水通道蛋白在内淋巴囊和肾脏的表达及加压素对水通道蛋白表达的影响

目的　从形态学上直接验证水通道蛋白 (aquaporin ,AQP) 2、3、4在内淋巴囊上皮的表达 ;观察加压素 (anti diuretichormone,AVP)及血管加压素受体 2 (V2receptor,V2 R)受体促效剂去精氨酸加压素 [(deamino Cys1,D Arg8) vasopressin ,dDAVP]对AQP2在内淋巴囊上表达的变化 ,并与肾脏比较 ,为耳肾相关提供参考。方法　成年大鼠 5只 ,经活体心脏灌注 ,取双侧颞骨 ,按石蜡包埋技术处理和切片。使用免疫组织化学方法标记确认AQP2、3、4在内淋巴囊的表达 ;使用成年大鼠 10只观察AVP及DDAVP对AQP2在内淋巴囊和肾脏上表达的变化。结果　荧光显微镜下 ,AQP 2、3、4一抗在内淋巴囊上皮胞膜胞浆显示荧光染色 ,肾脏集合管主细胞胞膜胞浆有稳定清晰染色反应。AVP组 (B组 )和DDAVP组 (C组 )的内淋巴囊上皮胞膜胞浆AQP 2的表达明显较阴性对照组 (A组 )降低 ,表现为荧光减弱 ,图像分析示灰度明显减弱 (P <0 0 1) ;B组和C组AQP 2的表达则差异无显著性。B组和C组肾脏集合管主细胞胞膜胞浆AQP 2的表达明显较A组增强 ,表现为棕黄色颗粒染色明显加深 ,图像分析示灰度明显减弱 (P <0 0 5 ) ,密度明显增加 (P <0 0 5 ) ,总积分密度 (totalintegrateddensity,IOD)明显提高 (P <0 0 5 ) ,面积明显增加 (P <0 0 5 ) ;B组和C     

豚鼠耳蜗和内淋巴囊水通道蛋白的表达

目的 研究豚鼠耳蜗和内淋巴囊组织中水通道蛋白(aquaporin，AQP)不同亚型的定位表达及其意义。方法 用兔抗大鼠AQP1、AQP2、AQP3、AQP4的多克隆抗体，采用免疫组化SP法分别检测相应AQP蛋白亚型在豚鼠耳蜗和内淋巴囊组织中的表达模式。结果 在耳蜗组织中，AQP1、4广泛分布于耳蜗的各个区域，如血管纹、螺旋韧带、Corti器、螺旋缘、螺旋神经节等，AQP3除了在血管纹表达呈弱阳性外，其余区域的表达与AQP1和AQP4相似，AQP2则仅表达于Reissner膜。在内淋巴囊组织中，AQP1、AQP3和AQP4在内淋巴囊上皮细胞和上皮下纤维组织均呈强阳性表达，只是AQP3的表达强度稍弱于AQP1和AQP4，而AQP2在内淋巴囊上皮细胞和上皮下组织均呈阴性表达。结论 水通道蛋白1、3、4以相似的方式广泛分布于豚鼠耳蜗和内淋巴囊组织中，而AQP2则仅表达于Reissner膜，提示不同亚型的AQP可能在不同区域协同作用参与内淋巴的调节，从而保持内耳内环境的稳定。     

豚鼠内淋巴囊Na—K—ATP酶亚基异构体的表达

目的 研究豚鼠内淋巴囊组织中Na,K-ATP酶α、 β亚基各个异构体的表达及其意义。方法 分别采用免疫组化汉和原位杂交法观察豚鼠内淋巴囊组织 Na,K-ATP酶α和β亚基异构体的分布。结果 Na,K-ATP酶不同的亚基异构体在豚鼠内淋巴囊上皮细胞和上皮下组织的表达有差异。内淋巴囊上皮细胞的胞膜主要表达α1、β1和β2异构体，且β2表达较β1表达更强，上皮下组织细胞的胞膜则主要表达α1和α2异构体，且α1表达较α2表达强，而上皮细胞和上皮下组织中均未见有α3 异构体表达。结论 内淋巴囊组织的Na,K-ATP酶由不同亚基异构体组成，它们协同作用参与保持内耳内环境的稳定。     

