您的位置:山东大学 -> 科技期刊社 -> 《山东大学学报(医学版)》

山东大学学报(医学版) ›› 2015, Vol. 53 ›› Issue (8): 16-22.doi: 10.6040/j.issn.1671-7554.0.2014.665

• 基础医学 • 上一篇    下一篇

Prestin在Spag6基因敲除小鼠外毛细胞中的表达

王镜涵1,2, 李霄飞1,3, 张志兵4, 王海波1,3, 李建峰2,3   

  1. 1. 山东大学附属省立医院耳鼻咽喉-头颈外科, 山东 济南 250021;
    2. 山东大学医学院病理学与病理生理学教研室, 山东 济南 250012;
    3. 山东省耳科学重点实验室, 山东 济南 250021;
    4. 宾夕法尼亚大学生殖医学及女性健康研究中心, 美国宾夕法尼亚州 费城 19104
  • 收稿日期:2014-10-08 发布日期:2015-08-10
  • 通讯作者: 李建峰.E-mail:lijianfeng@hotmail.com E-mail:lijianfeng@hotmail.com
  • 基金资助:
    国家自然科学基金(803300121, 81072200); 山东省自然科学基金(ZR2006GG2202)

Prestin expression in outer hair cells of Spag6 gene knockout mice

WANG Jinghan1,2, LI Xiaofei1,3, ZHANG Zhibing4, WANG Haibo1,3, LI Jianfeng2,3   

  1. 1. Department of Otolaryngology-Head and Neck Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong, China;
    2. Department of Pathology and Pathophysiology, School of Medicine, Shandong University, Jinan 250012, Shandong, China;
    3. Shandong Provincial Key Laboratory of Otology, Jinan 250021, Shandong, China;
    4. Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia 19104, Pennsylvania, USA
  • Received:2014-10-08 Published:2015-08-10

摘要: 目的 探究prestin在精子相关抗原6(Spag6)基因敲除小鼠中的表达及其与SPAG6之间的关系.方法 获取小鼠耳蜗基底膜组织,进行免疫染色,采用共聚焦显微镜观察prestin与SPAG6在毛细胞中的共同分布.比较野生小鼠(Spag6+/+) 、杂合型小鼠(Spag6+/-) 和同窝基因敲除小鼠(Spag6-/-) 外毛细胞prestin的荧光强度差异和毛细胞的形态改变,并以蛋白印记实验和实时定量聚合酶链反应检测3种基因型小鼠的prestin表达水平.结果 SPAG6存在于野生型和杂合型成年小鼠的外毛细胞中,在表皮板处呈阳性表达,与prestin共同分布于外毛细胞的外侧壁处.基因敲除小鼠prestin的荧光强度较野生小鼠更弱,并且外毛细胞出现形态异常.蛋白印迹实验和实时定量聚合酶链反应均显示,基因敲除小鼠prestin表达量显著降低.结论 SPAG6参与外毛细胞的构成,且影响外毛细胞prestin表达,提示两种蛋白之间存在相互作用.

关键词: 精子相关抗原6, 基因敲除小鼠, 外毛细胞, Prestin, 听觉障碍

Abstract: Objective To detect the prestin expression in sperm associated antigen 6 (Spag6) gene knockout mice and its possible relationship with SPAG6. Methods Through a confocal microscope, the isolated basilar membrane was immunostained for observing the distribution of prestin and SPAG6 in hair cells. In particular, the fluorescence intensity of prestin and the morphological alterations of hair cells were compared among wild type mice (Spag6+/+), heterozygous mice (Spag6+/-) and the corresponding Spag6 gene knockout littermates (Spag6-/-). The expressions of prestin protein and mRNA in mice of three genotypes were measured by Western blotting and real-time Q-PCR. Results SPAG6, which was positive at the cuticular plate and co-localized with prestin at the cell lateral wall, expressed in outer hair cells (OHCs) of both wild type (Spag6+/+) and heterozygous (Spag6+/-) adult mice. Compared with wild type (Spag6+/+) mice, gene knockout mice (Spag6-/-) showed the lower fluorescence intensity of prestin and apparent morphological abnormity of OHCs. The expressions of prestin at protein and mRNA levels in gene knockout mice (Spag6-/-) were significantly decreased as evidenced by Western blotting and real-timeQ-PCR. Conclusion SPAG6 participates in the formation of the cell membrane of OHCs and affects the expression of prestin in OHCs, which implies a possible interaction of these two proteins.

