【作者】 陈金；

【导师】 褚汉启；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉头颈外科学， 2013， 博士

【摘要】 第一部分Cav1.3钙离子通道蛋白在大鼠耳蜗中的表达模式目的：研究Cav1.3钙离子通道蛋白在成年大鼠耳蜗组织中的表达和分布，了解其在听觉生理和病理中的作用。方法：应用免疫组织化学的方法检测Cav1.3钙离子通道蛋白在耳蜗组织中的分布，采用免疫印迹技术（westernbloting，WB）以及逆转录聚合酶链反应（reversetranscription-polymerasechainreaction，RT-PCR）检测Cav1.3钙通道蛋白在耳蜗不同组织间的表达情况。结果：免疫化学方法显示Cav1.3钙通道蛋白主要分布在成年大鼠毛细胞、螺旋神经节细胞、螺旋神经板缘细胞、血管纹细胞以及螺旋韧带上。RT-PCR以及WB的结果显示Cav1.3钙通道蛋白表达具有一定的组织特异性，在耳蜗和肾脏组织中均有表达，其中耳蜗组织中基底膜上最多，螺旋神经节次之，血管纹和前两者相比其表达的量相对较少。结论：初步揭示了Cav1.3钙通道蛋白在成年大鼠耳蜗不同组织中的分布和表达差异，为进一步的研究Cav1.3钙离子通道蛋白在听觉生理和病理中重要的作用提供了理论依据。第二部分Cav1.3钙离子通道蛋白的年龄相关性表达下调与老年性耳聋的研究目的:研究Cav1.3钙离子通道蛋白在老年性耳聋小鼠耳蜗组织中的表达，探讨其在老年性耳聋发病中的作用。方法:培育4w,14w,24w,48w不同年龄段的C57/6J小鼠为实验对象，应用听性脑干反应（auditorybrainstemresponse，ABR）分别检测4、14、24、48周龄的C57BL/6小鼠的听力。免疫荧光方法观察Cav1.3在不同周龄小鼠内耳组织中的分布，采用免疫印迹技术（westernbloting，WB）检测Cav1.3蛋白在不同年龄小鼠内耳中表达。同时采用实时荧光定量PCR（Real-timeRT-PCR）检测其基因（CACNA1D）mRNA在内耳组织的年龄相关性表达。结果:Cav1.3主要分布在小鼠耳蜗内外毛细胞、螺旋神经节、螺旋韧带、血管纹、螺旋（板）缘细胞。14、24、48周龄小鼠与4周龄小鼠相比ABR反应阈值明显提高（p<0.05），48周龄C57小鼠同前三组小鼠相比，ABR反应阈值显著提高（p<0.01）。随着鼠龄的增长，Cav1.3表达含量逐渐减少（p<0.01）。结论：老年性耳聋小鼠Cav1.3钙离子通道蛋白表达含量随年龄增长而减少，其功能的异常和缺失可能在老年性耳聋的发病中起一定的作用。第三部分缝隙连蛋白26(Connexin26)在听觉功能中的作用及耳聋机制的研究目的:研究Connexin26在听觉功能以及内耳主动听觉机理中的作用，探讨Connexin26缺失引起耳聋的机制。方法:运用loxP-Cre技术敲除耳蜗DCs和OPCs上的缝隙连蛋白26(Cx26)的表达。由Cx26loxP/loxP转基因鼠和Prox1-CreERT2小鼠杂交培育缝隙连蛋白26选择性敲除小鼠（以下简称Cx26cKO小鼠）。分别检测野生型、杂合子以及纯合子不同基因型小鼠听性脑干反应（auditorybrainstemresponse，ABR）、微音器电位（CM）、畸变耳声发射（DPOAE）、内淋巴电位（EP）。免疫组织化学的方法检测Cx26、Cx30以及外毛细胞电能动性蛋白Prestin的表达。甲苯氨蓝染色观察不同基因型小鼠螺旋神经节差异。采用实时荧光定量PCR（Real-timeRT-PCR）检测Prestin在野生型和纯合子小鼠的表达。结果:在缝隙连蛋白26选择性敲除小鼠耳蜗顶回、中回及底回的Deiters’细胞和外柱细胞上均没有Cx26的表达。Cx26在耳蜗其他组织如螺旋缘以及外侧壁的表达正常。而另外一种连接蛋白Cx30在耳蜗组织中的表达也正常。Cx26cKO小鼠有听力损失，听性脑干反应阈值增加以及DPOAE降低，但是微音器电位和内淋巴电位均正常。外毛细胞电能动性蛋白Prestin的表达也并无异常，内耳的毛细胞和螺旋神经节也未发现明显的损失和退变。结论：耳蜗主动放大机理依赖于支持细胞的缝隙连接。缝隙连蛋白26功能的缺失可以减弱耳蜗放大器的功能从而引起听力的损失。更多还原

