AP-2蛋白在小鼠耳蜗毛细胞的表达及其调控突触囊泡胞吞的实验研究
发布时间：2018-07-18 15:57
【摘要】：一、研究背景和目的感音神经性耳聋是最常见的听觉残疾,听觉的产生和维持依赖于耳蜗内毛细胞(IHC)带状突触部释放谷氨酸神经递质,由此完成机械-电换能作用[1]。作为听觉传导第一级突触,耳蜗IHC突触发生病理改变是导致感音神经性耳聋的重要原因之一[2,3]。大量研究表明,听功能的减退与带状突触形态、结构和数量的异常变化密切相关[3]。每个IHC与螺旋神经元建立一个突触连接,单个IHC拥有10-30个带状突触。耳蜗IHC带状突触活性区域的快速可释放池(RRP)释放出的神经递质谷氨酸激活位于突触后膜的谷氨酸受体(AMPA),将听觉信息传递给蜗核的对应神经元[4]。可见,耳蜗感受到的绝大部分声信号是靠IHC及其突触完成的。每个RRP有数十个囊泡附着,可在几微秒内释放囊泡,大量囊泡的同步释放可通过兴奋性突触后电位(EPSPs)反映[5]。根据听觉信号传导的特点,IHC突触精确快速地传递神经递质需要正常有效的囊泡循环机制,即囊泡的胞吐和胞吞[6]。突触囊泡循环(recycling)是听觉神经递质持续释放的基础,而突触囊泡精确快速的内吞回收则是囊泡信息持续传递不被耗竭的重要保障。网格蛋白介导的内吞(CME)途径是中枢神经细胞突触囊泡膜回收的重要方式[7],AP-2蛋白为内吞起始阶段识别网格蛋白和协同其它辅助蛋白形成网格蛋白包被小泡(coated pits)的结构核心,它是由α、β2、μ2和σ2四个亚基组成的大的蛋白复合体,α亚基附件区域与质膜形成联系,同时通过DPF或者DPW分子信号与多种调控蛋白和辅助蛋白结合;β2亚基与网格蛋白β螺旋末端结构域结合,还与转运货物(cargo)的选择相关;μ2亚基通过酪氨酸分类信号识别胞质尾区受体[8]。AP-2蛋白在CME内吞起始阶段发挥着重要作用。耳蜗IHC及其传入神经元作为一种特殊的神经突触,AP-2蛋白调控CME途径理论上应该在此区域发挥着重要作用。目前关于AP-2蛋白的研究多局限于它的蛋白组学和它在中枢神经细胞的研究,AP-2蛋白在小鼠耳蜗中的定位表达及功能关系尚不清楚。本研究拟应用免疫荧光双重标记,激光共聚焦显微镜观察ap-2蛋白与带状突触前膜特异性蛋白rebeye/ctbp2在小鼠耳蜗毛细胞中的定位表达特点,探讨其在内耳听觉生理中的可能作用机制。再结合听性脑干反应(abr),荧光定量pcr(qrt-pcr)观察出生后不同发育阶段ap-2蛋白的表达,探讨ap-2蛋白与听功能的发生、形成和年龄相关性听力减退的相关性。最后通过膜片钳电生理方法,采用tyrphostina23(酪氨酸磷酸化抑制剂a23)抑制ap-2蛋白的衔接功能,进而影响cme的胞吞作用,通过研究ap-2蛋白的特异性抑制致小鼠耳蜗ihc电生理的改变情况,初步探讨ap-2蛋白在耳蜗ihc突触囊泡胞吞的可能作用机制,为研究感音神经性耳聋在蛋白质水平的发病机制提供参考。二、材料与方法1、选择健康成年c57bl/6j(8周龄)小鼠20只,耳廓反应灵敏,abr阈值正常。通过ap-2和dapi双重标记以及ap-2、ctbp2和dapi三重标记,应用免疫荧光标记技术结合激光共聚焦显微镜观察ap-2蛋白在耳蜗毛细胞的定位与表达。2、选用7、15、35日、及16月龄小鼠各20只,分别代表新生小鼠、听功能发育阶段小鼠、听功能发育成熟小鼠及老年性小鼠,通过免疫荧光激光共聚焦显微镜,qrt-pcr技术检测基底膜内毛细胞ap-2蛋白的表达特点,结合abr测定小鼠听阈等讨论听功能的发生、形成、以及年龄相关性听功能减退与小鼠耳蜗ap-2蛋白表达的相关性。3、通过tyrphostina23(酪氨酸磷酸化抑制剂a23)抑制yxxФ分子信号与ap-2μ2亚基的相互作用,分析内毛细胞ap-2蛋白被特异性抑制后小鼠ihc电生理的改变情况,从而反映ap-2蛋白对cme的影响。三、结果:1、ap-2蛋白属于细胞胞浆蛋白,主要表达于ihc突触活化部位,浓集于胞质基底外侧,靠近传入神经元区域。ap-2蛋白表达的形态学定位位点与其调控cme途径的功能学相映衬。2、abr阈值测定发现,p15、p35、16月龄小鼠听阈分别为18.67±1.21dbnhl,13.83±1.47dbnhl和37.83±7.68dbnhl,p7组小鼠听力尚未引出。软件计算p7、p15、p35及16月龄组荧光染色密度值(imv)分别为:190.91±17.27,494.06±27.63,838.41±38.23,682.65±72.22;qrt-pcr测定p7、p15、p35及16月龄组ap-2mrna相对表达量(rq)分别为0.53±0.09,1.03±0.02,1.00±0.09,1.03±0.06。p7与p15组比较,随着听功能的产生和形成,免疫荧光光密度值和ap-2mrna相对表达量均提示AP-2的表达水平明显升高,差异具有统计学意义(P0.05);P35与16月龄组比较,老年组AP-2蛋白荧光光密度值减少,然而通过q RT-PCR分析两者AP-2 mRNA的表达水平,差异无明显统计学意义(P0.05)。3、通过Tyrphostin A23的抑制作用,AP-2蛋白的衔接功能受阻碍,进而影响CME路径,IHC电压依赖性Ca2+通道开放和关闭延迟,ICa2+减小,△Cm降低,IHC膜电容及钙电流的改变表明AP-2蛋白调控的CME途径在IHC突触区域参与了囊泡的内吞作用。四、结论1、AP-2蛋白在成年小鼠耳蜗中主要表达于IHC突触活化区域,形态学的定位位点与其调控CME途径的功能学相符合,间接反映了AP-2蛋白与突触的关联及意义,它可能在IHC突触囊泡的内吞中发挥着重要作用,为深入探索AP-2蛋白在内耳听觉生理和病理中的作用奠定了基础。2、AP-2蛋白在小鼠内耳发育不同阶段均有不等强度的表达,新生小鼠至听力的产生和发育成熟阶段,耳蜗AP-2蛋白随小鼠日龄增大而表达增强,提示AP-2蛋白的表达水平可能与听功能的发生、形成和维持密切相关。3、特异性抑制AP-2蛋白的功能,能影响CME途径,突触间隙内神经递质不能被IHC突触前膜有效地内吞回收,影响了突触循环,最终影响了IHC神经递质的释放。说明AP-2蛋白在调控小鼠耳蜗IHC的突触囊泡回收中发挥着重要重要。
[Abstract]:First, research background and purpose sensorineural deafness is the most common auditory disability. The generation and maintenance of hearing depends on the release of glutamate neurotransmitters in the IHC band synapses, thus completing the mechanical and electrical energy transfer function [1]. as the first stage of auditory conduction, and the pathological changes in the cochlear IHC synapse cause the sensorineural nerve. One of the important causes of sexual deafness [2,3]. a large number of studies have shown that the hypofunction of auditory function is closely related to the abnormal changes in the form of banded synapses, structure and number of [3]., each IHC has a synaptic connection with the spiral neurons, and a single IHC has 10-30 banded synapses. The rapid release pool (RRP) of the