聚氰基丙烯酸正丁酯纳米粒载NF-κB圈套基因的制备与分析

Preparetion and analysis of polybutycyanocrylate nanoparticles loaded NF-κB decoy gene

目的制备携载NF-κB圈套DNA的聚氰基丙烯酸正丁酯纳米粒(PBCA-NPs),研究其在大鼠脑内的生物学活性。方法选用乳化聚合法制备PBCA-NPs,以激光粒度分析仪及透射电子显微镜分析纳米粒的形态和粒径。用阳离子表面活性剂十六烷基三乙基溴化铵(CTAB)对纳米粒进行表面修饰后连接DNA,并分析PBCA-NPs及CTAB修饰的PBCA-NPs的细胞毒性效应、DNA装载效率、PBCA-NPs-DNA抗超声破坏和抗DNaseI的降解作用及其在体内的生物活性。结果激光粒度分析仪及透射电子显微镜结果表明,PBCA-NPs粒形圆整,大小均匀,平均粒径90nm。琼脂糖凝胶电泳及紫外分光光度计测定DNA装载量达96%。该纳米粒能显著提高DNA抵抗核酸酶降解和超声波剪切的能力。在体试验证明PBCA-NPs-DNA通过侧脑室注射能以较高效能抑制大鼠皮质的NF-κB蛋白活性。结论PBCA-NPs能够与NF-κB圈套DNA结合组成基因转运体系,该体系可以作为研究NF-κB功能的有效工具。

[Objective] To prepare polybutycyanocrylate nanoparticles（PBCA-NPs ） loaded NF-kB decoy gene as the gene delivery system, its biological activity was simultaneously investigated in vivo, [Method] PBCA-NPs were prepared by the emulsion polymerization method. Surface of PBCA-NPs was surveyed by transmission electron micrographs （TEM）, the grain distribution and zeta potentials of PBCA-NPs were determined with the laser grain analyzer. Through surface medification of PBCA-NPs with the cationic surfactant cetyhrimethylammonium bromide （CTAB）, NF-kB decoy gene was loaded on the PBCA-NPs by adsorption. The cytotoxities effect of PBCA -NPs and CTAB modified PBCA -NPs was examined by MTT method. The loading efficiency was determined. The gene protection effect of the PBCA-NPs was investigated by sonication and DNase Ⅰ. NPs-NF-kB decoy gene were used to be injected in lateral cerebral ventricle to investigate its activity. [Result] The mean diameter of optimal PBCA-NPs was 90 nm by laser grain analyzer and TEM analysis, All NPs were uniformity sphericity, superficial smoothness and integrity .size distributing was normal distribution and well-distributed. The DNA loading ratio reached to 96% by agarose gel analysis and UV speetrophotometry analysis. The PBCA-NPs can protect DNA from digestion by DNase Ⅰ and sonication-induced shearing in vitro. PBCA-NPs NF-KB decoy gene can inhibit the activity of NF-KB in upper efficiency in animal experiment. [ Conclusion ] NPs gene transfer made with NF-kB decoy DNA and CTAB-PBCA- NP can inhibit high-potential activity of NF-kB protein, which is essential for NF-kB function study.