以克服的缺点，如具有免疫原性、细胞毒性、缺少组织特异性等等。因此对非病毒载体的研究受到人们的重视，而目前常用的非病毒载体，如多聚阳离子聚合物和脂质体，也存在着转染效率低，尤其在体内的转染效率低等缺点。因此寻找新的高效、安全、靶向的非病毒载体成为目前的研究热点。随着纳米技术的兴起和发展，在纳米尺度内的物质的纳米特性引起了人们的关注。纳米颗粒一般是指尺寸在1～100 nm间的粒子，表现出强烈的尺寸依赖性、表面效应、小尺寸效应和宏观量子隧道效应等特点，从而使其出现了许多不同于常规固体的新奇特性。由于纳米粒子表面原子数增多，表面原子配位数不足和高的表面能，使这些原子易与其它原子相结合而稳定下来，故方便在纳米颗粒的表面结合生物大分子，成为生物分子的高效载体。同时纳米颗粒的小粒径效应，使得它们可以有效的逃避脾滤过、延长血液循环时间、增强靶组织的累积，从而得以实现有效的基因转移。由于纳米颗粒作为基因转移载体具有如此众多的优越性，使其成为突破基因转移瓶颈最有希望的工具，是当今基因转移和纳米生物研究领域最热门的课题之一。目前研究较多的阳离子聚合物中发展最快的是树枝状聚合物(dendrimers)，其中又以聚酰胺-胺型树枝状聚合物(PAMAM dendrimers)研究的最为广泛。PAMAM作为基因载体具有很多优势，不会引起机体的免疫反应、无遗传毒性和细胞毒性、不会导致细胞的转化和细胞死亡、具有很高的基因转移效率、能介导外源基因在宿主细胞染色体DNA中的整合，从而获得转基因的长期、稳定表达，还可以保护转移基因不受机体血浆或组织细胞中各种补体以及各种酶的破坏，从而有利于目的基因在转移进入靶细胞后能更好、更稳定的发挥作用。因此PAMAM作为基因载体具有广阔的应用前景。【PAMAM纳米载体结合和保护DNA的能力】本研究项目通过体内外实验验证了PAMAM dendrimers作为基因载体的可行性，并将其应用于白血病的基因治疗研究。首先制备水溶性的PAMAM dendrimers纳米颗粒，原子力显微镜检测显示PAMAM直径约8nm±0.5nm左右，分布均匀。作为基因载体，与DNA的结合能力是必备条件之一，我们应用凝胶阻滞实验和DNA共沉淀实验来评价PAMAM的DNA结合能力。结果显示，该纳米颗粒在pH5～9时可以结合和保护DNA，但是在pH3时就丧失了这种能力。因此PAMAM具有在生理条件下(pH7.35～7.45)对DNA的结合和保护能力。Zeta电位测定说明PAMAM的DNA结合能力是基于一种电负性DNA磷酸骨架和PAMAM的正电性之间的静电结合。用MTT检测PAMAM、PAMAM／DNA复合物对不同细胞系COS7、SMF、K562和Jurkat细胞生长的影响，结果显示在质量比为2∶1～4∶1时形成的PAMAM／DNA复合物对细胞生长的影响很小，但质量比超过4∶1／时形成的复合物对细胞的生长有一定的影响。【PAMAM基因转运体系的体内外研究】以PAMAM为载体转染编码绿色荧光蛋白的报道基因pEGFP-C2到COS7细胞，直接在荧光显微镜下观察其转染效率，结果证实PAMAM可以有效的携带外源基因在细胞中分布并表达出目的蛋白。但是，荧光素酶分析结果显示PAMAM与目的DNA以不同比例结合时的转染效率不同，当PAMAM／DNA质量比为4∶1时，转染效率达到最高。扫描电子显微镜下直观观察细胞对PAMAM／DNA复合物的吞噬，结果显示细胞可以高效吞噬PAMAM及其复合体，并在细胞的胞浆和胞核中广泛分布，但在胞浆中的分布比胞核中要多。为观察携带外源质粒DNA的PAMAM在体内的分布状况，我们将PAMAM／EGFP-C2复合物从BALB／c小鼠尾静脉注射进入小鼠体内，在不同时间点处死小鼠，取其各组织器官用戊二醛固定，制备组织切片在扫描电子显微镜下观察PAMAM和PAMAM／DNA在体内的组织细胞分布。同时制备冰冻切片，直接在荧光显微镜下观察PAMAM携带DNA在体内的组织分布及表达。电镜检测显示，小鼠的肝、肾、肺、脾、胰、脑、淋巴结和骨髓中有较多的PAMAM及PAMAM／DNA复合物存在，而在肠、胃、心中分布较少或没有分布。PAMAM可以在小鼠的肾小管中分布，说明PAMAM可以通过肾脏排泄，不会在体内蓄积。在小鼠的淋巴结和骨髓中也有PAMAM的分布，说明PAMAM可以有效的到达造血器官。组织荧光检测结果显示，PAMAM／DNA复合物在小鼠的肝、肾、肺、脾中有很强的荧光表达，而在心、脑、胃、肠中未检测到明显的荧光信号。【PAMAM基因转运体系对白血病基因治疗的研究】PAMAM载体具备结合和保护DNA的能力、较高的转染效率、携带外源基因在体内组织的广泛分布和表达，为其作为基因载体进行基因治疗研究提供了实验基础。在肿瘤病理标本的获取、对治疗有直接意义的造血干／祖细胞的体外分离纯化、扩增以及骨髓移植等方面，白血病都有其在基因治疗领域中的独特优势，因而受到广大学者的重视，以期以白血病的基因治疗为突破口，开辟人类肿瘤治疗的新天地。p16基因是一个经典的抑癌基因，研究发现p16基因缺失在多种肿瘤的细胞株中存在，占已知肿瘤细胞株的30～50％，在白血病细胞株中占25～64％，说明p16基因异常与多种肿瘤有密切关系。LRRC4基因是我室克隆的基因，实验室前期工作发现LRRC4可能作为一个受体或受体激酶或粘附分子参与调控细胞因子或生长因子所介导的信号传导通路，参与神经系统的发育和肿瘤的形成。我们通过RT-PCR发现p16和LRRC4基因在K562细胞中的表达明显下调。为了明确其在体内对于引发慢性粒细胞性白血病的K562细胞是否具有治疗效果，我们构建了K562-SCID白血病模型，评价p16和LRRC4对于白血病基因治疗的效果，并为PAMAM作为基因载体用于体内实验提供了更加充分的实验依据。对SCID小鼠经尾静脉注射K562细胞的方式建立人慢性粒细胞性白血病细胞异体移植SCID小鼠白血病动物模型，以PAMAM为载体，携带目的治疗基因p16和LRRC4通过静脉注射治疗。结果表明PAMAM同时携带p16和LRRC4基因时能对白血病进行有效的治疗，降低外周血中原始粒细胞的比例；减轻白血病对肝、脾和骨髓的浸润转移；使白血病小鼠的存活率延长43％，其治疗效果明显。这些结果说明，PAMAM可以携带目的基因到达骨髓、肝脏、脾脏以及其他组织器官，并表达出相应的蛋白而发挥作用。LRRC4和p16基因的共同作用可以有效的抑制K562细胞在体内的增殖，从而减缓其诱导的慢性粒细胞性白血病的发生和发展。综上所述，PAMAM具有基因载体的必要条件，包括在生理条件下结合和保护DNA；能在体内外被细胞吞噬，并在细胞的胞浆和胞核分布。结合报道基因系统证实了PAMAM可以将外源基因携带至肝、肾、肺、脾，并且在上述组织中表达出目的蛋白。本研究结合纳米技术和分子生物学技术，创新性的应用PAMAM作为载体，评价了其携带p16和LRRC4抑癌基因对白血病动物模型的治疗效果，充分利用了纳米颗粒的优势，为血液系统疾病的基因治疗提供了良好的实验基础。更多还原

