KCNQ1基因缺失致小鼠听觉丧失和耳蜗形态异常

由KCNQ1基因编码的KCNQ1蛋白分布于内耳血管纹边缘细胞的顶膜，KCNQ1是参与维持耳蜗钾离子代谢的重要通道。我们应用听性脑干反应（auditory brainstem response，ABR）和耳蜗内电位（endococldear potential，EP）检测KCNQ1^＋/＋、KCNQ1^＋/- KCNQ1^-/- 3种小鼠的听觉生理功能，并观察其耳蜗形态变化，以了解KCNQ1基因和离子通道在维持耳蜗钾循环及听觉生理中的作用。     

小鼠耳蜗侧壁α2 Na,K-ATPase在听觉及年龄相关性听力下降中的作用

目的探讨耳蜗侧壁α2Na,K-ATPase通道在听觉功能和年龄相关性听力减退中的作用。方法利用半拷贝基因的α2Na,K-ATPase /-杂合子小鼠作为试验平台,采用听性脑干反应(ABR)和耳蜗内电位(endoco-chlear potential,EP)检测方法,对α2Na,K-ATPase /-杂合子小鼠和α2Na,K-ATPase / 野生型小鼠进行ABR和EP检测,并对各个鼠龄段的小鼠进行ABR检测。结果α2Na,K-ATPase /-杂合子小鼠的听力和EP值均低于α2Na,K-ATPase / 野生型鼠;α2Na,K-ATPase /-杂合子小鼠的听力随鼠龄增长而逐渐下降,高龄期小鼠的ABR阈值与其低龄时相比显著性升高(P<0.05)。结论耳蜗侧壁的α2Na,K-ATPase通道对听觉功能具有重要作用;携带半拷贝基因的αNa,K-ATPase /-杂合子小鼠表现出轻度年龄相关性听力减退。     

溶酶体保护蛋白/组织蛋白酶A基因敲除小鼠听力和耳形态学初步研究

目的研究溶酶体保护蛋白/组织蛋白酶A(protective protein/cathepsin A,PPCA)基因敲除小鼠听功能和耳形态学改变,探讨半乳糖唾液酸沉积症听力损害的病理生理机制。方法应用听性脑干反应(ABR)测试和颞骨连续切片,观察1月和2月龄的PPCA基因敲除纯合子(PPCA-/-)小鼠ABR反应阈和光镜下外耳、中耳及内耳形态改变,并以野生型(PPCA / )小鼠作对照。结果1月龄PPCA-/-小鼠ABR反应阈和耳形态无明显改变;2月龄时,短声和短音8、163、2 kHz反应阈较PPCA / 提高40~45 dB SPL,中耳黏膜增厚、听骨细胞囊泡化、变形和关节腔融合,血管纹增厚、螺旋神经节细胞、螺旋缘纤维细胞、前庭膜、基底膜及沿前庭阶外淋巴隙的间皮细胞囊泡化,但Corti器毛细胞及支持细胞正常。结论溶酶体保护蛋白/组织蛋白酶A缺乏可导致听力损害、中耳及耳蜗形态改变、中耳炎、听骨改变以及耳蜗螺旋神经节、血管纹、螺旋缘、前庭膜和基底膜等细胞的溶酶体储积,可能分别是传导性聋和感觉神经性聋的形成机制。     

