用于防龋DNA疫苗粘膜给药的阳离子脂质体载体的研究

发布时间：2018-01-02 01:04

本文关键词：用于防龋DNA疫苗粘膜给药的阳离子脂质体载体的研究　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：基因治疗成功的关键是需要高效、安全和靶向的载体系统,将DNA转移至靶细胞核内进行表达。阳离子脂质体是用于基因治疗中最有前景的非病毒载体系统之一。阳离子脂质体/DNA复合物,也叫脂质复合物,是自组装纳米体系,能将DNA输送至多种类型的细胞中。在本研究中,我们制备了DOTAP/DOPE脂质体,对其与DNA的相互作用进行了理化表征,并研究了它们的体外转染效率。完成的工作包括以下几个方面: (1)薄膜超声法制备DOTAP/DOPE阳离子脂质体,考察确定了此工艺中水化时间与超声时间,并对制得的脂质体的粒径分散性、zeta电位分布、形貌以及初步物理稳定性进行表征。结果表明此制备工艺有较好的重复性。最后制得的阳离子脂质体平均粒径为128.6±4.6 nm,PDI为0.168±0.016,zeta电位为68.7±4.6 mV。在4℃或37℃贮存1个月后,其胶体稳定性较好。 (2)制备了阳离子脂质体/DNA复合物,并考察了复合物的理化性质。粒径和电位测试、琼脂糖凝胶电泳的结果表明在N/P比为1.5以上时,DNA与阳离子脂质体形成了稳定且带正电的复合物。核酸酶酶切实验的结果表明复合物能保护DNA抵御核酸酶的降解。我们还对在生理离子强度下制备的复合物进行粒径和电位考察,结果表明在N/P比为3以上时,复合物带正电,且复合物颗粒有成簇聚集现象。此外,我们对动物实验要求的脂质复合物的制备进行摸索。 (3)通过对CHO和NIH-3T3细胞的MTT细胞毒性和转染实验表明,复合物的细胞毒性较小;阳离子脂质体可以将DNA转移到两种细胞内得到有效的表达,转染的最佳N/P比为1.5到2。在CHO细胞中的转染效率比NIH-3T3细胞中高。综上所述,DOTAP/DOPE阳离子脂质体与DNA通过静电作用自组装形成的复合物,能有效介导基因的体外转染,为后续的研究打下良好的基础。
[Abstract]:Gene therapy is the key to success needs to be efficient, safe and carrier system to the target, will be transferred to the DNA nuclei of the target cells. The cationic liposome is used as one of the most promising nonviral systems in gene therapy. The cationic liposome /DNA complexes, also called lipid complexes, self-assembly nanometer system and DNA can be transported to various types of cells. In this study, we prepared DOTAP/DOPE liposome was prepared, and its interaction on the DNA of physicochemical characterization, and study the transfection efficiency in vitro. Their work includes the following aspects:
(1) preparation of DOTAP/DOPE cationic liposome film ultrasonic method, investigate and determine the process of hydration time and ultrasonic time, and the liposomes prepared by particle size dispersion, zeta potential distribution, morphology and physical stability was investigated. The preliminary results show that the preparation process has good repeatability. Finally prepared the cationic liposomes with an average particle size of 128.6 + 4.6 nm, PDI was 0.168 + 0.016, zeta potential is 1 months 68.7 + 4.6 mV. storage at 4 degrees or 37 degrees Celsius, the colloidal stability is good.
(2) cationic liposome /DNA complexes were prepared, and the effects of physicochemical properties of the complexes. The particle size and zeta potential test, agarose gel electrophoresis results showed that the N/P ratio above 1.5, DNA and cationic liposomes formed stable and positively charged complexes nuclease digestion assay. The results show that the degradation compound can protect DNA against nuclease. We also prepared complexes at physiological ionic strength of the particle size and zeta potential investigation results show that in the N/P ratio above 3, the positively charged complexes, and composite particles aggregated to form. In addition, we on the lipid complex animal experiment requirements of the preparation were explored.
(3) by CHO and NIH-3T3 MTT cell cytotoxicity and transfection experiments showed that the cytotoxicity of small complexes; cationic liposome DNA can be transferred to the two kinds of effective expression in cells transfected with N/P, the best ratio of 1.5 to 2., the transfection efficiency in CHO cells than in NIH-3T3 cells.
In conclusion, DOTAP/DOPE cationic liposomes and DNA complex formed by electrostatic interaction can effectively mediate gene transfection in vitro, and lay a good foundation for subsequent research.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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