Key words: Sperm associated antigen 6, Gene knockout mice, Prestin, Auditory dysfunction, Outer hair cells

中图分类号: 

  • R764
[1] Neilson LI, Schneider PA, Van Deerlin PG, et al. Cdna cloning and characterization of a human sperm antigen (spag6) with homology to the product of the chlamydomonas pf16 locus[J]. Genomics, 1999, 60(3):272-280.
[2] Zhang Z, Sapiro R, Kapfhamer D, et al. A sperm-associated wd repeat protein orthologous to chlamydomonas pf20 associates with spag6, the mammalian orthologue of chlamydomonas pf16[J]. Mol Cell Biol, 2002, 22(22):7993-8004.
[3] Zhang Z, Tang W, Zhou R, et al. Accelerated mortality from hydrocephalus and pneumonia in mice with a combined deficiency of spag6 and spag16l reveals a functional interrelationship between the two central apparatus proteins[J]. Cell Motil Cytoskeleton, 2007, 64(5):360-376.
[4] Hamada T, Teraoka M, Imaki J, et al. Gene expression of spag6 in chick central nervous system[J]. Anat Histol Embryol, 2010, 39(3):227-232.
[5] Sapiro R, Kostetskii I, Olds-Clarke P, et al. Male infertility, impaired sperm motility, and hydrocephalus in mice deficient in sperm-associated antigen 6[J]. Mol Cell Biol, 2002, 22(17):6298-6305.
[6] Lonergan KM, Chari R, Deleeuw RJ, et al. Identification of novel lung genes in bronchial epithelium by serial analysis of gene expression[J]. Am J Respir Cell Mol Biol, 2006, 35(6):651-661.
[7] Steinbach D, Schramm A, Eggert A, et al. Identification of a set of seven genes for the monitoring of minimal residual disease in pediatric acute myeloid leukemia[J]. Clin Cancer Res, 2006, 12(8):2434-2441.
[8] Noushmehr H, Weisenberger DJ, Diefes K, et al. Identification of a cpg island methylator phenotype that defines a distinct subgroup of glioma[J]. Cancer Cell, 2010, 17(5):510-522.
[9] Silina K, Zayakin P, Kalnina Z, et al. Sperm-associated antigens as targets for cancer immunotherapy: expression pattern and humoral immune response in cancer patients[J]. J Immunother, 2011, 34(1):28-44.
[10] Yu N, Zhu ML, Zhao HB. Prestin is expressed on the whole outer hair cell basolateral surface[J]. Brain Res, 2006, 1095(1):51-58.
[11] Zheng J, Shen W, He DZ, et al. Prestin is the motor protein of cochlear outer hair cells[J]. Nature, 2000, 405(6783):149-155.
[12] Zheng J, Madison LD, Oliver D, et al. Prestin, the motor protein of outer hair cells[J]. Audiol Neurootol, 2002, 7(1):9-12.
[13] Liberman MC, Gao J, He DZ, et al. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier[J]. Nature, 2002, 419(6904):300-304.
[14] 张倩, 潭晓东, 贾淑萍, 等. 外毛细胞电致运动的分子基础[J]. 中华耳科学杂志, 2009,7(1):105-108.
[15] Sheppard A, Hayes SH, Chen GD, et al. Review of salicylate-induced hearing loss, neurotoxicity, tinnitus and neuropathophysiology[J]. Acta Otorhinolaryngol Ital, 2014, 34(2):79-93.
[16] Yu L, Jiang XH, Zhou Z, et al. A protective mechanism against antibiotic-induced ototoxicity: role of prestin[J]. PLoS One, 2011, 6(2):e17322. doi: 10.1371/journal.pone.0017322.
[17] 赵宁, 柳柯, 杨仕明, 等. 耳毒性药物对小鼠耳蜗外毛细胞prestin表达的影响[J]. 解剖科学进展, 2013,19(3): 250-254. ZHAO Ning, LIU Ke, YANG Shiming, et al. Ototoxic aminoglycoside exposures causes abnormal expression of prestin in cochlear outer hair cells of mice[J]. Progress of Anatomical Sciences, 2013, 19(3): 250-254.
[18] Xia A, Song Y, Wang R, et al. Prestin regulation and function in residual outer hair cells after noise-induced hearing loss[J]. PLoS one, 2013, 8(12):e82602. doi: 10.1371/journal.pone.0082602. eCollection 2013.
[19] Abrashkin KA, Izumikawa M, Miyazawa T, et al. The fate of outer hair cells after acoustic or ototoxic insults[J]. Hear Res, 2006, 218(1-2):20-29.
