COCH基因表达、调控及与内耳钾离子代谢的相关性
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摘要
COCH基因是人类常染色体显性遗传非综合症型感音神经性耳聋并前庭功能障碍(DFNA9)的疾病基因，有研究认为COCH基因突变，可能是部分梅尼埃病发生的分子基础。梅尼埃病是一种常见内耳疾病，典型病例表现为发作性眩晕、渐进波动性耳聋及耳闷胀感，其发病机理不明，膜迷路积水被认为是该疾病的病理基础，而内淋巴囊的吸收障碍是造成膜迷路积水的重要原因。COCH基因的功能不明，对基因突变引起耳聋及前庭功能障碍的机理有不同的观点，对梅尼埃病与COCH基因的关系也存在不同的认识，有必要进行进一步的研究。
为研究COCH基因功能，探讨COCH基因突变引起听觉及前庭功能障碍的机理及COCH基因与梅尼埃病的关系，分三部分进行了实验性研究。实验(一)体外转录标记COCH基因的cRNA探针及sRNA探针，以原位杂交方法观察小鼠内耳COCH基因的表达分布，重点观察既往报道没有涉及，而在膜迷路积水形成中具有重要作用的内淋巴囊的COCH基因的表达。实验(二)采用新生小鼠耳蜗螺旋韧带外植的方法，培养分离耳蜗螺旋韧带成纤维细胞，免疫细胞化学、酶细胞化学及培养细胞原位透射电镜技术观察、鉴别成纤维细胞的类型。RT-PCR、Western blot检测培养细胞COCH、POU3F4/brn4基因表达，建立观察COCH、POU3F4/brn4基因体外表达的细胞模型。实验(三)引入两种干预因素，观察体外培养的耳蜗螺旋韧带Ⅰ型成纤维细胞COCH、POU3F4/brn4基因的表达变化。①引入人工合成的针对POU3F4/brn4基因mRNA的反义硫代磷酸寡聚脱氧核苷酸，干扰耳蜗螺旋韧带Ⅰ型成纤维细胞POU3F4/brn4的表达，免疫印迹检测反义干扰细胞POU3F4/brn4蛋白条带的强弱，RT-PCR检测反义寡核苷酸干扰细胞COCH基因转录水平的变化，观察POU3F4/brn4对COCH基因的转录调控作用。②培养液中掺入氯化钾溶液，提高细胞外液中钾离子浓度，RT-PCR检测耳蜗螺旋韧带Ⅰ型成纤维细胞COCH、POU3F4/brn4基因的转录水平，观察钾离子浓度与基因表达的关系。实验的主要结果及结论如下：
1．成年小鼠内淋巴囊周围间质细胞COCH基因表达阳性，其原位杂交染色平均积分光密度为0.4±0.25((?)±SD，单位：U/mm~2)，与壶腹嵴感觉上皮下间质细胞的0.47±0.21相似，弱于耳蜗螺旋韧带成纤维细胞的0.77±0.19。小鼠内耳COCH基因原位
第三军医大学博士学位论文
表达分布的观察结果提示，基因的功能与维持内耳间质组织细胞外基质的结构稳定有
。关，不支持COCH基因的功能与内耳免疫应答反应有关的推论。
2．外植新生小鼠耳蜗螺旋韧带，培养分离得到螺旋韧带1型成纤维细胞。RTPCR、
Western blot检测，细胞表达 COCH、POU3F4／bm4基因，在有限传代次数内表达丰度
不变，可作为观察COCH、POU3F4／bm4基因体外表达变化的细胞模型。
3．反义寡核着酸干扰的耳蜗螺旋韧带1型成纤维细胞，POU3F4／bm4蛋白条带灰
度值下降至空白对照细胞的35．3％，COCH基因转录水平随之下降至26．3％。体外培
养耳蜗螺旋韧带1型成纤维细胞中COCH基因的转录水平受POU3F4／bm4表达的直接
“或间接调控。
4．体外培养细胞中COCH、POU3F4／bm4基因的转录水平，受细胞外液钾离子浓
度升高的影响而上调，与正常对照细胞差异有显著意义。提示COCH、POU3F4／bM
基因与内耳钾离子代谢过程有关。
5．COCH基因功能的初步推论，COCH基因可能编码内耳间质组织中一种分泌
型糖蛋白，通过与间质组织中其它细胞外基质成分的结合，共同形成稳定的细胞外基
质结构框架。内耳尤其耳蜗间质组织中，这种稳定的细胞外基质结构框架对维持钾离
。子稳态及蜗内电位水平具有重要意义。
6．人类COCH基因突变可能与POU3F4／bm4基因突变具有相似的听功能障碍机
理，COCH基因突变，引起螺旋韧带等问质组织细胞外基质成分变化，螺旋韧带等内
耳间质组织结构疏松，成纤维细胞间缝隙连接网络破坏，内耳钾离子循环障碍，蜗内
电位水平因而下降，导致听觉及前庭功能障碍。
7．梅尼埃病的发生是多基因遗传因素与环境因素相互作用的结果，COCH基因突
变不能解释多数梅尼埃病的病理过程。但某些病理情况下，COCH基因编码蛋白可能
成为诱发内耳自身免疫反应的靶抗原，或者受到炎症反应的攻击，使内耳间质组织细
、胞外基质的结构稳定性破坏，影响内耳钾离子代谢过程，而引起发作性听觉及前庭功
能障碍。寻找影响COCH基因功能的环境损伤因素，可能揭示出梅尼埃病的发生机理。
COCH is a deafness gene inwhich mutations are responsible for an autosomal dominant hereditary non-syndromic sensorineural hearing loss with vestibular dysfunction (DFNA9). It has been suggested that missense mutation in COCH gene might be related to the pathogenesis of Meniere disease. Meniere disease is a common inner ear disease characterized by a traid of symptoms: vestibular symptoms, auditory symptoms, and pressure. The etiopathogenesis of Meniere disease is unknown, and pathologic correlate of MD is endolymphic hydrops, which is mostly attributed to the deficiency of endolymphic sac's absorbtion. Role of COCH gene in the inner ear physiology as well as mutations in deafness and vestibular dysfuntion is unclear, moreover, there are different standpoints of pathogenesis of Meniere disease associated with COCH gene. It is necessary to do further investigations.
