| 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 genetranscription. 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.
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