本文關(guān)鍵詞：氨基葡萄糖陽離子脂質(zhì)體基因載體的合成和性能研究　出處：《湖南師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 基因治療 陽離子脂質(zhì)體 氨基葡萄糖 基因轉(zhuǎn)染效率

【摘要】：基因治療的核心是設(shè)計(jì)一類高效、安全的基因載體。作為非病毒載體的一類,陽離子脂質(zhì)體可與核酸藥物靜電吸引形成復(fù)合物,它與細(xì)胞膜的磷脂分子相互作用而附著在其表面,隨著細(xì)胞膜的內(nèi)陷,復(fù)合物進(jìn)入細(xì)胞并且釋放核酸藥物至細(xì)胞核。糖化學(xué)是生物化學(xué)的一個重要分支。糖類化合物承載了大量生物分子信息,在生命有機(jī)體中扮演重要角色,如識別和調(diào)控生物分子、確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類化合物對于生物結(jié)構(gòu)關(guān)系的認(rèn)知顯得尤為重要。本論文設(shè)計(jì)了兩個系列氨基葡萄糖陽離子脂質(zhì)體。一類以氨基葡萄糖鹽酸鹽為原料,通過全乙�；⒚�1-O-乙�；�、三氯乙酰亞胺酯化、糖苷化、疊氮化、脫全部乙酰基、4,6-O-異丙叉基保護(hù)、芐氧基保護(hù)、脫異丙叉基、醚化、還原氨化和季銨鹽化反應(yīng),合成不同疏水鏈長度的一系列氨基葡萄糖陽離子脂質(zhì)體:di-C12-GluNAc-TMA、di-C14-GluNAc-TMA、di-C16-GluNAc-TMA和di-C18-GluNAc-TMA;另一類以氨基葡萄糖鹽酸鹽為原料,通過全乙�；⒚�1-O-乙�；�、三氯乙酰亞胺酯化、糖苷化、疊氮化、脫全部乙�；�、還原氨化、叔胺化和季銨鹽化反應(yīng),合成不同疏水鏈長度和不同物理結(jié)構(gòu)的一系列氨基葡萄糖陽離子脂質(zhì)體:GluNAc-DiC12MA、GluNAc-DiC14MA、GluNAc-DiC16MA和GluNAc-DiC18MA。通過Zetasizer Nano ZS激光粒度儀激光粒度儀測試了陽離子脂質(zhì)體和脂質(zhì)體/dna二元復(fù)合物的zeta電勢、粒徑和pdi;凝膠阻滯實(shí)驗(yàn)檢測了陽離子脂質(zhì)體縮合質(zhì)粒dna的能力。原子力顯微鏡表征了陽離子脂質(zhì)體及二元復(fù)合物的表面形貌。激光粒度儀數(shù)據(jù)顯示di-c18-glunac-tma平均粒徑為182.4nm,大于其他陽離子脂質(zhì)體納米顆粒尺寸,分散系數(shù)(pdi)為0.43,粒徑分布不夠均一;其他七種陽離子脂質(zhì)體的粒徑范圍是80-150nm,脂質(zhì)體對應(yīng)復(fù)合物的粒徑范圍在65-140nm;陽離子脂質(zhì)體的zeta電勢在38-60mv之間,復(fù)合物的電勢在0.5-28mv之間。這些數(shù)據(jù)表明脂質(zhì)體及復(fù)合物的粒徑和zeta電勢適宜,符合體外轉(zhuǎn)染實(shí)驗(yàn)的要求。凝膠電泳實(shí)驗(yàn)證明陽離子脂質(zhì)體有能力與dna分子結(jié)合。當(dāng)脂質(zhì)體的量增多時,dna軌道的亮度減弱,當(dāng)n/p為3時,兩類陽離子脂質(zhì)體與dna分子結(jié)合完全。原子力顯微鏡結(jié)果表明單一脂質(zhì)體及脂質(zhì)體/dna復(fù)合物呈圓球狀、尺寸分布均一、表面光滑且直徑略大于脂質(zhì)體。核磁共振儀的圖譜結(jié)果證明陽離子脂質(zhì)體的化學(xué)結(jié)構(gòu)符合預(yù)期的設(shè)計(jì)。以上述兩類所合成的氨基葡萄糖陽離子脂質(zhì)體作為基因載體,檢測它們攜帶質(zhì)粒dna進(jìn)入hek293細(xì)胞系的轉(zhuǎn)染性能。經(jīng)24小時的細(xì)胞培養(yǎng)后,通過觀察綠色熒光蛋白的表達(dá)和細(xì)胞計(jì)數(shù)的方法評價陽離子脂質(zhì)體的轉(zhuǎn)染效率。體外轉(zhuǎn)染實(shí)驗(yàn)結(jié)果說明:di-c12-glunac-tma、di-c14-glunac-tma、di-c16-glunac-tma、di-c18-glunac-tma、glunac-dic12ma和glunac-dic18ma的轉(zhuǎn)染效果不理想,glunac-dic14ma和glunac-dic16ma的轉(zhuǎn)染效率可以與商用轉(zhuǎn)染試劑lipo2000媲美。細(xì)胞毒性實(shí)驗(yàn)結(jié)果表明:兩類氨基葡萄糖陽離子脂質(zhì)體的細(xì)胞毒性較小,生物兼容性良好。在整個基因傳遞過程中,GluNAc-Di C14MA和GluNAc-DiC16MA表現(xiàn)出低毒高效的轉(zhuǎn)染效果,具有作為商業(yè)基因載體的潛力。將商用脂質(zhì)體Lipo2000作為陽性對照組實(shí)驗(yàn),考察GluNAc-DiC14MA和GluNAc-DiC16MA在HeLa和HepG2兩種細(xì)胞系的體外轉(zhuǎn)染情況。結(jié)果發(fā)現(xiàn),這兩種陽離子脂質(zhì)體在HepG2細(xì)胞系中的轉(zhuǎn)染效果與HEK293細(xì)胞系類似,而在HeLa細(xì)胞系中幾乎沒有轉(zhuǎn)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.

【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R450;O629.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號：1398182