In this study the characteristics expression of COCH gene were systemically observed, in vivo and in vitro. In addition, the normal function of COCH gene, the underlying mechanism of COCH gene's mutation involves in hearing loss and vestibular dysfunction, and pathogenesis of Meniere disease associated with COCH gene were further discussed. In order to perform in situ hybridization, a complementary RNA probe to COCH mRNA were first labelled with digoxin by in vitro transcription; then with in situ hybridization, the localizations of COCH mRNA in adult mouse inner ears were detected. To facilitate observation, the postnatal day 0 mouse cochlear spiral ligaments (SPL) were dissectted and explanted to culture and purify the spiral ligament fibrocytes. Immunocytochemistry, enzymocytochemistry, and culturring cell in situ transmission electron microscopy were employed to identify fibrocyte type. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were used to detect the COCH and POU3F4/brn4
gene's expression. To investigate in vitro characteristics of COCH gene's expession, two factors were introduced in the culture medium of spiral ligament type I fibrocytes respectively. Firstly, phosphorothionate-modified antisense oligodeoxynucleiotides to POU3F4/brn4 mRNA was applied to study the regulation effects of POU3F4/brn4 to COCH gene
transcription. Secondly, 1M KCL was added in the normal culture medium, to rise the concentration of potassium in culture media. Then with RT-PCR and Western blot methods, the changes of gene expression levels were examined. The main results and conclusion were as following:
1. The positive hybridizations with COCH gene cRNA probe were detected in the mouse mesenchymic cell surroundding the endolymphic sac epithelium, the level of expression was similar to the fibrocytes underlying crista ampullaris sense epithellium, and inferior to the fibrocytes of spiral ligament. This expression distribution suggests that COCH gene may contribute to maintain inner ear connective tissue extracellular matrix architecture, and do not support the postulation that gene might play an inner ear immune defensive function.