cochlear IHC stripline contact area is released from the cochlea (RRP). The neurotransmitter glutamic acid activates the glutamate receptor (AMPA) in the postsynaptic membrane, which passes the auditory information to the corresponding neuron [4]. in the cochlear nucleus. Most of the acoustic signals are accomplished by IHC and its synapses. Ten vesicles per RRP are attached to each RRP, and the vesicles can be released in a few microseconds and the simultaneous release of a large number of vesicles can be obtained. Through the excitatory postsynaptic potential (EPSPs) reflecting the characteristics of [5]. based on auditory signal transduction, the precise and rapid transmission of neurotransmitters in IHC synapses requires a normal and effective vesicle cycle mechanism, namely, vesicular exocytosis and endocytosis of [6]. synaptic vesicle cycle (recycling), which is the basis for the continuous release of auditory nerve transmitters, and synaptic vesicles are accurate and fast within. The endocytosis recovery is an important guarantee for the continuous transmission of vesicle information. The CME pathway is an important way for the recovery of synaptic vesicles in central nervous cells, [7], and AP-2 protein is the structural core of the identification of gridin in the endocytic initial stage and the formation of a lrellin envelope (coated pits) with other auxiliary proteins. The heart, which is a large protein complex consisting of four subunits of alpha, beta 2, 2, and sigma 2, is associated with the plasma membrane of the appendage region of the alpha subunit, and is combined with a variety of regulatory proteins and auxiliary proteins by DPF or DPW molecular signals; the beta 2 subunit is associated with the helix terminal domain of the gridin beta, and is related to the selection of the transport goods (cargo); and the subunit of the mu 2 subunit. The identification of the cytoplasmic tail region receptor [8].AP-2 protein by the tyrosine classification signal plays an important role in the initial stage of the endocytic endocytosis. The cochlear IHC and its afferent neurons act as a special synapse, and the AP-2 protein regulation of the CME pathway should play an important role in this region. The study of the AP-2 protein before the eye is mostly limited to it. In the study of proteomics and its central nervous cells, the expression and function of AP-2 protein in the mouse cochlea are not clear. This study intends to use double immunofluorescence labeling and laser confocal microscopy to observe the localization and expression characteristics of AP-2 protein and the banded presynaptic specific protein rebeye/ctbp2 in the mouse cochlear hair cells To explore the possible mechanism of its possible action in the auditory physiology of the inner ear, the expression of AP-2 protein in different developmental stages after birth was observed with auditory brainstem response (ABR) and fluorescence quantitative PCR (qRT-PCR). The correlation between the occurrence of AP-2 protein and hearing function and the correlation of age related hearing loss was discussed. Finally, the electrophysiological method of patch clamp was used to use Tyr. Phostina23 (tyrosine phosphorylation inhibitor A23) inhibits the cohesive function of AP-2 protein and affects the endocytosis of CME. The possible mechanism of AP-2 protein in the cochlear IHC synaptic vesicle swallowing is preliminarily discussed by studying