Na-K-2Cl联合转运子1在小鼠耳蜗侧壁组织中的表达及意义

目的观察Na-K-2Cl联合转运子1(NKCC1)在小鼠耳蜗组织中的表达及分布,并探讨其与耳蜗听觉功能的关系。方法选用健康正常CBA/CaJ小鼠为实验组,NKCC1-/-(突变纯合子,全基因敲除)小鼠为对照组,利用听性脑干反应检测两组动物的听觉功能,取各组小鼠的耳蜗行冰冻切片,同时采用免疫组织化学技术和免疫荧光组织化学法检测Na-K-2Cl联合转运子1在实验组与对照组小鼠的耳蜗中的表达和分布。结果实验组小鼠的ABR反应阈值为18±3.50dBSPL、对照组小鼠在100dBSPL刺激时无反应。NKCC1阳性反应呈棕黄色,主要分布于实验组小鼠耳蜗血管纹边缘细胞和螺旋韧带下部,在耳蜗血管纹边缘细胞和螺旋韧带下部的纤维细胞、纹上区也有适度表达,而在对照组未见阳性表达。图像分析显示,两组灰度值差异有显著统计学意义(P<0.01)。结论在正常小鼠耳蜗,Na-K-2Cl联合转运子1主要在血管纹边缘细胞及螺旋韧带下部分布表达,并与耳蜗听觉生理功能密切相关。     

噪声对小鼠耳蜗外侧壁缝隙连接蛋白Connexin31表达的影响

目的观察噪声性聋小鼠耳蜗外侧壁缝隙连接蛋白Connexin31(Cx31)表达的变化。方法选用成年雄性Balb/c小鼠44只,随机分为噪声组和对照组,每组22只。两组小鼠均在实验前检测听性脑干反应(ABR),随后应用声刺激器给予噪声组小鼠高强度白噪声(115dB SPL,6h/d,共2天),并在噪声暴露结束后1h再检测噪声组小鼠ABR。对照组不予噪声刺激。于噪声暴露后4h,每组各取4只小鼠耳蜗做冰冻切片,其余18只小鼠提取耳蜗外侧壁组织总RNA,通过免疫荧光染色法观察小鼠耳蜗外侧壁Cx31蛋白的表达,荧光实时定量PCR检测小鼠耳蜗外侧壁Cx31mRNA的表达。结果对照组小鼠耳蜗螺旋韧带可见Cx31特异性荧光,血管纹处未见阳性荧光;噪声组小鼠耳蜗外侧壁免疫荧光染色阳性反应较对照组减弱,Cx31mRNA表达量明显低于对照组。结论噪声能下调Cx31在小鼠耳蜗外侧壁组织的表达,Cx31可能参与了噪声性聋的发病机制。     

年龄相关听力损失BALB／c小鼠耳蜗形态学观察

目的 探讨年龄相关听力损失小鼠耳蜗毛细胞形态学变化,建立年龄相关听力损失动物模型.方法 分别取3、6、12、18月龄BALB/c小鼠测定其听性脑干反应(ABR)阈值,应用耳蜗铺片和扫描电镜技术,观察不同月龄鼠耳蜗毛细胞计数和形态的变化.结果 3、6、12月龄鼠8 kHz ABR反应阈分别为(24.8±5.1)、(51.5±6.7)和(92.5±7.5)dB SPL.18月龄组120 dB SPL刺激声无诱发反应.耳蜗铺片毛细胞计数自6月龄组外毛细胞出现显著缺失,随月龄增加而加重,由底回逐渐向顶回发展,至12月龄时耳蜗底回和中回外毛细胞几乎完全丧失,内毛细胞显著缺失.扫描电镜显示6月龄组小鼠耳蜗毛细胞静纤毛可见不同程度的缺失、转位、散乱、倒伏、融合、变短现象,随月龄增加而逐渐加重.结论 BALB/c小鼠听力损失、耳蜗毛细胞缺失和纤毛损害随年龄增加而逐渐加重,可作为年龄相关听力损失研究的合适动物模型     

