本文選題：D-半乳糖　切入點(diǎn)：中樞聽覺系統(tǒng)　出處：《華中科技大學(xué)》2010年博士論文

【摘要】： 目的：利用D-半乳糖來建立中樞性老年性聾的動(dòng)物模型,為中樞聽覺系統(tǒng)老化機(jī)制的研究提供有效簡(jiǎn)便的途徑。 方法：用5%D-半乳糖(150mg/kg)對(duì)雄性Wistar大鼠進(jìn)行每日頸背部皮下注射,共8周,以同等劑量生理鹽水每日頸背部皮下注射作為對(duì)照組,HE染色和尼氏染色觀察聽皮層、下丘及耳蝸核的形態(tài)學(xué)改變,Taqman探針實(shí)時(shí)熒光定量多聚酶鏈反應(yīng)(real time qPCR)檢測(cè)線粒體DNA 4834 bp大片段缺失率。 結(jié)果：模型組大鼠聽皮層、下丘及耳蝸核神經(jīng)元數(shù)目(分別為322.58±35.25,344.08±40.50,207.33±22.01／mmm2)比對(duì)照組(278.13±41.67,292.83±43.91,145.41±25.07/μm2)顯著減少(P＜0.05),并出現(xiàn)細(xì)胞排列紊亂,層次不清,胞漿尼氏體減少,可見較多胞質(zhì)濃染的缺血神經(jīng)元,間質(zhì)結(jié)構(gòu)疏松,膠質(zhì)細(xì)胞增生,毛細(xì)血管增生、擴(kuò)張,并出現(xiàn)較多病理性形態(tài)改變,如迂曲、扭曲、球形及線形改變。模型組聽皮層、下丘、耳蝸核的線粒體DNA 4834 bp缺失率(16.22±1.29%,21.56±4.57%,13.96±2.47%)比對(duì)照組(11.24±1.09%,10.01±0.98%,8.78±0.94%)顯著升高(P＜0.05)。 結(jié)論：D-半乳糖可引起大鼠中樞聽覺通路上的老化改變。 目的：比較D-半乳糖模型大鼠和自然衰老大鼠聽覺中樞的改變,并探討其老化發(fā)生的可能機(jī)制。 方法：30只1月齡雄性Wistar大鼠隨機(jī)分為兩組：①D-半乳糖組(15只)：以5%D-半乳糖(150mg/kg)每日頸背部皮下注射,共8周；②對(duì)照組(15只)：同等劑量生理鹽水,每日頸背部皮下注射,共8周。另取22-24月齡同品系大鼠(15只)作為自然老化組。造模完畢后,每只動(dòng)物檢測(cè)聽性腦干反應(yīng)(auditory brainstem response,ABR)和中潛伏期電位(middle latency response, MLR);實(shí)時(shí)熒光定量PCR檢測(cè)聽皮層、下丘、蝸核的線粒體DNA 4834 bp缺失率；硫代戊巴比妥法檢測(cè)丙二醛(MDA)的含量；光鏡和透射電鏡下觀察各部位的病理學(xué)變化；TUNEL (terminal deoxynucleotidyltransferase-mediated UTP nick-end labeling)法檢測(cè)各部位細(xì)胞的凋亡情況。 結(jié)果：與對(duì)照組相比,自然老化組大鼠ABR閾值提高,各波潛伏期、Ⅰ-Ⅳ波間期及MLR Pa潛伏期均延長(zhǎng),而D-半乳糖組ABR閾值無改變,Ⅲ,Ⅳ，Ⅴ波潛伏期和Ⅰ-Ⅳ波間期延長(zhǎng),Pa波潛伏期延長(zhǎng),Ⅰ,Ⅱ波潛伏期雖有延長(zhǎng)趨勢(shì),但無統(tǒng)計(jì)學(xué)差異；兩組老化大鼠聽皮層、下丘、蝸核中MDA含量以及線粒體DNA 4834 bp缺失率比對(duì)照組顯著升高,凋亡細(xì)胞數(shù)增加,并出現(xiàn)細(xì)胞結(jié)構(gòu)排列紊亂,神經(jīng)元胞漿中脂褐素增多,線粒體腫脹,內(nèi)質(zhì)網(wǎng)擴(kuò)張,呈廣泛空泡化改變,有髓神經(jīng)纖維分離、斷裂等退行性改變。 結(jié)論：①年齡相關(guān)性中樞聽覺功能障礙以及退行性改變存在于D-半乳糖擬老化大鼠和自然老化大鼠中。②氧化應(yīng)激、mtDNA大片段缺失以及凋亡參與了中樞聽覺系統(tǒng)的老化過程。 目的：探討線粒體DNA損傷發(fā)生的機(jī)制及堿基切除修復(fù)系統(tǒng)在聽覺中樞老化中的作用。 方法：48只雄性wistar大鼠隨機(jī)分為4組：D-半乳糖低、中、高劑量組和生理鹽水對(duì)照組,分別于每日皮下注射5%D-半乳糖150、300、500mg/kg和等體積生理鹽水,共8周。采用實(shí)時(shí)熒光定量PCR和western blot方法檢測(cè)各組大鼠聽皮層、下丘和耳蝸核中線粒體DNA 4834 bp缺失率以及DNA修復(fù)酶8-氧鳥嘌呤糖苷酶(8-oxoguanine DNA glycosylase, OGG1)和DNA聚合酶γ(DNA polymeraseγ, DNA polγ)的基因和蛋白水平的變化；JC-1熒光探針法檢測(cè)各部位的線粒體膜電位。 結(jié)果：與對(duì)照組相比,三組D-gal組聽皮層、下丘及蝸核中線粒體DNA 4834 bp缺失率明顯增高(p0.05),高劑量D-gal組升高最明顯,其中聽皮層和耳蝸核中D-gal低劑量組與中劑量組CD缺失率無顯著性差異(p＞0.05),與高劑量組差異顯著(p＜0.05),而下丘中D-gal三個(gè)劑量組之間無顯著性差異(p0.05)。D-gal各劑量組三個(gè)部位OGG1和polγ的基因和蛋白表達(dá)水平與對(duì)照組均明顯降低(p＜0.05),并隨D-gal劑量增大而明顯降低,同時(shí)線粒體膜電位也顯著降低(p＜0.05)。 結(jié)論：在聽覺高級(jí)中樞老化的過程中線粒體DNA損傷可能是由于其修復(fù)功能減退造成的。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R764.436

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