氨基葡萄糖阳离子脂质体基因载体的合成和性能研究

发布时间：2018-01-08 18:19

本文关键词：氨基葡萄糖阳离子脂质体基因载体的合成和性能研究　出处：《湖南师范大学》2015年硕士论文　论文类型：学位论文

【摘要】：基因治疗的核心是设计一类高效、安全的基因载体。作为非病毒载体的一类,阳离子脂质体可与核酸药物静电吸引形成复合物,它与细胞膜的磷脂分子相互作用而附着在其表面,随着细胞膜的内陷,复合物进入细胞并且释放核酸药物至细胞核。糖化学是生物化学的一个重要分支。糖类化合物承载了大量生物分子信息,在生命有机体中扮演重要角色,如识别和调控生物分子、确定生物分子的表面形貌和提供能量等。因此,研究具有生物活性的糖类化合物对于生物结构关系的认知显得尤为重要。本论文设计了两个系列氨基葡萄糖阳离子脂质体。一类以氨基葡萄糖盐酸盐为原料,通过全乙酰化、脱1-O-乙酰基、三氯乙酰亚胺酯化、糖苷化、叠氮化、脱全部乙酰基、4,6-O-异丙叉基保护、苄氧基保护、脱异丙叉基、醚化、还原氨化和季铵盐化反应,合成不同疏水链长度的一系列氨基葡萄糖阳离子脂质体:di-C12-GluNAc-TMA、di-C14-GluNAc-TMA、di-C16-GluNAc-TMA和di-C18-GluNAc-TMA;另一类以氨基葡萄糖盐酸盐为原料,通过全乙酰化、脱1-O-乙酰基、三氯乙酰亚胺酯化、糖苷化、叠氮化、脱全部乙酰基、还原氨化、叔胺化和季铵盐化反应,合成不同疏水链长度和不同物理结构的一系列氨基葡萄糖阳离子脂质体:GluNAc-DiC12MA、GluNAc-DiC14MA、GluNAc-DiC16MA和GluNAc-DiC18MA。通过Zetasizer Nano ZS激光粒度仪激光粒度仪测试了阳离子脂质体和脂质体/dna二元复合物的zeta电势、粒径和pdi;凝胶阻滞实验检测了阳离子脂质体缩合质粒dna的能力。原子力显微镜表征了阳离子脂质体及二元复合物的表面形貌。激光粒度仪数据显示di-c18-glunac-tma平均粒径为182.4nm,大于其他阳离子脂质体纳米颗粒尺寸,分散系数(pdi)为0.43,粒径分布不够均一;其他七种阳离子脂质体的粒径范围是80-150nm,脂质体对应复合物的粒径范围在65-140nm;阳离子脂质体的zeta电势在38-60mv之间,复合物的电势在0.5-28mv之间。这些数据表明脂质体及复合物的粒径和zeta电势适宜,符合体外转染实验的要求。凝胶电泳实验证明阳离子脂质体有能力与dna分子结合。当脂质体的量增多时,dna轨道的亮度减弱,当n/p为3时,两类阳离子脂质体与dna分子结合完全。原子力显微镜结果表明单一脂质体及脂质体/dna复合物呈圆球状、尺寸分布均一、表面光滑且直径略大于脂质体。核磁共振仪的图谱结果证明阳离子脂质体的化学结构符合预期的设计。以上述两类所合成的氨基葡萄糖阳离子脂质体作为基因载体,检测它们携带质粒dna进入hek293细胞系的转染性能。经24小时的细胞培养后,通过观察绿色荧光蛋白的表达和细胞计数的方法评价阳离子脂质体的转染效率。体外转染实验结果说明:di-c12-glunac-tma、di-c14-glunac-tma、di-c16-glunac-tma、di-c18-glunac-tma、glunac-dic12ma和glunac-dic18ma的转染效果不理想,glunac-dic14ma和glunac-dic16ma的转染效率可以与商用转染试剂lipo2000媲美。细胞毒性实验结果表明:两类氨基葡萄糖阳离子脂质体的细胞毒性较小,生物兼容性良好。在整个基因传递过程中,GluNAc-Di C14MA和GluNAc-DiC16MA表现出低毒高效的转染效果,具有作为商业基因载体的潜力。将商用脂质体Lipo2000作为阳性对照组实验,考察GluNAc-DiC14MA和GluNAc-DiC16MA在HeLa和HepG2两种细胞系的体外转染情况。结果发现,这两种阳离子脂质体在HepG2细胞系中的转染效果与HEK293细胞系类似,而在HeLa细胞系中几乎没有转染效果。
[Abstract]:The core of gene therapy is to design a kind of high efficient, safe gene carrier. As a kind of non viral vector, cationic liposome can attract nucleic acid and drug electrostatic complex formation, cell membrane phospholipid molecules and its interaction with the cells attached to the surface, membrane invagination, complex into cells and release nucleic acid drugs to the nucleus. Sugar chemistry is an important branch of biochemistry. Carbohydrates carried a lot of biological molecules, play an important role in living organisms, such as the identification and control of biological molecules, determine the surface morphology of biological molecules and provide energy. Therefore, the research of cognitive carbohydrate compounds with biological activity for biological structure the relationship is particularly important. This paper designed two series of glucosamine cationic liposomes. A kind of glucosamine hydrochloride as raw material, through the peracetylated De 1-O-, acetyl, three chloro acetyl imine esterification, glycosylation, azide, and all acetyl 4,6-O- isopropylidene protection, benzyl protection, removal of isopropylidene, etherification, reductive amination and quaternization reaction, a series of glucosamine synthesis of different hydrophobic cationic liposomes chain length: di-C12-GluNAc-TMA, di-C14-GluNAc-TMA, di-C16-GluNAc-TMA and di-C18-GluNAc-TMA; the other on glucosamine hydrochloride as raw material, through acetylation, 1-O- and acetyl, three chloro acetyl imine esterification, glycosylation, azide, removing