2. In vitro, the culturing spiral ligament type I fibrocytes expressed COCH gene and POU3F4/brn4 gene, which were checked by RT-PCR and Western blot, and expression level did not chang with in 15 generations. This type cells may be in vitro model for investigatting the expression of COCH and POU3F4/brn4 gene.
3. The antisense oligonucleiotides to POU3F4/brn4 mRNA specially interfered in synthesis process of POU3F4/brn4 proteins, the quantity of proteins decreased to 35.3% that of control group cells. Along with it, the level of COCH gene mRNA also declined to 25.3%, compared to nomoral level. It suggests that POU3F4/brn4 may regulate the COCH gene's transcription by direct or indirect ways.
4. The expression of COCH as well as POU3F4/brn4 gene in cultured SPL type I fibrocytes were all upregulated when liftting concentration of potassium in culture media. It postulates that COCH gene and POU3F4/brn4 gene's function may be associated with metabolism of potassium in inner ear.
5.
为研究COCH基因功能，探讨COCH基因突变引起听觉及前庭功能障碍的机理及COCH基因与梅尼埃病的关系，分三部分进行了实验性研究。实验(一)体外转录标记COCH基因的cRNA探针及sRNA探针，以原位杂交方法观察小鼠内耳COCH基因的表达分布，重点观察既往报道没有涉及，而在膜迷路积水形成中具有重要作用的内淋巴囊的COCH基因的表达。实验(二)采用新生小鼠耳蜗螺旋韧带外植的方法，培养分离耳蜗螺旋韧带成纤维细胞，免疫细胞化学、酶细胞化学及培养细胞原位透射电镜技术观察、鉴别成纤维细胞的类型。RT-PCR、Western blot检测培养细胞COCH、POU3F4/brn4基因表达，建立观察COCH、POU3F4/brn4基因体外表达的细胞模型。实验(三)引入两种干预因素，观察体外培养的耳蜗螺旋韧带Ⅰ型成纤维细胞COCH、POU3F4/brn4基因的表达变化。①引入人工合成的针对POU3F4/brn4基因mRNA的反义硫代磷酸寡聚脱氧核苷酸，干扰耳蜗螺旋韧带Ⅰ型成纤维细胞POU3F4/brn4的表达，免疫印迹检测反义干扰细胞POU3F4/brn4蛋白条带的强弱，RT-PCR检测反义寡核苷酸干扰细胞COCH基因转录水平的变化，观察POU3F4/brn4对COCH基因的转录调控作用。②培养液中掺入氯化钾溶液，提高细胞外液中钾离子浓度，RT-PCR检测耳蜗螺旋韧带Ⅰ型成纤维细胞COCH、POU3F4/brn4基因的转录水平，观察钾离子浓度与基因表达的关系。实验的主要结果及结论如下：
1．成年小鼠内淋巴囊周围间质细胞COCH基因表达阳性，其原位杂交染色平均积分光密度为0.4±0.25((?)±SD，单位：U/mm~2)，与壶腹嵴感觉上皮下间质细胞的0.47±0.21相似，弱于耳蜗螺旋韧带成纤维细胞的0.77±0.19。小鼠内耳COCH基因原位
第三军医大学博士学位论文
表达分布的观察结果提示，基因的功能与维持内耳间质组织细胞外基质的结构稳定有
。关，不支持COCH基因的功能与内耳免疫应答反应有关的推论。
2．外植新生小鼠耳蜗螺旋韧带，培养分离得到螺旋韧带1型成纤维细胞。RTPCR、
Western blot检测，细胞表达 COCH、POU3F4／bm4基因，在有限传代次数内表达丰度
不变，可作为观察COCH、POU3F4／bm4基因体外表达变化的细胞模型。
3．反义寡核着酸干扰的耳蜗螺旋韧带1型成纤维细胞，POU3F4／bm4蛋白条带灰
度值下降至空白对照细胞的35．3％，COCH基因转录水平随之下降至26．3％。体外培
养耳蜗螺旋韧带1型成纤维细胞中COCH基因的转录水平受POU3F4／bm4表达的直接
“或间接调控。
4．体外培养细胞中COCH、POU3F4／bm4基因的转录水平，受细胞外液钾离子浓
度升高的影响而上调，与正常对照细胞差异有显著意义。提示COCH、POU3F4／bM
基因与内耳钾离子代谢过程有关。
5．COCH基因功能的初步推论，COCH基因可能编码内耳间质组织中一种分泌
型糖蛋白，通过与间质组织中其它细胞外基质成分的结合，共同形成稳定的细胞外基
质结构框架。内耳尤其耳蜗间质组织中，这种稳定的细胞外基质结构框架对维持钾离
。子稳态及蜗内电位水平具有重要意义。
6．人类COCH基因突变可能与POU3F4／bm4基因突变具有相似的听功能障碍机
理，COCH基因突变，引起螺旋韧带等问质组织细胞外基质成分变化，螺旋韧带等内
耳间质组织结构疏松，成纤维细胞间缝隙连接网络破坏，内耳钾离子循环障碍，蜗内
电位水平因而下降，导致听觉及前庭功能障碍。
7．梅尼埃病的发生是多基因遗传因素与环境因素相互作用的结果，COCH基因突
变不能解释多数梅尼埃病的病理过程。但某些病理情况下，COCH基因编码蛋白可能
成为诱发内耳自身免疫反应的靶抗原，或者受到炎症反应的攻击，使内耳间质组织细
、胞外基质的结构稳定性破坏，影响内耳钾离子代谢过程，而引起发作性听觉及前庭功
能障碍。寻找影响COCH基因功能的环境损伤因素，可能揭示出梅尼埃病的发生机理。
