壳聚糖-肠三叶因子纳米粒制备与体外转染的实验研究  

Experiment of preparation of chitosan-ITF nanoparticles and transfection in vitro

作　　者：孙勇[1,2] 王良喜[1,2] 孙曙光[1,2] 毛学飞[1,2] 邓向东[1,2] 潘晓峰[1,2] 张方[1,2] 陈宝君[1,2] 勒娟[1,2] 

机构地区：[1]徐州医学院附属淮海医院 [2]中国人民解放军第九七医院烧伤科,江苏徐州221004

出　　处：《南京医科大学学报（自然科学版）》2014年第8期1034-1039,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81100252);南京军区医学科研课题重点项目(09Z007);中国人民解放军第九七医院院管课题(YN2012027)

摘　　要：目的:构建hITF基因传递系统,检测其细胞转染效果。方法:首先构建肠三叶因子(intestinal trefoil factor,ITF)的真核表达载体,再利用复凝法制备壳聚糖纳米粒,用透射电镜观察纳米粒的形态及大小,并利用HEK293细胞检验纳米粒的转染能力,最后利用RT-PCR和Western blot检测目的基因的转录及表达情况。结果:成功构建出hITF真核表达载体,利用复凝法制备了不同N/P比的壳聚糖纳米粒,透射电镜观察到粒度<1 000 nm,粒径呈窄分布;荧光显微镜及流式细胞仪检测显示,壳聚糖纳米粒对HEK293细胞的转染率约为80%,和脂质体的转染率基本一致;RT-PCR和Western blot检测说明hITF被成功转录并分泌表达。结论:成功构建了hITF理想的基因传递系统,为壳聚糖介导hITF基因治疗的应用打下基础。Objective：To construct a hITF gene delivery system,and assess its gene transfection efficiency.Methods：ITF eukaryotic expression vector was constructed.Chitosan nanoparticles were prepared by a complex coacervation method,and its size and morphology were assessed using transmission electron microscope（TEM）.Gene transfer capability of nanoparticles was assessed in HEK293 cells.The transcription and expression of hITF were determined by RT-PCR and Western blot.Results：hITF eukaryotic expression vector was successfully constructed.The chitosan nanoparticles were prepared by complex coacervation method with different N / P ratio.Nanoparticle size less than 1 000 nm and narrow distribution of nanoparticle diameter were observed by TEM.The transfection efficiency assessed by fluorescence microscopy and flow cytometry was about 80%,similar to the transfection efficiency of Lipofectamine.RT-PCR and Western blot assay demonstrated that hITF was transfected and expressed successfully.Conclusion：An optimal hITF gene delivery system was successfully constructed.This research laid foundations for further application of hITF gene therapy.

关 键 词：人肠三叶因子 壳聚糖 基因治疗 转染 

分 类 号：Q789[生物学—分子生物学]