12-烷基化壳聚糖纳米粒-反义内皮素转换酶核酸表达质粒的制备及其性质研究  

The Study of 12-alkylated Chitosans/Plasmid-encoding Antisense ECE Complex Nanoparticles in Asthma

作　　者：周雨田[1] 徐军[1] 

机构地区：[1]广州呼吸疾病研究所,广州510120

出　　处：《中国生物工程杂志》2007年第11期20-26,共7页China Biotechnology

基　　金：国家自然科学基金重点课题资助项目(30230180)

摘　　要：目的:气道给予12-烷基化壳聚糖纳米粒(12-ACSs)包裹的反义内皮素转换酶(ECE)核酸表达质粒,观察对OVA致敏的小鼠变应性气道炎症的影响。方法:通过透射电镜观察12-ACSs/反义ECE质粒复合体纳米粒的形成、形态及大小;应用凝胶阻滞、结合平衡、DNA沉淀和DNA酶消化实验等检测12-ACSs对反义ECE核酸表达质粒的结合保护作用;通过MTT实验检测12-ACSs对细胞的毒性;通过离体培养细胞及活体动物转染实验观察12-ACSs能否携带反义ECE核酸表达质粒成功转染。结果:电镜观察显示纳米粒粒径在100～150nm之间。12-ACSs与反义ECE核酸表达质粒在质量比为1:1时,全部反义ECE质粒被结合。应用DNaseI消化后可见,12-ACSs可保护核酸免受破坏。MTT检测结果显示12-ACSs对16HBE细胞在低浓度下几乎没有毒性。12-ACSs包裹的反义ECE核酸表达质粒的纳米粒能成功转染离体培养的气道上皮细胞及活体动物。结论:12-ACSs能够成功包裹反义ECE质粒并且成功转染16HBE及小鼠,其有可能作为一种基因治疗的载体选择之一。vivo. Methods ： Aim： To prepare chitosan 12-ACSs was dissolved in 0. nanoparticlescarrying gene and study its characteristics in vitro and in 05mol/L sodium acetate buffer to form a solution of lmg/ml and a DNA（ plasmid-encoding antisense ECE ） solution of 0.1 mg/ml was dissolved in 25mmol/L Na2SO4. The charge ratio of components is selected as 2/1 for 12-ACSs/DNA complex. The complex was prepared by mixing 12-ACSs solution with DNA solution prior to observation by using transmission electron microscopy. Using Electrophoretic Retardation of DNA Migration detection, DNA precipitation, Binding Equilibration and DNase Resistivity Assay, the formation of 12-ACSs/DNA complex was determined and its stability was simultaneously evaluated. MTT Cell Proliferation Assays was performed on investigation of the cytotoxicity of 12-ACSs /DNA nanoparticles in bronchial epithelial cells （ 16HBE ）. The transfection efficiency of 12-ACSs/DNA nanoparticles in vitro and in vivo were from was investigated in 16HBE cells and Balb/c mice. Results： 12-ACSs/DNA nanoparticles （100 - 150nm） observed by transmission electron microscopy. 12-ACSs can protect the plasmid encoding antisence-ECE degradation by DNase I. 12-ACSs can transfer plasmid-encoding antisense ECE into 16HBE cells and into the airway epithelium in mice, As shown by fluorescent microscopy, green fluorescent protein reporter can be observed in the transfected cells as well as in the airway epithelium of the treated mice. And it showed a lower cell cytotocixity in cultured 16HBE cells and in mice treated with12-ACSs /DNA nanoparticles. Conclusion： In summery,the chitosan can be used as an effective protectant for DNA as well as an enhancer for in vitro gene transfection.

关 键 词：哮喘 壳聚糖 纳米粒 内皮素转换酶 基因治疗 

分 类 号：Q78[生物学—分子生物学]