内吞相关蛋白Myosin Ⅵ/Disabled-2在小鼠耳蜗毛细胞的表达及年龄相关性变化的研究

陈知己; 顾翔; 蔡婷; 宋锐; 周小清; 袁伟

doi:10.13201/j.issn.1001-1781.2015.07.018

内吞相关蛋白Myosin Ⅵ/Disabled-2在小鼠耳蜗毛细胞的表达及年龄相关性变化的研究

- 第三军医大学附属西南医院耳鼻咽喉-头颈外科(重庆, 400038)
基金项目:
国家自然科学基金资助项目(No:81470694

81271080

30973301)

重庆市自然基金资助项目(No:CSTC2009BB5170)

第三军医大学基金项目(No:2010XLC06)

详细信息

通讯作者: 袁伟,E-mail:weiyuan175@sina.com

中图分类号: R945.7

收稿日期: 2014-11-24

The relation between age and expression of Myosin Ⅵ and Disabled-2 in the cochlea of mice

- Department of Otolaryngology Head and Neck Surgery, Southwest Hospital, the 3rd Military Medical University, Chongqing, 400038, China

More Information

Corresponding author: YUAN Wei, E-mail: weiyuan175@sina.com

Received Date: 24 November 2014

摘要

摘要: 目的:研究不同年龄小鼠耳蜗毛细胞内吞相关蛋白MyosinⅥ和Disabled-2(Dab2)的表达及分布特点。方法:分别选取出生后2周、5周、9周及16月龄的昆明小鼠各10只,采用免疫荧光标记联合激光共聚焦显微镜扫描耳蜗基底膜铺片,观察MyosinⅥ及Dab2蛋白的表达定位,通过荧光定量逆转录PCR测定并比较不同年龄组小鼠耳蜗2种蛋白mRNA的表达水平。结果:MyosinⅥ及Dab2蛋白在内外毛细胞均有表达,支持细胞未见表达,且内毛细胞2种蛋白均存在局部高表达的亚细胞区域。比较不同年龄组MyosinⅥ荧光染色图像后发现,2周龄和16月龄小鼠荧光强度均低于5周龄和9周龄小鼠。比较不同年龄组小鼠2种蛋白mRNA表达后发现,2周龄小鼠mRNA相对表达水平均低于9周龄小鼠,差异有统计学意义(P<0.01),5周龄及16月龄组小鼠分别与9周龄小鼠相比,差异均无统计学意义(P>0.05)。结论:调控网格蛋白介导内吞过程的MyosinⅥ及Dab2蛋白表达于内外毛细胞,内毛细胞顶区表皮板及附近胞质可能是这一过程的主要部位。随着年龄的增长,MyosinⅥ及Dab2蛋白的表达可能会发生改变,网格蛋白介导内吞过程效率可能因此相继发生变化,内毛细胞功能损伤可能与此有关。
- 内毛细胞 /
- 网格蛋白介导内吞 /
- 囊泡 /
- 激光共聚焦显微镜扫描技术
Abstract: Objective:To investigate the expression of MyosinⅥ and Disabled-2 (Dab2) in the cochlea of mice at different ages.Method:Forty KM mice were divided into four groups according to age, named as postnatal 2 week (P2w), P5w, P9w, P16month. The localization of protein in the basilar membrane of mice cochlea was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). The mRNA expression level of protein in cochlear at different ages was evaluated by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Statistical analysis was performed by the SPSS18.0 software.Result:MyosinⅥ and Disabled-2 protein mainly expressed at the apical cytoplasm of hair cells. As for the inner hair cell, Dab2 labeling was abundant especially at the cuticular plate and nearby. Comparing four immunofluorescence staining images of MyosinⅥ, we found the fluorescence intensity of P2w and P16m were weaker than that of P5w and P9w. After setting P9w as the control group, qRT-PCR revealed that the mRNA expression of MyosinⅥ and Dab2 in P2w was less than that in the control group (P<0.01), while no significant difference was found between P5w and the control group, nor between P16m and the control group (P>0.05).Conclusion:MyosinⅥ and Dab2, two proteins which regulated the clathrin-mediated endocytosis, expressed at hair cells of mice cochlea. In the inner hair cell, this process of endocytosis may be more efficient at the cuticular plate and nearby. The expression level of protein may change in different ages, and this probably leads to a difference of CME, it also may cause a defect of inner hair cells function.
- inner hair cell /
- clathrin-mediated endocytosis /
- vesicle /
- laser scanning confocal microscope

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参考文献(16)

[1]	MCMAHON H T,BOUCROT E.Molecular mechanism and physiological functions of clathrin-mediated endocytosis[J].Nat Rev Mol Cell Biol,2011,12:517-533.
[2]	DANCE A L,MILLER M,SERAQAKI S,et al.Regulation of Myosin-Ⅵtargeting to endocytic compartments[J].Traffic,2004,5:798-813.
[3]	骆晓琴,郭旭尧,陈林,等.枸橼酸西地那非对小鼠听觉的影响[J].中华耳鼻咽喉头颈外科杂志,2014,49(6):483-487.
[4]	LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25:402-408.
[5]	GRIESINGER C B,RICHARD C D,ASHMORE J F.Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse[J].Nature,2005,435:212-215.
[6]	GRIESINGER C B,RICHARDS C D,ASHMORE J F.FM1-43reveals membrane recycling in adult inner hair cells of the mammalian cochlea[J].J Neurosci,2002,22:3939-3952.
[7]	TUMBARELLO D A,KENDRICK-JONES J,BUSS F.Myosin VI and its cargo adaptors-linking endocytosis and autophagy[J].J Cell Sci,2013,126(Pt 12):2561-2570.
[8]	KNEUSSEL M,WAGNER W.Myosin motors at neuronal synapses:drivers of membrane transport and actin dynamics[J].Nat Rev Neurosci,2013,14:233-247.
[9]	SEILER C,BEN-DAVID O,SIDI S,et al.Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish[J].Dev Biol,2004,272:328-338.
[10]	BUSS F,KENDRICK-JONES J.Multifunctional Myosin VI has a multitude of cargoes[J].Proc Natl Acad Sci USA,2011,108:5927-5928.
[11]	ROUX I,HOSIE S,SAFIEDDINE S,et al.Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses[J].Hum Mol Genet,2009,18:4615-4628.
[12]	李采,吴琍雯,迟放鲁.不同日龄小鼠听性脑干反应和静纤毛形态的发育特性[J].临床耳鼻咽喉科杂志,2003,17(4):224-225.
[13]	李春晖,杜波,丁大连.小鼠耳蜗的形态学发育过程[J].解剖科学进展,2008,14(2):148-150.
[14]	赵玉林,董明敏,董民声.小鼠听力发育学的研究[J].听力学及言语疾病杂志,1998,6(3):143-146.
[15]	JIANG Y,PRUNIER C,HOWE P H.The inhibitory effects of Disabled-2(Dab2)on Wnt signaling are mediated through Axin[J].Oncogene,2008,27:1865-1875.
[16]	MUNNAMALAI V,FEKETE D.Wnt signaling during cochlear development[J].Semin Cell Dev Biol,2013,24:480-489.