all acetyl, reductive amination, tertiary amine and quaternary ammonium salt chemical reaction, a series of glucosamine synthesis of cationic liposomes with different hydrophobic chain lengths and different physical structures: GluNAc-DiC12MA, GluNAc-DiC14MA, GluNAc-DiC16MA and GluNAc-DiC18MA. were tested by Zetasizer Nano cation ZS laser particle size analyzer and laser particle size analyzer Zeta potential of two yuan compound liposomes and liposome /dna, particle size and PDI; gel retardation experiments tested the ability of cationic liposome condensation of plasmid DNA. The atomic force microscope to characterize the surface morphology of cationic liposomes and two element complexes. The data of laser particle sizer di-c18-glunac-tma average particle size is 182.4nm larger than the other cationic liposome nano particle size, dispersion coefficient (PDI) was 0.43, the particle size distribution is not uniform; the other seven kinds of cationic liposome size is in the range of 80-150nm, the corresponding liposome complex particle size in the range of 65-140nm; cationic liposome zeta potential between 38-60mv potential of the complexes in the 0.5-28mv. These data suggest that liposomes and composite particle size and zeta potential suitable with in vitro transfection experiments. Gel electrophoresis experiments show that cationic liposomes with DNA binding ability when. The amount of liposomes increased when the brightness of the DNA orbit decreased when n/p was 3, two kinds of cationic liposomes with DNA molecules. Atomic force microscopy showed that single liposomes and liposome /dna complexes were spherical, uniform size, smooth surface and diameter slightly larger than the results of NMR liposomes. The instrument map proved the chemical structure of cationic liposomes in line with the expected design. With glucosamine cationic liposome of the above two kinds of synthesis as a gene vector, transfected into HEK293 DNA plasmid carrying properties of cells. After 24 hours of cell culture, through the observation of green fluorescent protein expression and cell counting the evaluation of cationic liposome transfection efficiency in vitro transfection. Experimental results show that: di-c12-glunac-tma, di-c14-glunac-tma, di-c16-glunac-tma, di-c18-glunac-tma, glunac-dic12ma and glunac- Dic18ma transfection effect is not ideal, and the transfection efficiency of glunac-dic16ma glunac-dic14ma can be comparable with the commercial transfection reagent lipo2000. The cytotoxicity experiment showed that the cytotoxicity of two kinds of small glucosamine cationic liposomes, good biological compatibility. The gene transfer process in GluNAc-Di, C14MA and GluNAc-DiC16MA showed low toxicity and high transfection efficiency, as with commercial gene vector potential. The commercial liposome Lipo2000 as positive control group of GluNAc-DiC14MA and GluNAc-DiC16MA in the experiment, HeLa and HepG2 two cell lines in vitro transfection. Results showed that the two kinds of cationic liposomes in HepG2 cells transfected with HEK293 cell lines with similar results, and almost in HeLa cell line no effect of transfection.

【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450;O629.1

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