壳聚糖季铵盐/累托石纳米粒作为基因载体的转染活性  

In vitro and in vivo studies on quaternized chitosan/rectorite nanocomposites as gene therapy vector

作　　者：裴小锋[1] 李雁[1] 王小英[2] 杜予民[2] 

机构地区：[1]武汉大学中南医院肿瘤科肿瘤生物学行为湖北省重点实验室,430071 [2]武汉大学环境与资源学院

出　　处：《中华实验外科杂志》2010年第1期35-37,共3页Chinese Journal of Experimental Surgery

基　　金：教育部新世纪优秀人才支持计划资助项目（NCET-04-0669）;全国优秀博士学位论文作者专项资金资助项目（200464）

摘　　要：目的 观察壳聚糖季铵盐/累托石纳米粒载体的体内外转染活性,探讨其作为基因治疗载体的可行性.方法 采用溶液插层法合成壳聚糖季铵盐/累托石复合物（QC/REC）纳米粒,包裹绿色荧光蛋向（GFP）基因质粒DNA.透射电镜、凝胶电泳和吸附实验研究其包裹效率;转染人肝癌细胞HCCLM6,测定其转染率.通过裸裸鼠胃肠道及肌肉注射途径给药,研究其体内转染效果及安全性.结果 QC/REC可有效包裹质粒DNA,形成纳米粒的直径小于100 nm.壳聚糖季铵盐、累托石、质粒DNA质量比为1：2：1.5时,120 h的转染率近100%.体内实验中,QC/REC-DNA纳米粒胃肠道和肌肉给药,均可见到明显荧光表达,主要在胃和十二指肠表达,裸鼠未出现明显毒性反应.结论 QC/REC纳米粒有较好的转染活性和安全性,有望作为基因治疗的非病毒载体.Objective To investigate the gene transfection activity of the quaternized chitosan/rectorite （QC/REC） nanocomposites in vitro and in vivo as gene therapy vector and to explore the optimal conditions of transfection.Methods Novel QC/REC nanocomposites were successfully prepared by using a simple technique of solution-mixing intercalation.QC/REC nanocomposites integrating plasmid DNA encoding green fluorescent protein （GFP） at different mass ratios were made by complex coacervation process.The QC/REC-DNA complexes were characterized by x-ray differentiation （XRD）,transmission electron microscopy（TEM）,gel electrophoresis and absorption assay.The optimal chitosan：DNA mass ratio for maximal in vitro and in vivo transfoetion Was studied on human hepatocellular carcinoma cell line HCCLM6 and on BALB/C-nu/nu nude mice via gastric tube foeding and muscular injection.Laser confocal microscopy and flow cytometry were used to detect the GFP expression.The nude mice fed on QC/REC were also observed for any adveme effects.Results QC/REC nanocomposites could effectively pack and condense GFP plasmid DNA and the nanoparticles were less than 100 nm in diameter.When the QC/REC/DNA ratio was 1：2：1.5.the nanoeomposites achieved the best transfoction efficiency of nearly 100% at 120 h.In vivo study showed the GFP expression in the gastrointestinal tract and the muscle tissue with no acute toxicity effects.Conclusion QC/REC nanocomposite is a relatively safe and efficient non-viral vector for gene therapy.

关 键 词：癌 肝细胞 纳米粒 DNA质粒 基因治疗 

分 类 号：R450[医药卫生—治疗学]