羟基磷灰石纳米颗粒介导的体外转染及其保存方式的研究
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摘要
目的:研究羟基磷灰石纳米颗粒结合DNA,体外转染细胞的能力。并分析作为基因载体的羟基磷灰石纳米颗粒胶体液的保存方式。
方法:向羟基磷灰石纳米颗粒胶体液中加入一定浓度的CaCl_2溶液,以提供二价阳离子Ca~(2+),修饰羟基磷灰石纳米颗粒;测定修饰后的羟基磷灰石纳米颗粒在不同pH值条件下的Zeta电位;用琼脂糖凝胶电泳和紫外分光光度计分析修饰后的羟基磷灰石纳米颗粒携带、保护DNA的能力,及与结合的DNA解离的方式;通过荧光显微镜的观察,计算羟基磷灰石纳米颗粒携带pEGFP-N3质粒,体外转染Bel7402细胞的转染率。将不同温度环境(-20℃、4℃及室温)保存下的羟基磷灰石用Ca~(2+)修饰,通过琼脂糖凝胶电泳、荧光显微镜,测定不同保存条件下的羟基磷灰石携带DNA的能力,体外转染Bel7402细胞的转染率。
结果:经过Ca~(2+)修饰的羟基磷灰石纳米颗粒在酸性、碱性环境下Zeta电位均为正值,并可有效结合DNA;结合到羟基磷灰石纳米颗粒上的DNA可以用磷酸缓冲液洗脱下来;经过Ca~(2+)修饰的羟基磷灰石纳米颗粒的转染效果明显好于没有修饰的羟基磷灰石纳米颗粒。羟基磷灰石纳米颗粒胶体液经-20℃冰冻保存后,不能通过Ca~(2+)的修饰结合DNA,亦不能进行转染。而未冰冻(4℃及室温)保存的羟基磷灰石纳米颗粒经Ca~(2+)修饰后有结合DNA的能力,并可体外转染细胞。
结论:经过Ca~(2+)修饰的羟基磷灰石纳米颗粒可以作为基因转导的载体。作为基因载体的羟基磷灰石纳米颗粒不能冰冻保存。
Objectives: To study the ability of hydroxyapatitie nanoparticles in binding DNA and transfection in vitro; and to explore the way of hydroxyapatitie preservation when it serves as gene carrier.
Method: CaCl_2 solution was added into hydroxyapatitie suspension (offering Ca(2+) for hydroxyapatitie modification), and the Zeta potentials were tested at different pH. The ability of modified hydroxyapatitie nanoparticles in absorbing and protecting DNA, and the way of dissociation from DNA were analyzed by agarose gel electrophoresis analysis and ultraviolet spectrophotometer. Subsequently, the modified hydroxyapatitie were used to transfer plasmid pEGFP-N3 into Bel7402 cells. Hydroxyapatitie gel preserved at different temperatures (-20℃, 4℃and room temperature) were modified with Ca~(2+), and then the abilities of carrying DNA were tested by agarose gel electrophoresis analysis and fluorescence microscope, finally they were used in Bel7402 cells transfection.
Results: The Zeta potentials of hydroxyapatitie nanoparticle with Ca~(2+) surface modification were positive in both acidic and alkaline environment, and the modified hydroxyapatitie can bind DNA efficiently. The absorbed DNA can be washed off by phosphate buffer solution. The transfection efficiency of modified hydroxyapatitie is much higher than that of the hydroxyapatitie without modification. The hydroxyapatitie suspension that were preserved in -20℃cannot bind DNA or transfer plasmid into cells by Ca~(2+) modification. However, preserved in 4℃and room temperature, hydroxyapatitie nanoparticles modified with Ca~(2+) can bind DNA and transfer target cells.
Conclusions: Hydroxyapatitie nanoparticles with Ca~(2+) surface modification can be utilized as DNA carrier for gene delivery. As the gene carriers, hydroxyapatitie nanoparticles cannot be preserved by freezing.
方法:向羟基磷灰石纳米颗粒胶体液中加入一定浓度的CaCl_2溶液,以提供二价阳离子Ca~(2+),修饰羟基磷灰石纳米颗粒;测定修饰后的羟基磷灰石纳米颗粒在不同pH值条件下的Zeta电位;用琼脂糖凝胶电泳和紫外分光光度计分析修饰后的羟基磷灰石纳米颗粒携带、保护DNA的能力,及与结合的DNA解离的方式;通过荧光显微镜的观察,计算羟基磷灰石纳米颗粒携带pEGFP-N3质粒,体外转染Bel7402细胞的转染率。将不同温度环境(-20℃、4℃及室温)保存下的羟基磷灰石用Ca~(2+)修饰,通过琼脂糖凝胶电泳、荧光显微镜,测定不同保存条件下的羟基磷灰石携带DNA的能力,体外转染Bel7402细胞的转染率。
结果:经过Ca~(2+)修饰的羟基磷灰石纳米颗粒在酸性、碱性环境下Zeta电位均为正值,并可有效结合DNA;结合到羟基磷灰石纳米颗粒上的DNA可以用磷酸缓冲液洗脱下来;经过Ca~(2+)修饰的羟基磷灰石纳米颗粒的转染效果明显好于没有修饰的羟基磷灰石纳米颗粒。羟基磷灰石纳米颗粒胶体液经-20℃冰冻保存后,不能通过Ca~(2+)的修饰结合DNA,亦不能进行转染。而未冰冻(4℃及室温)保存的羟基磷灰石纳米颗粒经Ca~(2+)修饰后有结合DNA的能力,并可体外转染细胞。
结论:经过Ca~(2+)修饰的羟基磷灰石纳米颗粒可以作为基因转导的载体。作为基因载体的羟基磷灰石纳米颗粒不能冰冻保存。
Objectives: To study the ability of hydroxyapatitie nanoparticles in binding DNA and transfection in vitro; and to explore the way of hydroxyapatitie preservation when it serves as gene carrier.
