目的:初步探索壳聚糖/siRNA纳米颗粒应用于炎症性肠病的可行性.方法:应用直接混合法将壳聚糖溶液与siRNA照N/P比为60混合形成壳聚糖/siRNA纳米颗粒,采用Zetasizer粒度仪对纳米颗粒直径进行检测,扫描电镜观察其形态以及透射电镜观察其结构;将纳米粒与RNA酶孵育后采用2%琼脂糖凝胶电泳分析其保护作用;采用细菌脂多糖(lipopolysaccharides,LPS)对小鼠巨噬细胞系RAW264.7进行炎症因子诱导后进行体外实验,荧光显微镜观察其转染效率,采用针对TNFα的siRNA进行转染,TaqMan荧光定量PCR和ELISA对TNFα的表达进行定量分析;利用MTT方法对转染后的细胞进行活力测试.结果:壳聚糖/siRNA纳米粒形态较为均一,粒径在200 nm左右;壳聚糖能够与siRNA形成可逆性结合,保护siRNA不被酶降解长达120 min以上;该纳米粒能够高效率的实现对巨噬细胞的转染;纳米粒转染后对LPS诱导巨噬细胞分泌大量的TNFα起到显著的抑制作用,效率高达50%以上,且细胞活性没有明显改变.结论:壳聚糖/siRNA纳米粒系统能够有效的转染巨噬细胞,实现对炎症因子TNFα的抑制作用,为炎症性肠病的分子治疗提供了一定的参考价值.%Objective: To explore the possible utilization of chitosan/siRNA in the inflammatory bowel disease. Methods: The chitosan molecules were mixed with siRNA directly at the N/P ratio of 60 to formulate the chitosan/siRNA nanoparticles (NPs). The hydro diameter was measured by Zetasizer while the morphology and structure were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM) respectively. The NPs were incubated with RNA enzyme followed separation in PAA and analyzed by 2% agarose electrophoresis. The LPS (10 μg/mL) was used to induce murine macrophages for 24 h and the NPs transfection was performed. The lfuorescence microscope (FM) was used to observe the transfection effciency. Atfer transfection of siRNA targeting TNFα, the TaqMan RT-PCR and ELISA kit were used to measure the TNFα expression. In the meanwhile, the cells viability was assessed by MTT assay.Results: The NPs were homogeneous with the average size of 200 nm. It bonded with siRNA reversibly and could protect siRNA from enzyme degradation for more than 120 minutes. A high transfection effciency was obtained in macrophages and TNFα expression was down regulated to more than 50% both in the mRNA level and protein level without apparent cytotoxicity.Conclusion:The chitosan/siRNA NPs can effectively transfect macrophages to suppress the TNFα expression, which may provide new insight into inlfammatory bowel disease molecule therapy.
展开▼
