【作者】 王燕；

【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， 2011， 博士

【摘要】 第一部分成年小鼠耳蜗血管纹缘细胞的原代分离培养及鉴定目的：建立成年小鼠耳蜗血管纹边缘细胞(marginal cell, MC)的体外培养及鉴定方法,为进一步研究成年期MC的生理功能提供实验细胞。方法：显微分离成年期小鼠耳蜗血管纹组织,以Ⅱ型胶原酶消化原代培养边缘细胞,将其接种于六孔板中贴壁生长,加入含有15%胎牛血清、表皮生长因子、氢化可的松、胰岛素、三碘甲状腺原氨酸等促进细胞生长的完全培养基,在5%CO2/95%空气的37℃恒温培养箱中培养,每天观察细胞生长情况,换液一周两次或培养基颜色变浑浊。倒置显微镜下观察细胞形态,噻唑蓝(MTT)法测细胞的生长曲线,透射电镜观察细胞的超微结构,免疫荧光法检测上皮细胞的标志物-中间丝角蛋白18(CK18)和耳蜗血管纹的独特标志物KCNQ1的表达,RT-PCR检测CK18和KCNQ1mRNA的表达。结果：倒置显微镜下可见接种24-48h后细胞贴壁增殖,形成大小不等的细胞岛,一周后,细胞迅速生长,相互融合,紧密连接,形成单层极性上皮层,呈典型的“铺路石”样外观。透射电镜观察可见多角形细胞表面有较多微绒毛,胞浆内线粒体、内质网等细胞器丰富。免疫荧光细胞化学技术检测显示CK18和KCNQ1蛋白表达阳性,RT-PCR结果示CK18和KCNQ1的mRNA均有表达。结论：采用原代消化培养技术,成功建立成年小鼠耳蜗血管纹MC的体外原代培养,为进一步研究成年期MC的功能和某些内耳疾病的发病机制提供了实验细胞基础。第二部分α1Na,K-ATPase通道蛋白在C57BL/6J小鼠耳蜗的年龄相关性表达及其与AHL的关系目的：研究α1Na, K-ATPase离子转运蛋白在不同年龄段C57BL/6J(C57)小鼠耳蜗血管纹的表达及其意义。方法：选择3M,6M,9M,12M月龄C57J小鼠各12只,采用听性脑干反应(Auditory Brainstem Response, ABR)分别检测其听力变化。采用免疫组织荧光法和免疫印迹杂交Western blot (WB)检测α1Na,K-ATPase蛋白在小鼠耳蜗的表达变化,逆转录聚合酶链反应法(RT-PCR)检测各年龄段小鼠耳蜗α1Na,K-ATPase mRNA在不同鼠龄小鼠耳蜗中的表达水平。结果：随着鼠龄的增长,C57小鼠各频率ABR闽值逐渐升高,自6月龄小鼠的ABR反应闽逐渐增加,其数值与低龄组小鼠相比其差异均具有统计学意义(P<0.05)。免疫荧光检测显示α1Na,K-ATPase主要分布于血管纹底侧膜和螺旋韧带,表达量也随着鼠龄的增加呈现减少的趋势,Real-Time PCR检测显示α1Na,K-ATPase mRNA随着小鼠年龄的增长其表达逐渐下调,与其蛋白变化趋势相一致。结论：C57小鼠耳蜗α1Na,K-ATPase的表达随着年龄的增加而逐渐下降,可能与年龄相关性听力损失具有一定联系。第三部分α1Na,K-ATPas通道蛋白在成年C57BL/6J小鼠血管纹边缘细胞的表达和意义目的：初步研究α1Na,K-ATPase在血管纹缘细胞的表达和意义,为进一步探索耳蜗K+循环提供实验研究基础。方法：采用胶原酶消化法体外分离培养成年小鼠耳蜗血管纹边缘细胞。采用免疫组织荧光法检测α1Na,K-ATPase在血管纹边缘细胞的表达和分布,并运用RT-PCR检测α1Na,K-ATPase mRNA在边缘细胞的表达情况。结果：体外成功培养和纯化了血管纹边缘细胞,免疫荧光检测可见有α1Na,K-ATPase阳性表达,主要位于细胞膜,RT-PCR也检测到α1Na,K-ATPase mRNA的表达。结论：α1Na,K-ATPase在血管纹缘细胞表达,可能参与了耳蜗K+循环形成,在维持正常耳蜗听力中起重要作用。第四部分凋亡相关蛋白PDCD5在C57BL/6J小鼠耳蜗毛细胞和螺旋神经节细胞的年龄相关性表达及与老年性的关系目的：研究PDCD5和caspase-3在不同年龄段C57BL/6J(C57)小鼠耳蜗毛细胞、血管纹及螺旋神经节细胞的表达,初步探讨其在年龄相关性听力下降发生、发展中的作用。方法：选择C57小鼠3M,6M,9M,12M月龄段各15只,分为四组。对各组小鼠双侧click、6、8 kHz ABR阈值检测。采用免疫组织化学法和Western blotting(WB)检测各月龄段小鼠耳蜗PDCD5和caspase-3蛋白的表达,实时荧光定量PCR(Real-Time PCR)检测各月龄段小鼠耳蜗PDCD5和Caspase-3基因mRNA的表达变化。结果：随着年龄的增长,C57小鼠各频率ABR阈值逐渐提高,耳蜗PDCD5和caspase-3蛋白的表达亦逐渐增强。3月龄和6月龄小鼠耳蜗毛细胞和血管纹细胞及螺旋神经节细胞仅出现少量PDCD5和caspase-3蛋白表达,9月龄时表达有明显增加,至12月龄时表达最强,各组间相比有显著性差异(P<0.05)。Real-Time PCR检测显示PDCD5和Caspase-3基因mRNA随着小鼠年龄的增长其表达逐渐增强,与其蛋白变化趋势相一致。结论：C57小鼠耳蜗PDCD5和caspase-3的表达随着年龄的增长而明显增高,说明参与了小鼠耳蜗毛细胞和血管纹细胞及螺旋神经节细胞的凋亡调控过程,与耳蜗的老化密切相关,可能是老年性聋发病机制中的一个重要因素。更多还原

【Abstract】 Part I Establishment of marginal cell culture from stria vascularis of adult mouse and the expression of KCNQ1 in MCObjective:To provide an in vitro cell model for further study of the function of marginal cell(MC), primary cultures of marginal cells of adult mouse were established.Methods:The marginal cells were isolated from the stria vascularis of adult mice by CollagenaseⅡto culture. The morphologic