【Abstract】 【The research status of nanoparticles】Gene therapy is an efficient and economical approach to disease prevention and therapy. In order to succeed in gene therapy however, efficient delivery and expression of exogenous genes into target cells are of critical importance. To date, about three quarters of gene therapy trials undertaken are for cancer, mostly based on recombinant viral vectors to carry the gene of interest. Although recombinant viral vectors have high transfection efficiency, they are associated with immunogenicity, toxicity, lack of tissue specificity, unknown long-term effects et al. Non-viral vectors have become attractive alternatives, but most non-viral vectors are limited for their low efficiency of transfection, especially in vivo. The recent topic has been focused on new kinds of efficient, safe and targeting non-viral vectors.With the development of nanotechnology, recent interest has been focused on a new kind of non-viral vector, nanoparticles, which have the characteristics of surface effect, small size effect and macroscopic quantum tunneling effect. Nanoparticles have low cytotoxicity, delivering the interesting DNA to cells efficiently, attaching gene therapy molecular, such as DNA, ODN, PNA, et al., in their surface and are taken up by endocytosis, then nanoparticles are degraded by lysosome and the targeting genes are released and enter the nucleus to play a role of gene therapy. Among these nanoparticles, polyamidoamine (PAMAM) dendrimers have significant advantages over others. PAMAM dendrimers are new class of highly branched spherical polymers that are highly soluble in aqueous solution and have unique surface of positively charged primary amine groups, which make them form stable complexes with plasmid DNA, oligonucleotides, antibodies or drugs. The molecular weight of the dendrimer doubles, the number of surface amine groups exactly doubles. So PAMAM dendrimers are effective in gene transfer or applications in different areas of biology and medMne with high transfection efficiencies and minimal cytotoxicity.【The ability of PAMAM binding and protecting DNA】This study detailed some biological characteristics of G5 PAMAM dendfimers, their distribution in vivo and gene therapy of chronic myelogeneous leukemia. Atomic force microscopy indicated that the diameter of PAMAM is about 8 nm±0.5nm. As a gene vector, the DNA binding ability is essential requirement. DNA binding ability of PAMAM has been determined by observing whether DNA mobility was retarded on gel analysis and co-sedimentation assay. Results showed that from pH 5 to pH 9, PAMAM can bind DNA, but at pH 3, PAMAM couldn’t bind