COCH is a deafness gene inwhich mutations are responsible for an autosomal dominant hereditary non-syndromic sensorineural hearing loss with vestibular dysfunction (DFNA9). It has been suggested that missense mutation in COCH gene might be related to the pathogenesis of Meniere disease. Meniere disease is a common inner ear disease characterized by a traid of symptoms: vestibular symptoms, auditory symptoms, and pressure. The etiopathogenesis of Meniere disease is unknown, and pathologic correlate of MD is endolymphic hydrops, which is mostly attributed to the deficiency of endolymphic sac's absorbtion. Role of COCH gene in the inner ear physiology as well as mutations in deafness and vestibular dysfuntion is unclear, moreover, there are different standpoints of pathogenesis of Meniere disease associated with COCH gene. It is necessary to do further investigations.
In this study the characteristics expression of COCH gene were systemically observed, in vivo and in vitro. In addition, the normal function of COCH gene, the underlying mechanism of COCH gene's mutation involves in hearing loss and vestibular dysfunction, and pathogenesis of Meniere disease associated with COCH gene were further discussed. In order to perform in situ hybridization, a complementary RNA probe to COCH mRNA were first labelled with digoxin by in vitro transcription; then with in situ hybridization, the localizations of COCH mRNA in adult mouse inner ears were detected. To facilitate observation, the postnatal day 0 mouse cochlear spiral ligaments (SPL) were dissectted and explanted to culture and purify the spiral ligament fibrocytes. Immunocytochemistry, enzymocytochemistry, and culturring cell in situ transmission electron microscopy were employed to identify fibrocyte type. Reverse transcription polymerase chain reaction (RT-PCR) and western blot were used to detect the COCH and POU3F4/brn4
gene's expression. To investigate in vitro characteristics of COCH gene's expession, two factors were introduced in the culture medium of spiral ligament type I fibrocytes respectively. Firstly, phosphorothionate-modified antisense oligodeoxynucleiotides to POU3F4/brn4 mRNA was applied to study the regulation effects of POU3F4/brn4 to COCH gene
transcription. Secondly, 1M KCL was added in the normal culture medium, to rise the concentration of potassium in culture media. Then with RT-PCR and Western blot methods, the changes of gene expression levels were examined. The main results and conclusion were as following:
1. The positive hybridizations with COCH gene cRNA probe were detected in the mouse mesenchymic cell surroundding the endolymphic sac epithelium, the level of expression was similar to the fibrocytes underlying crista ampullaris sense epithellium, and inferior to the fibrocytes of spiral ligament. This expression distribution suggests that COCH gene may contribute to maintain inner ear connective tissue extracellular matrix architecture, and do not support the postulation that gene might play an inner ear immune defensive function.
