中国生物工程杂志

Objective: Aim: To prepare chitosan nanoparticlescarrying gene and study its characteristics in vitro and vivo. Methods: 12-ACSs was dissolved in 0.05 M sodium acetate buffer to form a solution of 1 mg/mL and a DNA(plasmid-encoding antisense ECE ) solution of 0.1 mg/mL was dissolved in 25mM 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 was investigated in 16HBE cells and Balb/c mice. Results: 12-ACSs /DNA nanoparticles (100~150nm) were observed by transmission electron microscopy.12-ACSs can protect the plasmid encoding antisence-ECE from 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. Key words: asthma, chitosan, nanoparticle, Endothelin converting enzyme, gene therapy