the specific inhibition of the IHC electrophysiology of the mouse cochlea by the specific inhibition of the AP-2 protein, so as to study the sensorineural deafness in the egg. Two, materials and methods 1, materials and methods 1, select 20 healthy adult c57bl/6j (8 weeks old) mice, the auricle is sensitive and the ABR threshold is normal. By AP-2 and DAPI double markers and AP-2, ctbp2 and DAPI three markers, the immunofluorescent labeling technique combined with laser confocal microscope to observe the cochlear capillary of AP-2 protein The location and expression of.2, 7,15,35 day, and 20 mice in 16 month old mice, respectively representing newborn mice, hearing functional development mice, hearing functional mature mice and senile mice, using immunofluorescent laser confocal microscopy, qRT-PCR technique to detect the expression of AP-2 protein in the basal membrane of the hair cells, and ABR for the determination of the mouse hearing. Threshold, etc., to discuss the occurrence, formation of auditory function, and the correlation of age related impairment of auditory function with the expression of AP-2 protein in the cochlea of mice, and the inhibition of the interaction between the yxx molecular signal and AP-2 2 subunit by tyrphostina23 (tyrosine phosphorylation inhibitor A23), and the analysis of the electrophysiological changes of IHC in the mouse after the specific inhibition of the internal capillary cell AP-2 protein. Change the situation to reflect the effect of AP-2 protein on CME. Three, results: 1, AP-2 protein belongs to cytoplasmic protein, mainly expressed in the activation site of IHC synapse, which is concentrated in the lateral of cytoplasm basement. The morphological loci of the.Ap-2 protein expression near the afferent neuron region reflect the function of CME pathway, and the ABR threshold determination is found, p15 The hearing threshold of p35,16 months of age in mice was 18.67 + 1.21dbnhl, 13.83 + 1.47dbnhl and 37.83 + 7.68dbnhl, and the hearing loss in P7 group was not elicited. The software P7, p15, p35 and 16 month old groups of fluorescent staining density values (IMV) were 190.91 + 17.27494.06 + 27.63838.41 + 38.23682.65 72.22, respectively. The amount of RQ was 0.53 + 0.09,1.03 + 0.02,1.00 + 0.09,1.03 + 0.06.p7 and p15 group respectively. With the emergence and formation of auditory function, the expression of immunofluorescence light density and ap-2mrna relative expression suggested that the expression level of AP-2 increased significantly, and the difference was statistically significant (P0.05); P35 and the 16 month old groups were compared with the fluorescent light density of the AP-2 protein in the elderly group. However, the expression level of AP-2 mRNA was analyzed by Q RT-PCR, and the difference was not statistically significant (P0.05).3. The cohesive function of AP-2 protein was hindered by the inhibition of Tyrphostin A23, and then the CME path was hindered, the IHC voltage dependent Ca2+ channel opening and closing delay, the decrease of delta, the capacitance and the calcium current were reduced. The changes indicated that the CME pathway regulated by AP-2 protein participates in the endocytosis of vesicles in the IHC synaptic region. Four, conclusion 1, AP-2 protein is mainly expressed in the IHC synaptic activation region in adult mouse cochlea. The location of the morphologic loci is in accordance with the function of the CME pathway, which reflects