2. In vitro, the culturing spiral ligament type I fibrocytes expressed COCH gene and POU3F4/brn4 gene, which were checked by RT-PCR and Western blot, and expression level did not chang with in 15 generations. This type cells may be in vitro model for investigatting the expression of COCH and POU3F4/brn4 gene.
3. The antisense oligonucleiotides to POU3F4/brn4 mRNA specially interfered in synthesis process of POU3F4/brn4 proteins, the quantity of proteins decreased to 35.3% that of control group cells. Along with it, the level of COCH gene mRNA also declined to 25.3%, compared to nomoral level. It suggests that POU3F4/brn4 may regulate the COCH gene's transcription by direct or indirect ways.
4. The expression of COCH as well as POU3F4/brn4 gene in cultured SPL type I fibrocytes were all upregulated when liftting concentration of potassium in culture media. It postulates that COCH gene and POU3F4/brn4 gene's function may be associated with metabolism of potassium in inner ear.
5.
引文
1．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁、黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；16-34
2．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁、黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；956
3．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁、黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；304-340
4．吉昌华，王伯沄，张建中．核酸分子杂交技术概述，见蔡文琴，王伯沄主编．实用免疫细胞化学与核酸分子杂交技术．成都，四川科学技术出版社，1994年，第一版：354-393
5．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁，黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；510-515
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7. Spicer SS, Schulte BA. The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency. Hear Res. 1996; 100:80-100
8. Robertson NG, Skvorak AB, Yin Y. et al. Mapping and characterization of a novel cochlear gene in human and in mouse: a positional candidate gene for a deafness disorder, DFNA9. Genomics 1997; 46(3): 345-54
9. Robertson NG, Lu L, Heller S. et al. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nat Genet 1998; 20(3): 299-303
10. Manolis EN, Khetarpal U. A gene for non-syndromic antosomal dominant progressive postlingual sensorineural hearing loss maps to chromosome 14q12-13.Hum Mol Genet 1996; 5:1047-1050
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24．姜泗长 翟所强 主编，耳鼻咽喉科实验技术．北京，人民军医出版社，1999年，第一版：42-49
25．Dieffenbach CW,Dveksler GS主编，黄培堂等译，PCR技术实验指南．北京，科学出版社，1997年，第一版：451-498
26．Ausubel FM,Kingston RF,Seidman JG等主编，颜子颖、王海林翻译，精编分子生物学实验指南．北京，科学出版社，1998年，第一版：329-373
27. Rarery KE, Patterson K. Establishment of inner ear epithelial cell culture: isolation, growth and characerization. Hear Res 1989; 38:277-288
28. Suko T, Ichimiya I, Yoshida K. et al. Classification and culture of spial ligament fibrocytes from mice. Hear Res. 2000; 140:137-144
29. Kim HN, Chang MH, Chung MH. et al. Establishment of primary cell culture from stria vascularis explants. Acta Otolaryngol(stockh)1996;116:805-811
30. Gratton MA, Schulte BA, Hazen-Martin DJ. Characterization and development of an inner ear type Ⅰ fibrocyte cell culture. Hear Res. 1996; 99:71-78
31．鄂征 主编，组织培养和分子细胞技术．北京出版社，1995年，第一版：9-11
32. Spicer SS, Schulte BA. Differentiation of inner ear fibrocytes according to their ion transport related activity. Hear Res. 1991; 56:53-64
33. Spicer SS, Schulte BA. Evidence for K recircling pathway from inner hair cells. Hear Res. 1998; 118:1-12
34. Spicer SS, Schulte BA. Differences along the place frequency map in the structure of supporting cells in the gerbil cochlea. Hear Res. 1994; 79:161-177
35. Schulte BA. Immunohistochemical localization of intracellular Ca~(2+)-ATPase in outer hairs cell, neurons and fibrocytes in the adult and developing inner ear. Hear Res. 1993; 65:262-273
36. Minowa O, Ikeda K, Sugitani Y. et al. Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness. Science. 1999; 285(5432):1408-1411.