changes of the cells were observed under an inverted lightmicroscope and the growth curve of the cell was determined by MTT assay. The ultrastructure of the cultured cells was observed by transmission electron microscopy. The expression of CK18 and KCNQ1 were detected by immunofluorescence and reverse transcription- polymerase chain reaction (RT-PCR).Results:The primary cultured cells attached within 24-48 h of seeding and proliferated in an island-like monolayer. After one week, more and more epithelial-like cells had a tendency to become confluent and became tightly packed, growing into a monocellular layer,"cobblestone-like appearance. Transmission electron microscope showed typical epithelia with microvilli on the cells surface, massive mitochondria and rough endoplasmic reticulum in the cells. Expression of CK18 and KCNQ1 in MC was confirmed by immunofluorescence staining, and expression of CK18 mRNA and KCNQ1 mRNA was also detected by RT-PCR.Conclusions:The cell culture system of marginal cells of adult mouse has been successfully established by enzyme digestion which can provide a stable source of MC for investigation of the ongoing function of the marginal cell.PartⅡExpression ofα1Na,K-ATPase in the cochlea of the C57BL/6J mouse at different ages and relationship with presbycusisObjective:To investigate the age related changes of the expression ofα1Na,K-ATPase in the cochlea of the C57BL/6 (C57) mice, and also discusses the relationship ofα1Na,K-ATPase and Presbycusis.Method:Auditory function of C57 mice t different ages between 3 and 12 months old was measured by auditory brainstem response (ABR) respectively. The changes ofα1Na, K-ATPase protein in the cochlea was detected by immunofluorescence and Western blot, the expression ofα1Na, K-ATPase mRNA and caspase-3 mRNA were detected using RT-PCR.Result:The average ABR threshold value from 6-month-old C57 mice was not significantly different from that of 3-month-old mice (P>0.05), but the difference between the average ABR threshold value from mice of 12 months old than 9 months and that of 3-month-old mice was highly significant (P<0.01), and the average value appeared to progressively increase with age. The localization ofα1Na, K-ATPase was found mainly in the stria vascularis and the spiral ligament, and the expression ofα1Na, K-ATPase protein was decrease in the stria vascularis and the spiral ligament with age. The change ofα1Na, K-ATPase mRNA expression was in accordance with that ofα1Na,K-ATPase protein expression by the real-time PCR.Conclusion:Expression ofα1Na, K-ATPase in the cochlea of C57 mice gradually decreased with increasing age, and might play an important role in pathogenic mechanism of Age-related