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
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摘要
目的研究半胱氨酸链蛋白β(cysteine string proteinβ,CSPβ)在巴马小香猪及出生后不同日龄的C57小鼠内耳组织中的表达,探讨其与听功能的相关性。方法应用耳蜗冰冻切片免疫荧光染色观察CSPβ在内耳中的表达分布,应用基底膜铺片观察CSPβ随生长发育的表达变化。应用ABR检测C57小鼠发育过程中的听力变化。结果采用SPSS23.0统计学软件进行各组间单因素方差分析。结果 CSPβ在成年C57小鼠的螺旋神经节以及内毛细胞中均有表达,而在外毛细胞中没有表达;出生12天之前的C57小鼠耳蜗基底膜内毛细胞中都没有CSPβ的表达,直到出生后第12d,内毛细胞中才出现CSPβ的表达,且随着小鼠日龄的增加,CSPβ的表达信号增强,而在外毛细胞中始终没有出现CSPβ的表达。与C57小鼠不同的是,出生1天的小型猪内毛细胞胞中即有CSPβ表达,但在3排外毛细胞中没有表达;ABR结果显示自小鼠出生第14d开始检测到ABR,随着日龄的增加听力越来越成熟。结论我们推测CSPβ与内毛细胞释放神经递质之间可能存在密切联系,可能是神经递质释放,信息传递以及听觉发生的前提条件之一。
Objective To investigate the expression of cysteine string protein beta(CSPβ) in the inner ear in Bama miniature pigs and its dynamic changes during the development of C57 mice; and to study its correlation with hearing function. Methods The distribution of CSPβ was detected by immunofluorescence on frozen sections and on whole cochlear basilar membrane preparations during different developmental stages of cochlear maturation. Auditory brainstem responses(ABRs) were recorded in developing C57 mice. One-way analysis of variance using the SPSS23.0 software was employed in results analysis. Results CSPβ was localized in the cytoplasm of spiral ganglion cells and inner hair cells rather than in outer hair cells in adult C57 mice. During cochlear maturation, there was no expression of CSPβ in inner hair cells in C57 mice until postnatal day 12. In addition, fluorescence signals increased during development while outer hair cells lacked CSPβ signals all the time. Unlike C57 mice, CSPβ expression was seen in inner hair cells in miniature pigs within 1 day of birth. ABRs were first recorded by postnatal day 14, and a decrease in latency and threshold was observed in the weeks following hearing onset. Conclusion We believe that there may be a close link between CSPβ and neurotransmitter release from inner hair cells, probably a prerequisite for neurotransmitter release, information transmission and hearing onset.
Objective To investigate the expression of cysteine string protein beta(CSPβ) in the inner ear in Bama miniature pigs and its dynamic changes during the development of C57 mice; and to study its correlation with hearing function. Methods The distribution of CSPβ was detected by immunofluorescence on frozen sections and on whole cochlear basilar membrane preparations during different developmental stages of cochlear maturation. Auditory brainstem responses(ABRs) were recorded in developing C57 mice. One-way analysis of variance using the SPSS23.0 software was employed in results analysis. Results CSPβ was localized in the cytoplasm of spiral ganglion cells and inner hair cells rather than in outer hair cells in adult C57 mice. During cochlear maturation, there was no expression of CSPβ in inner hair cells in C57 mice until postnatal day 12. In addition, fluorescence signals increased during development while outer hair cells lacked CSPβ signals all the time. Unlike C57 mice, CSPβ expression was seen in inner hair cells in miniature pigs within 1 day of birth. ABRs were first recorded by postnatal day 14, and a decrease in latency and threshold was observed in the weeks following hearing onset. Conclusion We believe that there may be a close link between CSPβ and neurotransmitter release from inner hair cells, probably a prerequisite for neurotransmitter release, information transmission and hearing onset.
引文
1陈丽平,商秀丽,内毛细胞带状突触结构及功能的研究进展[J].医学综述.2011,17(19):2899-2902Chen LP,Shang XL.Progress of Research on the Structure and Function of Inner Hair Cell Ribbon Synapse[J].Medical Recapitulate,2011,17(19):2899-2902.