37. Wangemann P. K~+ cycling and the endocochlear patential. Hear Res. 2002; 165:1-9
38. Ichimiya I, Kurono Y, Hirano T et al. Changes immunostaining of inner ears after antigen challenge into the scala tympani. Laryngoscope. 1998;108:589-591
39. Phippard D, Lu LH, Lee D. et al. Targeted Mutagenesis of the POU-domain gene POU3F4/brn4/Pou3f4 caused developmental defects in the inner ear. J Neurosci. 1999; 19(14):5980-5989
40. Herweijer H, Wolff JA. Progress and prospects: naked DNA gene transfer and therapy. Gene Ther 2003;10(6):453-458
41. Sugimoto N, Yasumatsu I. A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability. Curr Med Chem Anti-Canc Agents 2001;1(1): 95-112
42. Mercatante DR, Sazani P, Kole R. Modification of alternative splicing by antisense oligonucleotides as a potential chemotherapy for cancer and other diseases. Curr Cancer Drug Targets 2001; 1(3):211-230
43. Brantl S. Antisense-RNA regulation and RNA interference. Biochim Biophys Acta 2002 May 3; 1575(1-3): 15-25
44. Phippard D, Heydemann A, Lechner M et al. Changes in the subcellular localization of
the POU3F4/brn4 gene product precede mesenchymal remodeling of the otic capsule.Hear Res. 1998;120(1-2):77-85
45. de Kok Y J, van der Maarel SM, Bitner-Glindzicz M et al. Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science 1995;267(5198):685-688
46. Friedman RA, Bykhovskaya Y, Tu G, Talbot JM et al. Molecular analysis of the POU3F4 gene in patients with clinical and radiographic evidence of X-linked mixed deafness with perilymphatic gusher. Ann Otol Rhinol Laryngol 1997;106(4):320-325
47. Hara Y, Rovescalli AC, Kim Y et al. Structure and evolution of four POU domain genes expressed in mouse brain. Proc Natl Acad Sci USA. 1992;89:3280-3284
48．姜泗长，顾瑞，王正敏 主编，耳鼻咽喉科全书 耳科学．上海科学技术出版社，2002年，第2版：951-968
49. Khetarpal U. Autosomal dominant sensorineural hearing loss-further temporal bone findings. Arch otolaryngol head neck surg. 1993;119:106-108
50. Verhagen W I M, Bom S J H, Huygen P L M et al. Familial progressive vestibulocochlear dysfunction caused by a coch mutation(DFNA9). Arch Neuro. 2000; 57:1045-1050
51. da Costa SS, de Sousa LC, Piza MR. Meniere's disease: overview, epidemiology, and natural history. Otolaryngol Clin North Am 2002; 35(3):455-495
52. Mancini F, Catalani M, Caru M, Monti B. History of Meniere's disease and its clinical presentation. Otolaryngol Clin North Am 2002; 35(3):565-580
53. Morton CC. Genetics genomics and gene discovery in the auditory system. Hum Mol Genet. 2002; 11(10): 1229-1240
54. Ikezono T, Omori A, Ichinose S. et al. Identification of the protein product of the COCH gene (hereditary deafness gene) as the major component of bovine inner ear protein. Biochim Biophys Acta 2001;1535(3):258-265
55. Paparella MM, Djalilian HR. Etiology, pathophysiology of symptoms, and pathogenesis of Meniere's disease. Otolaryngol Clin North Am 2002; 35(3):529-545
2．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁、黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；956
3．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁、黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；304-340
4．吉昌华，王伯沄，张建中．核酸分子杂交技术概述，见蔡文琴，王伯沄主编．实用免疫细胞化学与核酸分子杂交技术．成都，四川科学技术出版社，1994年，第一版：354-393
5．Sambrook J,Fritsch EF,Maniatis T主编；金冬雁，黎孟枫等译．分子克隆实验指南．北京，科学出版社，1999年，第2版；510-515
6. Robertson NG, Resendes BL, Lin JS. et al. Inner ear localization of mRNA and protein products of COCH, mutated in the sensorineural deafness and vestibular disorder, DFNA9. Hum Mol Genet 2001; 10(22):2493-2500
7. Spicer SS, Schulte BA. The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency. Hear Res. 1996; 100:80-100
8. Robertson NG, Skvorak AB, Yin Y. et al. Mapping and characterization of a novel cochlear gene in human and in mouse: a positional candidate gene for a deafness disorder, DFNA9. Genomics 1997; 46(3): 345-54
9. Robertson NG, Lu L, Heller S. et al. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nat Genet 1998; 20(3): 299-303
10. Manolis EN, Khetarpal U. A gene for non-syndromic antosomal dominant progressive postlingual sensorineural hearing loss maps to chromosome 14q12-13.Hum Mol Genet 1996; 5:1047-1050
11. Khetarpal U, Schuknecht HF, Gacek RR. et al. Autosomal dominant sensorineural hearing loss. Arch otolaryngol head neck surg. 1991; 117:1032-1042
12. Khetarpal U. DFNA9 is a progressive audiovestibular dysfunction with a microfibrillar deposit in the inner ear. Laryngoscope 2000; 110(8): 1379-1384.