不同月龄BALB/c小鼠耳蜗miR-96表达与ABR阈值及耳蜗形态的关系

目的　研究不同月龄BALB/c小鼠ABR阈值、耳蜗形态学改变及miR-96的表达,明确miR-96对老年性聋小鼠听力的调节作用。方法　通过听性脑干反应测试、荧光染色、扫描电镜,观察3、6、12、18月龄BALB/c小鼠8 kHz听反应阈值及耳蜗形态学改变。实时定量PCR定量检测miR-96在各月龄BALB/c小鼠耳蜗内的表达。SPSS13.0统计软件进行 统计分析。结果　3、6、12月龄组小鼠8 kHz听反应阈值分别为（18.5±8.3）、（45.8±7.8）、（85.6±15.6）dB SPL,18月龄组120 dB SPL刺激声基本测不出听觉反应。荧光染色及扫描电镜发现,自6月龄起毛细胞出现显著缺失,静纤毛出现不同程度缺失、倒伏、融合、变短和转位等改变,并随月龄增大病变逐渐加重。miR-96在3、6、12、18月龄BALB/c小鼠耳蜗中的相对表达量（2-△CT）分别为0.0225±0.0073、0.0162±0.0048、0.0116±0.0048和0.0050±0.0014,与3月龄小鼠相比,差异有统计学意义（P <0.05）。结论　BALB/c小鼠听力损失、耳蜗毛细胞缺失及纤毛损害随月龄增长而逐渐加重,小鼠耳蜗中miR-96表达随月龄增加而减少,提示miR-96可能在老年性聋的发病机制中起重要用。     

小鼠耳蜗Na-K-2Cl联合转运子-1的定位及其缺失后的耳蜗组织学改变

目的探讨耳蜗钾循环途径中Na-K-2Cl联合转运子-1(Na-K-2Clcotransporter-1,NKCC1)在小鼠耳蜗的分布及NKCC1基因敲除后耳蜗组织学的改变。方法选用10只C57BL/6J小鼠((NCKK1 / )和5只NKCC1基因敲除小鼠(NKCC1-/-),应用听性脑干反应(auditorybrainstemresponse,ABR)分别检测NCKK1 / 小鼠和NKCC1-/-小鼠的听功能,采用免疫组织化学及甲苯胺蓝染色的方法观察NKCC1在NCKK1 / 小鼠耳蜗的定位及NKCC1-/-小鼠耳蜗组织学的变化。结果NCKK1 / 小鼠ABR平均阈值为31±5.36dBSPL,而NKCC1-/-小鼠听力完全丧失。NKCC1在NCKK1 / 型小鼠耳蜗主要分布在血管纹上皮(边缘细胞)和螺旋韧带下部纤维细胞,在纹上区和螺旋缘处的纤维细胞中也有适度表达;NKCC1-/-小鼠耳蜗前庭膜塌陷,中阶完全消失,内毛细胞、外毛细胞、支持细胞减少,Corti隧道消失。结论NKCC1在耳蜗的定位与耳蜗钾循环密切相关,NKCC1缺失会导致耳蜗正常结构的破坏,继而影响耳蜗生理功能。     

X-连锁低磷酸盐血症Pug小鼠的听力学和耳形态学研究

目的Phex基因点突变Pug小鼠是典型的人类X-连锁低磷酸盐血症小鼠模型。通过研究Pug小鼠的听力学表型,探讨Phex基因点突变对小鼠听觉功能与内耳形态的影响。方法运用听性脑干诱发电位（ABR）对4月龄Pug小鼠和野生型小鼠进行听功能测试,取小鼠耳蜗进行大体形态观察,运用石蜡切片观察小鼠耳蜗骨壁、前庭膜、血管纹、螺旋韧带、盖膜、Corti器、螺旋神经节等结构。运用扫描电镜观察小鼠耳蜗Corti器上的内、外毛细胞及其表皮板等结构。结果Pug小鼠的ABR在短声和8kHz、16kHz、32kHz短纯音刺激的反应阈值均有提高,在短声和16kHz、32kHz频率,其差异具有统计学意义（P〈0.05）。小鼠耳蜗骨壁增厚,骨质过度增长,有大量骨质矿化不完全区域;耳蜗底转部位螺旋神经节出现明显退化,神经元数目大量减少;耳蜗顶转到底转内、外毛细胞静纤毛散在性缺失,静纤毛形态异常。结论Phex基因点突变引起的基因功能缺失导致了Pug小鼠的听觉功能减退和耳蜗形态异常。     

