CAJ | 학술논문

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.
목적 관찰각취당계안염/루탁석납미립재체적체내외전염활성,탐토기작위기인치료재체적가행성.방법 채용용액삽층법합성각취당계안염/루탁석복합물(QC/REC)납미립,포과록색형광단향(GFP)기인질립DNA.투사전경、응효전영화흡부실험연구기포과효솔;전염인간암세포HCCLM6,측정기전염솔.통과라라서위장도급기육주사도경급약,연구기체내전염효과급안전성.결과 QC/REC가유효포과질립DNA,형성납미립적직경소우100 nm.각취당계안염、루탁석、질립DNA질량비위1:2:1.5시,120 h적전염솔근100%.체내실험중,QC/REC-DNA납미립위장도화기육급약,균가견도명현형광표체,주요재위화십이지장표체,라서미출현명현독성반응.결론 QC/REC납미립유교호적전염활성화안전성,유망작위기인치료적비병독재체.
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.