13. Fransen E, Verstreken M, Verhagen WI. et al. High prevalence of symptoms of Meniere's disease in three families with a mutation in the COCH gene. Hum Mol Genet
1999; 8(8): 1425-1429
14. Steel KP. The benefits of recycling. Science 1999:285(5432):1363-1364
15. Rinkwitz S, Bober E, Baker R. Development of the vertebrate inner ear. Ann N Y Acad Sci. 2001; 942:1-14.
16. de Kok YJM, Bom SJH, Brunt TM. et al. A pro51ser mutation of COCH gene is associated with late onset autosomal dominant progressive sensorineural hearing loss with vestibular defects. Hum Mol Genet 1999; 8:345-354
17. Kamarinos M, Mcgill J, Lynch M. et al. Identification of a novel COCH mutation I109N, highlight the similar clinical features observed in DFNA9 families. Hum Mutat. 2001; 17:351-357
18. Trexler M, Banyai L, Patthy L. The LCCL module. Eur J Biochem 2000; 267(18):5751-5757
19. Ruckenstein MJ. Immunologic aspects of Meniere's disease. Am J Otolaryngol. 20(3):161-165
20. Ichimiya I, Suzuki M, Hirano T. et al. The influence pneumococcal otitis media on the cochlear lateral wall. Hear Res. 1999; 131:128-134.
21. Ichimiya I, Adams JC, Kimura RS. Immunolocalization of Na-K-ATPase. Ca-ATPase. Calcium-binding proteins and carbonic anhydrase in the guinea pig inner ear. Acta Otolaryngol.(Stockh) 1994; 114:167-176
22．姜泗长 主编，耳解剖学与颞骨组织病理学．北京，人民军医出版社，1999年，第一版：274-276
23．司徒镇强，吴军正 主编，细胞培养．西安，世界图书出版公司，1996年，第一版：69-71，77-79，206-209
24．姜泗长 翟所强 主编，耳鼻咽喉科实验技术．北京，人民军医出版社，1999年，第一版：42-49
25．Dieffenbach CW,Dveksler GS主编，黄培堂等译，PCR技术实验指南．北京，科学出版社，1997年，第一版：451-498
26．Ausubel FM,Kingston RF,Seidman JG等主编，颜子颖、王海林翻译，精编分子生物学实验指南．北京，科学出版社，1998年，第一版：329-373
27. Rarery KE, Patterson K. Establishment of inner ear epithelial cell culture: isolation, growth and characerization. Hear Res 1989; 38:277-288
28. Suko T, Ichimiya I, Yoshida K. et al. Classification and culture of spial ligament fibrocytes from mice. Hear Res. 2000; 140:137-144
29. Kim HN, Chang MH, Chung MH. et al. Establishment of primary cell culture from stria vascularis explants. Acta Otolaryngol(stockh)1996;116:805-811
30. Gratton MA, Schulte BA, Hazen-Martin DJ. Characterization and development of an inner ear type Ⅰ fibrocyte cell culture. Hear Res. 1996; 99:71-78
31．鄂征 主编，组织培养和分子细胞技术．北京出版社，1995年，第一版：9-11
32. Spicer SS, Schulte BA. Differentiation of inner ear fibrocytes according to their ion transport related activity. Hear Res. 1991; 56:53-64
33. Spicer SS, Schulte BA. Evidence for K recircling pathway from inner hair cells. Hear Res. 1998; 118:1-12
34. Spicer SS, Schulte BA. Differences along the place frequency map in the structure of supporting cells in the gerbil cochlea. Hear Res. 1994; 79:161-177
35. Schulte BA. Immunohistochemical localization of intracellular Ca~(2+)-ATPase in outer hairs cell, neurons and fibrocytes in the adult and developing inner ear. Hear Res. 1993; 65:262-273
36. Minowa O, Ikeda K, Sugitani Y. et al. Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness. Science. 1999; 285(5432):1408-1411.
