缝隙连接蛋白Connexin26在拟老化大鼠耳蜗中的表达及基因启动子区域甲基化的研究
发布时间：2018-09-18 19:26
【摘要】：第一部分D-半乳糖诱导拟老化伴线粒体普遍缺失大鼠模型的建立 目的建立内耳拟老化伴mtDNA4834bp缺失大鼠模型,为进一步研究老年性耳聋的发病机制奠定基础。 方法Wistar大鼠48只,随机分为2组,A组：生理盐水组,24只,每天颈部皮下注射同等剂量生理盐水,连续注射8周,随后继续饲养6个月。B组：D-半乳糖组,24只,每天颈部皮下注射D-gal500mg/kg,连续注射8周,随后继续饲养6个月。监测两组大鼠造模前后体重变化,利用听性脑干反应ABR检测两组大鼠造模前后听力反应阈,实时荧光定量PCR检测两组大鼠耳蜗线粒体DNA4834bp缺失率,并将所得产物测序检测。HE染色观察两组大鼠内耳耳蜗形态学改变。 结果：造模前后两组之间大鼠体重无显著性差异；ABR检测结果显示两组大鼠听阈无显著性差异,D-半乳糖组大鼠耳蜗线粒体DNA4834bp缺失较对照组升高,两者有统计学差异。HE染色显示两组大鼠耳蜗形态学基本正常,无明显差异性改变。 结论：D-半乳糖诱导内耳拟老化伴mtDNA4834bp缺失大鼠模型建立成功。 第二部分缝隙连接蛋白Connexin26在拟老化大鼠耳蜗中的表达以及基因启动子区域甲基化的研究 目的探索性研究缝隙连接蛋白Connexin26与老年性耳聋的关系以及其在老年性耳聋中的可能作用机制。 方法利用D-半乳糖诱导内耳拟老化伴mtDNA4834bp缺失大鼠模型,采用实时定量RT-PCR及Western Blot免疫印迹法检测造模完成后两组大鼠耳蜗内Cx26在mRNA和蛋白水平的表达情况,亚硫酸氢盐测序法(BSP)检测大鼠耳蜗内Cx26基因启动子区域甲基化状态。相关性分析两组大鼠耳蜗中Cx26在mRNA、蛋白水平的表达差异以及GJB2基因启动子区域甲基化程度差异。 结果：D-半乳糖内耳拟老化组大鼠耳蜗Cx26在mRNA水平及蛋白水平较生理盐水对照组降低,两组间有统计学差异(P0.05)；BSP法检测GJB2基因启动子区域甲基化状态提示D-半乳糖组耳蜗中启动子区域甲基化显著高于生理盐水对照组(P0.001)。 结论：拟老化内耳中缝隙连接蛋白Connexin26基因启动子区域甲基化率升高与其表达降低相关,推测缝隙连接蛋白Connexin26的降低可能参与老年性耳聋的发生发展过程,且Cx26基因启动子区域甲基化水平增高可能是导致老年性耳聋中Connexin26表达水平降低的原因。
[Abstract]:Part I the establishment of a rat model of mimic aging with common mitochondrial deletion induced by D-galactose objective to establish the model of inner ear pseudo aging with mtDNA4834bp deletion in order to lay a foundation for further study on the pathogenesis of presbycusis. Methods Forty-eight Wistar rats were randomly divided into two groups: saline group (n = 24) and normal saline group (n = 24). Each day, the same dose of normal saline was injected subcutaneously to the neck for 8 weeks, and then continued to be fed for 6 months. Group B: D-galactose group (n = 24). D-gal500 mg / kg was injected subcutaneously to the neck every day for 8 weeks and then continued for 6 months. The changes of body weight before and after modeling were monitored, hearing threshold was measured by auditory brainstem response (ABR) and cochlear mitochondrial DNA4834bp deletion rate was measured by real-time fluorescence quantitative PCR. The morphologic changes of cochlea were observed by HE staining. Results: there was no significant difference in body weight between the two groups before and after modeling. The results showed that there was no significant difference in auditory threshold between the two groups. The mitochondrial DNA4834bp deletion in the Dgalactose group was higher than that in the control group. There was statistical difference between the two groups. He staining showed that the cochlea morphology of the two groups was basically normal, and there was no significant difference between the two groups. Conclusion the rat model of pseudo aging of inner ear with mtDNA4834bp deficiency induced by w D-galactose was successfully established. Part two the expression of gap junction protein Connexin26 in cochlea of aging rats and the methylation of gene promoter region objective to explore the relationship between gap junction protein Connexin26 and presbycusis and its role in aging rat cochlea The possible mechanism of presbycusis. Methods the rat model of pseudo aging of inner ear with mtDNA4834bp deletion was induced by D-galactose. The expression of Cx26 in cochlea of the two groups was detected by real-time quantitative RT-PCR and Western Blot Western blotting. The methylation status of the promoter region of Cx26 gene in rat cochlea was detected by (BSP) with bisulfite sequencing. The expression of Cx26 in the cochlea of the two groups was analyzed. The difference of methylation in the promoter region of the GJB2 gene was also found in the cochlea of the two groups. Results the level of mRNA and protein in the cochlea of the aged rats in the WW D-galactose-induced aging group was lower than that in the saline control group (P0.05), and there was a significant difference between the two groups (P0.05). The methylation of the promoter region of GJB2 gene in D- galactose group was significantly higher than that in normal saline group (P0.001). Conclusion: the increased methylation rate of gap junction protein (Connexin26) promoter region is associated with the decreased expression of gap junction protein (Connexin26) gene in the aged inner ear. It is speculated that the decrease of gap junction protein Connexin26 may be involved in the occurrence and development of presbycusis. The increase of methylation in promoter region of Cx26 gene may lead to the decrease of Connexin26 expression in presbycusis.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.436
