Unique expression of connexins in the human cochlea

Mutations in the genes GJB2 and GJB6, which encode the proteins Connexin 26 (Cx26) and Connexin 30 (Cx30), have been linked to nonsyndromic prelingual deafness in humans. These proteins may form so-called gap junctions (GJ) or transcellular pathways between cells. The pathogenesis of deafness due to GJ Connexin mutations remains unclear partly because examinations performed in the human ear are infrequent. Here we analysed the expression and distribution of Cx26 and Cx30 in five fresh normal human cochleae taken out at occasional surgery. Immunohistochemistry including confocal microscopy in decalcified specimen showed that these proteins are widely expressed in the human cochlea. In the lateral wall there was strong antibody co-labeling for Cx26 and Cx30 that support the existence of channels comprising heteromeric Cx26/Cx30 connexons. In the organ of Corti there were some co-labeling in the supporting cell area including mainly the Claudius cells and Deiter cells of these two Cxs, apart from isolated Cx26 and Cx30 labeling in the same area, suggestive of both homomeric/homotypic pattern and hybrid pattern (heteromeric or heterotypic). Cx30, Cx26 and Connexin 36 (Cx36) immunoreactivity was also associated with spiral ganglion type I neurons, the latter being a gap junction protein specific to neurons. Gap–junction-based electrical synapses are not known to occur in mammalian auditory system other than in bats where they may play a role for fast electrical nerve transmission useful for echolocation. Their potential role in the processing of human auditory nerve signaling as well as non-GJ roles of the connexins in human cochlea is discussed.     

Distribution of pejvakin in human spiral ganglion: An immunohistochemical study

Abstract

Up to 10% of permanent hearing impairments in children originate from lesions in the neuronal auditory pathway. This form of auditory neuron injury called auditory neuropathy features a preservation of outer hair cell integrity but an impaired inner hair cell function and/or neuronal transmission. DFNB59 gene encodes the protein pejvakin (PJVK) and its mutations cause autosomal recessive auditory neuropathy as well as other forms of sensorineural hearing loss. The finding of distinct forms of hearing anomalies was based on studies of consanguineous families from different ethnic groups as well as studies in mice with PJVK gene mutations. In the present immunohistochemical study, the distribution of pejvakin protein in surgically obtained human cochleae was for the first time investigated. The human cochleae had normal hearing thresholds before the operation. The expression of pejvakin was located in the cell bodies of all spiral ganglion neurons rather than the nerve fibers that were labeled with Tuj 1 antibody. As Tuj 1 antibody stained the cytoplasm of Type 1 cells, pejvakin antibody labeled both type 1 and type 2 cells. The nuclei of the neurons were also PJVK-positive. No labeling was seen in the structures within the organ of Corti and the stria vascularis. In the previous study, PJVK had been detected in the hair cells, the spiral ganglion, the cochlear nuclei, the superior olivary nucleus, and the inferior colliculus in mouse. Our study demonstrated for the first time the expression of PJVK in human spiral ganglion neurons. Its functional role in neural signal propagation and synchrony needs further elucidation.     

Regional variations in the expression of cytokeratin proteins in the adult human cochlea

Summary In the adult human cochlea, a cytokeratin (Ck) network exists along the entire surface of the organ of Corti, enclosing it like a shell. Only the surfaces of the outer and inner hair cells are not integrated in this network. In temporal bone specimens, Ck filaments in Hensen's cells were found to be arranged parallel with and closely apposed to the plasma membrane. In the stria vascularis, Cks were identified only in the marginal cells. Cells in Reissner's membrane and spiral prominence showed varying degrees of immunoreactivity to different monoclonal antibodies directed against Cks. A distinct positivity for Cks was found in most spiral ganglion cells, indicating their presence in all cells. The principal pattern of immunoreactivity was the same in the organ of Corti of the entire cochlea. However, a quantitative gradient in the expression of Cks was observed, with more Cks at the apex than at the base. This was correlated to a difference in the number of Hensen's cells between the two regions. The distinct shell configuration of the Ck network in Corti's organ gives it a tonotopically related difference in rigidity which must be of considerable importance for the perception of sound in the cochlea. The absence of Cks in inner and outer sulcus cells gives them cytoskeletal characteristics of mesenchymal cells with a possible regenerative potential.Supported by grants from the Swedish Medical Research Council (M. A.: 12X-7305; L.-E. T.: 12X-3934), the Ragnar and Torsten Söderberg Foundation (M. A.), the Foundation Tysta Skolan (M. A.), the Swiss National Funds (W. A., C. R. P.: 3.997-1.86), the Finnish Cancer Research Fund (I. V.), the Sigrid Juselius Foundation (I. V.), the Dutch Cancer Foundation (K. W. F.) and the Medical Faculty University of Ume»     

