肝靶向免疫基因载体的研究

发布时间：2017-12-31 18:41

本文关键词：肝靶向免疫基因载体的研究　出处：《郑州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 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。
[Abstract]:This study developed a novel non viral gene delivery system. By using polycation PEI compression plasmid DNA, then liposome PEI/pDNA with PEG of a compression body, formation of lipid composite carrier LPD, and monoclonal antibody to human insulin receptor (8314-SA) and lipid in DSPE-PEG2000-biotin biotin groups (biotin) conjugate formed by monoclonal antibody (MAb) targeting composite carrier group (8314-LPD), MAb improved the composite carrier targeting ability of.8314-LPD is a liver targeting gene vector four yuan long circulating preparation. The specific research contents and results such as (?)
First transformed Escherichia coli amplification of plasmid DNA containing reporter gene (PEGFP-C1), then QIAGEN endotoxin free Maxi kit extraction of plasmid DNA, and to evaluate its quality and concentration. Then prepared liposomes of PEG (lipid formulation for POPC 94%, DDAB 2%, DSPE-PEG2000 3% and DSPE-PEG2000-biotin 1%) PEI/pDNA and polycation compression body, the formation of LPD complexes with the liposome PEI/pDNA compression. The single factor experiment combined with the optimization of prescription LPD uniform design experiments, single factor experiments results are as follows: the plasmid concentration at 40 g below /ml, the particle size changes little, the increase of the concentration of composite particle size increased more than N/P; 1.5, plasmid encapsulation completely, with the increase of N/P composite particle size decreases, the potential increase in N/P is equal to 15, the smallest particle size, increasing the particle size of N/P, basically no change; the molar ratio of lipid /pDNA In 50:1, enzyme digestion electrophoresis showed that the lipid can be completely encapsulated with PEI/pDNA compression, lipid /pDNA ratio increased, the grain size of LPD decreases, the 170:1 potential decreased, the smallest particle size and the molar ratio of lipid /pDNA and then increase the complex size began to become larger. The optimization of LPD compound prescription uniform design experiment results: in HEPES 7 pH buffer, plasmid concentration was 40 g/ml, N/P=15, lipid /pDNA=170:1, LPD prepared by the average particle size is 130 nm, the average zeta potential of 1.5mV. LPD for further structure modification in the outer lipid membrane PEG chain receptor monoclonal antibody conjugated anti human insulin (8314-SA), Lipo/PEI/pDNA complexes modified by monoclonal antibody targeting of the liver (8314-LPD). Transmission electron microscope, the liver targeting of 8314-LPD complex spherical, regular shape, no adhesion; the particle size of potential analysis, the average particle size At about 150nm, the potential is about 4.5mv. The green fluorescent protein gene PEGFP-C1 as a reporter gene study complex on plasmid protection ability analysis experiments prove that this gene vector can effectively protect plasmid DNA from DNA enzyme gel electrophoresis (?) Nase 1) degradation; experimental serum stability indicated that 8314-LPD complex can exist stably in the serum; long-term stability experiment results, 4 degrees and 25 degrees of nitrogen sealed storage, complex size has increased, but the extent of different description of temperature on stability of composites with obvious effect, 25 C storage will make the complex size occurred within a month of very large increases.
Analysis of cell transfection experiments in vitro cytotoxicity and transfection efficiency of LPD vector, less than PEI/pDNA compression, but the monoclonal antibody modified 8314-LPD gene vector can well be human hepatocellular carcinoma cell SMMC-7721 uptake, compared with the previous two group gene vector, transfection rate showed that LPD and 8314.-LPD complexes increased obviously.MTT experiment the toxicity of less than PEI/pDNA polyoxocations compression body.
In order to improve the long-term stability of the 8314-LPD complex, by freeze drying method to prepare freeze-dried powder. The specific steps, the first selection of quality cryoprotector, through the investigation of freeze-dried powder of appearance, color, surface refinement, dispersing ability, found that the lyophilized preparation of trehalose appearance does not collapse, no bubble, uniform color, delicate texture; after rehydration, dispersing time is short; after reconstitution, composite particle size and Zeta potential did not change, is the best protection agent carrier. Secondly, to evaluate the quality of freeze-dried powder, the freeze-dried powder dissolved after anti enzyme digestion experiment, found by lyophilization preparation after rehydration can still maintain the nuclease resistance, indicating vector structure in freeze drying process have not been damaged. Transfection experiments showed that the freeze-dried carrier dissolved after the transfection activity is basically no change.
The results of this study show that the 8314-LPD composite preparation method is simple and feasible and can effectively protect the plasmid DNA by nuclease degradation, serum complexes stability and cell transfection rate are high, low cytotoxicity, and can be prepared by freeze drying properties of stable long term preservation of freeze-dried powder of long cycle. The liver immune gene carrier to the active target, comprehensive utilization of the advantages of cationic polymer and liposome, overcomes two shortcomings, I believe in the gene therapy of liver cancer, there will be good prospects for development.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【引证文献】