基于胆固醇的阳离子脂质材料的设计合成及其在基因转染中的应用研究
发布时间：2018-09-18 18:00
【摘要】：目的:基因治疗是目前癌症和遗传性相关疾病治疗中很有前途的一种新技术,其关键问题在于构建有效携载治疗基因并使其发挥作用的载体。非病毒载体作为新兴的基因转导系统,具有低毒、低免疫原性、无基因插入片段大小的限制,以及制备方便、便于保存和使用等优势。阳离子脂质体是非病毒载体中最常用的载体,具有易于生产、低免疫原性、无致癌性和保护基因免受核酸酶降解等优点,在介导基因转染上具有广阔的发展前景。本论文综合考虑胆固醇疏水基团、不同种类胺基和碱性氨基酸头部的片段优势,通过连接键的桥连,在其侧链引入不同的阳离子头部,设计合成了三个系列含有不同胺基或碱性氨基酸残基的胆固醇阳离子脂质材料,为阳离子脂质体的制备及基因治疗提供载体材料。方法:以胆固醇,1,6-己二醇,碱性氨基酸(赖氨酸、组氨酸和精氨酸),二甲胺盐酸盐,二乙胺,三乙胺,1,4-二溴乙醚,1,4-二溴丁烷,1,6-二溴己烷,1,12-二溴十二烷等为原料,设计合成了单胺基头部(M1-M6)、碱性氨基酸头部(A1-A6)和双生脂质(G1-G4)三个系列的阳离子脂质材料。通过质谱、核磁、红外等方法对合成的材料进行结构鉴定。采用薄膜分散法将上述阳离子脂质材料与辅助脂质DOPE混合制备得到阳离子脂质体,并进行表征。采用MTT比色法对不同空白阳离子脂质体的细胞毒性进行检测。采用凝胶电泳阻滞实验考察不同阳离子脂质体与p EGFP质粒的结合能力,筛选出DNA与阳离子脂质体完全复合时的N/P比,并进行体外转染活性的筛选。采用流式细胞仪定量检测细胞转染效率,并用荧光显微镜定性观察p EGFP在细胞内的表达情况。对初筛转染活性较好的阳离子脂质材料进行处方的优化,筛选出的最优处方进一步考察血清存在对其细胞转染活性的影响。结果:设计合成了三系列共16种基于胆固醇的阳离子脂质材料,经1H NMR,MS,IR等方法鉴定,所得化合物的结构与目标物的结构一致。经纳米粒度及Zeta电位分析仪测定,所制备的16种阳离子脂质体粒径均小于240 nm,Zeta电位均在35 m V以上,满足基因载体的要求。MTT细胞毒性实验结果表明,上述阳离子脂质体IC50均大于阳性对照Lipofectamine 2000(IC50=33μM);与DC-Chol(IC50=51μM)相比,除G1(IC50=44μM)、G4(IC50=35μM)、A4(IC50=48μM)和A5(IC50=49μM)外,其它12种阳离子脂质材料所得脂质体IC50均大于DC-Chol,安全性和生物相容性好。凝胶阻滞实验结果显示,在低N/P比(小于3)的情况下,所合成的阳离子脂质材料都具有较强的携载DNA的能力。基因转染实验结果表明,除M3、M5和G1外,其余13种阳离子脂质材料所得脂质体均具有良好的转染活性。M1、M6和A1-A6的转染活性优于DC-Chol(p0.05或0.01),其中M6、A4和A6的转染活性最好,M6和A4转染效率与Lipofectamine 2000相当(p0.05),A6明显优于Lipofectamine2000(p0.05)。脂质材料M1,M6和A1-A3的处方优化和血清存在下的转染实验结果表明,即使在血清存在下M1和M6仍具有很好的转染活性,优于Lipofectamine2000(p0.05或0.01)。结论:本论文在胆固醇骨架中引入不同胺基头部构建了三个系列16种阳离子脂质材料并对其细胞毒性和基因转染活性进行研究。研究结果显示,上述阳离子脂质材料所得基因载体具有粒径均一,细胞毒性小,携载DNA能力强等优点。除M3、M5和G1外,所有阳离子脂质体均具有较好的转染活性,其中M1和M6的活性最好,在血清存在下仍优于DC-Chol和Lipofectamine 2000。通过进一步的体内活性研究,可望为基因治疗提供安全有效的载体。
[Abstract]:Objective: Gene therapy is a promising new technique in the treatment of cancer and hereditary related diseases. The key problem is to construct effective carriers for carrying therapeutic genes and making them work. Cationic liposomes are the most commonly used carriers in non-viral vectors. They have the advantages of easy production, low immunogenicity, non-carcinogenicity and protection of genes from nuclease degradation. They have broad prospects for gene transfection. Three series of cholesterol cationic lipids containing different amino or basic amino acid residues were designed and synthesized by bridging the amino and basic amino acid heads and introducing different cationic heads into their side chains. Three series of amino acids (lysine, histidine and arginine), dimethylamine hydrochloride, diethylamine, triethylamine, 1,4-dibromoethyl ether, 1,4-dibromobutane, 1,6-dibromohexane, 1,12-dibromododecane) were designed and synthesized from cholesterol, 1,6-hexanediol, basic amino acid head (A1-A6) and Gemini lipid (G1-G4). Cationic Liposomes. Structural identification of the synthesized materials was carried out by mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. Cationic liposomes were prepared by mixing the cationic liposomes with auxiliary liposomes DOPE by membrane dispersion method and characterized. The cytotoxicity of different blank cationic liposomes was detected by MTT colorimetry. The binding ability of different cationic liposomes to P EGFP plasmid was examined by gel electrophoresis block test. The N/P ratio of DNA completely combined with cationic liposomes was screened, and the transfection activity was screened. The transfection efficiency was quantitatively detected by flow cytometry, and the expression of P EGFP in cells was qualitatively observed by fluorescence microscope. Results: Three series of 16 cholesterol-based cationic lipid materials were designed and synthesized and identified by 1H NMR, MS and IR. The sixteen cationic liposomes were found to be smaller than 240 nm in size and above 35 m V in Zeta potential. The results of MTT cytotoxicity test showed that the IC50 of these liposomes was higher than that of Lipofectamine 2000 (IC50 = 33 mu M). Compared with DC-Chol (IC50=51 M), except G1 (IC50=44 M M), G4 (IC50=35 M), A4 (IC50=48 mul), and (12), the liposomes of other 3 cationic liposomes were all larger than that of the liposomes, and their safety and biocompatibility were good. The results of gel retardation test showed that the synthesized cationic lipids were low to low ratio (less than 3). The transfection activity of M1, M6 and A 1-A6 was better than that of DC-Chol (p0.05 or 0.01). The transfection activity of M6, A4 and A6 was the best, and the transfection efficiency of M6 and A4 was the same as that of Lipofectamine 2000. When (p0.05), A6 was significantly superior to Lipofectamine 2000 (p0.05). The results of formulation optimization and serum transfection of lipid materials M1, M6 and A1-A3 showed that M1 and M6 had good transfection activity even in the presence of serum, superior to Lipofectamine 2000 (p0.05 or 0.01). Conclusion: Different amino groups were introduced into the cholesterol skeleton in this study. Three series of 16 cationic liposomes were constructed and their cytotoxicity and gene transfection activity were studied. The results showed that the cationic liposomes had the advantages of uniform particle size, low cytotoxicity and strong ability to carry DNA. Except M3, M5 and G1, all cationic liposomes had good transfection activity. Among them, M1 and M6 have the best activity and are still superior to DC-Chol and Lipofectamine 2000 in the presence of serum.