人Bcl-2基因重组腺病毒的构建及其在螺旋神经节细胞中的表达

目的构建表达人Bcl-2基因的重组腺病毒，并研究其在原代培养螺旋神经节细胞(spiral ganglion cells，SGC)的表达特性。方法采用细菌内同源重组法，从pUC-CAGGS,／Bcl-2质粒中获得人Bcl-2 cDNA，克隆到腺病毒穿梭质粒pAdTrack-CMV上，后者和腺病毒骨架质粒pAdEasy-1在细菌内同源重组，在HEK293细胞中包装成为重组Ad增强型绿色荧光蛋白／Bcl-2腺病毒，电镜观察病毒颗粒，用病毒感染原代培养的大鼠螺旋神经节细胞，在荧光显微镜下观察感染效率，用逆转录聚合酶链反应方法检测Bcl-2基因在螺旋神经节细胞的转录表达，用Western Blot检测其蛋白的表达。结果经酶切鉴定证实重组腺病毒质粒构建成功，电镜显示包装细胞中有大量病毒颗粒存在，呈规则六边形。荧光显微镜下观测到感染病毒的SGC细胞发出明亮的绿色荧光。分别在mRNA水平及蛋白水平证实有外源性Bcl-2基因的表达。结论细菌内同源重组法是一种简便、高效的制备方法。构建的重组Ad增强型绿色荧光蛋白／Bcl-2腺病毒能有效的感染SGC，并可在SGC中正确地转录和翻译，为进一步研究腺病毒介导的Bcl-2基因对螺旋神经节损伤的保护作用奠定基础。     

Structural maturation of the interface region between the stria vascularis and spiral ligament in the neonatal rat cochlea

The ultrastructural morphology of the interface region between the stria vascularis (SV) and spiral ligament (SL) was examined in the neonatal rat cochlea via transmission electron microscopy. At postnatal day (PND) 3, morphology of both basal cells and fibrocytes was simple and immature. Only a small number of fibrocytes was observed in the SL. Intercellular junctions between basal cells and fibrocytes, and between adjacent fibrocytes, were few. At PND 7, the number of fibrocytes increased, and more organelles appeared within their cytoplasm. From PND 11 to 14, nuclei of the basal cells appeared to be more spindle-shaped and contained more heterochromatin. The cytoplasm of the fibrocytes was pale, and a greater number of cytoplasmic vesicles and mitochondria emerged. More intercellular junctions were observed between basal cells and fibrocytes at the interface region and between fibrocytes in the SL. By PND 21, the morphology of basal cells and fibrocytes and their intercellular junctionsappeared to be adult-like. These morphological observations correlate with previous reports on the functional maturation of the developing rat cochlea.     

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCII and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy (SR-SIM) were used in the study. CD4⁺ and CD8⁺ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons, CD4⁺ and CD8⁺ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus, macrophages, CD4⁺ and CD8⁺ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4⁺ and CD8⁺ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.     

Effects of aminoglycoside administration on cochlear elements in human temporal bones

OBJECTIVE: Although there have been numerous reports on the relationship between the period of aminoglycoside administration and cochlear damage in animals, to date there have been no such studies in humans. The purpose of this study is to observe the early and late cochlear effects of aminoglycoside administration on hair cells, spiral ganglion cells, stria vascularis, and spiral ligament. METHODS: Specimens were divided into three groups. Group I included "normal" temporal bones with no histopathologic findings of otitis media and no history of otologic or ototoxic drug administration. Group II consisted of temporal bones that received aminoglycosides within 2 weeks before death and group III of temporal bones that had aminoglycosides from 2 weeks to 6 months prior to death. Patients in groups II and III received gentamycin, kanamycin or tobramycin. Temporal bones were excluded from groups II and III if patients had a history of otologic disease or other ototoxic drugs. All temporal bones were examined under light microscopy. Standard cytocochleograms and spiral ganglion cell reconstructions were done on all temporal bones. Morphometric measurements of areas of stria vascularis were made in all turns of the cochlea on mid-modiolar sections. Spiral ligament was divided into four segments according to the locations of different types of fibrocytes. The mean loss of fibrocytes in each segment was estimated. RESULTS: The percentages of intact outer hair cells in the basal turn were significantly greater in group I compared to groups II and III. The mean area of the stria vascularis in the apical turn was significantly less in groups II and III compared to group I. CONCLUSION: This study demonstrates that in a short period (within 2 weeks) after aminoglycoside administration, a decrease in hair cells and in the area of the stria vascularis occurred.     