the Association and significance of the AP-2 protein with the synapse. It may play an important role in the endocytosis of IHC synaptic vesicles, which lays a foundation for the in-depth exploration of the role of AP-2 protein in the auditory physiology and pathology of the inner ear. The AP-2 protein has unequal intensity expression in different stages of the development of the inner ear of mice, the generation of newborn mice to hearing and the mature stage, and the AP-2 protein of the cochlea with the age of mice. The expression of AP-2 protein may be enhanced, suggesting that the expression level of the protein may be related to the occurrence of auditory function, the formation and maintenance of the closely related.3, the specific inhibition of the function of the AP-2 protein and the influence of the CME pathway. The neurotransmitters in the synaptic gap can not be effectively recovered from the IHC presynaptic membrane, which affects the synaptic cycle and ultimately affects the release of the IHC neurotransmitter. It indicates that AP-2 protein plays an important role in regulating the recovery of synaptic vesicles in mouse cochlea IHC.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R764.35
本文编号：2132404
[Abstract]:First, research background and purpose sensorineural deafness is the most common auditory disability. The generation and maintenance of hearing depends on the release of glutamate neurotransmitters in the IHC band synapses, thus completing the mechanical and electrical energy transfer function [1]. as the first stage of auditory conduction, and the pathological changes in the cochlear IHC synapse cause the sensorineural nerve. One of the important causes of sexual deafness [2,3]. a large number of studies have shown that the hypofunction of auditory function is closely related to the abnormal changes in the form of banded synapses, structure and number of [3]., each IHC has a synaptic connection with the spiral neurons, and a single IHC has 10-30 banded synapses. The rapid release pool (RRP) of the cochlear IHC stripline contact area is released from the cochlea (RRP). The neurotransmitter glutamic acid activates the glutamate receptor (AMPA) in the postsynaptic membrane, which passes the auditory information to the corresponding neuron [4]. in the cochlear nucleus. Most of the acoustic signals are accomplished by IHC and its synapses. Ten vesicles per RRP are attached to each RRP, and the vesicles can be released in a few microseconds and the simultaneous release of a large number of vesicles can be obtained. Through the excitatory postsynaptic potential (EPSPs) reflecting the characteristics of [5]. based on auditory signal transduction, the precise and rapid transmission of neurotransmitters in IHC synapses requires a normal and effective vesicle cycle mechanism, namely, vesicular exocytosis and endocytosis of [6]. synaptic vesicle cycle (recycling), which is the basis for the continuous release of auditory nerve transmitters, and synaptic vesicles are accurate and fast within. The endocytosis recovery is an important guarantee for the continuous transmission of vesicle information. The CME pathway is an important way for the recovery of synaptic vesicles in central nervous cells, [7], and AP-2 protein is the structural core of the identification of gridin in the endocytic initial stage and the formation of a lrellin envelope (coated pits) with other auxiliary proteins. The