9型腺相关病毒经圆窗膜转导小鼠耳蜗两种方式的对比研究
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摘要
目的通过圆窗膜显微注射和完整圆窗膜途径转导小鼠内耳携带GFP的9型腺相关病毒(adeno-asso-ciated virus,AAV),比较两种方式的可行性、转染效率以及对听力的影响。方法 18只C57BL/6小鼠,随机分为圆窗膜显微注射组和完整圆窗膜途径组,术前均行听性脑干反应(auditory brain response, ABR)测听,术后两周行ABR测听后取双侧耳蜗基底膜铺片及冰冻切片观察GFP表达情况。结果两种转导方法对小鼠听力均无显著影响,圆窗膜显微注射组转染耳蜗内毛细胞GFP可见表达,转染效率由底转到顶转逐渐降低,无毛细胞损伤,经完整圆窗膜途径转染耳蜗未见GFP表达。结论 9型AAV难以通过完整圆窗膜,通过显微注射方式能够将目的基因高效转导内耳,对内毛细胞有选择特异性,且对听力影响较小,是一种安全有效的内耳转导方式。
Objective To compare the feasibility, transfection efficiency and impact on hearing between trans-round window membrane(RWM) microinjection and intact RWM delivery of GFP into the inner ear via adeno-associated virus(AAV) type 9. Methods Eighteen C57 BL/6 mice were randomly chosen to receive trans-RWM microinjection or intact RWM delivery. Auditory brain response(ABR) thresholds were determined before and two weeks after operation. Animals were sacrificed and specimens collected for examination of GFP expression. Results Neither method had deleterious effect on hearing. High levels of GFP expression in inner hair cells were seen only in the microinjection group, with transfection efficiency gradually decreasing from the base to the apex. There was no hair cell damage. Conclusion AAV type 9 is hard to deliver through the intact RWM. Trans-round window membrane microinjection is a safe and efficient delivery approach into the inner ear. AAV type 9 shows high specificity to inner hair cells with little impact on hearing.
Objective To compare the feasibility, transfection efficiency and impact on hearing between trans-round window membrane(RWM) microinjection and intact RWM delivery of GFP into the inner ear via adeno-associated virus(AAV) type 9. Methods Eighteen C57 BL/6 mice were randomly chosen to receive trans-RWM microinjection or intact RWM delivery. Auditory brain response(ABR) thresholds were determined before and two weeks after operation. Animals were sacrificed and specimens collected for examination of GFP expression. Results Neither method had deleterious effect on hearing. High levels of GFP expression in inner hair cells were seen only in the microinjection group, with transfection efficiency gradually decreasing from the base to the apex. There was no hair cell damage. Conclusion AAV type 9 is hard to deliver through the intact RWM. Trans-round window membrane microinjection is a safe and efficient delivery approach into the inner ear. AAV type 9 shows high specificity to inner hair cells with little impact on hearing.
引文
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19杨仕明.小型猪动物模型在耳科学领域的应用[J].中华耳科学杂志,2016,14(1):1-5.YANG SM.The Miniature Pig as an Animal Model in Otological Research.Chinese Journal of Otology,2016,14(1):1-5.
20任丽丽.白化荣昌猪耳聋的分子病理机制研究[D].中国人民解放军医学院.Ren Lili.Probing the Molecular Pathological Mechanism Underlying the Deafness in Albino Rongchang Swine[D].Chinese PLAMedical School,2013.
2 Dalkara D,Goureau O,Marazova K,et al.Let There be Light:Gene and Cell Therapy for Blindness[J].Human Gene Therapy,2016,27(2).
3 Lalwani A K,Walsh B J,Reilly P G,et al.Development of in Vivo Gene Therapy for Hearing Disorders:Introduction of Adeno-associated Virus Into the Cochlea of the Guinea Pig.[J].Gene Therapy,1996,3(7):588-92.
4 Ryan A F,Mullen L M,Doherty J K.Cellular Targeting for Cochlear Gene Therapy[J].Adv Otorhinolaryngol,2009,66(66):99-115.
