本文關(guān)鍵詞： 年齡相關(guān)性聽力損失 線粒體 NADPH氧化酶3 氧化性應(yīng)激 激活型caspase-3 凋亡 高脂飲食 D-半乳糖 聽力損傷 氧化性應(yīng)激 線粒體 凋亡　出處：《華中科技大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分D-半乳糖誘導(dǎo)大鼠內(nèi)耳氧化性應(yīng)激、線粒體損傷和凋亡 目的：線粒體DNA (mitochondrial DNA,mtDNA)的氧化性損傷和凋亡是老化的重要特征。我們之前利用連續(xù)8周注射D-半乳糖(D-galactose, D-gal)的方法建立了大鼠老化模型,并且發(fā)現(xiàn)在D-gal誘導(dǎo)的老化大鼠內(nèi)耳中氧化性應(yīng)激的增加和mtDNA常見缺失(common deletion, CD)的增加。但是,在D-gal誘導(dǎo)的老化大鼠內(nèi)耳中,目前沒有直接的證據(jù)表明mtDNA CD的累積是由氧化性損傷引起的；同時此模型內(nèi)耳中活性氧(reactive oxygen species, ROS)的來源以及凋亡情況仍不清楚。 方法：聽力正常,無中耳疾患的40只5周齡雄性Spragua-Dawley大鼠隨機分成2組(各20只)：①D-半乳糖組(D-gal group):每日頸背部皮下注射D-gal (500mg/kg),連續(xù)8周；②對照組(control group):每日頸背部皮下注射同體積的生理鹽水,連續(xù)8周。聽性腦干反應(yīng)(auditory brainstem response, ABR)檢測每組大鼠聽功能。比色法檢測大鼠血清H202,總超氧化物歧化酶(total superoxide dismutase, T-SOD)活性以及丙二醛(malondialdehyde, MDA)含量。實時定量PCR檢測大鼠內(nèi)耳mtDNA CD的累積以及NADPH氧化酶3(NOX3)和P22phox]mRNA水平的變化。透射電子顯微鏡檢術(shù)(Transmission Electron Microscopy, TEM)觀察大鼠內(nèi)耳組織細胞中線粒體超微結(jié)構(gòu)的改變。免疫組織化學(xué)法檢測大鼠耳蝸8-羥基-2-脫氧鳥苷(8-hydroxy-2-deoxyguanosine,8-OHdG)以及NOX3和P22phox蛋白水平的變化。免疫印跡法檢測大鼠內(nèi)耳組織中激活型caspase-3(cleaved caspase-3)的表達。原位末端轉(zhuǎn)移酶標(biāo)記(terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, TUNEL染色檢測大鼠耳蝸細胞凋亡發(fā)生的情況。 結(jié)果：D-半乳糖組大鼠血清H202和MDA含量比對照組明顯增加,T-SOD水平比對照組明顯降低,差異有統(tǒng)計學(xué)意義(P0.01)。D-半乳糖組大鼠內(nèi)耳組織中mtDNACD累積比對照組明顯增加,差異有統(tǒng)計學(xué)意義(P0.01)。TEM發(fā)現(xiàn)D-半乳糖組大鼠耳蝸組織細胞中線粒體表現(xiàn)為基質(zhì)密度降低,甚至發(fā)生嚴重變性。D-半乳糖組大鼠耳蝸組織中8-OHdG、NOX3、P22phox和cleaved caspase-3的表達比對照組明顯升高,差異有統(tǒng)計學(xué)意義(P0.01)。TUNEL染色發(fā)現(xiàn)少量凋亡細胞局限于D-半乳糖組大鼠耳蝸血管紋,而對照組大鼠耳蝸未發(fā)現(xiàn)凋亡細胞。兩組大鼠ABR閾值差異無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：在內(nèi)耳老化過程中mtDNACD的積累可能是NADPH氧化酶相關(guān)的氧化性應(yīng)激造成的,并且caspase-3介導(dǎo)的細胞凋亡可能參與了內(nèi)耳的老化過程。 第二部分長期高脂飲食加重D-半乳糖誘導(dǎo)的老化大鼠內(nèi)耳氧化性應(yīng)激,線粒體損傷和凋亡 目的：研究12個月的高脂飲食對Sprague-Dawley大鼠內(nèi)耳及D-半乳糖誘導(dǎo)的老化大鼠內(nèi)耳的老化過程的作用。 方法：104只5周齡雄性Spragua-Dawley大鼠隨機分成4組(各26只)：①對照組(control group):飼喂基礎(chǔ)飲食12個月,前8周每日頸背部皮下注射和D-半乳糖組同體積的生理鹽水；②D-半乳糖組(D-gal group):飼喂基礎(chǔ)飲食12個月,前8周每日頸背部皮下注射D-gal (500mg/kg);③高脂飲食組(HFD group):飼喂高脂飲食12個月,前8周每日頸背部皮下注射和D-半乳糖組同體積的生理鹽水；④D-半乳糖+高脂飲食組(D-gal+HFD group):飼喂高脂飲食12個月,前8周每日頸背部皮下注射D-gal (500mg/kg)。