hearing loss (AHL).PartⅢExpression and significance ofα1Na, K-ATPase in the marginal cell from stria vascularis of adult C57BL/6J miceObjective:To study the expression and significance of ofα1Na, K-ATPase in the marginal cell from stria vascularis of adult C57BL/6J (C57) mice, and provides an experimental basis for explored K+ ion recycling.Method:The marginal cell isolated by CollagenaseⅡto culture. The expression ofα1Na, K-ATPase ion transporters in the marginal cell of C57 mice was detected by immunofluorescence and the expression ofα1Na, K-ATPase by RT-PCRResult:Theα1Na, K-ATPase were cultured and purified in vitro. The immunofluorescence results showedα1Na, K-ATPase only expressed in membrane of marginal cell, and the mRNA expression ofα1Na, K-ATPase was also detected by RT-PCR.Conclusion:The present results suggest that theα1Na,K-ATPase expression in membrane of of marginal cell may play an important role in the K+ ion recycling of the inner ear.Part IV Expression of PDCD5 and caspase-3 in the cochlea of different age C57BL/6J mice and relationship with presbycusisObjective:To investigate the age related changes of the expression of PDCD5 and caspase-3 in the cochlea of the C57BL/6(C57)mice. This paper also discusses the relationship of PDCD5 and caspase-3 and the possible role in the pathogenesis of Presbycusis.Method:C57 mice of 3,6,9 and 12 months old were selected and divided into 4 groups (fifteen mice each). Auditory function of C57 mice was measured by auditory brainstem response (ABR) respectively. The changes of PDCD5 and caspase-3 protein in the cochlea was detected by immunohistochemistry and Western blot, the changes of PDCD5 mRNA and caspase-3 mRNA were detected using RT-PCR.Result:With an increase of age,The mean value for ABR thresholds in response to click,4 kHz and 8 kHz sound stimulus of C57 mice gradually increased, the expression of PDCD5 and caspase-3 was increased also. At 3 months and 6 months of age in the cochlea of C57, all sorts of expression of PDCD5 and caspase-3 and the expression was enhanced with age. There was an evident expression at 9 months of age, but the highest expression was detected at 12 months of age. The PDCD5 and caspase-3 expression was statistically different in each group (P<0.05).The change of PDCD5 and caspase-3 mRNA expression was in accordance with that of PDCD5 and caspase-3 protein expression by the real-time PCR.Conclusion:The increased expression levels of PDCD5 and caspase-3 in the cochlea of C57 mice with age, the result s suggested that the expression of PDCD5 and caspase-3 participated in the regulatory procedure of apoptosis and might play an important role in pathogenic mechanism of Presbycusis.更多还原