中华耳科学杂志2016，Vol.14Issue(6)：803-807,5.DOI:10.3969/j.issn.1672-2922.2016.06.020
HA-bPEI纳米颗粒携带Atoh1质粒豚鼠耳蜗转染观察
In vivo transduction of Atoh1 gene into cochlear cells mediated by HA-PEI nanoparticle vectors
摘要
Abstract
Objective To test in vivo transfection efficiency of Atoh1-EGFP plasmids using hyaluronic acid modified branched polyethylenimine nanoparticles as a novel non-viral gene vector. Methods Plasmids were extracted following kit instructions. The gene-vector nanoparticles were synthesized (COOH/N/P=4:10:1) and then characterized before being trans-duced into the inner ear through intact round window membrane. At 7 days after transduction operation, transfection effi-ciency was evaluated by examination of both basilar membrane surface preparation and frozen sections under a confocal mi-croscope. Transfection results were finally verified by Western blot and RT-PCR at protein and nucleic acid levels respec-tively. Results We successfully synthesized a non-viral gene vector which loaded Atoh1 gene well and had a negative-ly-charged surface to reduce possible cytotoxicity. At 7 days after transfection, confocal microscope observation of basilar membrane showed EGFP in OHCs and IHCs. OHC transfection efficiency was 81.7 ± 4.71% in the basal turn and 33.8 ± 9.02%in the middle turn. Frozen section results revealed expression of EGFP mainly in the basal turn and only part of the middle turn of the cochlea. No signs of EGFP transduction were seen in either the top of the cochlea or in cells other than hair cells. Furthermore, Basilar membrane cells showed no obvious damage after operation. Both Western Blot and RT-PCR results verified successful transfection of the Atoh1 gene on the basilar membrane. Conclusion The Atoh1 gene can be suc-cessfully transduced into the inner ear by the HA modified bPEI nanoparticles and there is no obvious cytotoxicity to cochle-ar cells. The new nanoparticles are easy to produce at a low cost, which makes it an ideal non-viral vector for inner ear gene transfection.关键词
透明质酸/分支型聚乙烯亚胺/Atoh1基因/内耳基因转染Key words
Hyaluronic acid/branched Polyetherimide/Atoh1 gene/inner ear gene delivery分类
医药卫生引用本文复制引用
王方园,陈志婷,邓雄威,吴雁,张悦,杨风波,杨仕明,吴南..HA-bPEI纳米颗粒携带Atoh1质粒豚鼠耳蜗转染观察[J].中华耳科学杂志,2016,14(6):803-807,5.基金项目
国家863青年科学家项目(2014AA020510)；国家973计划重大科学研究计划干细胞项目（2012CB967900)；国家科技部新药创制重大专项（2014ZX09J14101-06C）；国家自然科学基金项目（NSFC 81470700）；中国科协创新驱动助力工程（2016CXQD01）Funds by The National High-tech R$D Program of China for young Scientists (2014AA020510), National Basic Research Program of China (973 Program)(#2012CB967900), National Ministry of science and technology new drug creation major project (2014ZX09J14101-06C) （2014AA020510）
National Natural Science Foundation of China (NSFC 81470700)；Innovation and drive power engineering project of China association for science and technology(2016CXQD01) （NSFC 81470700）
