縫隙連接蛋白Connexin26在擬老化大鼠耳蝸中的表達(dá)及基因啟動子區(qū)域甲基化的研究
發(fā)布時(shí)間：2018-09-18 19:26
【摘要】：第一部分D-半乳糖誘導(dǎo)擬老化伴線粒體普遍缺失大鼠模型的建立 目的建立內(nèi)耳擬老化伴mtDNA4834bp缺失大鼠模型,為進(jìn)一步研究老年性耳聾的發(fā)病機(jī)制奠定基礎(chǔ)。 方法Wistar大鼠48只,隨機(jī)分為2組,A組：生理鹽水組,24只,每天頸部皮下注射同等劑量生理鹽水,連續(xù)注射8周,隨后繼續(xù)飼養(yǎng)6個(gè)月。B組：D-半乳糖組,24只,每天頸部皮下注射D-gal500mg/kg,連續(xù)注射8周,隨后繼續(xù)飼養(yǎng)6個(gè)月。監(jiān)測兩組大鼠造模前后體重變化,利用聽性腦干反應(yīng)ABR檢測兩組大鼠造模前后聽力反應(yīng)閾,實(shí)時(shí)熒光定量PCR檢測兩組大鼠耳蝸線粒體DNA4834bp缺失率,并將所得產(chǎn)物測序檢測。HE染色觀察兩組大鼠內(nèi)耳耳蝸形態(tài)學(xué)改變。 結(jié)果：造模前后兩組之間大鼠體重?zé)o顯著性差異；ABR檢測結(jié)果顯示兩組大鼠聽閾無顯著性差異,D-半乳糖組大鼠耳蝸線粒體DNA4834bp缺失較對照組升高,兩者有統(tǒng)計(jì)學(xué)差異。HE染色顯示兩組大鼠耳蝸形態(tài)學(xué)基本正常,無明顯差異性改變。 結(jié)論：D-半乳糖誘導(dǎo)內(nèi)耳擬老化伴mtDNA4834bp缺失大鼠模型建立成功。 第二部分縫隙連接蛋白Connexin26在擬老化大鼠耳蝸中的表達(dá)以及基因啟動子區(qū)域甲基化的研究 目的探索性研究縫隙連接蛋白Connexin26與老年性耳聾的關(guān)系以及其在老年性耳聾中的可能作用機(jī)制。 方法利用D-半乳糖誘導(dǎo)內(nèi)耳擬老化伴mtDNA4834bp缺失大鼠模型,采用實(shí)時(shí)定量RT-PCR及Western Blot免疫印跡法檢測造模完成后兩組大鼠耳蝸內(nèi)Cx26在mRNA和蛋白水平的表達(dá)情況,亞硫酸氫鹽測序法(BSP)檢測大鼠耳蝸內(nèi)Cx26基因啟動子區(qū)域甲基化狀態(tài)。相關(guān)性分析兩組大鼠耳蝸中Cx26在mRNA、蛋白水平的表達(dá)差異以及GJB2基因啟動子區(qū)域甲基化程度差異。 結(jié)果：D-半乳糖內(nèi)耳擬老化組大鼠耳蝸Cx26在mRNA水平及蛋白水平較生理鹽水對照組降低,兩組間有統(tǒng)計(jì)學(xué)差異(P0.05)；BSP法檢測GJB2基因啟動子區(qū)域甲基化狀態(tài)提示D-半乳糖組耳蝸中啟動子區(qū)域甲基化顯著高于生理鹽水對照組(P0.001)。 結(jié)論：擬老化內(nèi)耳中縫隙連接蛋白Connexin26基因啟動子區(qū)域甲基化率升高與其表達(dá)降低相關(guān),推測縫隙連接蛋白Connexin26的降低可能參與老年性耳聾的發(fā)生發(fā)展過程,且Cx26基因啟動子區(qū)域甲基化水平增高可能是導(dǎo)致老年性耳聾中Connexin26表達(dá)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.436
本文編號：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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R764.436
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 孔維佳;胡鈺娟;王瓊;許黎;王瑩;韓月臣;李雋;劉波;孔雯;;大鼠內(nèi)耳擬老化伴線粒體突變模型的建立[J];臨床耳鼻咽喉科雜志;2006年19期
，本文編號：2248890
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