本文编号：2248890
[Abstract]:Part I the establishment of a rat model of mimic aging with common mitochondrial deletion induced by D-galactose objective to establish the model of inner ear pseudo aging with mtDNA4834bp deletion in order to lay a foundation for further study on the pathogenesis of presbycusis. Methods Forty-eight Wistar rats were randomly divided into two groups: saline group (n = 24) and normal saline group (n = 24). Each day, the same dose of normal saline was injected subcutaneously to the neck for 8 weeks, and then continued to be fed for 6 months. Group B: D-galactose group (n = 24). D-gal500 mg / kg was injected subcutaneously to the neck every day for 8 weeks and then continued for 6 months. The changes of body weight before and after modeling were monitored, hearing threshold was measured by auditory brainstem response (ABR) and cochlear mitochondrial DNA4834bp deletion rate was measured by real-time fluorescence quantitative PCR. The morphologic changes of cochlea were observed by HE staining. Results: there was no significant difference in body weight between the two groups before and after modeling. The results showed that there was no significant difference in auditory threshold between the two groups. The mitochondrial DNA4834bp deletion in the Dgalactose group was higher than that in the control group. There was statistical difference between the two groups. He staining showed that the cochlea morphology of the two groups was basically normal, and there was no significant difference between the two groups. Conclusion the rat model of pseudo aging of inner ear with mtDNA4834bp deficiency induced by w D-galactose was successfully established. Part two the expression of gap junction protein Connexin26 in cochlea of aging rats and the methylation of gene promoter region objective to explore the relationship between gap junction protein Connexin26 and presbycusis and its role in aging rat cochlea The possible mechanism of presbycusis. Methods the rat model of pseudo aging of inner ear with mtDNA4834bp deletion was induced by D-galactose. The expression of Cx26 in cochlea of the two groups was detected by real-time quantitative RT-PCR and Western Blot Western blotting. The methylation status of the promoter region of Cx26 gene in rat cochlea was detected by (BSP) with bisulfite sequencing. The expression of Cx26 in the cochlea of the two groups was analyzed. The difference of methylation in the promoter region of the GJB2 gene was also found in the cochlea of the two groups. Results the level of mRNA and protein in the cochlea of the aged rats in the WW D-galactose-induced aging group was lower than that in the saline control group (P0.05), and there was a significant difference between the two groups (P0.05). The methylation of the promoter region of GJB2 gene in D- galactose group was significantly higher than that in normal saline group (P0.001). Conclusion: the increased methylation rate of gap junction protein (Connexin26) promoter region is associated with the decreased expression of gap junction protein (Connexin26) gene in the aged inner ear. It is speculated that the decrease of gap junction protein Connexin26 may be involved in the occurrence and development of presbycusis. The increase of methylation in promoter region of Cx26 gene may lead to the decrease of Connexin26 expression in presbycusis.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.436
【参考文献】
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1 孔维佳;胡钰娟;王琼;许黎;王莹;韩月臣;李隽;刘波;孔雯;;大鼠内耳拟老化伴线粒体突变模型的建立[J];临床耳鼻咽喉科杂志;2006年19期
，本文编号：2248890
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