heart, which is a large protein complex consisting of four subunits of alpha, beta 2, 2, and sigma 2, is associated with the plasma membrane of the appendage region of the alpha subunit, and is combined with a variety of regulatory proteins and auxiliary proteins by DPF or DPW molecular signals; the beta 2 subunit is associated with the helix terminal domain of the gridin beta, and is related to the selection of the transport goods (cargo); and the subunit of the mu 2 subunit. The identification of the cytoplasmic tail region receptor [8].AP-2 protein by the tyrosine classification signal plays an important role in the initial stage of the endocytic endocytosis. The cochlear IHC and its afferent neurons act as a special synapse, and the AP-2 protein regulation of the CME pathway should play an important role in this region. The study of the AP-2 protein before the eye is mostly limited to it. In the study of proteomics and its central nervous cells, the expression and function of AP-2 protein in the mouse cochlea are not clear. This study intends to use double immunofluorescence labeling and laser confocal microscopy to observe the localization and expression characteristics of AP-2 protein and the banded presynaptic specific protein rebeye/ctbp2 in the mouse cochlear hair cells To explore the possible mechanism of its possible action in the auditory physiology of the inner ear, the expression of AP-2 protein in different developmental stages after birth was observed with auditory brainstem response (ABR) and fluorescence quantitative PCR (qRT-PCR). The correlation between the occurrence of AP-2 protein and hearing function and the correlation of age related hearing loss was discussed. Finally, the electrophysiological method of patch clamp was used to use Tyr. Phostina23 (tyrosine phosphorylation inhibitor A23) inhibits the cohesive function of AP-2 protein and affects the endocytosis of CME. The possible mechanism of AP-2 protein in the cochlear IHC synaptic vesicle swallowing is preliminarily discussed by studying the specific inhibition of the IHC electrophysiology of the mouse cochlea by the specific inhibition of the AP-2 protein, so as to study the sensorineural deafness in the egg. Two, materials and methods 1, materials and methods 1, select 20 healthy adult c57bl/6j (8 weeks old) mice, the auricle is sensitive and the ABR threshold is normal. By AP-2 and DAPI double markers and AP-2, ctbp2 and DAPI three markers, the immunofluorescent labeling technique combined with laser confocal microscope to observe the cochlear capillary of AP-2 protein The location and expression of.2, 7,15,35 day, and 20 mice in 16 month old mice, respectively representing newborn mice, hearing functional development mice, hearing functional mature mice and senile mice, using immunofluorescent laser confocal microscopy, qRT-PCR technique to detect the expression of AP-2 protein in the basal membrane of the hair cells, and ABR for the determination of the mouse hearing. Threshold, etc., to discuss the occurrence, formation of auditory function, and the correlation of age related impairment of auditory function with the expression of AP-2 protein in the cochlea of mice, and the inhibition of the interaction between the yxx molecular signal and AP-2 2 subunit by tyrphostina23 (tyrosine phosphorylation inhibitor A23), and the analysis of the electrophysiological changes of IHC in the mouse after the specific inhibition