功能化的第五代树状大分子包裹纳米金颗粒的制备及其在基因传递中的应用

发布时间：2018-08-03 20:15

【摘要】：随着癌症以及其它基因类疾病发病率的升高,基因治疗在癌症以及其它疾病治疗中的应用引起人们的广泛关注。基因治疗(gene therapy)是指将外源正常基因导入靶细胞,以纠正或补偿因基因缺陷和异常引起的疾病,以达到治疗目的的一种治疗手段。基因治疗的关键在于通过安全、高效的载体将治疗基因传递到靶细胞。基因传递常用的载体一般分为病毒载体和非病毒载体。虽然病毒载体的基因传递效率高,但它的免疫源性、致癌性和细胞毒性较高等缺陷限制了其在体内基因治疗中的应用。近年来,研究者开始致力于探索非病毒载体在基因治疗中的应用潜能。在本研究中,我们报道了两类基于第五代树状大分子(G5.NH2)的新型非病毒基因传递载体,分别是乙酰基修饰的第五代树状大分子包裹纳米金颗粒(Au DENPs-Ac)和聚乙二醇修饰的第五代树状大分子包裹纳米金颗粒(Au DENPs-PEG)。通过核磁、紫外分光光度计以及透射电镜对材料的物理化学特征进行表征；琼脂糖凝胶电泳技术确定载体完全结合基因的N/P；水动力粒径大小和电势正负的测量初步研究在不同的N/P下,载体／基因是否形成了有利于进入细胞的复合物。绿色荧光蛋白和荧光素酶的表达用来研究载体传递质粒DNA的效率；Western blot技术用来研究载体传递Bcl-2-siRNA的效率；能够持续表达EGFP的Hela细胞用来研究材料传递ssDNA的效率。共聚焦显微镜技术以及流式细胞技术也被用来研究载体／基因复合物的细胞内吞效率。结果表明：Au DENPs-Ac能够显著地降低Au DENPs的细胞生物学毒性,但不影响Au DENPs的基因传递效率。Au DENPs-PEG不仅能高效地压缩pDNA、siRNA和ssDNA形成有利于基因传递的复合物,而且显著地提高了GS.NH2的基因传递效率。在氯金酸与树状大分子比相同的条件下,低分子量的聚乙二醇(mPEG,分子量为2000)修饰的Au DENPs比高分子量的mPEG(分子量为5000)修饰的Au DENPs传递质粒DNA的效率高。同时,在N/P=5时,分子量为2000的mPEG修饰的AuDENPs (Au0)50-G5.NH2-mPEG2k,0)表现出最高的传递siRNA和质粒DNA的效率。同时,Au DENPs-PEG在传递ssDNA方面也有潜在的应用价值。上述研究为功能化树状大分子包金材料在基因传递领域的应用奠定了基础。
[Abstract]:With the increasing incidence of cancer and other genetic diseases, the application of gene therapy in the treatment of cancer and other diseases has attracted wide attention. Gene therapy (gene therapy) is a therapeutic method that inducts exogenous normal genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities. The key to gene therapy is to transfer therapeutic genes to target cells through safe and efficient vectors. The vectors commonly used for gene transmission are generally divided into viral vectors and non-viral vectors. Although the gene transfer efficiency of virus vector is high, its defects such as immunogenicity, carcinogenicity and cytotoxicity limit its application in gene therapy in vivo. In recent years, researchers have begun to explore the potential of non-viral vectors in gene therapy. In this study, we reported two novel non-viral gene transfer vectors based on the fifth generation dendritic macromolecule (G5.NH2). The fifth generation dendrimer coated gold nanoparticles (au DENPs-Ac) were modified by acetyl group and the au DENPs-PEG particles (au DENPs-PEG) by polyethylene glycol modified dendrimer nanoparticles. The physical and chemical characteristics of the material were characterized by NMR, UV spectrophotometer and transmission electron microscope. Preliminary study on the measurement of hydrodynamic particle size and potential positive or negative whether the vector / gene formed a complex favourable to cell entry under different N / P conditions. The expression of green fluorescent protein and luciferase was used to study the efficiency of vector transfer plasmid DNA. Western blot technique was used to study the efficiency of vector delivery of Bcl-2-siRNA, and Hela cells, which could continuously express EGFP, were used to study the efficiency of ssDNA transfer. Confocal microscopy and flow cytometry have also been used to study the endocytosis efficiency of vector / gene complexes. The results showed that DENPs-Ac could significantly reduce the cytotoxicity of au DENPs, but did not affect the gene transfer efficiency of au DENPs. Au DENPs-PEG could not only efficiently compress DENPs siRNA and ssDNA to form a gene transfer complex. Moreover, the gene transmission efficiency of GS.NH2 was improved significantly. Under the same ratio of chloruronic acid to dendritic macromolecule, au DENPs modified with low molecular weight polyethylene glycol (mPEG, molecular weight 2000) is more efficient than au DENPs transfer plasmid DNA modified by high molecular weight mPEG (molecular weight 5000). At N / P = 5, mPEG modified AuDENPs (Au0) 50-G5.NH2-mPEG2kU _ 0 with molecular weight of 2000 exhibited the highest transfer efficiency of siRNA and plasmid DNA. At the same time, au DENPs-PEG also has potential application value in ssDNA transmission. These studies have laid a foundation for the application of functional dendritic macromolecular gold inclusion materials in gene transfer.
【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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