【作者】 韩磊；

【导师】 常津； 康春生；

【作者基本信息】 天津大学 ， 材料学， 2008， 博士

【摘要】 细菌磁小体(BMPs)是趋磁性细菌体内依靠生物矿化法合成的磁性纳米颗粒,主要由磁核和包被磁核的脂质膜两部分组成。磁小体本身所具有的独特性质,使其在许多领域得到了广泛的应用,但作为基因载体的应用研究还很少。本文通过对磁小体的修饰和改性,将其构建成具有磁靶向和跨膜复合功能的基因投递系统,并在此基础上使用该基因投递系统对人脑胶质瘤进行体内外基因治疗实验,并探讨治疗效果。本文通过FTIR、XPS、TEM和HRTEM对磁小体脂质膜的组分、官能团和微观形貌进行观察和分析,发现磁小体具有独特的微观排列形貌和粒径分布,平均粒径为45nm,饱和磁化强度为74.33emu/g,并采用荧光胺的方法对磁小体脂质膜上氨基含量进行了定量分析,发现每毫克磁小体的脂质膜上含有58.58 nmol的氨基基团;通过XRD、SQUID和紫外-可见分光光度计对磁小体磁核的组分和磁性能进行分析,发现磁小体的磁核由四氧化三铁晶体组成,具有较强的磁响应性和分散稳定性,并通过ICP-MS分析了磁小体中铁元素的含量为每毫克磁小体含有0.605mg的铁元素,含有约3.3549×1012个磁小体颗粒;细胞毒性实验的结果证实了磁小体具有很好的生物相容性。通过碳二亚胺(EDC·HCl)偶合法,制备出具有磁靶向的基因投递系统(BMPs-PAMAM),并优化了制备条件,在此基础上将跨膜多肽(Tat)借助偶联剂(Sulfo-LC-SPDP)连接到BMPs-PAMAM磁性粒子表面,制备出具有磁靶向和跨膜复合功能的基因投递系统(Tat-BMPs-PAMAM)。通过FTIR、XPS、Zeta Potential Analyzer、TEM、SQUID和紫外-可见分光光度计对复合功能基因投递系统的结构、组成、微观形貌和磁性能进行了分析表征。定量了每毫克BMPs表面接枝有0.056mg的PAMAM大分子和0.027mg的Tat多肽,大约每个BMPs上接枝了1472个PAMAM大分子和3190个Tat多肽分子。该投递系统粒径均匀,约为50nm;具有较强的磁响应性和分散稳定性,其饱和磁化强度为62.46emu/g。凝胶电泳阻滞实验证明了两种基因投递系统(BMPs-PAMAM和Tat-BMPs- PAMAM)均能够与报告质粒(pEGFP与pGL-3)形成非常稳定的复合物,并且对报告质粒具有很好的酶切保护作用;细胞毒性实验证实了两种投递系统具有较好的生物相容性。使用两种基因投递系统将两种报告质粒转染U251细胞,结果显示,当BMPs-PAMAM与报告质粒的质量比为20:1、Tat-BMPs-PAMAM与报告质粒的质量比为12.5:1时,均具有最高的转染效率,分别为16.65%和20.56%,并且外加磁场的靶向性作用能够使两种基因投递系统的转染效率提高25%。体外磁靶向跨膜定位实验证明所制备的两种复合功能基因投递系统(BMPs-PAMAM和Tat-BMPs- PAMAM)具有较好的磁靶向和跨膜复合功能,其转染效率分别为为11.56%,13.28%,高于商用转染试剂Lipofectamine 2000的转染效率9.86%。对两种基因投递系统进行放射性元素99mTc标记,用SPECT考察了两种投递系统荷载pGL-3质粒的复合物在正常SD大鼠体内分布情况,并详细考察了注射方式、Tat多肽和外加磁场对复合物在各脏器中分布和跨血脑屏障能力的影响,显示其磁靶向和跨膜的复合功能。使用U251胶质瘤细胞模型研究了两种基因投递系统的体外基因转染对肿瘤的抑制效果,并与Lipofectamine 2000进行了对比。通过细胞免疫荧光、细胞免疫组化和Western blot检测相关靶蛋白的表达,证明了Tat-BMPs-PAMAM与Lipofectamine 2000对靶蛋白具有相似的治疗效果。体外细胞生物学特性实验证明,经两种基因投递系统基因治疗后的U251细胞的增殖活性和侵袭能力均显著降低,凋亡细胞数显著上升,并且Tat-BMPs-PAMAM/psiRNA比BMPs-PAMAM /psiRNA具有更好的体外抑制效果,与Lipofectamine 2000/psiRNA组相比没有显著性的差异。以荷U251胶质瘤裸鼠为动物模型研究了两种基因投递系统的体内基因转染对肿瘤的抑制效果。裸鼠体内肿瘤生长速率曲线、肿瘤组织标本的免疫组化和原位凋亡检测(TUNEL法)的结果与体外实验结果相一致:经体内基因治疗后,Tat-BMPs-PAMAM/psiRNA比BMPs-PAMAM/psiRNA具有更好的体内治疗效果,与Lipofectamine 2000/psiRNA组相比没有显著性的差异。综上所述,本文将纳米生物技术与肿瘤分子生物学技术相结合,将制备的BMPs-PAMAM和Tat-BMPs-PAMAM作为非病毒基因投递系统进行基因治疗的研究,充分利用磁小体的磁靶向性和Tat多肽的跨膜功能构建出具有复合功能的非病毒基因投递系统,为疾病的基因治疗尤其是中枢神经系统疾病的基因治疗奠定了基础。更多还原