豚鼠内淋巴囊Na-K-ATP酶亚基异构体的表达

目的研究豚鼠内淋巴囊组织中Na,K-ATP酶α、β亚基各个异构体的表达及其意义。方法分别采用免疫组化法和原位杂交法观察豚鼠内淋巴囊组织中Na,K-ATP酶α和β亚基异构体的分布。结果Na,K-ATP酶不同的亚基异构体在豚鼠内淋巴囊上皮细胞和上皮下组织的表达有差异。内淋巴囊上皮细胞的胞膜主要表达α1、β1和β2异构体,且β2表达较β1表达更强,上皮下组织细胞的胞膜则主要表达α1和α2异构体,且α1表达较α2表达强,而上皮细胞和上皮下组织中均未见有α3异构体表达。结论内淋巴囊组织的Na,K-ATP酶由不同亚基异构体组成,它们协同作用参与保持内耳内环境的稳定。     

两种内淋巴积水动物模型中水通道蛋白1的表达

目的了解水通道蛋白1（aquaporin 1，AQP1）在手术及醛固酮引起的内淋巴积水豚鼠耳蜗及内淋巴囊中的表达情况。方法30只健康豚鼠随机分为手术组、醛固酮注射组和对照组，每组10只。通过手术阻塞内淋巴囊和腹腔注射醛固酮的方法制造出两种内淋巴积水动物模型，采用免疫组化染色及蛋白质印迹法检测豚鼠耳蜗及内淋巴囊中AQP1的表达，通过图像处理软件进行半定量分析。结果手术组出现中、重度内淋巴积水，以顶回最为明显，自顶回向底回减轻；醛固酮注射组出现轻、中度积水，积水多位于底回。两组积水动物模型中AQP1表达的部位与对照组相同，手术组耳蜗与对照组相比AQP1表达差异无统计学意义（t=0．718，P〉0．05）；醛固酮组耳蜗AQP1表达低于对照组（t=6．609，P〈0．01）；对照组与醛固酮组内淋巴囊AQP1表达差异无统计学意义（t=0．998，P〉0．05）。醛固酮组耳蜗外侧壁组织中AQP1蛋白定量低于对照组（t=13．626，P〈0．01）。结论AQP1在手术引起的内淋巴积水中表达没有改变，在醛固酮引起的内淋巴积水中表达下降。AQP1在豚鼠内耳中的表达可能受离子浓度的调节。     

前庭学

20050401脉鼠耳蜗和内淋巴囊水通道蛋白的表达/钟时勋…//听力学及言语疾病杂志.2004,12(5).337～339目的:研究脉鼠耳蜗和内淋巴囊组织中水通道蛋白(AQP)不同亚型的定位表达及其意义。方法:用兔抗大鼠AQP1、AQP2、AQP3、AQP4的多克隆抗体,采用免疫组化SP法分别检测相应AQP蛋白亚型在豚鼠耳蜗和内淋巴囊组织中的表达模式。结果:在耳蜗组织中,AQP1、4广泛分布于耳蜗的各个区域,如血管纹、螺旋韧带、Corit器、螺旋缘、螺旋神经节等,AQP3除了在血管纹表达呈弱阳性外,其余区域的表达与AQP1和AQP4相似,AQP2则仅表达于Reissner膜。…     

水通道蛋白-2及加压素的调控——水通道蛋白-2在内淋巴囊的表达和调控

水通道蛋白-2(AQP2)是一种加压素调控的四聚体通道蛋白，是水转运的特异性通道，主要表达于肾脏集合管主细管腔侧(顶端)的细胞膜上和细胞质内。研究发现不论是人类还是大鼠的内耳组织，水通道蛋白-2仅在内淋巴囊表达，其它内耳组织则未见表达；而AQP2是肾脏尿液的重吸收，充血性心衰的水潴留等生理、病理机制的重要环节。这个发现无疑对有关内耳水代谢疾病的研究，如梅尼埃病，开辟了一个新的方向。AQP2在肾脏和内淋巴囊的特异性表达，进一步揭示了耳肾相关的机制，也提示了内淋巴囊在耳肾相关中的重要地位。     

