【摘要】：第一部分D-半乳糖誘導內耳老化小鼠模型的建立 目的：利用D-半乳糖建立小鼠內耳老化模型。 方法：用800mg/kg、1000mg/kg D-半乳糖生理鹽水水溶液分別對兩組雄性昆明小鼠進行每日一次的頸背部皮下注射,每個劑量組小鼠為6只,共十周。以同等體積的生理鹽水給予相同處理的6只雄性小鼠作為對照組。ABR檢測小鼠聽閾；分光光度法檢測小鼠血漿T-SOD活性及MDA水平；實時熒光定量多聚酶鏈反應(real timePCR)檢測各組小鼠耳蝸外側壁DNA 3867 bp大片段缺失率,并且所得產(chǎn)物進行測序。 結果：ABR測得結果顯示各組之間并無明顯差異；兩組D-半乳糖組的血漿T-SOD活性較對照組降低,且高劑量組低于低劑量組；兩組D-半乳糖組的血漿MDA水平增高,且高劑量組低于低劑量組；半乳糖處理組線粒體大片段缺失率較對照組顯著升高(p0.01)。 結論：D-半乳糖可引起小鼠內耳老化改變 第二部分p66shc在正常成年小鼠及不同年齡段小鼠內耳中的表達 目的：研究p66shc在正常小鼠內耳中的表達部位及表達水平。 方法：①正常成年昆明小鼠10只。免疫熒光檢測SHC蛋白在小鼠耳蝸中的表達部位。實時熒光定量多聚酶鏈反應檢測p66shc小鼠耳蝸及肝臟中的表達水平。 ②年齡為3天、1個月、3個月、12個月的小鼠各8只,Western blot檢測p66shc及Ser36-P-p66shc在不同年齡段小鼠中耳蝸中的表達水平。 結果：①SHC蛋白在成年小鼠耳蝸中主要表達在血管紋處；p66shc在成年小鼠中耳蝸表達量較肝臟高達40倍。 ②SHC蛋白在3天小鼠表達量明顯較1、3、12個月小鼠高。Ser36-P-p66shc在1、3、12個月小鼠表達水平明顯較3天小鼠高。 結論：p66shc在內耳血管紋中高表達,SHC及Ser36-P-p66shc蛋白的表達與年齡相關。 第三部分p66shc在D-半乳糖誘導內耳線粒體DNA損傷中的作用及其作用機制 目的：探討p66shc在D-半乳糖誘導內耳線粒體DNA損傷中的作用及其作用機制 方法：60只昆明小鼠隨機分為3組：D-半乳糖低、高劑量組及生理鹽水對照組,分別每日于皮下注射D-半乳糖水溶劑800mg/kg,1000mg/kg及等體積的生理鹽水,共十周。實時定量PCR檢測各組小鼠耳蝸p66shc、FOXO3a mRNA水平。Western blot檢測各組小鼠耳蝸中p66shc、FOXO3a、p-FOXO3a、Ser36-P-p66shc總蛋白水平以及p66shc線粒體蛋白水平。免疫熒光檢測Ser36-P-p66shc在各組小鼠耳蝸外側壁中的表達。 結果：與對照組相比,低高劑量組p66shc、FOXO3amRNA表達水平增高；p66shc、FOXO3a、p-FOXO3a. Ser36磷酸化p66shc總蛋白水平以及p66shc線粒體蛋白水平均升高(p0.05)。 結論：p66shc可能參與D-半乳糖誘導內耳線粒體DNA損傷。
[Abstract]:The first part is the establishment of mouse model of inner ear aging induced by D-galactose. Objective: to establish the aging model of inner ear of mice by using D-galactose. Methods: two groups of male Kunming mice were subcutaneously injected with 800 mg 路kg ~ (-1) D ~ (-1) of D-galactose saline solution once a day for 10 weeks. Six male mice treated with the same volume of normal saline were used as control group. ABR was used to detect the hearing threshold of mice, and the activity of T-SOD and the level of MDA in plasma were measured by spectrophotometry. The deletion rate of large DNA 3867 bp fragment in the lateral wall of cochlea was detected by real-time quantitative polymerase chain reaction (real timePCR), and the products were sequenced. Results: the results of ABR showed that there was no significant difference between the two groups, the plasma T-SOD activity of the two groups was lower than that of the control group, and the activity of plasma T-SOD in the high dose group was lower than that in the low dose group. The plasma MDA level in the two groups was higher than that in the low dose group, and the deletion rate of large mitochondrial fragments in the galactose-treated group was significantly higher than that in the control group (p0.01). Conclusion: D-galactose can induce aging changes of mouse inner ear the second part is the expression of p66shc in the inner ear of normal adult mice and mice of different ages. Objective: to study the expression site and expression level of p66shc in the inner ear of normal mice. Methods: 1 Ten normal adult Kunming mice. The expression of SHC protein in mouse cochlea was detected by immunofluorescence. Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the expression level in cochlea and liver of p66shc mice. 2The expressions of p66shc and Ser36-P-p66shc in cochlea of 8 mice aged 3 days, 1 month, 3 months and 12 months were detected by, Western blot. Results: 1SHC protein was mainly expressed in the stria vascularis of adult mouse cochlea, and the expression of p66shc in cochlea of adult mice was 40 times higher than that in liver. The expression of 2SHC protein in mice was significantly higher than that in mice at 3 days and 12 months, while the expression of Ser36-P-p66shc in mice at 1 day and 12 months was significantly higher than that in mice at day 3. Conclusion: p66shc is highly expressed in stria vascularis of inner ear, and the expression of SHC and Ser36-P-p66shc is related to age. The role of p66shc in the damage of mitochondrial DNA in inner ear induced by D-galactose objective: to investigate the role and mechanism of p66shc in the damage of mitochondrial DNA in inner ear induced by D-galactose. Sixty Kunming mice were randomly divided into three groups: low D-galactose, The high-dose group and the saline control group were subcutaneously injected with D- galactose water solvent 800 mg / kg and the same volume of saline for 10 weeks. Real-time quantitative PCR was used to detect the level of FOXO3a mRNA in cochlea of each group. Western blot was used to detect the total protein level of p66shcpFOXO3aPFOXO3aPFOXO3aSc Ser36-P-p66shc in cochlea of each group and the protein level of p66shc mitochondria in cochlea of each group. The expression of Ser36-P-p66shc in the lateral wall of cochlea was detected by immunofluorescence. Results: compared with the control group, the expression of FOXO3a mRNA in low and high dose group was higher than that in control group, and the expression level of FOXO3a in low dose group was higher than that in control group. The total protein level of Ser36 phosphorylated p66shc and the mitochondrial protein level of p66shc were increased (p0.05). Conclusion: p66shc may be involved in the damage of mitochondrial DNA induced by D-galactose.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R764

【參考文獻】

相關期刊論文 前4條

1 趙玉林,董明敏,李曉萍,董民聲;小鼠耳蝸發(fā)育的形態(tài)學研究[J];解剖學雜志;2000年01期

2 趙玉林,董明敏,董民聲;小鼠聽力發(fā)育學的研究[J];聽力學及言語疾病雜志;1998年03期

3 張欣欣,韓東一,丁大連,戴樸,楊偉炎,姜泗長,Richard J.Salvi;Cu/ZnSOD基因敲除小鼠耳蝸易于出現(xiàn)mtDNA缺失突變—ROS對組織損傷的特異性研究(英文)[J];Chinese Medical Journal;2002年02期

4 張欣欣,韓東一,丁大連,戴樸,楊偉炎,姜泗長,Richard J.Salvi;老齡小鼠耳蝸mtDNA3867bp大片缺失(英文)[J];Chinese Medical Journal;2002年09期

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