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R943
本文编号：2248683
[Abstract]:Objective: Gene therapy is a promising new technique in the treatment of cancer and hereditary related diseases. The key problem is to construct effective carriers for carrying therapeutic genes and making them work. Cationic liposomes are the most commonly used carriers in non-viral vectors. They have the advantages of easy production, low immunogenicity, non-carcinogenicity and protection of genes from nuclease degradation. They have broad prospects for gene transfection. Three series of cholesterol cationic lipids containing different amino or basic amino acid residues were designed and synthesized by bridging the amino and basic amino acid heads and introducing different cationic heads into their side chains. Three series of amino acids (lysine, histidine and arginine), dimethylamine hydrochloride, diethylamine, triethylamine, 1,4-dibromoethyl ether, 1,4-dibromobutane, 1,6-dibromohexane, 1,12-dibromododecane) were designed and synthesized from cholesterol, 1,6-hexanediol, basic amino acid head (A1-A6) and Gemini lipid (G1-G4). Cationic Liposomes. Structural identification of the synthesized materials was carried out by mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. Cationic liposomes were prepared by mixing the cationic liposomes with auxiliary liposomes DOPE by membrane dispersion method and characterized. The cytotoxicity of different blank cationic liposomes was detected by MTT colorimetry. The binding ability of different cationic liposomes to P EGFP plasmid was examined by gel electrophoresis block test. The N/P ratio of DNA completely combined with cationic liposomes was screened, and the transfection activity was screened. The transfection efficiency was quantitatively detected by flow cytometry, and the expression of P EGFP in cells was qualitatively observed by fluorescence microscope. Results: Three series of 16 cholesterol-based cationic lipid materials were designed and synthesized and identified by 1H NMR, MS and IR. The sixteen cationic liposomes were found to be smaller than 240 nm in size and above 35 m V in Zeta potential. The results of MTT cytotoxicity test showed that the IC50 of these liposomes was higher than that of Lipofectamine 2000 (IC50 = 33 mu M). Compared with DC-Chol (IC50=51 M), except G1 (IC50=44 M M), G4 (IC50=35 M), A4 (IC50=48 mul), and (12), the liposomes of other 3 cationic liposomes were all larger than that of the liposomes, and their safety and biocompatibility were good. The results of gel retardation test showed that the synthesized cationic lipids were low to low ratio (less than 3). The transfection activity of M1, M6 and A 1-A6 was better than that of DC-Chol (p0.05 or 0.01). The transfection activity of M6, A4 and A6 was the best, and the transfection efficiency of M6 and A4 was the same as that of Lipofectamine 2000. When (p0.05), A6 was significantly superior to Lipofectamine 2000 (p0.05). The results of formulation optimization and serum transfection of lipid materials M1, M6 and A1-A3 showed that M1 and M6 had good transfection activity even in the presence of serum, superior to Lipofectamine 2000 (p0.05 or 0.01). Conclusion: Different amino groups were introduced into the cholesterol skeleton in this study. Three series of 16 cationic liposomes were constructed and their cytotoxicity and gene transfection activity were studied. The results showed that the cationic liposomes had the advantages of uniform particle size, low cytotoxicity and strong ability to carry DNA. Except M3, M5 and G1, all cationic liposomes had good transfection activity. Among them, M1 and M6 have the best activity and are still superior to DC-Chol and Lipofectamine 2000 in the presence of serum.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R943
【参考文献】
相关期刊论文 前1条
1 王冰;张树彪;周集体;赵不凋;杨宝灵;崔绍辉;赵轶男;;阳离子脂质体介导的基因转移机制[J];中国组织工程研究与临床康复;2011年08期
，本文编号：2248683
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