支持细胞在哺乳动物耳蜗发育及功能中的作用

耳蜗的感觉上皮细胞有毛细胞和支持细胞两种类型.许多研究旨在探讨听力损失的基本机制,重点针对毛细胞和螺旋神经元,较少关注支持细胞.目前对支持细胞作用的了解已从单纯的结构支持扩展到耳蜗毛细胞分化发育、耳蜗离子稳态、毛细胞感音功能的调节以及突触发生发育、毛细胞损伤修复的多个方面.本文对支持细胞在哺乳动物耳蜗发育及功能中的关键作用做一综述.     

缺氧对体外培养耳蜗螺旋神经节细胞及神经纤维的影响

目的:建立耳蜗器官体外培养模型,详细观察缺氧对体外培养耳蜗螺旋神经节细胞(SGC)及神经纤维的影响。方法:分离生后3 d Wistar大鼠耳蜗基底膜,平铺在含有DMEM培养液的培养基内(每盘6条基底膜),2盘为1组,随机分为5组。4组作为实验组,按不同时间段(6 h、12 h、24 h、48 h)进行缺氧(37℃,90%N2,5%CO2,5%O2)培养。1组作为对照组放置在37℃、5%CO2培养箱。用抗神经丝蛋白作为一抗,对耳蜗SGC及神经纤维进行免疫荧光染色。激光共聚焦显微镜下观察缺氧时耳蜗SGC及神经纤维形态变化并计数单位面积(24 mm×36 mm)SGC数即细胞密度。结果:缺氧早期(6 h)神经纤维局灶性水肿,SGC变化不明显。12 h神经纤维局灶性断裂及崩解,SGC减少,细胞密度与对照组相比差异有显著性(P<0.01)。随缺氧时间延长,神经纤维崩解和SGC缺失逐渐加重,48 h神经纤维完全崩解破坏,SGC严重缺失,细胞密度与对照组比较差异有显著性(P<0.01)。结论:缺氧造成体外培养耳蜗SGC及神经纤维损伤,神经纤维对缺氧更敏感。     

EGCG对阿米卡星诱导大鼠螺旋神经元诱导型一氧化氮合酶表达的影响

目的探讨表没食子儿茶素没食子酸酯［(-)-epigallocatechin-3-gallate,EGCG］对阿米卡星（amikacin,Amk）诱导的大鼠耳蜗螺旋神经元(spiral ganglion neuron;SGN) 诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）表达的影响。方法将30只SD大鼠分为生理盐水对照组、Amk（450?mg/kg·d×14d）损伤组和Amk加EGCG（50?mg/kg·d×14d）保护组。各组动物均经深度麻醉后,半身灌注固定处死,取右侧耳蜗标本固定、脱钙、石蜡轴位包埋、轴位切片,近中轴位切片行iNOS免疫组化染色,光学显微镜下检测耳蜗SGN表达情况,并通过切片图像分析进行半定量处理比较。结果对照组SGN胞浆及细胞核基本上不着色（SGN iNOS染色平均灰度值为111.04±3.15）,损伤组SGN部分神经元胞浆内有棕黄色或黄色颗粒或斑片（SGN iNOS染色平均灰度值为90.32±5.26）,保护组胞浆及细胞核无明显着色（SGN iNOS染色平均灰度值为109.35±4.89）,图像分析结果显示,损伤组与对照组、损伤组与保护组之间的SGN iNOS染色平均灰度值差异有统计学意义。结论iNOS参与Amk耳中毒时耳蜗螺旋神经元的氧化损伤,EGCG可以减弱Amk耳中毒时耳蜗螺旋神经元iNOS的表达,从而减轻Amk耳毒性。     

