【作者】 刘英鹏；

【导师】 龚树生； Zhao，Hong-Bo；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 目的:研究缝隙连接Cx26和Cx30在耳蜗侧壁中的表达和细胞分布,同时比较Cx26和Cx30在不同哺乳动物耳蜗中表达的差异。方法:采用免疫荧光染色方法和激光共聚焦显微镜检测大鼠、小鼠和豚鼠耳蜗侧壁血管纹和螺旋韧带中缝隙连接Cx26和Cx30表达和细胞分布情况。结果:耳蜗侧壁由血管纹和螺旋韧带组成,通过血管纹中间细胞的特异性细胞标记Kir4.1,我们检测到在血管纹中间细胞层,Cx26和Cx30均有广泛表达,并且呈树叶状分布;而在血管纹的基底细胞层,Cx26和Cx30表达呈六角形分布在细胞周围;Cx26和Cx30在血管纹的边缘细胞没有表达。在螺旋韧带中,Cx26和Cx30的表达与血管纹中不一样,Cx26和Cx30表达是沿着与螺旋韧带的纵轴垂直的方向排列分布的。Cx26主要表达在螺旋韧带的边缘部位,在螺旋韧带中间部位,即在螺旋韧带的血管纹侧表达较弱;而在螺旋韧带中间部位主要以Cx30表达为主。结论:Cx26和Cx30表达在耳蜗侧壁血管纹和螺旋韧带不同,在血管纹和螺旋韧带不同细胞中的表达也不一样,Cx26与Cx30在耳蜗侧壁的分布差异,提示Cx26和Cx30在耳蜗内淋巴电位和离子梯度的形成中发挥不同的作用。目的:研究不同哺乳动物耳蜗侧壁中缝隙连接Cx26和Cx30蛋白和mRNA表达情况及定量分析。方法:首先,采用Western-blot方法检测不同的动物(包括小鼠、大鼠和豚鼠)耳蜗侧壁血管纹和螺旋韧带,以及顶回和低回的血管纹和螺旋韧带中缝隙连接Cx26和Cx30蛋白表达情况及定量分析。其次,RT-PCR方法检测小鼠耳蜗血管纹和螺旋韧带中Cx26和Cx30的mRNA表达情况及定量分析。结果:耳蜗血管纹和螺旋韧带的Western-blot结果分析,显示三种动物的螺旋韧带中Cx26和Cx30蛋白表达量是高于血管纹Cx26和Cx30的蛋白表达量,在豚鼠、大鼠和小鼠螺旋韧带中的Cx26蛋白表达量分别是血管纹中的5.1958、1.7708和1.7570倍;三种动物Cx26蛋白表达量不同具有统计学差异(P< 0.05,ANOVA)。Cx30蛋白表达量在豚鼠、大鼠和小鼠螺旋韧带和血管纹的比例分别是4.2973、3.6205和3.5554,三种动物Cx30蛋白表达量不同没有显著统计学差异(P > 0.05,ANOVA)。此外,三种动物耳蜗侧壁顶回和底回Cx26和Cx30蛋白表达量比较结果,显示顶回和底回Cx26和Cx30蛋白表达量没有显著统计学差异(P > 0.05,T-test)。RT-PCR结果显示小鼠耳蜗螺旋韧带中Cx26和Cx30的mRNA表达要高于血管纹,Cx26和Cx30在螺旋韧带与血管纹的比值分别是1.8812和1.1643倍。结论: Cx26和Cx30的蛋白和mRNA表达在耳蜗螺旋韧带总是要高于血管纹,在不同动物耳蜗血管纹和螺旋韧带的表达不一样;Cx26和Cx30在侧壁表达的差异提示Cx26和Cx30在内耳侧壁中的作用可能不完全一样。目的:研究耳蜗基底膜组织中Cx26、Cx30和紧密连接ZO1的表达和相互关系。方法:采用免疫荧光双标记和激光共聚焦显微镜方法观察耳蜗基底膜组织中缝隙连接Cx26和Cx30与紧密连接ZO1的分布和相互关系。结果:在耳蜗基底膜非感觉上皮细胞,Cx26和Cx30共同表达在Hensen细胞、内外支柱细胞和Claudius细胞,Cx26在基底膜的表达部位大部分与Cx30的表达重合;在蜗轴螺旋缘也检测到Cx26和Cx30表达。在耳蜗内、外毛细胞没有Cx26和Cx30的表达。Cx26和ZO1在基底膜表达不重合,在基底膜的不同层面,Cx26和ZO1的表达逐渐发生改变;在基底膜网状板面,ZO1广泛分布在与内、外毛细胞和与之相邻的支持细胞之间的细胞连接中,Cx26在基底膜网状板面没有表达。而在基底膜深面, Cx26广泛分布在基底膜组织的各种支持细胞,在该层面没有发现的ZO1表达。结论:Cx26和Cx30分布在基底膜的各种支持细胞,在感觉毛细胞没有表达;此外,该研究发现Cx26和ZO1在耳蜗内具有不同的表达方式和表达部位,紧密连接ZO1在网状板面构成了毛细胞之间或与其支持细胞之间的离子屏障,阻止内、外淋巴之间的相互流通,保持了各自之间的离子平衡;缝隙连接则表达在基底膜的深层,参与离子转运。更多还原

【Abstract】 Object: To study the expressions and cellular distributions of Cx26 and Cx30 in the cochlea lateral wall in different mammalian cochlea.Methods: We used double immunofluorescent staining and confocal microscopy to examine the expressions and cellular distributions of Cx26 and Cx30 in the stria vascularis (SV) and spiral ligament (SPL) in the cochlear lateral wall of rats,mice and guinea pigs.Results: By using specific cell marker (Kir4.1), we have firstly found that the intermediate cell has strong expressions of Cx26 and Cx30. Cx26 and Cx30 labeling appeared to be irregular and leaf-like in the intermediate cell. However, Cx26 and Cx30 in the basal cell layer showed intense labeling surrounding the basal cells, demonstrating a hexagonal distribution pattern. In the dissociated-cell preparation, Cx26 and Cx30 have labeling at the basal cell and the intermediate cell. The marginal cells were negative to Cx26 and Cx30 labeling. Cx26 and Cx30 in SPL showed a different pattern. The labeled puncta were distributed vertical to the long axis of the SPL. Cx26 labeling was intense at the edge region of the SPL and little at the middle region. Cx30 had intense labeling at the middle region in SPL.Conclusion: The data demonstrate that Cx26 and Cx30 in the SV and SPL have distinct, cell-specific expressions, implying that they achieve different functions in the cochlear lateral wall. Object: Quantitatively analysed the expressions of Cx26 and Cx30 proteins in the cochlear lateral wall.Methods: First, using Western Blot to examine the expressions of Cx26 and Cx30 in the SV and SPL, and in the apical and basal turns in the cochlear lateral wall in different species (mice, rats, and guinea pigs). Second, using RT-PCR to analyse the expressions of mRNA of Cx26 and Cx30 in the SV and SPL in the cochlear lateral wall of rats,mice and guinea pigs.Results: From Western-blot of SV and SPL, we found that the expressions of Cx26 and Cx30 in the SPL were higher than in the SV. The level of Cx26 protein in SPL was 5.1958,1.7708 and 1.7570 fold higher than in SV in guinea pigs, rats and mice respectively. There was a statistically significant difference among different species (P < 0.05, ANOVA). However, Cx30 protein level of SPL was 4.2973,3.6205 and 3.5554 fold compare to that of SV in guinea pigs, rats and mice, respectively. These was no statistically significant difference among different species(P >0.05,ANOVA). The analysis of Cx26 and Cx30 protein expressions in different turns indicated that there was no a significant difference between the apical and basal turns. We also found that the mRNA of Cx26 and Cx30 in the SPL of mice were higher than in the SV, the ratios between SPL to SV for Cx26 and Cx30 were 1.8812 and 1.1643, respectively.Conclusion: Our date indicate that the expressions of Cx26 and Cx30 in SPL are higher than those in the SV at mRNA and protein levels. The data also reveal that the expressions of Cx26 and Cx30 in the cochlea have differently expressive levels in different species. Objective: To study the expressions of Cx26, Cx30 and ZO-1 and relationship in the cochlear sensory epithelium.Methods: In this study, we used double immunofluorescent staining and confocal microscopy to examine the distrubutions Cx26, Cx30 and ZO-1 and relationship in the cochlear sensory epithelium.Results: Cx26 and Cx30 were both expressed in Hensen cell, inner and outer pillar cell and Claudius cell. Cx26 labeling largely overlapped that of Cx30 in these regions. Cx26 and Cx30 were also coexpressed in the spiral limbus. Neither Cx26 nor Cx30 labeling was seen in the hair cells. The expressions of Cx26 and ZO1 demonstrated distinct expressions between the surface layer and the deep layer in the cochlear sensory epithelium. In the surface layer of cochlear sensory epithelium, ZO1-positive labling was mainly localized between senseory hair cells and suporting cells, there were fewer or no Cx26-postive spots. In the deep layer of cochlear sensory epithelium, Cx26 was widely expressed. However, ZO1 staining was undetectable.Conclusion: ZO1 and Cx26 expressions in the sensory epithelium have different location and were not overlapped. Tight junctions maintain the ion barrier between the endolymph and perilymph,and gap junctions take part in ion transportation.更多还原