树状大分子包裹的纳米金颗粒载体人铁蛋白重链转染自然杀伤细胞92和磁共振成像

发布时间：2018-03-21 17:11

本文选题：树状大分子　切入点：基因转染　出处：《上海医学》2017年09期 　论文类型：期刊论文

【摘要】：目的以聚乙二醇化树状大分子包裹的纳米金颗粒作为非病毒载体,将人铁蛋白重链(FTH1)转染到自然杀伤细胞92(NK92细胞)中,并进行体外细胞MRI检查。方法将第5代聚酰胺-胺树状大分子用聚乙二醇(PEG)修饰之后包裹纳米金颗粒作为非病毒载体,压缩FTH1报告基因然后转染到NK92细胞内。应用磁共振氢谱、紫外分光光度计和透射电子显微镜等对合成的载体材料进行表征;采用琼脂糖凝胶电泳实验确定载体材料与相应基因之间最佳压缩的氮磷比;应用细胞计数试剂盒-8(CCK-8)检测载体材料的毒性;通过测定水动力粒径来确定不同氮磷比的条件下载体材料和基因形成的复合物能否顺利通过细胞膜;应用荧光显微镜和流式细胞仪测定转染效率;应用3.0T的MRI对转染FTH1的NK92细胞进行体外成像。结果合成的聚乙二醇化的包裹纳米金颗粒的树状大分子{(Au~0)_(25)-G5.NH_2-mPEG_(17)}DENPs具有较好的生物相容性,材料细胞毒性较小,在浓度达到3 000nmol/L时细胞存活率仍然能达到60%以上。荧光显微镜和流式细胞仪测定显示,在氮磷比为1∶1、2∶1、5:1、10:1时,转染效率分别为57.8%、64.0%、80.2%、38.3%。在200和500μmol/L枸橼酸铁铵的条件下,MRI检查发现T2加权像(T2WI)的信号均降低,后者更为明显。结论聚乙二醇化树状大分子包裹的纳米金颗粒能够成功地转染目的基因,MRI检查能有效地在体外检测FTH1转染之后的NK92细胞,为MRI活体示踪自然杀伤细胞过继免疫肿瘤治疗奠定了基础。
[Abstract]:Objective to transfect human ferritin heavy chain FTH _ 1 into natural killer 92(NK92 cells by using nano-gold particles encapsulated by polyethylene glycol dendrimers as non-viral carriers. Methods the 5th generation polyamide-amine dendrimers were modified with polyethylene glycol (PEG) and coated with gold nanoparticles as non-viral carriers, then the FTH1 reporter gene was compressed and transfected into NK92 cells. The synthesized carrier materials were characterized by UV spectrophotometer and transmission electron microscope, and the optimum ratio of nitrogen to phosphorus was determined by agarose gel electrophoresis. The cytotoxicity of carrier material was detected by using cell count kit -8 (CCK-8), and whether the download material with different nitrogen and phosphorus ratio and the complex formed by gene could pass through the cell membrane smoothly, the hydrodynamic particle size was measured to determine whether the download material and the complex formed by gene were able to pass through the cell membrane successfully. The transfection efficiency was determined by fluorescence microscope and flow cytometry, and the NK92 cells transfected with FTH1 were imaged by 3.0T MRI in vitro. Results the synthesized poly (ethylene glycol) coated dendrimer {Austenized gold nanoparticles {{Auroethyleneuryleneglycolated] G5.NH2-mPEGG 17} DENPs had good biocompatibility. The cytotoxicity of the material was relatively small, and the cell survival rate was still over 60% when the concentration reached 3,000 nmol / L. fluorescence microscopy and flow cytometry showed that when the ratio of nitrogen to phosphorus was 1: 1, 2: 2, 1: 5: 1 10: 1, The transfection efficiency was 57.8% and 64.0%, respectively. Under the condition of 200 渭 mol/L and 500 渭 mol/L ammonium citrate, the signal of T2-weighted T2WI was decreased. Conclusion Polyethylene Glycol dendrimers encapsulated nanoparticles can be successfully transfected into NK92 cells after transfection of target gene and can effectively detect NK92 cells after FTH1 transfection in vitro. It lays a foundation for the adoptive immunotherapy of MRI tracer natural killer cells.
【作者单位】：上海交通大学附属第一人民医院放射科;东华大学化学化工学院;
【基金】：上海市浦江人才计划(14PJD028) 上海交通大学医工交叉合作项目(YG2015MS31) 市级医院临床技能与临床创新三年行动(16CR3091B) 上海市科学技术委员会科技发展项目资助
【分类号】：R445.2;R730.3

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