水通道蛋白2在内淋巴囊上皮和肾脏中表达的研究

目的 探讨水通道蛋白2(Aquaporins 2,AQP-2)和加压素(anti-diuretic hormone,AVP)在内耳水代谢中的作有机制.方法 15只(30耳)成年大鼠,随机分为三组,每组5只(10耳).A组(对照组):腹腔注射生理盐水;B组(AVP组):腹腔注射AVP(0.2 U/g),C组(DDAVP组):尾静脉注射V2受体促效剂去精氨酸加压素([deamino-Cys1,D-Arg8]-Vasopressin,DDAVP)1 ng/只.采用间接免疫荧光法标记内淋巴囊(endolymphatic sac,ES)中AQP-2的表达,ABC法标记肾脏中AQP-2的表达.结果 AQP-2-抗在内淋巴囊上皮胞膜胞浆显示荧光染色,B组和C组的内淋巴囊上皮胞膜胞浆AQP-2的表达明显较A组降低(P＜0.01);B组和C组AQP-2的表达则差异无显著统计学意义(P＞0.05).B组和C组肾脏集合管主细胞胞膜胞浆AQP-2的表达明显较A组增强(P＜0.05),B组和C组AQP-2表达则差异无显著统计学意义(P＞0.05).结论 AVP促进肾脏表达AQP-2,提高肾脏重吸收的功能,同时抑制内淋巴囊上皮表达AQP-2,从而降低内淋巴囊上皮对水的重吸收;AVP对肾脏和内淋巴囊AQP-2表达的调节可能是通过AVP-V2R-cAMP-AQP2途径实现.     

内淋巴囊的神经分布

目的：通过实验从形态学上直接验证内淋巴囊上存在神经纤维，为梅尼埃病的病因病理学研究提供新的思路。方法：使用成年SD大鼠15只，经活体心脏灌注，取双侧颞骨，常规石蜡包埋处理并切片。用免疫组织化学方法标记，确认抗体神经特异性烯醇化酶（neuronal specific enolase, NSE）、胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）、神经微丝（neurofilament, NF）在内淋巴囊(Endolymphatic Sac, ES)上的表达。结果：在光学显微镜下，NSE、GFAP、NF一抗在内淋巴囊上皮及上皮下表达为棕黄色阳性着色颗粒。结论：内淋巴囊近侧端、中间段和远侧段均有神经纤维分布，以近侧段最为丰富。     

水通道蛋白—2及加压素的调控——水通道蛋白—2在内淋巴囊的表达和调控

水通道蛋白－2（AQP2）是一种加压素调控的四聚体通道蛋白，是水转运的特异性通道，主要表达于肾脏集合管主细管腔铡（顶端）的细胞膜上和细菌质内。研究发现不论是人类还是大鼠的内耳组织，水通道蛋白－2仅在内淋巴囊表达，其它内耳组织则未见表达；而AQP2是肾脏尿液的重吸收，充血性心衰的水潴留等生理、病理机制的重要环节。这个发现无疑对有关内耳水代谢疾病的研究，如梅尼埃病，开辟了一个新的方向。APQ2在肾脏和肉淋巴囊的特异性表达，进一步揭示了耳肾相关的机制，也提示了要巴囊在耳肾相关中的重要地位。     

Expressions of aquaporin-2, vasopressin type 2 receptor, transient receptor potential channel vanilloid (TRPV)1, and TRPV4 in the human endolymphatic sac

OBJECTIVE: To localize aquaporin (AQP)2, vasopressin type 2 receptor (V2-R), and transient receptor potential channel vanilloid subfamily 1, 4 (TRPV1, TRPV4) in the human endolymphatic sac (ES). METHODS: Three samples of human ES were sampled during the removal of vestibular schwannoma by way of the translabyrinthine approach. The samples were immediately fixed in 4% paraformaldehyde and embedded in OCT compound; immunohistochemistry was performed with AQP2, V2-R, TRPV1, and TRPV4 polyclonal antibodies. RESULTS: AQP2, V2-R, TRPV1, and TRPV4 proteins were detected in the epithelial layer of the ES but were not observed in connective tissue around the ES. TRPV1 was also expressed in blood vascular endothelial cells of the connective tissue of ES. CONCLUSIONS: AQP2, V2-R, and TRPV4 were expressed in the luminal epithelium of human ES. The same characteristic distribution of water and ion channels is seen in the kidney, where a significant amount of fluid is filtrated and resorbed. ES probably plays an active role in the homeostasis of the endolymph.     