Method: CaCl_2 solution was added into hydroxyapatitie suspension (offering Ca(2+) for hydroxyapatitie modification), and the Zeta potentials were tested at different pH. The ability of modified hydroxyapatitie nanoparticles in absorbing and protecting DNA, and the way of dissociation from DNA were analyzed by agarose gel electrophoresis analysis and ultraviolet spectrophotometer. Subsequently, the modified hydroxyapatitie were used to transfer plasmid pEGFP-N3 into Bel7402 cells. Hydroxyapatitie gel preserved at different temperatures (-20℃, 4℃and room temperature) were modified with Ca~(2+), and then the abilities of carrying DNA were tested by agarose gel electrophoresis analysis and fluorescence microscope, finally they were used in Bel7402 cells transfection.
Results: The Zeta potentials of hydroxyapatitie nanoparticle with Ca~(2+) surface modification were positive in both acidic and alkaline environment, and the modified hydroxyapatitie can bind DNA efficiently. The absorbed DNA can be washed off by phosphate buffer solution. The transfection efficiency of modified hydroxyapatitie is much higher than that of the hydroxyapatitie without modification. The hydroxyapatitie suspension that were preserved in -20℃cannot bind DNA or transfer plasmid into cells by Ca~(2+) modification. However, preserved in 4℃and room temperature, hydroxyapatitie nanoparticles modified with Ca~(2+) can bind DNA and transfer target cells.
Conclusions: Hydroxyapatitie nanoparticles with Ca~(2+) surface modification can be utilized as DNA carrier for gene delivery. As the gene carriers, hydroxyapatitie nanoparticles cannot be preserved by freezing.
引文
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6.Vijayanathan V,Thomas T,Thomas TJ.DNA nanoparticles and development of DNA delivery vehicles for gene therapy[J].Biochem,2002,41(48):14085-14094.
7.He XX,Wang K,Tan W,et al.Bioconjugated nanoparticles for DNA protection from cleavage[J].J Am Chem Soc,2003,125(24):7168-7169.
8.Dobson J.Gone therapy progress and prospects:magnetic nanoparticle-based gone delivery[J].Gone Ther,2006,13(4):283-287.
9.Bisht S,Bhakta G,Mitra S,et al.pDNA loaded calcium phosphate nanoparticles:highly efficient non-viral vector for gene delivery[J].Int J Pharm,2005,288(1):157-168.
10.Zhu shaihong,Huang Boyun,Zhou Kechao,et al.Hydroxyapatite Nanoparticles as a Novel Gene Carrier[J].Journal of Nanoparticle Research,2004,6(2):307-311.
11.MT Madigan,JM Martinko,J Parker.Biology of Microorganisms.8th.New Jersey:Prentice-Hall,Inc.,1997.
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15.Dower WJ,Miller JF,Ragsdale CW.High efficiency transformation of E.coli by high voltage electroporation[J].Nucl Acids Res,1988,16:6127-6145.
16.Davies J,Smith DI.Plasmid-determined resistance to antimicrobial agents[J].Annd Rev Microbiol,1978,32:469-518.
17.J 萨姆布鲁克,EF 弗里奇,T 曼尼阿蒂斯,著.黄培堂,等译.分子克隆实验指南.第三版,北京:科学出版社,2002:26-35.
18.Kneuer C,Sameti M,Haltner EG,et al.Silica nanoparticles modified with aminosilanes as carriers for plasmid DNA[J].Int J Pharm,2000,196:257-261.
19.Zhang Y,Kohler N,Zhang M.Surface modification of super-paramagnetic magnetite nanoparticles and their intracellular uptake[J].Biomaterials,2002,23:1553-1561.
20.Roy I,Mitra S,Maitra A,et al.Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery[J].Int J Pharm,2003,250:25-33.
21.曹宁,李木森,沈翔,等.纳米羟基磷灰石粉体的合成与分散技术进展[J].生物骨科材料与临床研究,2006,3(6):48-50.
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