造模結(jié)束后,聽性腦干反應(yīng)(auditory brainstem response, ABR)檢測每組大鼠聽功能；每組大鼠稱重,比色法檢測大鼠血清甘油三脂(triglycerides,TG),總膽固醇(total cholesterol, TC),游離脂肪酸(nonesterified fatty acids, NEFA)和H202含量；實時定量PCR檢測大鼠內(nèi)耳mtDNACD的累積以及NADPH氧化酶3(NOX3), P22phox, uncoupling protein2(UCP2)和uncoupling protein3(UCP3) mRNA水平的變化；免疫組織化學(xué)法檢測大鼠耳蝸NOX3蛋白水平的表達；免疫印跡法檢測大鼠內(nèi)耳組織中NOX3,P22phox,UCP2,UCP3和激活型caspase-3(cleaved caspase-3)的蛋白水平的表達；透射電子顯微鏡檢術(shù)(Transmission Electron Microscopy, TEM)觀察大鼠耳蝸血管紋邊緣細胞線粒體超微結(jié)構(gòu)的改變；原位末端轉(zhuǎn)移酶標(biāo)記(terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, TUNEL染色檢測大鼠耳蝸細胞凋亡情況。 結(jié)果：D-半乳糖組和D-半乳糖+高脂飲食組大鼠ABR閾值在4,8,16和32kHz比對照組升高,差異有統(tǒng)計學(xué)意義(P0.05)；高脂飲食組大鼠ABR閾值數(shù)值上在上述4個頻率均比對照組升高,但僅在32kHz差異有統(tǒng)計學(xué)意義(P0.05)；同時,D-半乳糖+高脂飲食組大鼠ABR閾值在16和32kHz比D-半乳糖組升高,差異有統(tǒng)計學(xué)意義(P0.05)。高脂飲食組和D-半乳糖+高脂飲食組大鼠血清TG、TC和NEFA水平比對照組和D-半乳糖組升高,差異有統(tǒng)計學(xué)意義(P0.01)；D-半乳糖組、高脂飲食組和D-半乳糖+高脂飲食組大鼠血清H202水平比對照組升高,差異有統(tǒng)計學(xué)意義(P0.01),其中D-半乳糖+高脂飲食組大鼠血清H202水平最高,差異有統(tǒng)計學(xué)意義(P0.01)。D-半乳糖組、高脂飲食組和D-半乳糖+高脂飲食組大鼠內(nèi)耳組織NOX3、P22phox、UCP2、UCP3和cleaved caspase-3的表達比對照組升高,其中D-半乳糖+高脂飲食組最高,差異有統(tǒng)計學(xué)意義(P0.01)。D-半乳糖組、高脂飲食組和D-半乳糖+高脂飲食組大鼠內(nèi)耳組織mtDNA CD的累積比對照組增加,差異有統(tǒng)計學(xué)意義(P0.05)。D-半乳糖組、高脂飲食組和D-半乳糖+高脂飲食組大鼠耳蝸血管紋邊緣細胞線粒體有不同程度的腫脹,伴有線粒體基質(zhì)密度降低。TUNEL染色發(fā)現(xiàn)D-半乳糖組、高脂飲食組和D-半乳糖+高脂飲食組凋亡細胞比對照組明顯增多,且凋亡細胞主要局限于血管紋；同時,在D-半乳糖+高脂飲食組大鼠耳蝸Corti器發(fā)現(xiàn)少量凋亡細胞。 結(jié)論：長期高脂飲食可誘導(dǎo)內(nèi)耳氧化性應(yīng)激、線粒體損傷和凋亡。長期高脂飲食可加速年齡相關(guān)性聽力損失的進展。
[Abstract]:The first part of D- galactose induced oxidative stress, mitochondrial damage and apoptosis in the inner ear of rats
Objective: mitochondrial DNA (mitochondrial DNA mtDNA) on the oxidative damage and apoptosis is an important feature of aging. We use before 8 weeks of continuous injection of D- galactose (D-galactose, D-gal) method to establish the model of aging rats, and found that in D-gal induced aging increased oxidative stress in the inner ear of rats and mtDNA common deletion (common deletion, CD) increased. However, in aging rats induced by D-gal in the inner ear, there is no direct evidence that the accumulation of mtDNA CD caused by oxidative damage; active oxygen at the same time, this model in the inner ear (reactive oxygen species, ROS) as well as the source of apoptosis remains unclear.
Methods: normal hearing, no middle ear diseases of the 40 5 week old male Spragua-Dawley rats were randomly divided into 2 groups (20 each): D- group (D-gal group) galactose: daily subcutaneous injection of D-gal (500mg/kg), for 8 weeks; the control group (control group): saline daily journal subcutaneous injection of the same volume for 8 consecutive weeks. Auditory brainstem response (auditory brainstem, response, ABR) function to detect the rats. Detect rats