噪声暴露对大鼠耳蜗碳酸酐酶活性及mRNA表达的影响

目的：探讨噪声暴露后耳蜗碳酸酐酶（CA）活性及其mRNA表达的变化。方法：选用健康白色大鼠24只,随机分为4组,1组为正常对照组,另外3组为实验组。实验组暴露在4kHz、110dBSPL窄带白噪声环境中4h／d,分别测试噪声暴露1d、1周、3周和对照组大鼠脑干反应（ABR）。采用免疫组织化学法观察耳蜗外侧壁CA的活性,RT-PCR检测大鼠耳蜗CAIImRNA的表达。结果：噪声暴露后大鼠ABR阈值较对照组显著增加;而耳CA活性及CAIImRNA的表达较对照组显著下降;随着噪声暴露时间的延长,ABR阈值增加而cA活性及CAIImRNA的表达呈下降趋势。结论：噪声刺激能降低耳蜗CA的活性及CAIImRNA的表达,CA可能参与了噪声性聋的发病机制。     

脑源性神经营养因子基因修饰的骨髓间充质干细胞在药物致聋豚鼠内耳的表达及保护作用

目的 探讨脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因修饰的骨髓间充质干细胞(mesenchymal stem cell,MSC)在药物致聋豚鼠内耳的表达及对螺旋神经节细胞(spiral ganglion cell,SGC)的保护作用.方法 阿米卡星致聋的豚鼠随机数字表法分为两组,治疗组经鼓阶开窗注射BDNF基因修饰的MSC,对照组注射外淋巴液.各组分别在术后7 d及28 d处死动物,荧光定量反转录聚合酶链反应(RT-PCR)检测耳蜗组织中BDNF mRNA的表达,耳蜗切片计算SGC细胞密度,末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling,TUNEL)检测SGC凋亡情况.结果 治疗组在术后7 d和28 d的BDNF mRNA相对表达量均明显高于对照组,差异具有统计学意义(P值均＜0.01).治疗组术后7 d及28 d的SGC密度均高于对照组,并且治疗组术后7 d及28 d的SGC凋亡指数也比对照组明显下降,差异具有统计学意义(P值均＜0.01).结论 BDNF基因修饰的MSC在致聋豚鼠内耳中的表达时间超过28 d,对SGC具有保护作用.     

New Developments in Understanding the Mechanisms and Function of Spontaneous Electrical Activity in the Developing Mammalian Auditory System

In the mature mammalian auditory system, inner hair cells are responsible for converting sound-evoked vibrations into graded electrical responses, resulting in release of neurotransmitter and neuronal transmission via the VIIIth cranial nerve to auditory centres in the central nervous system. Before the cochlea can reliably respond to sound, inner hair cells are not merely immature quiescent pre-hearing cells, but instead are capable of generating 'spontaneous' calcium-based action potentials. The resulting calcium signal promotes transmitter release that drives action potential firing in developing spiral ganglion neurones. These early signalling events that occur before sound-evoked activity are thought to be important in guiding and refining the initial phases of development of the auditory circuits. This review will summarise our current knowledge of the mechanisms that underlie spontaneous action potentials in developing inner hair cells and how these events are triggered and regulated.     

Expression and significance of endothelin 1 in spiral ganglion cells of guinea pig

OBJECTIVES: Endothelin 1 has many biological activities including actions in the nervous system. This study aimed to investigate the expression of the endothelin 1 in spiral ganglion cells of guinea pig and its significance in the auditory transmission. METHODS: Healthy guinea pigs were sacrificed and cardiac perfused with saline followed by 4% paraformaldehyde. Temporal bones were removed and fixed, decalcified in 10% EDTA, embedded in paraffin block and serially sectioned in 5 microm thick slice. Rabbit anti-endothelin 1 polyclonal antibody was used as primary antibody to examine the expression of endothelin 1 in the spiral ganglion by immunohistochemistry. RESULTS: Endothelin 1 expression was detected in spiral ganglion cells from the basal turn to the apical turn of the cochlea. CONCLUSIONS: The endothelin 1 presents in spiral ganglions cells of the guinea pig and might play a role in the auditory transmission.     