KCNQ1/KCNE1 potassium channels in mammalian vestibular dark cells

The high [K(+)] in the inner ear endolymph is essential for mechanosensory transduction in hearing and balance. Several ion channels, including a slowly activating, voltage-dependent, outwardly conducting K(+) channel composed of the KCNQ1 (KvLQT1) and KCNE1 (IsK/minK) subunits, are expressed at the apical surface of vestibular dark cells. We investigated the underlying molecular mechanisms of this conductance using in situ hybridization, RT-PCR, and immunocytochemistry and by tracking the ultrastructural changes of vestibular structures in kcne1(-/-) mice. In the wild type mice, the KCNE1 and KCNQ1 proteins are expressed specifically at the apical membrane of dark cells, as early as gestational day (GD) 17 for KCNE1 while KCNQ1 mRNAs can be detected at GD 18. This is the first demonstration that the two protein components of this potassium channel co-localize in a polarized fashion at the cellular level. Although the vestibular end-organs are normal at birth in kcne1(-/-) mice, they begin to show modifications during postnatal development: we observed an increase in the height of the dark cells, in their number of mitochondria, and in basolateral membrane infoldings. Subsequently, the epithelium degenerates and the endolymphatic space collapses. Similar changes are known to occur in the cardio-auditory Jervell--Lange-Nielsen syndrome which is caused by mutations in the same channel.     

Expression and localization of K channels KCNQ2 and KCNQ3 in the mammalian cochlea

KCNQ1 and KCNQ4 voltage-gated potassium channel subunits play key roles in hearing. Other members of the KCNQ family also encode slow, low voltage-activated K(+) M currents. We have previously reported the presence of M-like K(+) currents in sensory hair cells, and expression of Kcnq family genes in the cochlea. Here, we describe Kcnq2/3 gene expression and distribution of M channel subunits KCNQ2 and 3 in the cochlea. By using RT-PCR, we found expression of Kcnq2 in the modiolus and organ of Corti, while Kcnq3 expression was also detected in the cochlear lateral wall. Five alternative splice variants of the Kcnq2 gene, one of which has not been reported previously, were identified in the rat cochlea. KCNQ2 and KCNQ3 immunoreactivities were observed in spiral ganglion auditory neurons. In addition, the unmyelinated parts of the nerve fibers innervating hair cells and synaptic regions under hair cells showed KCNQ2 immunoreactivity. KCNQ3 immunoreactivity was also prominent in spiral ganglion satellite cells. These findings suggest that cochlear M channels play important roles in regulation of cellular excitability and maintenance of cochlear K(+) homeostasis in the auditory system.     

Identification of KCNQ1 compound heterozygous mutations in three Chinese families with Jervell and Lange-Nielsen Syndrome

Conclusion: Besides expanding the spectrum of KCNQ1 mutations causing Jervell and Lange-Nielsen Syndrome (JLNS), the results showed diversity of its phenotypes, and emphasized the importance of molecular genetic analysis in confirming clinical diagnosis and making diagnosis possible before the emergency symptoms for deaf individuals.

Objectives: This study aimed to investigate four patients from three Chinese families with congenital hearing loss clinically and genetically.

Method: Genetic analysis of previously reported deafness genes based on massively parallel sequencing was conducted in more than five thousand Chinese hearing loss patients. Detailed clinical features of the patients with compound heterozygous or homozygous mutations of KCNQ1 gene were collected and analyzed.