抗分泌因子与水通道蛋白1和2在大鼠内耳中的表达及其相互作用

目的研究抗分泌因子（antisecretory factor，AF）与水通道蛋白1、2（aquaporin-1，2，AQP1，2）在大鼠内耳中的表达及其相互作用。方法选取6只健康雄性SD大鼠，采用Envision二步法免疫组化技术，观察AF、AQP1和AQP2在大鼠内耳中的表达情况；选取20只健康雄性SD大鼠，分别取其前庭及耳蜗组织，运用免疫共沉淀结合蛋白质印记方法，用抗AQP1的单克隆抗体和抗AQP2多克隆抗体分别特异性地沉淀前庭和耳蜗组织中的蛋白抗原，用抗AF的特异性抗体检测沉淀物。结果AF在内耳分布广泛，耳蜗血管纹边缘细胞、螺旋韧带Ⅰ-Ⅴ型纤维细胞、前庭膜、基底膜、壶腹嵴等部位呈轻中度阳性反应，圆窗膜呈中强阳性反应，耳蜗螺旋神经节及前庭、耳蜗神经纤维均有阳性分布；AQP1主要分布于血管纹的中间细胞、螺旋韧带Ⅲ型纤维细胞、基底膜以及圆窗膜，染色强度为中重度；AQP2主要表达于螺旋韧带Ⅱ型、Ⅳ及Ⅴ型纤维细胞，呈中重度阳性染色反应，圆窗膜也有轻度表达。以AF特异性抗体分别检测AQP1及AQP2特异性抗体沉淀物中有清晰的阳性条带，相对分子质量约60000，与AF的相对分子质量吻合，而未加AQP1及AQP2特异性抗体的对照实验中无反应条带出现。结论AF、AQP1及AQP2在内耳组织中的分布有一定的规律，多位于与内淋巴关系密切的部位，提示三种蛋白可能参与内淋巴中水的调节。AF的分布区与AQP1及AQP2均有重叠，AQP1、AQP2与AF之间均存在相互结合作用。     

水通道蛋白-1,2在大鼠内耳中表达的研究

目的 通过研究水通道蛋白1（aquaporin 1,AQP1）和水通道蛋白2（aquaporin 2,AQP2）在大鼠内耳组织中的表达及分布,为临床治疗梅尼埃病提供实验依据.方法选取6只健康雄性SD大鼠,断头后行内耳组织切片,用兔抗大鼠AQP1和AQP2的特异性多克隆抗体分别进行免疫组织化学染色,观察AQP1和AQP2在内耳组织中的表达情况.结果 AQP1在内耳组织中的分布有一定的规律,主要分布于血管纹的中间细胞,螺旋韧带Ⅲ型纤维细胞,基底膜以及圆窗膜,染色强度为中重度,前庭阶及鼓阶的外淋巴表面的细胞呈现较弱的阳性反应,其余部位为阴性反应.AQP2主要表达于螺旋韧带Ⅱ、Ⅳ及Ⅴ型纤维细胞,呈中重度染色反应,圆窗膜也有轻度表达,其余部位为阴性反应.结论 AQP1和AQP2分布于内耳中水及离子代谢的重要部位,提示两种蛋白可能参与内耳内环境稳态的调节.     

Ultrastructural studies of the round-window membrane of the cat

An ultrastructural study of eight round-window membranes of four normal cats disclosed three basic layers: an outer epithelium (middle ear), a middle core of connective tissue, and an inner epithelium (inner ear). Morphologic evidence suggests that these layers participate in absorption and secretion of substances to and from the inner ear, such that the entire membrane could play a role in an inner ear "defense system." A comparison of morphologic features of round-window membranes from cats, rodents, and human beings suggests that the cat is a superior animal model for experimental studies. Cationic ferritin, placed for two hours in the round-window niche of four normal cats, was observed to traverse the round-window membrane through pinocytotic vesicles into the connective tissue layer. Evidence of exocytosis of tracer by the inner epithelial layer into the scala tympani is presented. When placed in perilymph, this same tracer was incorporated by inner epithelial cells, suggesting absorptive capabilities of the round-window membrane.     

Expression of histamine receptors (H(1), H(2), and H(3)) in the rabbit endolymphatic sac: an immunohistochemical study

OBJECTIVE: The endolymphatic sac (ES) is part of the membranous labyrinth of the inner ear. Its central role in immunologic activity within the inner ear has been confirmed by numerous studies. The aim of this study was to investigate the expression of histamine receptors (H(1), H(2), H(3)) in the rabbit ES. METHODS: A total of 10 healthy male New Zealand white rabbits weighing 2 to 3 kg were used in the experiments. For immunohistochemical studies, immunostaining was performed according to the avidin-biotin-peroxidase complex technique. RESULTS: Serial sections of the ES of rabbits revealed the presence of H(1), H(2), and H(3) receptor immunoreactivity. Immunoreactive cells for all H(1), H(2), and H(3) were found in the epithelial and subepithelial layers of the duct and the proximal ES. In conclusion, this study showed the immunohistochemical localization of H(1), H(2), and H(3) receptors in the ES of rabbits. These receptors may be important in the homeostasis of the inner ear. In addition, they may be target receptors in the medical treatment of inner ear disorders such as endolymphatic hydrops.     