2 Dallos P,Corey ME.The role of outer hair cell motility in cochlear tuning[J].Curr Opin Neurobiol,1991,1(2):215-220.
3 Issa NP,Hudspeth AJ.The entry and clearance of Ca2+at individual presynaptic active zones of hair cells from the bullfrog's sacculus[J].Proc Natl Acad Sci USA,1996;93:9527-9532
4 Burgoyne RD,Morgan A.Cysteine string protein(CSP)and its role in preventing neurodegeneration[J].Seminars in Cell&Developmental Biology,2015,40:153-159.
5 Zinsmaier KE.Cysteine-String Protein’s Neuroprotective Role[J].J Neurogenetics,2010,24:120-132.
6 Schmitz F,Tabares L,Khimich D,et al.CSPalpha-deficiency causes massive and rapid photoreceptor degeneration[J].Proc Nat Acad Sci USA,2006,10(3):2926-2931.
7 Chamberlain LH,Burgoyne RD.Cysteine-String Protein:The Chaperone at the Synapse[J].J Neurochem,2000,74:1781-1789.
8 Evans GJ,Morgan A,Burgoyne RD.Tying everything together the multiple roles of cysteine string protein(CSP)in regulated exocytosis[J].Traffic,2003,4(10):653-659.
9 Gundersen CB,Kohan SA,Souda P,et al.Cysteine string proteinβis prominently associated with nerve terminals and secretory organelles in mouse brain[J].Brain Reseacch,2010,1332:1-11.
10 Eybalin M,Renard N,Aure F,et al.Cysteine-string protein in inner hair cells of the organ of Corti:synaptic expression and upregulation at the onset of hearing[J].Eur J Neurosci,2002,15(9)1409-1420.
11 Robertson D.Horseradish peroxidase injection of physiologically characterized afferent and efferent neurones in the guinea pig spiral ganglion[J].Hear Research,1984,15:113-121.
12 Delacroix L,Malgrange B.Cochlear afferent innervation development[J].Hearing Research,2015,330:157-169.
13 Stamataki S,Francis HW,Lehar M,et al.Synaptic alterations a inner hair cells precede spiral ganglion cell loss in aging C57BL6J mice[J].Hearing Research,2006,221(1-2):104-118.
14 Fernandez-Chacon R,Wolfel M,Nishimune H,et al.The synaptic vesicle protein CSP alpha prevents presynaptic degeneration[J].Neuron,2004,42(2):237-251.
15 Huang LC,Barclay M,Lee K,et al.Synaptic profiles during neurite extension,refinement and retraction in the developing cochlea[J].Neural development,2012,7(1):38-55.
16 Puel JL,Uziel A.Correlative development of cochlear actionpotential sensitivity,latency,and frequency selectivity[J].Dev Brain Res,1987,37:179-188.
17赵玉林,董明敏,董民声等.小鼠胚胎及出生后耳蜗发育的组织学研究[J].中华耳鼻咽喉科杂志.1999,34(1):57-58.Zhao YL,Dong MM,Dong MS,et al.Histological study of developmental changes of cochlear in mice embryo and after birth[J].Chinese Journal of Otorhinolaryngology,1999,34(1):57-58.
18杨乐,柳柯,杨仕明等.C57小鼠毛细胞突触带的发育观察[J].中华耳科学杂志.2017,1.15(2):234-239.Yang L,Liu K,Yang SM,et al.Observation of developmental changes of hair cell synaptic ribbons in C57 mice[J].Chinese Journal of Otology,2017,1.15(2):234-239.
19侯赟,郭维维,杨仕明等.小型猪内耳发育形态学观察[J].中华耳科学杂志.2012,10(4):485-488.Hou Y,Guo WW,Yang SM,et al.Developmental of Inner Ear in Miniature pigs[J].Chinese Journal of Otology,2012,10(4):485-488.
20 Guo W,Yi H,Ren LL,Chen L,Zhao L,Sun W,Yang SM.The Morphology and Electrophysiology of the Cochlea of the Miniature Pig.[J].The Anatomicai Record,2015,298:494-500.
21 Gorleku OA,Chamberlain LH.Palmitoylation and testis-enriched expressionof the cysteine-string protein beta isoform.[J].Biochemistry,2010,49(25):5308-5313.
22 Ramakrishnan NA,Drescher MJ,Drescher DG.Direct interaction otoferlin with syntaxin1A,SNAP25,and the L-type voltage-gated calcium channel Cavl1.3[J].JBiol Chem,2009,284(1):1364-1372.
