D-半乳糖老年性聋动物模型听觉中枢的改变及机制研究

发布时间：2018-04-02 02:37

本文选题：D-半乳糖　切入点：中枢听觉系统　出处：《华中科技大学》2010年博士论文

【摘要】： 目的：利用D-半乳糖来建立中枢性老年性聋的动物模型,为中枢听觉系统老化机制的研究提供有效简便的途径。方法：用5%D-半乳糖(150mg/kg)对雄性Wistar大鼠进行每日颈背部皮下注射,共8周,以同等剂量生理盐水每日颈背部皮下注射作为对照组,HE染色和尼氏染色观察听皮层、下丘及耳蜗核的形态学改变,Taqman探针实时荧光定量多聚酶链反应(real time qPCR)检测线粒体DNA 4834 bp大片段缺失率。结果：模型组大鼠听皮层、下丘及耳蜗核神经元数目(分别为322.58±35.25,344.08±40.50,207.33±22.01／mmm2)比对照组(278.13±41.67,292.83±43.91,145.41±25.07/μm2)显著减少(P＜0.05),并出现细胞排列紊乱,层次不清,胞浆尼氏体减少,可见较多胞质浓染的缺血神经元,间质结构疏松,胶质细胞增生,毛细血管增生、扩张,并出现较多病理性形态改变,如迂曲、扭曲、球形及线形改变。模型组听皮层、下丘、耳蜗核的线粒体DNA 4834 bp缺失率(16.22±1.29%,21.56±4.57%,13.96±2.47%)比对照组(11.24±1.09%,10.01±0.98%,8.78±0.94%)显著升高(P＜0.05)。结论：D-半乳糖可引起大鼠中枢听觉通路上的老化改变。目的：比较D-半乳糖模型大鼠和自然衰老大鼠听觉中枢的改变,并探讨其老化发生的可能机制。方法：30只1月龄雄性Wistar大鼠随机分为两组：①D-半乳糖组(15只)：以5%D-半乳糖(150mg/kg)每日颈背部皮下注射,共8周；②对照组(15只)：同等剂量生理盐水,每日颈背部皮下注射,共8周。另取22-24月龄同品系大鼠(15只)作为自然老化组。造模完毕后,每只动物检测听性脑干反应(auditory brainstem response,ABR)和中潜伏期电位(middle latency response, MLR);实时荧光定量PCR检测听皮层、下丘、蜗核的线粒体DNA 4834 bp缺失率；硫代戊巴比妥法检测丙二醛(MDA)的含量；光镜和透射电镜下观察各部位的病理学变化；TUNEL (terminal deoxynucleotidyltransferase-mediated UTP nick-end labeling)法检测各部位细胞的凋亡情况。结果：与对照组相比,自然老化组大鼠ABR阈值提高,各波潜伏期、Ⅰ-Ⅳ波间期及MLR Pa潜伏期均延长,而D-半乳糖组ABR阈值无改变,Ⅲ,Ⅳ，Ⅴ波潜伏期和Ⅰ-Ⅳ波间期延长,Pa波潜伏期延长,Ⅰ,Ⅱ波潜伏期虽有延长趋势,但无统计学差异；两组老化大鼠听皮层、下丘、蜗核中MDA含量以及线粒体DNA 4834 bp缺失率比对照组显著升高,凋亡细胞数增加,并出现细胞结构排列紊乱,神经元胞浆中脂褐素增多,线粒体肿胀,内质网扩张,呈广泛空泡化改变,有髓神经纤维分离、断裂等退行性改变。结论：①年龄相关性中枢听觉功能障碍以及退行性改变存在于D-半乳糖拟老化大鼠和自然老化大鼠中。②氧化应激、mtDNA大片段缺失以及凋亡参与了中枢听觉系统的老化过程。目的：探讨线粒体DNA损伤发生的机制及碱基切除修复系统在听觉中枢老化中的作用。方法：48只雄性wistar大鼠随机分为4组：D-半乳糖低、中、高剂量组和生理盐水对照组,分别于每日皮下注射5%D-半乳糖150、300、500mg/kg和等体积生理盐水,共8周。采用实时荧光定量PCR和western blot方法检测各组大鼠听皮层、下丘和耳蜗核中线粒体DNA 4834 bp缺失率以及DNA修复酶8-氧鸟嘌呤糖苷酶(8-oxoguanine DNA glycosylase, OGG1)和DNA聚合酶γ(DNA polymeraseγ, DNA polγ)的基因和蛋白水平的变化；JC-1荧光探针法检测各部位的线粒体膜电位。结果：与对照组相比,三组D-gal组听皮层、下丘及蜗核中线粒体DNA 4834 bp缺失率明显增高(p0.05),高剂量D-gal组升高最明显,其中听皮层和耳蜗核中D-gal低剂量组与中剂量组CD缺失率无显著性差异(p＞0.05),与高剂量组差异显著(p＜0.05),而下丘中D-gal三个剂量组之间无显著性差异(p0.05)。D-gal各剂量组三个部位OGG1和polγ的基因和蛋白表达水平与对照组均明显降低(p＜0.05),并随D-gal剂量增大而明显降低,同时线粒体膜电位也显著降低(p＜0.05)。结论：在听觉高级中枢老化的过程中线粒体DNA损伤可能是由于其修复功能减退造成的。
[Abstract]:Objective: to establish an animal model of central senile deafness by using D- galactose to provide an effective and simple way for the study of the mechanism of the aging of the central auditory system.
Methods: 5%D- galactose (150mg/kg) daily subcutaneous injection of male Wistar rats for 8 weeks, with the same dose of saline daily subcutaneous injection as control group, to observe the auditory cortex by HE staining and Nissl staining, the morphological changes of nucleus of the inferior colliculus and cochlea, Taqman probe real-time fluorescence quantitative polymerase chain the reaction (real time qPCR) to detect the mitochondrial DNA 4834 bp deletion rate.
Results: the auditory cortex of rats in the model group, the number of neurons in the cochlear nucleus and inferior colliculus (322.58 + 35.25344.08 + 40.50207.33 + 22.01 / mmm2) than the control group (278.13 + 41.67292.83 + 43.91145.41 + 25.07/ m2) decreased significantly (P < 0.05), and the emergence of cell disorder, level is not clear, the cytoplasm of Nigeria bainite decreases, more visible cytoplasmic stain of ischemic neurons, interstitial loose structure, glial cell proliferation, capillary proliferation, expansion, and the emergence of more pathological change, such as tortuosity, twisted, spherical and linear change of model group. The auditory cortex, the cochlear nucleus of the inferior colliculus, mitochondrial DNA 4834 bp deletion rate (16.22 + 1.29%, 21.56 + 4.57%, 13.96 + 2.47%) than the control group (11.24 + 1.09%, 10.01 + 0.98%, 8.78 + 0.94%) increased significantly (P < 0.05).
Conclusion: D- galactose can induce aging changes in the central auditory pathway of rats.
Objective: To compare the changes in the auditory center of D- galactose model rats and natural aging rats, and to explore the possible mechanism of its aging.