Mechanical tuning of free-standing stereociliary bundles and frequency analysis in the alligator lizard cochlea

Excised cochleae of alligator lizards were prepared to permit microscopic observations of motion of hair-cell free-standing stereociliary bundles and of the underlying basilar papilla during acoustic stimulation, using stroboscopic illumination. In response to tones of frequency from 0.2 to 5 kHz, the papilla rocks about an axis parallel to its length, displacing stereociliary bundles in the morphologically predicted direction of hair cell sensitivity. The papilla moves in phase along its entire length; for frequencies above about 3 kHz, the amplitude of motion of the most basal region is several times larger than that of the rest of the papilla.Over the basal two-thirds of the organ, stereociliary bundles stand freely in endolymph. In this region, maximum bundle height gradually decreases from about 30 to 12 μm; phase vs. frequency characteristics of bundle displacement with respect to the underlying papilla are those of nearly critically damped mechanical resonators. Resonant frequencies measured along the papilla vary inversely with a power (between $\text{3}\text{2}$ and 2) of bundle height and are close in value to auditory nerve fiber CFs measured in vivo at corresponding locations across the nerve. We suggest that length-dependent mechanical tuning of stereociliary bundles determines neural frequency selectivity and tonotopic organization in this part of the organ.     

Expression of p75NTR and its associated protein NADE in the rat cochlea

OBJECTIVES/HYPOTHESIS: To investigate the expression of the low-affinity neurotrophin receptor p75 (p75NTR) and its associated protein NADE in the cochlea of the developing and the adult rat. Studies such as this one will help to predict the functional role of p75NTR and NADE in cochlear development. STUDY DESIGN: Histochemical evaluation of p75NTR and NADE in the rat cochlea was performed. METHODS: Immunohistochemical analysis was used to localize p75NTR and NADE in the rat cochlea at postnatal (PN) days PN0, PN2, PN4, PN6, PN8, PN10, and PN13 and in the adult. Confocal laser scanning microscopy was used to analyze whole-mount specimens. RESULTS: Immunoreactivity of both p75NTR and NADE was observed in pillar cells. However, these proteins displayed reciprocal expression patterns. Expression of p75NTR was detected at PN0 and PN2, but disappeared after PN4. In contrast, NADE expression was initially detected at PN2 and persisted into adulthood. CONCLUSIONS: The neurotrophin receptor p75NTR and NADE have distinct and independent roles in developing and mature cochlea.     

喹啉酸对大鼠螺旋神经节细胞的神经兴奋毒性作用及其机制探讨

目的 探讨喹啉酸(quinolinic acid,QA)对大鼠螺旋神经节细胞(spiral ganglion cell,SGC)的神经兴奋毒性作用,观察N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体拮抗剂5-甲基二氢丙环庚烯业胺马来酸(MK-801)对QA神经兴奋毒性的拮抗作用,同时观察镁离子对SGC的保护作用.方法 新生SD大鼠SGC原代培养72 h后,更换培养基,分为空白对照组、QA损伤组(1 mmol/L QA)、MK-801拮抗组(1 mmol/L QA+20 μmol/L MK-801)、MgCl2保护组(1 mmoL/L QA+1 mmol/L MgCl2),继续培养24 h后,通过磷脂酰丝氨酸外翻分析法在荧光显微镜下测定SGC凋亡率.SGC原代培养72 h后,分为四组:低浓度QA组(100 μmol/L QA),高浓度QA组(1 mmnol/LQA),MK-801拮抗组(20 μmol/L MK-801+l mmol/QA),MgCl2保护组(1 mmol/L MgCl2+l mmol/L QA);激光共聚焦显微镜动态检测各组SGC胞内瞬时钙离子浓度的变化.结果 与空白对照组(凋亡率9.2%±0.9%,(x)±s,下同)相比,QA损伤组可见大量SGC凋亡(凋亡率59.1%±7.5%),差异具有统计学意义(q=11.9,P<0.05);MgCl2保护组凋亡细胞比QA组明显减少,凋亡率为27.5%±8.3%,二者筹异具有统计学意义(q=7.5,P<0.05);MK-801组细胞凋亡率(12.8%±5.7%)与空白对照组差异无统计学意义(q=0.9,P>0.05).在高浓度QA(1 mmoL/L)的作用下,SGC内钙离子浓度明显升高,190 s时达高峰,随后逐渐下降,450 s时基本恢复加药前水平;低浓度QA(100 μmol/L)对SGC胞内钙离子浓度没有明赤影响;Mgcl2组SGC胞内钙离子浓度曲线峰值降低,时程缩短;MK-801组未见明显SGC胞内钙离子浓度变化.结论 QA可过度激活细胞膜上的NMDA受体而造成大鼠离体培养SGC的损伤,镁离子可以降低QA对SGC的神经兴奋毒性作用.     

