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CMC-TAT蛋白转导肽纳米载体体外转染效果评估

ISSN号：1672-2922
期刊名称：《中华耳科学杂志》
时间：0
分类：R764.35[医药卫生—耳鼻咽喉科;医药卫生—临床医学]
作者机构：[1]中国人民解放军总医院耳鼻咽喉头颈外科聋病教育部重点实验室,北京100853, [2]中国科学院国家纳米科学中心,北京100190
相关基金：国家863青年科学家项目（2014AA020510）; 国家自然科学基金面上项目（81470683）; 中国科协创新驱动助力工程（2016CX-QD01）; 国家973计划重大科学研究计划干细胞项目（2012CB967900,其子课题：2012CB967901,2012CB967903）

作者：唐朝颖, 韩维举[1], 邓雄威[2], 王方园[1], 袁硕龙, 杨仕明[1], 吴南[1]

关键词：羧甲基壳聚糖-TAT蛋白转导肽纳米载体, MIRNA, miR96, 内耳转染, O-carboxymethy chitosan-TAT Nanocarriers, miRNA, miR96, Transfection in cochlea

中文摘要：

目的构建羧甲基壳聚糖-TAT蛋白转导肽纳米载体（CTNs）,探究CTNs介导miR96转染293细胞和耳蜗基底膜的效率及安全性,为mi R96在内耳的功能研究奠定基础。方法应用羧甲基化的壳聚糖（CMC）与TAT蛋白转导肽合成CTNs,携带标记花青染料荧光（cy3）的mi R96模拟物（mi R96 mimics）,转染293细胞和耳蜗基底膜,利用激光共聚焦荧光显微镜对293细胞和耳蜗基底膜铺片进行荧光阳性细胞计数,以阳离子脂质体作为对照,评价转染效率,采用MTT试验评价CTNs的安全性。结果成功制备了CTNs,纳米颗粒平均直径约186.6nm,粒径分布集中,无明显聚集现象;CTNs-mi R96-cy3转染293细胞的平均阳性细胞率为32.6%,转染耳蜗基底膜毛细胞的平均阳性细胞率为24.4%,均低于阳离子脂质体-mi R96-cy3;MTT试验示CTNs细胞毒性低于阳离子脂质体。结论 CTNs作为一种新型的纳米载体,可携带miR96成功转染耳蜗基底膜,提示了纳米载体作为miRNA载体的可行性,并且安全性较高,但转染效率不足。

英文摘要：

Objective To construct O-carboxymethy chitosan-TAT nanoparticles（CTNs）, and to explore the efficiency and security of CTNs for mi R96 by transfect 293 cells and cochlear basilar membrane. Method The CTNs were generated with the optimal weight ratio of 1.5：1（CMC： TAT）, which were used for the incorporation with mi R-96 mimisc marked cy3.Transfect 293 cells and cochlear basilar membrane with Lipofectamine-mi R96-cy3 and CTNs-mi R96-cy3 at the concentration of 150 n M with control. The red fluorescence positive cells were counted to evaluate the efficiency of CTNs. The security of CTNs was evaluated by MTT assay. Result CTNs with a size of 186.8 nm and a zeta potential of-30.9 m V were successfully prepared. The transduction efficiency of CTNs-mi R96-cy3 was lower than that of Lipofectamine-mi R96-cy3 in both 293 cells and cochlear basilar membrane. MTT assay showed that the cytotoxicity of CTNs was significantly lower than that of Lipofectamine. Conclusion CTNs can successfully transduct mi R96 in vitro, which reveals the feasibility and security of CTNs as delivery vector for mi RNA in cochlea,however, it＇s transduction efficiency is low.

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