DNA, which means that PAMAM dendrimers could bind DNA and guarantee its integrity at a certain wide range ofpH values. The binding is achieved by interaction between the negatively charged DNA phosphate groups and positively charged gene vectors. DNase I digestion assay showed that PAMAM dendrimer/DNA complexes could protect DNA from digestion at different pH values and in serum. Whether PAMAM and PAMAM/DNA complexes influenced cell viability was investigated in several cell lines, including COS7, SMF, K562 and Jurkat cells through MTT assays, which showed that PAMAM/DNA complexes showed no obvious cytotoxicity from ratio of 2:1 to 4:1, but at ratio beyond 6:1, these complexes had some cytotoxicity.【Study of PAMAM and PAMAM/DNA complexes in vitro and in vivo】Besides the requirements for gene delivery mentioned above such as high affinity for nucleic acids and small size, the ability to cross the cell membrane is necessary too. In this study, PAMAM was shown to internalize and accumulate at the cytoplasm and nucleus of the treated cells by transmission electron microscopy. To test the ability of PAMAM to transfer gene in vitro, assays of delivery of reporter plasmid DNA into cells were performed, pEGFP-C2, encoding green fluorescent protein, reporter gene system was chosed to evaluate the efficiency of transfection. PAMAM transfer exogenous gene into COS7 cells. Maximal transfection and expression of GFP were obtained when the gram ratio of PAMAM to DNA was 4:1.To define the tissue distribution of PAMAM/DNA in vivo, tissue frozen section assay, western blotting and transmission electron microscopy ass were analyzed in many organs after the intravenous injection of PAMAM/DNA to BALB/c mice. PAMAM mediated reporter plasmid EGFP-C2 encoding green fluorescent protein in vivo was analyzed after intravenous injection of PAMAM/DNA complexes to BALB/c mice by fluorescent microscopy. Fluorescent micrographs showed fluorescent signal apperared in cells of liver, kidney, lung and spleen compared with the control, but in heart, brain, stomach and intestine, there were only very weak or no GFP expression. It was further verified by Western blot that GFP could expressed in heart, brain, lung, liver, kidney, spleen, stomach and intestine tissues, and the expression was stronger in liver, kidney, lung and spleen. With electron micrographs, it could be seen that PAMAM and PAMAM/DNA complexes can be distributed in the liver, kidney, lung, spleen, pancreas, marrow, lymphonode and brain. After intravenous injection, PAMAM was distributed in the renal tubule, indicating that PAMAM can be excreted from the kidney, not resulting in cumulation.