Methods: 30 1 month old male Wistar rats were randomly divided into two groups: D- galactose group (15 rats): 5%D- galactose (150mg/kg) daily subcutaneous injection, a total of 8 weeks; the control group (15 rats): the same dose of saline, daily subcutaneous injection for 8 weeks. Another 22-24 months of age. The same strain rats (15 rats) as natural aging model group. After each animal detection of auditory brainstem response (auditory brainstem, response, ABR) and middle latency potentials (middle latency response, MLR); real time fluorescence quantitative PCR detection of auditory cortex, inferior colliculus, mitochondrial DNA cochlear nucleus 4834 bp deletion rate; malondialdehyde (MDA) method by pentobarbital content; under light microscope and transmission electron microscope to observe the pathological changes of the various parts of the TUNEL (terminal deoxynucleotidyltransferase-mediated UTP; nick-end labeling) apoptosis was detected by parts of the cell.
Results: compared with control group, ABR group, natural aging threshold of rats increased, the latency of each wave, I - IV interval and MLR wave latency of Pa was prolonged, and the D- galactose group ABR threshold unchanged, III, IV, V and prolong the latency of IV wave interval, Pa wave latency was prolonged, I. although the II latency prolonged trend, but no significant difference; two groups of aging rat auditory cortex, inferior colliculus, cochlear nucleus MDA content, mitochondrial DNA 4834 bp deletion rate is significantly higher than the control group, the number of apoptotic cells increased, and the cell structure was disordered, lipofuscin in the cytoplasm of neurons increased, mitochondria swelling, endoplasmic reticulum network expansion, showed extensive vacuolization, myelinated nerve fiber separation, fracture and other degenerative changes.
Conclusion: (1) age-related central auditory dysfunction and degenerative changes are present in D- galactose induced aging rats and natural aging rats. 2. Oxidative stress, mtDNA deletion and apoptosis are involved in the aging process of central auditory system.
Objective: To investigate the mechanism of mitochondrial DNA damage and the role of base excision repair system in the aging of auditory center.
Methods: 48 male Wistar rats were randomly divided into 4 groups: D- galactose, low, high dose group and saline control group respectively in the subcutaneous injection of 5%D- galactose and 150300500mg/kg normal saline for 8 weeks. By using real-time quantitative PCR and Western blot were measured in rat auditory the cortex, inferior colliculus and mitochondrial DNA 4834 BP deletion in the cochlear nucleus and the rate of DNA repair enzyme 8- yguanine glycosidase (8-oxoguanine DNA glycosylase, OGG1) and DNA (DNA polymerase polymerase gamma gamma gamma, DNA POL) changes in gene and protein level; mitochondrial MembranePotential detection JC-1 fluorescent probe method.
Results: compared with the control group, three group of D-gal group of auditory cortex, inferior colliculus and cochlear nuclei in the mitochondrial DNA 4834 bp deletion rate was significantly higher (P0.05), high dose of D-gal group was increased significantly, the auditory cortex and cochlear nucleus D-gal in low dose group and middle dose group CD deletion rate had no significant difference (P > 0.05), and high dose group had significant difference (P < 0.05), but there is no significant difference between the inferior colliculus three dosages of D-gal (P0.05).D-gal in each dose group of three parts OGG1 and Pol gamma gene and protein expression level and the control group were significantly lower (P < 0.05), and with the dose of D-gal decreases, mitochondrial membrane potential decreased significantly at the same time (P < 0.05).
Conclusion: the damage of mitochondrial DNA may be caused by the hypogonadism during the aging of the advanced auditory center.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764.436

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