2 Dallos P,Corey ME.The role of outer hair cell motility in cochlear tuning[J].Curr Opin Neurobiol,1991,1(2):215-220.
3 Issa NP,Hudspeth AJ.The entry and clearance of Ca2+at individual presynaptic active zones of hair cells from the bullfrog's sacculus[J].Proc Natl Acad Sci USA,1996;93:9527-9532
4 Burgoyne RD,Morgan A.Cysteine string protein(CSP)and its role in preventing neurodegeneration[J].Seminars in Cell&Developmental Biology,2015,40:153-159.
5 Zinsmaier KE.Cysteine-String Protein’s Neuroprotective Role[J].J Neurogenetics,2010,24:120-132.
6 Schmitz F,Tabares L,Khimich D,et al.CSPalpha-deficiency causes massive and rapid photoreceptor degeneration[J].Proc Nat Acad Sci USA,2006,10(3):2926-2931.
7 Chamberlain LH,Burgoyne RD.Cysteine-String Protein:The Chaperone at the Synapse[J].J Neurochem,2000,74:1781-1789.
8 Evans GJ,Morgan A,Burgoyne RD.Tying everything together the multiple roles of cysteine string protein(CSP)in regulated exocytosis[J].Traffic,2003,4(10):653-659.
9 Gundersen CB,Kohan SA,Souda P,et al.Cysteine string proteinβis prominently associated with nerve terminals and secretory organelles in mouse brain[J].Brain Reseacch,2010,1332:1-11.
10 Eybalin M,Renard N,Aure F,et al.Cysteine-string protein in inner hair cells of the organ of Corti:synaptic expression and upregulation at the onset of hearing[J].Eur J Neurosci,2002,15(9)1409-1420.
11 Robertson D.Horseradish peroxidase injection of physiologically characterized afferent and efferent neurones in the guinea pig spiral ganglion[J].Hear Research,1984,15:113-121.
12 Delacroix L,Malgrange B.Cochlear afferent innervation development[J].Hearing Research,2015,330:157-169.
13 Stamataki S,Francis HW,Lehar M,et al.Synaptic alterations a inner hair cells precede spiral ganglion cell loss in aging C57BL6J mice[J].Hearing Research,2006,221(1-2):104-118.
14 Fernandez-Chacon R,Wolfel M,Nishimune H,et al.The synaptic vesicle protein CSP alpha prevents presynaptic degeneration[J].Neuron,2004,42(2):237-251.
15 Huang LC,Barclay M,Lee K,et al.Synaptic profiles during neurite extension,refinement and retraction in the developing cochlea[J].Neural development,2012,7(1):38-55.
16 Puel JL,Uziel A.Correlative development of cochlear actionpotential sensitivity,latency,and frequency selectivity[J].Dev Brain Res,1987,37:179-188.
17赵玉林,董明敏,董民声等.小鼠胚胎及出生后耳蜗发育的组织学研究[J].中华耳鼻咽喉科杂志.1999,34(1):57-58.Zhao YL,Dong MM,Dong MS,et al.Histological study of developmental changes of cochlear in mice embryo and after birth[J].Chinese Journal of Otorhinolaryngology,1999,34(1):57-58.
18杨乐,柳柯,杨仕明等.C57小鼠毛细胞突触带的发育观察[J].中华耳科学杂志.2017,1.15(2):234-239.Yang L,Liu K,Yang SM,et al.Observation of developmental changes of hair cell synaptic ribbons in C57 mice[J].Chinese Journal of Otology,2017,1.15(2):234-239.
19侯赟,郭维维,杨仕明等.小型猪内耳发育形态学观察[J].中华耳科学杂志.2012,10(4):485-488.Hou Y,Guo WW,Yang SM,et al.Developmental of Inner Ear in Miniature pigs[J].Chinese Journal of Otology,2012,10(4):485-488.
20 Guo W,Yi H,Ren LL,Chen L,Zhao L,Sun W,Yang SM.The Morphology and Electrophysiology of the Cochlea of the Miniature Pig.[J].The Anatomicai Record,2015,298:494-500.
21 Gorleku OA,Chamberlain LH.Palmitoylation and testis-enriched expressionof the cysteine-string protein beta isoform.[J].Biochemistry,2010,49(25):5308-5313.
22 Ramakrishnan NA,Drescher MJ,Drescher DG.Direct interaction otoferlin with syntaxin1A,SNAP25,and the L-type voltage-gated calcium channel Cavl1.3[J].JBiol Chem,2009,284(1):1364-1372.