serum H202 assay, total superoxide dismutase (total superoxide, dismutase, T-SOD) activity and malondialdehyde (malondialdehyde, MDA). Real time quantitative content detection of PCR accumulation in the rat inner ear and mtDNA CD NADPH (NOX3) oxidase 3 changes and P22phox]mRNA level. Microscopy transmission electron microscopy (Transmission Electron, Microscopy, TEM) to observe the mitochondrial ultrastructure of cells of the inner ear tissues of rats without change. Immunohistochemical method was used to detect the rat cochlea 8- -2- hydroxy deoxyguanosine (8-hydroxy-2-deoxyguanosine, 8-OHdG) and the changes of NOX3 and P22phox protein levels. Caspase-3 activation in rats were detected by Western blotting in inner ear tissues (cleaved caspase-3). The expression of TDT mediated dUTP nick end labeling (terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP nick-end-labelling), TUNEL staining of rat cochlear cell apoptosis detection occurs.
Results: D- D-galactose rats serum H202 and MDA contents were significantly higher than control group, T-SOD levels were significantly lower than the control group, the difference was statistically significant (P0.01) the inner ear tissue in rats of.D- galactose in mtDNACD accumulation increased significantly than the control group, the difference was statistically significant (P0.01.TEM) found that mitochondrial cochlear tissue group rat D- cells showed galactose matrix density decreased, or even 8-OHdG, serious degeneration of.D- in D-galactose rats cochlea tissues NOX3, expression of P22phox and cleaved caspase-3 were significantly higher than the control group, the difference was statistically significant (P0.01).TUNEL staining showed a few apoptotic cells confined to D- galactose group in rat cochlea the vascular pattern, while the control group rat cochlea no apoptotic cells were found. There was no significant difference between two groups of rats ABR threshold (P0.05).
Conclusion: the accumulation of mtDNACD in the aging process of the inner ear may be caused by NADPH oxidase related oxidative stress, and caspase-3 mediated apoptosis may be involved in the aging process of the inner ear.
The second part of long term high fat diet aggravates the oxidative stress, mitochondrial damage and apoptosis of the aged rats with D- galactose induced aging
Objective: To study the effect of high fat diet for 12 months on the aging process of inner ear of Sprague-Dawley rats and D- galactose induced aging rats.