【Effect of PAMAM/DNA complexes on gene therapy of chronic myelogeneous leukemia】Leukemia is one of the most threatening hematological malignant cancers currently, and has distinct advantage in gene therapy field, so many researchers pay more attention to the gene therapy of hematological system disease, p16 gene is an antioncogene, and deleted in 25～64 % leukemia cell lines. LRRC4 gene is cloned by our laboratory, which involves in signal pathway as a receptor, receptor kinase, or adhesion molecule.To determine the possibility of the anti-tumor effect of p16 gene and LRRC4 gene, and the application of PAMAM as a vector for gene therapy, a series of experiments had been carried out 24 hrs after mice injected cyclophosphamide (2 mg) for two days, K562 cells were inoculated i.v. into the SCID mice. Treatment was given once a week from the 2nd day to the 28th day. Results indicated that PAMAM/pl6＋LRRC4 could decrease the percentage of immature granular leudocytes and monocytes among the peripheral blood, alleviate the infiltration of tumor cells into the liver, spleen and marrow. The average survival time of PAMAM/pl6＋LRRC4 treated groups were prolonged for 30 % compared with the PAMAM treated group. The effect of PAMAM/pl 6＋LRRC4 treated group was more effective than that of PAMAM/p16 and PAMAM/LRRC4 treated group. These results suggest that p16 and LRRC4 have in vivo anti-tumor activity in the human leukemia xenograft models when used together and PAMAM was an efficient vector for in vivo application.In conclusion, PAMAM can bind and protection DNA effectively, be uptaken by cells, and distributed in cytoplasm and nuclear of cells in vitro and in vivo. Using reporter gene system, PAMAM was demonstrated to transfer exogenous genes to cells and express encoded protein in vitro. PAMAM can transfer exogenous genes to organs, such as liver, kdney, lung, and spleen, and express encoded protein. In this study, PAMAM was firstly used as non-viral gene vector for gene therapy of leukemia. Our studies also demonstrate potent antitumor activity of p 16 and LRRC4 used together in K562 xenograft models, p16 and LRRC4 used together could decrease the percentage of immature granular leudocytes and monocytes among the peripheral blood, alleviate the infiltration of tumor cells into the liver, spleen and marrow. The average survival time of p16 and LRRC4 co-treated groups were longer than that of the PAMAM treated group. A novel non-viral gene vector which is somewhat characteristic of high efficiency, safety, targetting was developed using the advantage of nanoparticles. This study has also provided foundation for gene therapy, espacially for gene therapy of CML disease. Thus, our findings provide new insights into the therapy of leukemia, and polyamidoamine dendrimers, high-efficiency, low-cytotoxicity gene vector, appear to have potential for fundamental research and gene therapy in vitro and in vivo.更多还原