衰老蛋白p66shc在D-半乳糖诱导小鼠内耳老化中的作用及其机制的研究
发布时间：2018-11-12 17:32
【摘要】：第一部分D-半乳糖诱导内耳老化小鼠模型的建立 目的：利用D-半乳糖建立小鼠内耳老化模型。 方法：用800mg/kg、1000mg/kg D-半乳糖生理盐水水溶液分别对两组雄性昆明小鼠进行每日一次的颈背部皮下注射,每个剂量组小鼠为6只,共十周。以同等体积的生理盐水给予相同处理的6只雄性小鼠作为对照组。ABR检测小鼠听阈；分光光度法检测小鼠血浆T-SOD活性及MDA水平；实时荧光定量多聚酶链反应(real timePCR)检测各组小鼠耳蜗外侧壁DNA 3867 bp大片段缺失率,并且所得产物进行测序。 结果：ABR测得结果显示各组之间并无明显差异；两组D-半乳糖组的血浆T-SOD活性较对照组降低,且高剂量组低于低剂量组；两组D-半乳糖组的血浆MDA水平增高,且高剂量组低于低剂量组；半乳糖处理组线粒体大片段缺失率较对照组显著升高(p0.01)。 结论：D-半乳糖可引起小鼠内耳老化改变 第二部分p66shc在正常成年小鼠及不同年龄段小鼠内耳中的表达 目的：研究p66shc在正常小鼠内耳中的表达部位及表达水平。 方法：①正常成年昆明小鼠10只。免疫荧光检测SHC蛋白在小鼠耳蜗中的表达部位。实时荧光定量多聚酶链反应检测p66shc小鼠耳蜗及肝脏中的表达水平。 ②年龄为3天、1个月、3个月、12个月的小鼠各8只,Western blot检测p66shc及Ser36-P-p66shc在不同年龄段小鼠中耳蜗中的表达水平。 结果：①SHC蛋白在成年小鼠耳蜗中主要表达在血管纹处；p66shc在成年小鼠中耳蜗表达量较肝脏高达40倍。 ②SHC蛋白在3天小鼠表达量明显较1、3、12个月小鼠高。Ser36-P-p66shc在1、3、12个月小鼠表达水平明显较3天小鼠高。 结论：p66shc在内耳血管纹中高表达,SHC及Ser36-P-p66shc蛋白的表达与年龄相关。 第三部分p66shc在D-半乳糖诱导内耳线粒体DNA损伤中的作用及其作用机制 目的：探讨p66shc在D-半乳糖诱导内耳线粒体DNA损伤中的作用及其作用机制 方法：60只昆明小鼠随机分为3组：D-半乳糖低、高剂量组及生理盐水对照组,分别每日于皮下注射D-半乳糖水溶剂800mg/kg,1000mg/kg及等体积的生理盐水,共十周。实时定量PCR检测各组小鼠耳蜗p66shc、FOXO3a mRNA水平。Western blot检测各组小鼠耳蜗中p66shc、FOXO3a、p-FOXO3a、Ser36-P-p66shc总蛋白水平以及p66shc线粒体蛋白水平。免疫荧光检测Ser36-P-p66shc在各组小鼠耳蜗外侧壁中的表达。 结果：与对照组相比,低高剂量组p66shc、FOXO3amRNA表达水平增高；p66shc、FOXO3a、p-FOXO3a. Ser36磷酸化p66shc总蛋白水平以及p66shc线粒体蛋白水平均升高(p0.05)。 结论：p66shc可能参与D-半乳糖诱导内耳线粒体DNA损伤。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764
本文编号：2327744
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R764
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