一种用于内耳siRNA转染的新型蛋白载体  

A Novel Protein Carrier for Inner Ear SiRNA Transfection

作　　者：亓卫东 丁大连[2] 曹轶倓 袁芳 QI Weidong;DING Dalian;CAO Yitan;YUAN Fang(Department of Otolaryngology Head and Neck Surgery,Huashan Hospital Fudan University,Shanghai 20040,China;Center for Hearing and Deafness,Department of Communicative Disorders and Sciences,State University of New York at Buffalo,NY 14214,USA)

机构地区：[1]复旦大学附属华山医院耳鼻咽喉头颈外科,上海200040 [2]美国纽约州立大学布法罗分校耳聋及听力研究中心,NY 14214 USA

出　　处：《中华耳科学杂志》2020年第5期932-937,共6页Chinese Journal of Otology

基　　金：国家自然科学基金81470706。

摘　　要：目的通过体外实验与脂质载体LipoFiter^(TM)进行对比,验证小干扰RNA(siRNA)蛋白载体TAT-DRBD载体在耳蜗的转染效率及安全性。并在卡铂南美栗鼠内耳损害实验模型中,通过TAT-TRBD载体携带siRNA特异性下调铜转运蛋白Slc31a1基因的表达,验证对毛细胞的保护。方法离体条件下,应用TAT-DRBD携带Cy3标记的siRNA转染新生SD大鼠耳蜗基底膜,12小时后激光共聚焦显微镜观察;TAT-DRBD及LipoFiter^(TM)载体转染新生SD大鼠耳蜗基底膜48小时后计数毛细胞丢失及损伤情况。南美栗鼠在接受单次腹腔内卡铂注射前24小时左耳通过TAT-DRBD载体行Slc31a1基因特异性siRNA转染,右耳为对照,12天后处死并通过行全耳蜗基底膜铺片计数毛细胞丢失比例。结果在离体耳蜗器官型培养中,TAT-DRBD可高效转染Cy3标记的siRNA进入毛细胞,效率几达100%。新生SD大鼠耳蜗基底膜中段在加入脂质体LipoFiterTM48小时后会导致严重的耳基底膜内、外毛细胞损害及纤毛排列紊乱;TAT-DRBD组却无明显毛细胞丢失及纤毛排列紊乱。在活体南美栗鼠,治疗耳的内毛细胞(IHC)仅在距蜗尖(Apex)45%~85%距离处有不超过20%的毛细胞丢失。而对照组全程均有IHC损害,在距蜗尖65%~75%处有近40%的IHC丢失。结论 TAT-DRBD作为一种新型的基因重组蛋白载体,在离体条件下可有很高的转染效率且无明显耳毒性作用,在内耳转染安全性方面较脂质体有明显优势。通过RNAi手段特异性抑制铜离子跨膜转运蛋白表达可以有效降低卡铂耳毒性。Objective By comparison with liposome transfection vector LipoFiterTM,to verify the efficiency and biosafety of small interfering RNA(siRNA)protein vector TAT-DRBD in cochlea in vitro.In addition,targeted siRNA was transfected by TAT-DRBD into inner ear to down-regulate the expression of the copper transporter Slc31a1 gene to verify the anti-ototoxic effect in carboplatin-induced chinchilla inner ear damage.Methods For in vitro testing,Cy3-labeled siRNA was transfected into the cochlear basilar membrane(BM)in newborn SD rats using TAT-DRBD,and specimens were examined under a confocal laser microscope after 12 hours.TAT-DRBD and LipoFiterTM vectors were used to transfect in the newborn SD rats cochlear organotypic culture followed by counting hair cell loss after 48 hours.In in vivo experiments,siRNA targeted Slc31a1 was transfected into the left inner ear through the round window membrane in chinchillas 24 hours before receiving a single intraperitoneal carboplatin injection(right ear without siRNA treatment as control).Animals were sacrificed 12 days later and hair cell loss was examined.Results TAT-DRBD transfected Cy3 labeled siRNA into hair cells in vitro with an efficiency of almost 100%.Severe inner and outer hair cells damage was seen in the middle turn BM in newborn SD rats 48 hours after transfection,with disorders in cilia arrangement,in animals transfected using LipoFiterTM;but no hair cell loss those transfected using TAT-DRBD.In vivo,IHC loss in the treatment ear was no more than 20%at 45%-85%distance from apex,while IHCs in the control ear were damaged throughout the entire BM with 40%of IHCs lost at 65%-75%distance from apex.Conclusions As a new gene recombination protein carrier,TAT-DRBD shows high in vitro transfection efficiency without obvious ototoxicity,and has obvious advantages over liposomes in the inner ear.Inhibition of copper transporter expression by RNAi can effectively reduce carboplatin ototoxicity.

关 键 词：内耳 小干扰RNA 转染 

分 类 号：R764[医药卫生—耳鼻咽喉科]