of the internal capillary cell AP-2 protein. Change the situation to reflect the effect of AP-2 protein on CME. Three, results: 1, AP-2 protein belongs to cytoplasmic protein, mainly expressed in the activation site of IHC synapse, which is concentrated in the lateral of cytoplasm basement. The morphological loci of the.Ap-2 protein expression near the afferent neuron region reflect the function of CME pathway, and the ABR threshold determination is found, p15 The hearing threshold of p35,16 months of age in mice was 18.67 + 1.21dbnhl, 13.83 + 1.47dbnhl and 37.83 + 7.68dbnhl, and the hearing loss in P7 group was not elicited. The software P7, p15, p35 and 16 month old groups of fluorescent staining density values (IMV) were 190.91 + 17.27494.06 + 27.63838.41 + 38.23682.65 72.22, respectively. The amount of RQ was 0.53 + 0.09,1.03 + 0.02,1.00 + 0.09,1.03 + 0.06.p7 and p15 group respectively. With the emergence and formation of auditory function, the expression of immunofluorescence light density and ap-2mrna relative expression suggested that the expression level of AP-2 increased significantly, and the difference was statistically significant (P0.05); P35 and the 16 month old groups were compared with the fluorescent light density of the AP-2 protein in the elderly group. However, the expression level of AP-2 mRNA was analyzed by Q RT-PCR, and the difference was not statistically significant (P0.05).3. The cohesive function of AP-2 protein was hindered by the inhibition of Tyrphostin A23, and then the CME path was hindered, the IHC voltage dependent Ca2+ channel opening and closing delay, the decrease of delta, the capacitance and the calcium current were reduced. The changes indicated that the CME pathway regulated by AP-2 protein participates in the endocytosis of vesicles in the IHC synaptic region. Four, conclusion 1, AP-2 protein is mainly expressed in the IHC synaptic activation region in adult mouse cochlea. The location of the morphologic loci is in accordance with the function of the CME pathway, which reflects the Association and significance of the AP-2 protein with the synapse. It may play an important role in the endocytosis of IHC synaptic vesicles, which lays a foundation for the in-depth exploration of the role of AP-2 protein in the auditory physiology and pathology of the inner ear. The AP-2 protein has unequal intensity expression in different stages of the development of the inner ear of mice, the generation of newborn mice to hearing and the mature stage, and the AP-2 protein of the cochlea with the age of mice. The expression of AP-2 protein may be enhanced, suggesting that the expression level of the protein may be related to the occurrence of auditory function, the formation and maintenance of the closely related.3, the specific inhibition of the function of the AP-2 protein and the influence of the CME pathway. The neurotransmitters in the synaptic gap can not be effectively recovered from the IHC presynaptic membrane, which affects the synaptic cycle and ultimately affects the release of the IHC neurotransmitter. It indicates that AP-2 protein plays an important role in regulating the recovery of synaptic vesicles in mouse cochlea IHC.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R764.35
【参考文献】
相关期刊论文 前2条
1 李春晖;杜波;丁大连;杜宝东;;小鼠耳蜗的形态学发育过程[J];解剖科学进展;2008年02期
2 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年03期
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