37. Wangemann P. K~+ cycling and the endocochlear patential. Hear Res. 2002; 165:1-9
38. Ichimiya I, Kurono Y, Hirano T et al. Changes immunostaining of inner ears after antigen challenge into the scala tympani. Laryngoscope. 1998;108:589-591
39. Phippard D, Lu LH, Lee D. et al. Targeted Mutagenesis of the POU-domain gene POU3F4/brn4/Pou3f4 caused developmental defects in the inner ear. J Neurosci. 1999; 19(14):5980-5989
40. Herweijer H, Wolff JA. Progress and prospects: naked DNA gene transfer and therapy. Gene Ther 2003;10(6):453-458
41. Sugimoto N, Yasumatsu I. A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability. Curr Med Chem Anti-Canc Agents 2001;1(1): 95-112
42. Mercatante DR, Sazani P, Kole R. Modification of alternative splicing by antisense oligonucleotides as a potential chemotherapy for cancer and other diseases. Curr Cancer Drug Targets 2001; 1(3):211-230
43. Brantl S. Antisense-RNA regulation and RNA interference. Biochim Biophys Acta 2002 May 3; 1575(1-3): 15-25
44. Phippard D, Heydemann A, Lechner M et al. Changes in the subcellular localization of
the POU3F4/brn4 gene product precede mesenchymal remodeling of the otic capsule.Hear Res. 1998;120(1-2):77-85
45. de Kok Y J, van der Maarel SM, Bitner-Glindzicz M et al. Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science 1995;267(5198):685-688
46. Friedman RA, Bykhovskaya Y, Tu G, Talbot JM et al. Molecular analysis of the POU3F4 gene in patients with clinical and radiographic evidence of X-linked mixed deafness with perilymphatic gusher. Ann Otol Rhinol Laryngol 1997;106(4):320-325
47. Hara Y, Rovescalli AC, Kim Y et al. Structure and evolution of four POU domain genes expressed in mouse brain. Proc Natl Acad Sci USA. 1992;89:3280-3284
48．姜泗长，顾瑞，王正敏 主编，耳鼻咽喉科全书 耳科学．上海科学技术出版社，2002年，第2版：951-968
49. Khetarpal U. Autosomal dominant sensorineural hearing loss-further temporal bone findings. Arch otolaryngol head neck surg. 1993;119:106-108
50. Verhagen W I M, Bom S J H, Huygen P L M et al. Familial progressive vestibulocochlear dysfunction caused by a coch mutation(DFNA9). Arch Neuro. 2000; 57:1045-1050
51. da Costa SS, de Sousa LC, Piza MR. Meniere's disease: overview, epidemiology, and natural history. Otolaryngol Clin North Am 2002; 35(3):455-495
52. Mancini F, Catalani M, Caru M, Monti B. History of Meniere's disease and its clinical presentation. Otolaryngol Clin North Am 2002; 35(3):565-580
53. Morton CC. Genetics genomics and gene discovery in the auditory system. Hum Mol Genet. 2002; 11(10): 1229-1240
54. Ikezono T, Omori A, Ichinose S. et al. Identification of the protein product of the COCH gene (hereditary deafness gene) as the major component of bovine inner ear protein. Biochim Biophys Acta 2001;1535(3):258-265
55. Paparella MM, Djalilian HR. Etiology, pathophysiology of symptoms, and pathogenesis of Meniere's disease. Otolaryngol Clin North Am 2002; 35(3):529-545