[20] 赵向东, 梁勇, 任陈, 等. 不同剂量放射线对小鼠畸变产物耳声发射和外毛细胞prestin蛋白表达的影响[J]. 听力学及言语疾病杂志, 2013, 21(3): 267-271. ZHAO Xiangdong, LIANG Yong, REN Chen, et al. Experimental observation of influence of different doses of radiation on distortion product otoacoustic emission and the prestin expression in mouse outer hair cells[J]. Journal of Audiology and Speech Pathology, 2013, 21(3): 267-271.
[21] Deak L, Zheng J, Orem A, et al. Effects of cyclic nucleotides on the function of prestin[J]. J Physiol, 2005, 563(Pt 2):483-496.
[22] Bai JP, Surguchev A, Ogando Y, et al. Prestin surface expression and activity are augmented by interaction with map1s, a microtubule-associated protein[J]. J Biol Chem, 2010, 285(27):20834-20843.
[23] Wu X, Currall B, Yamashita T, et al. Prestin-prestin and prestin-glut5 interactions in hek293t cells[J]. Dev Neurobiol, 2007, 67(4):483-497.
[24] Holzmann D, Ott PM, Felix H. Diagnostic approach to primary ciliary dyskinesia: a review[J]. Eur J Pediatr, 2000, 159(1-2):95-98.
[25] Yoon H, Lee DJ, Kim MH, et al. Identification of genes concordantly expressed with atoh1 during inner ear development[J]. Anat Cell Biol, 2011, 44(1):69-78.
[26] Li X, Xu L, Li J, et al. Otitis media in sperm-associated antigen 6 (spag6)-deficient mice[J]. PLoS one, 2014, 9(11):e112879. doi: 10.1371/journal.pone.0112879. eCollection 2014.
[27] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method[J]. Methods, 2001, 25(4):402-408.
[28] Hasson T, Heintzelman MB, Santos-Sacchi J, et al. Expression in cochlea and retina of myosin vii a, the gene product defective in usher syndrome type 1b[J]. Proc Natl Acad Sci U S A, 1995, 92(21):9815-9819.
[29] Schwander M, Kachar B, Muller U. Review series: the cell biology of hearing[J]. J Cell Biol, 2010, 190(1):9-20.
[30] Lopez I, Ishiyama G, Tang Y, et al. Regional estimates of hair cells and supporting cells in the human crista ampullaris[J]. J Neurosci Res, 2005, 82(3):421-431.
[31] Raphael Y, Athey BD, Wang Y, et al. F-actin, tubulin and spectrin in the organ of corti: comparative distribution in different cell types and mammalian species[J]. Hear Res, 1994,76(1-2):173-187.
[32] Rajagopalan L, Organ-Darling LE, Liu H, et al. Glycosylation regulates prestin cellular activity[J]. J Assoc Res Otolaryngol, 2010, 11(1):39-51.
[33] Mulaw MA, Krause A, Deshpande AJ, et al. Calm/af10-positive leukemias show upregulation of genes involved in chromatin assembly and DNA repair processes and of genes adjacent to the breakpoint at 10p12[J]. Leukemia, 2012, 26(5):1012-1019.
[1] 马新军. 原发性皮肤腺样囊性癌1例[J]. 山东大学学报(医学版), 2016, 54(12): 95-96.
[2] 杨倩倩,孙高英,曹智新,尹海燕,王海波,李建峰. NLRX1在C57BL/6小鼠耳蜗毛细胞内的表达[J]. 山东大学学报(医学版), 2016, 54(1): 11-16.
[3] 朱发梅, 郑家法, 谢鼎华, 胡鹏. 5个大前庭水管综合征家系SLC26A4基因的检测分析[J]. 山东大学学报(医学版), 2014, 52(10): 90-95.
[4] 张道宫1,樊兆民1,韩月臣1,刘闻闻2,3,白晓卉2,3,李亚伟1,徐磊1,王明明1,罗建芬1,李建峰2,3,王海波1,2,3. AIF和PARP-1在庆大霉素致前庭毛细胞凋亡中的作用[J]. 山东大学学报(医学版), 2013, 51(8): 49-52.
[5] 朱荣誉, 孙勍, 彭新,李娜, 单希征. 梅尼埃病患者的垂直半规管功能分析[J]. 山东大学学报(医学版), 2013, 51(8): 89-94.
[6] 王福俊. 联合鼓室内用药治疗糖尿病伴急性低频感音神经性听力损失[J]. 山东大学学报(医学版), 2012, 50(8): 100-102.
[7] 李树峰,梁琴. 慢性非胆脂瘤中耳炎并耳蜗瘘管[J]. 山东大学学报(医学版), 2011, 49(4): 95-.
[8] 李晓1,王启荣1,刘树伟2,韩飞1,古林涛1,梁辉1,崔朝阳1. 咽鼓管薄层断层解剖及临床应用[J]. 山东大学学报(医学版), 2010, 48(10): 101-104.
[9] 杨颖,夏明,边志强,许安廷 . 高分辨CT正常内耳解剖结构的测量研究[J]. 山东大学学报(医学版), 2008, 46(11): 1075-1079.
[10] 边致强,刘亚群,李笃民,崔凤玉. 多层螺旋CT对婴幼儿先天性感音性耳聋的诊断价值[J]. 山东大学学报(医学版), 2007, 45(12): 1257-1260.
[11] 丁寿玲,夏明,张寒冰,张菊红,杨颖,刘昉,许安廷. RANKL和OPG在胆脂瘤型中耳炎中的表达及其意义[J]. 山东大学学报(医学版), 2007, 45(10): 1034-1037.
[12] . 耳后筋膜-骨膜瓣在乳突充填术中的应用[J]. 山东大学学报(医学版), 2009, 47(7): 107-110.
[13] . 大前庭水管综合征婴幼儿患者ASSR和ABR听力学特点[J]. 山东大学学报(医学版), 2009, 47(8): 89-92.
[14] 张延平1 ,佟明望1,李丽娜1,张晓强1,孙建和2,林曦3. GJB2基因条件敲除小鼠耳蜗扫描电镜观察[J]. 山东大学学报(医学版), 2010, 48(12): 19-22.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!