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肝靶向免疫基因载体的研究

Investigation on Liver-Targeted Immune Gene Delivery

作者: 专业:药物分析 导师:胡玉荣 年度:2010 学位:硕士  院校: 郑州大学

Keywords

Liver-targeting, Liposome-polycation-pDNA Complexes, Monoclonal Antibody, Freeze-drying

        本研究开发了一种新型的非病毒基因传递系统。通过利用多聚阳离子PEI压缩质粒DNA,然后用PEG化的脂质体包裹PEI/pDNA压缩体,形成脂质复合载体LPD,并将人胰岛素受体的单克隆抗体(8314-SA)与脂质中的DSPE-PEG2000-biotin中的生物素基团(biotin)共轭,形成以单克隆抗体(MAb)为靶向基团的复合载体(8314-LPD),MAb提高了复合载体的靶向能力。8314-LPD是一种肝主动靶向四元长循环基因载体制剂。其具体研究内容及结果如(?)首先用转化的大肠杆菌扩增含有报告基因的质粒DNA (PEGFP-C1),再用QIAGEN无内毒素大提试剂盒提取质粒DNA,并对其浓度和质量进行评定。然后分别制备出PEG化的脂质体(脂质处方为POPC 94%, DDAB 2%, DSPE-PEG2000 3%和DSPE-PEG2000-biotin 1%)和PEI/pDNA聚阳离子压缩体,用所得脂质体包裹PEI/pDNA压缩体形成了LPD复合物。单因素实验结合均匀设计实验优化LPD处方,单因素实验考察结果为:质粒浓度在40μg/ml以下,粒径变化不大,浓度增大,复合物粒径增大;N/P超过1.5时,质粒的包封完全,随着N/P的增大,复合物粒径减小,电位增大,在N/P等于15时,粒径最小,再增大N/P,粒径基本不再发生变化;脂质/pDNA的摩尔比在50:1时,酶切电泳显示脂质可以完全包封PEI/pDNA压缩体,随着脂质/pDNA的比例增大,LPD的粒径减小,电位降低,在170:1时粒径最小,脂质/pDNA的摩尔比再增大,复合物粒径开始变大。均匀设计实验优化LPD复合物处方的结果为:在pH 7.0的HEPES缓冲液中,质粒浓度为40μg/ml,N/P=15,脂质/pDNA=170:1时,制备的LPD平均粒径为130 nm,平均zeta电位为1.5mV。进一步对LPD进行结构修饰,在外层脂质膜的PEG链上偶联抗人胰岛素受体的单克隆抗体(8314-SA),形成肝主动靶向的单克隆抗体修饰的Lipo/PEI/pDNA复合物(8314-LPD)。透射电镜下观察,肝靶向8314-LPD复合物的近似球形,形态规整,无粘连;对其粒径电位分析,平均粒径在150nm左右,电位在4.5mv左右。以绿色荧光蛋白基因PEGFP-C1作为报告基因考察复合物对质粒的酶切保护能力,凝胶电泳分析实验证明这种基因载体能有效地保护质粒DNA免受DNA酶(?)NaseⅠ)降解;血清稳定性实验表明8314-LPD复合物可以在血清中稳定存在;长期稳定性实验考察结果,4℃和25℃氮气密封贮存,复合物粒径都有所增大,但变化幅度不同,说明温度对复合物的稳定性有明显的作用,25℃贮存会使复合物粒径在一个月内发生非常大的增大。体外的细胞转染实验和细胞毒性实验分析,LPD载体的转染效率小于PEI/pDNA压缩体,但是经单克隆抗体修饰后,8314-LPD基因载体能够很好的被人肝癌细胞SMMC-7721摄取,与前两组基因载体相比,转染率有明显的提高。MTT实验中显示LPD和8314.-LPD复合物的毒性都明显小于PEI/pDNA多聚阳离子压缩体。为了提高8314-LPD复合物的长期稳定性,通过冷冻干燥法将其制备成冻干粉针剂。具体步骤是,首先筛选优质冻干保护剂,通过考察冻干粉的外观,色泽,表面细化程度,再分散能力,发现海藻糖制备的冻干粉外观不塌陷,不起泡,色泽均匀,质地细腻;复溶后,再分散时间短;复溶后,复合物的粒径和Zeta电位基本没有变化,是载体的最优保护剂。其次,对冻干粉进行质量评价,通过对冻干粉复溶后抗酶切实验,发现经冻干复溶后的制剂仍能保持抗核酸酶能力,说明载体结构在冻干过程中没有受到破坏。转染实验证明,经冻干复溶的载体的转染活性也基本没有变化。本研究结果表明,8314-LPD复合物制备方法简单易行,能够有效保护质粒DNA不被核酸酶降解,复合物的血清中稳定性和细胞转染率都比较高,细胞毒性小,并可通过冷冻干燥法制备出性质稳定可长期保存的冻干粉针剂。这种肝主动靶向的长循环免疫基因载体,综合利用了阳离子多聚物和脂质体的优点,克服了二者的缺点,相信在肝癌的基因治疗中,将会有很好的发展前景。
    In this study, a novel non-viral gene delivery system was developed. We used the cationic polymer PEI to compress plasmid DNA, Then,added PEG-liposomes to coated PEI/pDNA compression.the liposome-PEI-pDNA complexes was called lipoplexes(LPD).Through the hydrosulfide group conjugating with biotin, The anti-human insulin receptor Monoclonal antibody(8314-SA) connect with DSPE-PEG2000-biotin. Monoclonal antibodies are active targeting group of LPD and improve the targeting capacity of this carrier. This is a long cycling and active targeting of liver gene vector.The research contents and results are as follows:In the first step,Escherichia coli was used to amplify the reporter gene and Plasmid DNA was isolated and purified from the escherichia coli cells using the Qiagen Endo-Free Maxi Plasmid Purification kit. DNA concentration and Purity were quantified by ultraviolet absorption at 260nm and 280nm. Further analysis of purified DNA was by agarose gel electrophoresis. The second step, Preparated liposomes and DNA/PFI respectively.Using the film-vibration to prepare liposomes. The Lipid composition is 94% of POPC,2% of DDAB,3% of DSPE-PEG2000 and 1% of DSPE-PEG2000-biotin. The third step, added liposomes to PEI/pDNA, vortexed 30 second, the LPD was completed. Single-factor experiment results are:Plasmid concentration below 40μg/ml, the particle size changed little, as the DNA concentration increasing, complex particle size increases; N/P ratio exceeds 1.5, the plasmid is encapsulated entirely, with the N/P increasing, the PEI/pDNA particle size decreases,zeta potential increase,at the N/P equal to 15, the particle size of PEI/pDNA was smallest, and then increasing N/P ratio, particle size did not