Results: Compound mutations of KCNQ1 were found to be the genetic etiology of four patients from three families. Among the six KCNQ1 mutations, c.546C?>?A was identified as a novel mutation, c.965C?>?T had been reported in JLNS, while c.683?+?5G?>?A, c.1484_1485delCT, c.905C?>?T and c.1831G?>?A were previously reported in LQT1. In addition to congenital profound hearing loss in all subjects, two sibling subjects showed typical JLNS cardiac phenotype of prolonged QTc and recurrent syncopal episodes. One subject presented not only JLNS, but also iron-deficiency anemia and epilepsy. The other subject did not present any cardiac phenotype.     

Transient Delivery of a KCNQ2/3-Specific Channel Activator 1 Week After Noise Trauma Mitigates Noise-Induced Tinnitus

Exposure to loud noise can cause hearing loss and tinnitus in mice and humans. In mice, one major underlying mechanism of noise-induced tinnitus is hyperactivity of auditory brainstem neurons, due at least in part, to decreased Kv7.2/3 (KCNQ2/3) potassium channel activity. In our previous studies, we used a reflex-based mouse model of tinnitus and showed that administration of a non-specific KCNQ channel activator, immediately after noise trauma, prevented the development of noise-induced tinnitus, assessed 1 week after trauma. Subsequently, we developed RL-81, a very potent and highly specific activator of KCNQ2/3 channels. Here, to test the timing window within which RL-81 prevents tinnitus in mice, we modified and employed an operant animal model of tinnitus, where mice are trained to move in response to sound but not move in silence. Mice with behavioral evidence of tinnitus are expected to move in silence. We validated this mouse model by testing the effect of salicylate, which is known to induce tinnitus. We found that transient administration of RL-81 1 week after noise exposure did not affect hearing loss but reduced significantly the percentage of mice with behavioral evidence of tinnitus, assessed 2 weeks after noise exposure. Our results indicate that RL-81 is a promising drug candidate for further development for the treatment of noise-induced tinnitus.     

KCNQ1/KCNE1 K+ channel and P2Y4 receptor are co-expressed from the time of birth in the apical membrane of rat strial marginal cells

CONCLUSION: KCNQ1/KCNE1 K(+) channels and P2Y(4) receptors are expressed in the apical membrane of rat strial marginal cells from postnatal day 1 (P1) and maintained throughout development. OBJECTIVES: The purpose of the present study was to investigate the developmental expression of KCNQ1/KCNE1 K(+) channel and of P2Y(4), which is an important metabotropic regulator of KCNQ1/KCNE1 K(+) channel in strial marginal cells. MATERIALS AND METHODS: Sprague-Dawley rats at different stages of development (P1, P3, P5, P7, P14, and P21) were studied. The spiral ligament with the stria vascularis was detached from the cartilaginous or bony cochlea and prepared for a voltage-sensitive vibrating probe and immunohistochemistry. RESULTS: Chromanol 293B, a blocker of KCNQ1/KCNE1 K(+) channel, inhibited short-circuit currents (I ( sc )) from P1 to P21. Similarly, I ( sc ) were found to be decreased by uridine 5'-triphosphate at all ages. The antagonist profiles indicated that the apical P2Y receptor is P2Y(4) subtype. KCNQ1, KCNE1, and P2Y(4) were immunolocalized in the apical region of stria vascularis at P1.     

散发高频听力下降人群中KCNQ4基因的突变筛查

目的 了解高频听力下降型感音性神经聋患者是否存在KcNQ4基因突变，并检测其是否存在与已知的KCNQ4相同的或者是新的基因突变，了解它们对高频听力下降发病机制的影响。方法 在门诊收集散发高频听力下降型感音神经性聋患者71例，另外选取正常对照者40例。采取PCR扩增、直接测序的方法检测KCNQ4基因突变。结果 KCNQ4基因中已经报道的和高频感音神经性听力损失有关的位点在实验组中未发现类似的突变，也没有发现可以引起氨基酸改变的新突变，只发现了几个多态性改变；在对照组中只发现了47bp的插入，没有发现47bp的缺失，也没有发现其他的多态性改变。结论 KCNQ4基因的突变可能并不是引起散发高频听力下降的主要原因。