Methods: 104 5 week old male Spragua-Dawley rats were randomly divided into 4 groups (26 each): control group (control group): fed the basal diet for 12 months, 8 weeks before the daily subcutaneous injection of D- galactose group and the same volume of normal saline; the D- galactose group (D-gal group): fed the basal diet for 12 months, 8 weeks before the daily subcutaneous injection of D-gal (500mg/kg); high fat diet group (HFD group): fed with high fat diet for 12 months, 8 weeks before the daily subcutaneous injection of D- galactose group and the same volume of normal saline; the D- galactose + high fat diet group (D-gal+HFD group): fed with high fat diet for 12 months, 8 weeks before the daily subcutaneous injection of D-gal (500mg/kg). After the modeling, auditory brainstem response (auditory brainstem, response, ABR) function to detect the rats; the rats weighing, than the detection of rat serum glycerol color three fat (triglyceride S, TG), total cholesterol (total, cholesterol, TC), free fatty acids (nonesterified fatty, acids, NEFA) and H202 content accumulated; real time quantitative PCR detection of rat inner ear mtDNACD and NADPH oxidase 3 (NOX3), P22phox uncoupling protein2 (UCP2) and uncoupling protein3 (UCP3) mRNA levels; immunohistochemistry was used to detect the expression level of NOX3 protein in rat cochlea; NOX3 rats were detected by Western blot in the inner ear tissues of P22phox, UCP2, UCP3 and caspase-3 (cleaved caspase-3) activated protein level expression; microscopy transmission electron microscopy (Transmission Electron, Microscopy, TEM) to observe the rat cochlea mitochondria the edge of cell ultrastructure change; TDT mediated dUTP nick end labeling (terminal deoxynucleotidyl transferase (TdT) -mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, TUNEL staining The apoptosis of cochlear cells in rats was detected by color.
Results: D- galactose group and D- galactose + high fat diet group rats ABR threshold in 4,8,16 and 32kHz than the control group increased, the difference was statistically significant (P0.05); numerical threshold of ABR in high fat diet group rats in each of the 4 frequencies were higher than control group, but the difference is only in the 32kHz statistics significance (P0.05); at the same time, D- galactose + high fat diet rats in group ABR and 32kHz than the 16 threshold in D- galactose group increased, the difference was statistically significant (P0.05). Serum TG D- galactose diet group and high-fat + high-fat diet group rats, TC and NEFA levels than the control group galactose and D- group increased, the difference was statistically significant (P0.01); D- galactose group, the serum level of H202 diet group and D- galactose + high fat high-fat diet group rats is higher than that of the control group, the difference was statistically significant (P0.01), the level of serum H202 in D- galactose + high fat diet group were the highest, the difference was statistically significant ( P0.01).D- galactose group, high fat diet group and inner ear tissue D- galactose + high fat diet group rats NOX3, P22phox, UCP2, UCP3 and cleaved expression of Caspase-3 was higher than control, which D- galactose + high fat diet group was the highest, the difference was statistically significant (P0.01).D- galactose group. Cumulative increase in high fat diet group and inner ear tissue D- galactose + high fat diet group rats mtDNA CD than in the control group, the difference was statistically significant (P0.05).D- galactose group, mitochondria in marginal cells of the stria vascularis D- galactose + diet group and high-fat diet group rats with high fat have different degrees of swelling that is associated with decreased mitochondrial matrix density of.TUNEL staining indicated that D- galactose group, high fat diet group and D- galactose + high fat diet group than the control group, the apoptotic cells increased significantly, and the apoptosis was mainly confined to the vascular pattern; at the same time, in D- galactose + high fat diet rats cochlear Corti A small number of apoptotic cells were found.
Conclusion: long term high fat diet can induce oxidative stress in the inner ear, mitochondrial damage and apoptosis. Long term high fat diet can accelerate the progress of age related hearing loss.

【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】：2012
【分類號】：R363

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本文編號：1464123