【Abstract】 PART ONE Expression Patterns of Cav1.3calcium channels in the rat cochleaObjective: To investigate the expression of Cav1.3calcium channels in the rat cochlea and study its role in auditory physiology and pathology.Methods:The distribution of Cav1.3calcium channels in the rat cochlea was detected by immunohistochemistry. The expression of Cav1.3calcium channels was measured with western bloting (WB) and reverse transcription-polymerase chain reaction (RT-PCR).Results:Immunohistochemistry revealed that Cav1.3calcium channels were localized in the outer hair cells (OHCs), inner hair cells (IHCs), limbus laminae spiralis, spiral ganglion cell, spiral ligament and stria vascularis. The results of RT-PCR and Western blotting demonstrated Cav1.3calcium channels had a tissue-specific expression pattern. Cavl.3mRNA and protein were intensively expressed in the basilar membrane and spiral ganglion while moderate level of Cavl.3was observed in spiral ligament and stria vascularis.Conclusion:The study preliminary reveals the expression patterns of Cavl.3calcium channels in the rat cochlea, providing a theoretical basis for further research on the role of Cav1.3calcium channels in the periphery auditory system. PART Two Downregulation of Cav1.3calcium channel expression in the cochlea is associated with Presbycusis in C57BL/6miceObjective:To investigate the expression of Cav1.3calcium channel in C57BL/6mice cochlea and study its correlation with presbycusis.Methods:Auditory function was measured with auditory brainstem response (ABR) in C57BL/6mice at4,14,24and48weeks. The expression of Cav1.3calcium channels was detected by immunofluorescence,Western Blotting and Real-time RT-PCR at various ages.Results:Immunofluorescence photographs reveal that Cav1.3channels were mainly localized in the hair cell, spiral ganglion cell, spiral ligment, stria vascularis, spiral limbus. Comparing with4-week group, the ABR threshold of4-,24-, and48-week increased (p<0.05). The ABR threshold at48-week group was significantly higher than others (p<0.01). Along with age, the missing hair cells and spiral ganglion cells increased, stria vascularis and spiral ligament atrophied, the expression of Cav1.3channels was gradually decrease d(p<0.01).Conclusion:The expression of Cav1.3channels decreased along with age. Dysfunction or missing of Cavl.3calcium channels may be related to presbycusis. PART Three Contribution of connexin26to active cochlear amplification and underlying mechanisms for connexin26deficiency induced hearing lossObjective:To investigate Connexin26hearing function and contribution to active cochlear amplification and deafness mechanisms underlying Cx26deficiency induced hearing loss.Methods:We used loxP-Cre technique to delete Cx26expression in Deiter cells (DCs) and outer pillar cells (OPCs). The Cx26conditional knockout (cKO) mouse was generated by crossing Cx26loxP/loxP transgenic mice with a Proxl-CreERT2mouse line. Auditory function was measured with auditory brainstem response (ABR), DPOAE, CM and EP in Cx26cKO mice. The expression of Cx26, Cx30and Prestein were detected by immunofluorescence staining. Consecutive cochlear paraffin sections were stained with toluidine blue to account SG neuron. Prestin expression at the transcriptional level was measured by real-time RT-PCR.Results:Cx26labeling was absent in DCs and OPCs in Cx26cKO mice. The deletion of Cx26is visible in all three apical, middle, and basal turns. Cx26labeling in other cochlear structures, including the spiral limbus (SLM) and the lateral wall, remained normal. Cx30, which is another predominant connexin isoform in the cochlea, also retained normal expression. Targeted-deletion of Cx26in DCs and OPCs induced hearing loss The ABR threshold in homozygous mice was increased significantly and DPOAE was significantly reduced. CM and EP in Cx26cKO mice were normal. No reduction in prestin. No apparent cell degeneration in the Cx26cKO mouse cochlea. Conclusion:The study demonstrates that active cochlear amplification in vivo is dependent on supporting cell gap junctions. These new findings also show that connexin26deficiency can reduce active cochlear amplification to induce hearing loss.更多还原