change significantly;Lipid/pDNA molar ratio of 50:1, liposome can encapsulate PEI/DNA compression completely. with the lipid/pDNA ratio of increasing, LPD particle size decreases, potential reduce at 170:1, the particle size was smallest, as the lipid/ pDNA ratio further increasing,the complexes particle size began to get bigger. LPD uniform design optimization of experimental results are:At pH 7.0 of HEPES buffer, the plasmid concentration of 40μg/ml, N/P=15, lipid/pDNA=170:1,LPD mean diameter was about 130 nm, the average zeta potential is around 1.5mV. further modificated LPD structure, the outer lipid membrane of PEG chain conjugated with anti-human insulin receptor monoclonal antibody, the formation of liver active targeting monoclonal antibody-modified Lipo/PEI/pDNA complexes was completed.which is called 8314-LPD. By TEM observation.8314-LPD complexes approximate spherical shape, neat and not adhesions; analysis of their particle size and zeta potential, the particle has an average diameter about 150nm, and has an average zeta potential 4.5mv. We use green fluorescent protein gene as a reporter gene to study of the this complexes protective effect of the plasmid, gel electrophoresis result show that the gene vector could effectively protect the plasmid DNA do not degraded by DNase I; Serum stability experiment results show that 8314-LPD complexes are stable in the serum; long-term stability experiment results show, both 4℃and 25℃nitrogen sealed storage condition, complexes size would increase, but the degree of change is not the same, which indicated that the temperature has an obvious effect on the stability of the 8314-LPD complexes, at 25℃storage condition,8314-LPD particle size will occur a obviously increasing within one month.In vitro cell transfection and cell toxicity experiments, LPD vector transfection efficiency is lower than PEI/DNA compression, but after modified by the monoclonal antibody,8314-LPD gene vector can be good at human hepatocellular carcinoma cell SMMC-7721 intaking, compared with the PEI/pDNA and LPD vectors,8314-LPD transfection rate was increased significantly. MTT experiments show toxicity of LPD and 8314-LPD complexes are lower than the PEI/DNA compression.In order to improve the long-term stability of the 8314-LPD,we used freeze-drying method. firstly, Select a suitable freeze-dried agent, freeze-dried powder. at their appearance, color, surface refinement level, and dispersing ability, we found freeze-dried powder prepared with trehalose, the appearance does not collapse, non-foaming, color uniform, fine texture, re-dissolved within a short time after they dispersed.after re-dissolved,the complex particle size and zeta potential have no significant change. Trehalose is the optimal freeze-dried agent. Secondly quality evaluation of freeze-dried powder, through the re-dissolved anti-enzyme experiments, the freeze-dried formulations can still maintain the ability of anti-nuclease, this shows that the carrier structure in freeze-drying process has not been destroied, transfection experiments show that the re-dissolved complexes transfection activity was unchanged.The results of this study show,8314-LPD complexes preparation method is simple, can protect plasmid DNA from being nuclease degradation effectively, have a high stability in serum and high cells transfection, low cell toxicity, last one,this carrerier can be prepared as freeze-dried powder by freeze-drying for long-term preservation. This liver active targeting of long-circulating non-viral gene vector, utilize the advantages of cationic polymers and liposomes, overcome the shortcomings of both, in the future of gene therapy of liver cancer, will have a good development prospects.
        