Localization of melatonin and its receptors (melatonin 1a and 1b receptors) in the mouse inner ear

Abstract

Background: In the inner ear, evidence has been gathered indicating that melatonin plays important roles in inner ear physiology and pathophysiology. However, no attempt has been made previously to investigate the localization or expression of melatonin and its receptors in the whole inner ear.

Aims/objectives: To analyze the presence of melatonin and its receptors in the normal mouse inner ear.

Material and methods: C57BL6/J mice were used in this study. The localizations of melatonin, MT1a and MT1b in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion and endolymphatic sac (ES), were studied by immunohistochemistry.

Results: The organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory cells, vestibular dark and transitional cells, and ES epithelial cells showed an immunofluorescence reaction to melatonin, MT1a and MT1b.

Conclusion and significance: The present findings show that melatonin and its receptors (MT1a and MT1b) are present in the inner ear, thus supporting the hypothesis that melatonin plays a physiological role in the inner ear.     

Expression patterns of aquaporins in the inner ear: evidence for concerted actions of multiple types of aquaporins to facilitate water transport in the cochlea

Water transport between the perilymph and endolymph is important in regulations of volume and osmotic pressure of the inner ear labyrinth. It is now known that expression of water channels (aquaporins or AQPs) in the cell membrane dramatically increases the ability of water to cross epithelial cells. The aims of the current study were to investigate the cellular localization of AQPs by immunolabeling, and to study the developmental expression and relative abundance of various subtypes of AQPs. We report here that AQP3, AQP7 and AQP9 were expressed in the inner ear. Specific subtypes of AQPs were found in discrete regions expressed by both epithelial cells and fibrocytes in cochlear and vestibular organs. Semi-quantitative measurements showed that AQP4 and AQP1 were the two most abundantly expressed AQP subtypes in the inner ear, and their expressions were dramatically upregulated during development. These data showed a highly localized and largely non-overlapping distribution pattern for different subtypes of AQPs in the inner ear, suggesting the existence of regional subtype-specific water transport pathways, and global regulation of water transport in the inner ear may require concerted actions of multiple types of AQPs.     

Efficient transfer of embryonic stem cells into the cochlea via a non-invasive vestibular route

CONCLUSION: Cell transplantation into the utriculus provides an efficient and non-invasive route to introduce embryonic stem (ES) cells into the vestibular and cochlear portions of the inner ear. OBJECTIVE: The transfer of stem cells into the inner ear for therapeutic purposes is an important approach to cure damage to the cochlea and vestibulum. A key issue is to provide an entry point for cell transplants into the inner ear that does not affect its physiologic functions. The aim of this study was to examine the feasibility of transferring ES cells into the inner ear via the utriculus. MATERIALS AND METHODS: ES cells were injected via utriculostomy into the mouse inner ear. The distribution of the injected cells was determined using a beta-galactosidase marker gene expressed by the ES cells. RESULTS: Injected ES cells were found within the perilymph of the scala tympani and vestibuli. Moreover, ES cells were detected close to the cochlear sensory epithelium and spiral limbus.     

Anatomic distribution and localization of immunocompetent cells in normal mouse endolymphatic sac

The distribution and anatomic localization of immunocompetent cells in normal mouse endolymphatic sac (ES) were examined by immunohistochemical methods. Antibodies against T cells (anti-Thy-1, -Lyt-1, -Lyt-2), macrophage (anti-Mac-1) and immunoglobulins (anti-IgM, -IgG, -IgA) were employed in an indirect technique utilizing strept-avidin-biotin complex. In the normal mouse ES, each of these cells could be detected by a difference in its frequency and distribution within and around the ES. Thy-1 positive (Thy-1+) cells are the most predominant in this tissue and can be seen throughout the ES. Lyt-1+ cells were found within the ES epithelium and in the perisaccular region, whereas Lyt-2+ cells were rarely present. Mac-1+ cells were present primarily in the lumen of the distal portion of the ES. IgM-bearing (IgM+) cells were seen in the subepithelial region, IgG+ cells were occasionally detected in the lumen and only a few IgA+ cells were present in the perisaccular region. This study revealed that the ES has the necessary immunological components for antigen processing and the generation of local immune responses within the inner ear.