ATP和乙酰胆碱诱发豚鼠耳蜗外毛细胞内CICR的激光共聚焦研究

目的：运用激光共聚焦研究ATP和乙酰胆碱（ACh）对豚鼠耳蜗外毛细胞内游离钙离子浓度（[Ca^2＋]i）的影响以及诱发Ca^2＋介导的Ca^2＋释放（CICR）的可能机制。方法：用酶孵育机械分离法,分离豚鼠耳蜗外毛细胞（OHC）,钙敏荧光探针Fluo-3染色后,用激光扫描共聚焦显微镜分别记录在细胞外液有钙或无钙条件下,加入ATP、ACh、斯里兰卡肉桂碱（Ryanodine）＋ATP（或ACh）和毒胡萝卜素（Thapsigargin）＋ATP（或ACh）后的OHC的[Ca^2＋],的变化。结果：在含钙的细胞外液中,ATP、Ryanodine＋ATP、Thapsigargin＋ATP、ACh、Ryanodine＋ACh和Thapsigargin＋ACh均可引起[Ca^2＋],升高,引起明显的波峰,荧光强度相对峰值分别为1．60±0．01（ATP）、1．644±0．005（Ryanodine＋ATP）、1．491±0．005（Thapsigargin＋ATP）、1．43±0．01（ACh）、1．58±0．02（Ryanodine＋ACh）、1．398±0．003（Thapsigargin＋ACh）;而在不含钙的细胞外液中,ATP和Ryanodine＋ATP仍可引起[Ca^2＋]。出现幅度较小的波峰,分别为1．341±0．006和1．386±0．008,而ACh,Ryanodine＋ACh、Thapsigargin＋ACh和Thapsigargin＋ATP未引起明显[Ca^2＋],波峰出现,其中ACh不能引起[Ca^2＋]．的升高,Ryanodine＋ACh、Thapsigargin＋ACh和Thapsigargin＋ATP分别引起[Ca^2＋]。缓慢升高。结论：在细胞外液有Ca^2＋的条件下．ATP和ACh引起OHC的[Ca^2＋]。升高,除了通过离子通道引起胞外Ca^2＋内流,还有IP。敏感钙库的释放和诱发CICR。在无Ca^2＋的条件下,ATP仍能诱发CICR,其机制可能是ATP促进IP3敏感钙库释放Ca^2＋,Ca^2＋又诱发了CICR,而ACh的反应是Ca^2＋依赖性的,在细胞外液无Ca^2＋的条件下,ACh不能引起IP。敏感钙库的释放和诱发CICR。     

重组腺病毒构建及转染培养耳蜗Corti器和螺旋神经节细胞的实验研究

目的构建携带绿色荧光蛋白（GFP）基因的重组腺病毒,观察GFP基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞的表达。方法通过细菌内同源重组方法构建携带有绿色荧光蛋白基因的重组腺病毒（Ad-GFP）,观察重组腺病毒转染培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,不同时间内绿色荧光蛋白基因的表达情况、表达部位及病毒对培养耳蜗Corti器和螺旋神经节细胞的影响。结果构建的重组腺病毒经酶切电泳鉴定正确;荧光显微镜下观察腺病毒转染体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,12 h即开始表达绿色荧光蛋白基因,24 h达到高峰,表达时间可持续1周;倒置显微镜观察转染后的Corti器和螺旋神经节细胞形态结构及生长无明显改变。结论本实验成功构建了携带绿色荧光蛋白（GFP）基因的重组腺病毒,通过该方法构建的腺病毒可以介导绿色荧光蛋白（GFP）基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞中表达,但腺病毒本身对体外培养的耳蜗Corti器和螺旋神经节细胞生长无明显影响,为通过腺病毒进行内耳基因治疗提供了理论依据。