肝靶向免疫基因载体的研究

摘要4-6
Abstract6-8
第一章 前言13-16
第二章 质粒DNA的扩增、提取和质量鉴定16-25
    2.1 实验材料16-18
        2.1.1 仪器设备16
        2.1.2 试剂16-17
        2.1.3 试剂配制17-18
        2.1.4 质粒和菌株18
        2.1.5 细菌培养基18
    2.2 实验方法18-21
        2.2.1 质粒DNA的扩增19-20
        2.2.2 质粒DNA的提取20-21
        2.2.3 质粒DNA的产量和纯度分析21
    2.3 实验结果21-22
        2.3.1 质粒DNA的扩增结果22
        2.3.2 质粒DNA提取产量测定22
        2.3.3 质粒DNA的凝胶电泳结果22
    2.4 讨论22-24
        2.4.1 质粒载体扩增结果分析22-23
        2.4.2 质粒提取结果分析23
        2.4.3 质粒DNA的产量测定分析23
        2.4.4 质粒DNA的纯度分析23-24
    2.5 结论24-25
第三章 单克隆抗体修饰的LPD的制备及性质考察25-42
    3.1 实验材料25-27
        3.1.1 仪器设备25
        3.1.2 试剂25-26
        3.1.3 试剂配制26-27
    3.2 实验方法27-30
        3.2.1 脂质体的制备27
        3.2.2 PEI/DNA压缩体的制备27-28
        3.2.3 LPD的制备及脂质包封考察28
        3.2.4 LPD的处方优化28-29
        3.2.5 LPD的靶向修饰29
        3.2.6 8314-LPD的形态学观察29
        3.2.7 8314-LPD粒径和电位的测定29
        3.2.8 8314-LPD复合物的抗酶切能力考察29-30
        3.2.9 8314-LPD复合物在血清中的稳定性考察30
        3.2.10 8314-LPD的长期稳定性的考察30
    3.3 实验结果30-38
        3.3.1 脂质体的制备结果30
        3.3.2 PEI/DNA压缩体制备结果30-31
        3.3.3 LPD凝胶电泳结果31
        3.3.4 LPD的处方优化31-35
        3.3.5 8314-LPD粒径电位测定结果35
        3.3.6 8314-LPD形态学观察结果35-36
        3.3.7 8314-LPD抗酶切能力考察结果36-37
        3.3.8 8314-LPD血清中的稳定性考察结果37-38
        3.3.9 8314-LPD复合物长期稳定性考察结果38
    3.4 讨论38-41
        3.4.1 脂质体制备分析38-39
        3.4.2 PEI/DNA压缩体制备分析39
        3.4.3 LPD的制备分析39-40
        3.4.4 LPD的靶向修饰分析40
        3.4.5 8314-LPD抗酶切能力结果分析40
        3.4.6 8314-LPD血清稳定性结果分析40
        3.4.7 8314-LPD长期稳定性结果分析40-41
    3.5 结论41-42
第四章 8314-LPD的体外细胞实验42-48
    4.1 实验材料42-43
        4.1.1 仪器设备42
        4.1.2 试剂42
        4.1.3 细胞株42-43
    4.2 实验方法43-45
        4.2.1 细胞复苏与传代43
        4.2.2 细胞转染实验43-44
        4.2.3 MTT法检测细胞活性44-45
    4.3 实验结果45-46
        4.3.1 细胞转染实验结果45
        4.3.2 MTT法检测细胞活性结果45-46
    4.4 讨论46-47
        4.4.1 细胞转染实验结果分析46
        4.4.2 MTT法检测细胞活性结果分析46-47
    4.5 结论47-48
第五章 8314-LPD的冷冻干燥实验48-53
    5.1 实验材料48
        5.1.1 仪器设备48
        5.1.2 试剂48
    5.2 实验方法48-49
        5.2.1 冻干保护剂的选择48-49
        5.2.2 冻干粉针剂的制备49
        5.2.3 冻干粉的质量考察49
    5.3 实验结果49-51
        5.3.1 冻干保护剂的选择结果49-50
        5.3.2 冻干粉复溶后粒径电位考察结果50
        5.3.3 冻干粉复溶后抗酶切能力考察结果50
        5.3.4 冻干粉复溶后转染率考察结果50-51
    5.4 讨论51-52
        5.4.1 冻干保护剂选择结果分析51
        5.4.2 冻干前后粒径电位结果分析51
        5.4.3 冻干粉复溶后抗酶切能力结果分析51
        5.4.4 冻干前后转染效能分析51-52
    5.5 结论52-53
第六章 结论53-55
参考文献55-59
综述59-67
个人简历67-68
致谢68
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