5 Isgrig K,Shteamer J W,Belyantseva I A,et al.Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome[J].Molecular Therapy,2017,25(3):780-791.
6张世丽,祖勉,冀飞,等.半胱氨酸链蛋白β在内耳中的表达及其与听功能的相关性研究[J].中华耳科学杂志,2017(6).ZHANG SL,ZU M,JI FEI,et al.Expression of Cysteine String Protein Beta in the Inner Ear and Its Correlation with Hearing Function.Chinese Journal of Otology,2017(6).
7徐延军,胡吟燕,翟所强,等.耳后入路圆窗膜显微注射小鼠耳蜗基因转染新途径的研究[J].听力学及言语疾病杂志,2009,17(3):279-282.XU YJ,HU YY,ZHAI SQ et al.The Study of a New Approach to Postauricular Microinjection Via the Round Window Membrane for Cochlear Gene Transfection in Mouse.Joural of Audiology and Speech Pathology,2009,17(3):279-282.
8 Akil O,Rouse S L,Chan D K,et al.Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear Through the Round Window Membrane[J].Journal of Visualized Experiments Jove,2015,2015(97):e52187-e52187.
9陈伟,杨仕明,郭维,等.腺病毒携带EGFP基因经完整圆窗膜转导豚鼠耳蜗的实验研究[J].中华耳科学杂志,2007,5(2):231-234.CHEN W,YANG SM,GUO W et al.Cochlear Ad-EGFP Expression by Transferred Gene Through an Intact Round Window Membrane in Guinea Pigs.Chinese Journal of Otology,2007,5(2):231-234.
10 Yilai Shu,Yong Tao,Zhengmin Wang,et al.Identification of Adeno-Associated Viral Vectors That Target Neonatal and Adult Mammalian Inner Ear Cell Subtypes[J].Human Gene Therapy,2016,27(9):687.
11 Jero J,Mhatre A N,Tseng C J,et al.Cochlear Gene Delivery Through an Intact Round Window Membrane in Mouse[J].Human Gene Therapy,2001,12(5):539-548.
12 Qiu J,Handa A,Kirby M,et al.The Interaction of Heparin Sulfate and Adeno-associated Virus 2.[J].Virology,2000,269(1):137-147.
13 Nam H J,Lane M D,Padron E,et al.Structure of Adeno-Associated Virus Serotype 8,a Gene Therapy Vector[J].Journal of Virology,2007,81(22):12260.
14 A.N.Salt and K.Hirose.Communication Pathways to and from the Inner Ear and Their Contributions to Drug Delivery.Hearing Research,2017.
15 A.N.Salt,C.Kellner,and S.Hale.Contamination of Perilymph Sampled from the Basal Cochlear Turn with Cerebrospinal Fluid.Hearing Research,Vol.182,no.1,pp.24-33,2003.
16 T.St?ver and M.Y.Y.Raphael.Transduction of the Contralateral Ear After Adenovirus-mediated Cochlear Gene Transfer.Gene Therapy,Vol.7,no.5,pp.377-83,2000.
17 R.Sacheli,L.Delacroix,P.Vandenackerveken,et al.Gene Transfer in Inner Ear Cells:A Challenging Race.Gene Ther.,vol.20,no.3,pp.237-247,2013.
18 J.Suzuki,K.Hashimoto,R.Xiao,et al.Cochlear Gene Therapy with Ancestral AAV in Adult Mice:Complete Transduction of Inner Hair Cells without Cochlear Dysfunction.Sci.Rep.,vol.7,p.45524,2017.
19杨仕明.小型猪动物模型在耳科学领域的应用[J].中华耳科学杂志,2016,14(1):1-5.YANG SM.The Miniature Pig as an Animal Model in Otological Research.Chinese Journal of Otology,2016,14(1):1-5.
20任丽丽.白化荣昌猪耳聋的分子病理机制研究[D].中国人民解放军医学院.Ren Lili.Probing the Molecular Pathological Mechanism Underlying the Deafness in Albino Rongchang Swine[D].Chinese PLAMedical School,2013.