【Abstract】 Magnetotactic bacteria synthesized intracellular magnetic nanoparticles by biomineralization, also called magnetosomes or bacterial magnetic particles (BMPs), which were consisted of magnetic core enveloped by stable lipid membrane which contained some lipids and proteins. Magnetosomes with the special characteristics possessed the broad applicable prospects in many fields. After being modified, the BMPs were constructed to a novel gene delivery system with the composite function of magnetic targeting and translocational capability which were used to delivery gene to glioma and investigated inhibition effect in vitro and in vivo in this article.In this article, the crystalline, morphology, crystal-size distributions, iron element content and magnetic properties of the magnetosomes were researched by XRD, TEM, HRTEM, ICP-MS and SQUID. Furthermore, in order to connect magnetosomes with small biomolecule to serve as a new magnetic delivery system, we characterized the functional groups on its surface by FTIR and XPS analysis and quantified the primary amino groups on its surface by fluorescamine assay for the first time, the primary amino groups in the membrane of magnetosomes is 58.58 nmol/mg and its saturation magnetization was 74.33 emu/g. Iron element content in the magnetosomes was 0.605mg/mg, which meaned one milligram of magnetosomes contained 3.3549×1012 magnetosomes nanoparticles. Finally, the cytotoxicity of magnetosome tested with MTT method showed that lipid biomembrane on magnetosome surface endowed them with better biocompatibility.BMPs-PAMAM gene delivery system was synthesized by linking PAMAM to the surface of BMPs using EDC·HCl. On this basis, Tat-BMPs-PAMAM was constructed by connecting tat peptide to the surface of BMPs-PAMAM using Sulfo-LC-SPDP. The configuration, components, magnetic properties and morphology of BMPs-PAMAM and Tat- BMPs-PAMAM were characterized and reaesrched by FTIR、XPS、Zeta Potential Analyzer、TEM、SQUID. The mean size of Tat-BMPs-PAMAM nanoparticle was 50nm and its saturation magnetization was 62.46 emu/g. One milligram of magnetosomes contained 0.056mg PAMAM and 0.027 mg Tat peptides that meaned every magnetosome contained 1472 PAMAM and 3190 Tat peptides.The stable complexes of vectors (BMPs-PAMAM or Tat-BMPs-PAMAM) and plasmid (pEGFP-N1 and pGL-3) at different mass ratios were investigated via agarose gel electrophoresis. The complexes could protect plasmid from digested by DNase-I, and the MTT assay showed that vectors had better biocompatibility which suggested their advantages for gene delivery in vitro. Maximal transfection efficiency of the vectors was obtained at BMPs-PAMAM to plasmid mass ratio of 20:1 and at Tat-BMPs-PAMAM to plasmid mass ratio of 12.5:1. Maximal transfection efficiency of BMPs-PAMAM and Tat-BMPs-PAMAM were 16.65% and 20.56% respectively. In addition, orientation experiment in vitro with the magnetic field, transfection efficiency of BMPs-PAMAM and Tat-BMPs-PAMAM, which were 11.56% and 13.28% respectively would be higher than that of Lipofectamine 2000 which was 9.86%.The biodistribution of 99mTc-labeled complexes of BMPs-PAMAM/pGL-3 and Tat-BMPs-PAMAM/pGL-3 in SD rat was investigated using SPECT. The effections of injection approaches, tat peptide and magnetic field on the biodistribution had also been further investigated in detail.The gene transfection and tumor inhibition assay in vitro were investigated using U251 glioma cell line as cell model. Cell immunofluorescence, cell immuno- histochemistry and western blot were used to analyze the expression of target proteins.And the cell experiments in vitro showed the lowered cell proliferation activity, attenuated cell invasive ability and induced apoptosis of glioma cell after the psiRNA delivery to the U251 cell by BMPs-PAMAM, Tat-BMPs-PAMAM and Lipofectamine 2000. However, Tat-BMPs-PAMAM showed more significant tumor inhibition effect than BMPs-PAMAM and with no difference with that of Lipofectamine 2000.The gene transfection and tumor inhibition assay in vivo were investigated using subcutaneous U251 glioma model established in nude mice. The tumor growth was measured every other day and cell apoptosis and expression of target proteins after gene therapy were detected by TUNEL method and tissue immunochemistry. The results of research in vivo showed Tat-BMPs-PAMAM owned more significant tumor inhibition effect than BMPs-PAMAM and with no difference with that of Lipofectamine 2000, which were in accordance with those in vitro.In this study, the newly nanotechnology and molecular biology were combined to develop a novel kind of non-viral gene delivery system, BMPs-PAMAM and Tat-BMPs-PAMAM. This novel non-viral gene delivery system with high transfection efficiency, safety, magnetic targeting and translocational capability was developed to provide foundation for gene therapy, especially for gene therapy of CNS disease.更多还原