线粒体DNA4834缺失突变在内耳拟老化模型中的作用及其机制研究
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摘要
实验一大鼠内耳拟老化伴线粒体突变模型的建立
【摘要】目的探讨建立大鼠内耳拟老化伴线粒体突变模型,为进一步揭示老年性聋发病机制及其防治策略奠定基础。方法Wistar大鼠24只,随机分为2组,A组(D-半乳糖组,14只),5%D-半乳糖颈部皮下注射(150mg*kg-1*d-1),共8周,继以生理盐水腹腔注射10天;B组(空白对照组,10只)全程仅给予生理盐水注射。听性脑干反应(auditory brainstem respones , ABR)检测两组大鼠反应阈,比色法检测两组大鼠膜迷路谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)活力。利用巢氏聚合酶链反应技术检测内耳组织线粒体DNA 4834 bp缺失突变的发生情况。结果用药后A组大鼠线粒体DNA 4834bp缺失突变发生率为100 %(28耳/28耳) ,B组无线粒体DNA 4834 bp缺失突变检出。A组GSH-PX活力(59.07±2.32U)明显低于B组(142.10±2.21U),其差异具有显著统计学意义(t检验,双侧检验,P=0.000<0.01)。而A组ABR反应阈平均提高5.36±3.08 dB peSPL,B组为6.50±3.37 dB peSPL。经t检验,A组和B组之间差异无显著性意义(t检验,双侧检验,pAD=0.508)。结论D-半乳糖可以诱导大鼠内耳组织拟老化,此模型大鼠内耳组织线粒体DNA4834bp缺失突变率极高,但是ABR反应阈无明显提高。比较,差异无统计学意义(χ=0.296,P >0.05)。提取核DNA成功率,SDS-蛋白酶K法(60.00%)分别与PCR缓冲液法(90.00%)和Chelex-100法(90.83%)比较,差异均有统计学意义(χ1=49.091,χ2=30.767,均P<0.001);PCR缓冲液法与Chelex-100法比较,差异无统计学意义(χ=0.048,P >0.05)。PCR缓冲液法不能从毛干中提取核酸,SDS-蛋白酶K法不能从1、2根鼠毛中提取mtDNA,而Chelex-100法可从毛干和单根鼠毛中提取mtDNA和DNA。结论Chelex-100法对从毛发中提取核酸较为合适。
实验三大鼠线粒体DNA4834bp缺失突变在不同组织器官中的差异表现
【摘要】目的探讨在模型大鼠内耳、肾脏、大脑、血、骨骼肌、脾脏、心脏、肝脏、肺和毛发等不同的组织中,线粒体DNA4834bp缺失突变的发生情况及其可能的病理意义。同时探讨毛发代替血标本用于临床检测线粒体缺失突变的可行性。方法Wistar大鼠24只,随机分为2组,A组(D-半乳糖组,14只),5%D-半乳糖颈部皮下注射(150mg*kg-1*d-1),共8周,继以生理盐水腹腔注射10天;B组(空白对照组,10只)仅给予生理盐水注射。利用巢氏聚合酶链反应技术检测各组织线粒体DNA 4834 bp缺失突变的发生情况。结果用药后A组大鼠各器官均可检出CD,其中内耳、颞肌、肝脏CD检出率最高,为100%,肾脏和大脑次之,为92.86%(a),脾脏85.71%(b),心脏、肺脏和毛发组织为71.43%,血液中检出率最低,仅为35.71%。其中除血液CD检出率与内耳CD检出率比较,差异具有显著统计学意义外(p=0.001<0.01,双尾检验,Fisher’s精确概率检验),其他组织和内耳组织CD检出率之间的差异无统计学意义(pa=1.000,pb=0.481,pc=0.098,p均>0.05,双尾检验,Fisher’s精确概率检验)。而在对照组中,仅肝脏(20%),大脑和肾脏(10%)检出了CD突变,其他组织均未检出CD的存在。结论提示D-半乳糖除了诱导大鼠内耳4834bp缺失突变以外,可以诱导模型多器官CD突变。CD缺失率高低可能与组织的有丝分裂程度和氧化磷酸化活跃程度有关。同时提示毛发CD检出率更能代表内耳CD缺失情况,可以代替血标本用于临床检测线粒体缺失突变。
实验四大鼠内耳拟老化模型对氨基糖甙类抗生素耳毒性的易感性
【摘要】目的研究大鼠内耳拟老化模型对氨基糖甙类抗生素的易感性。方法Wistar大鼠50只,随机分为4组,A组(半乳糖组,14只)5%D-半乳糖颈部皮下注射(150mg.kg-1.d-1),共8周,继以生理盐水腹腔注射10天;B组(半乳糖加卡那霉素组,14只)皮下注射半乳糖同A组,8周后,腹腔注射硫酸卡那霉素(500mg.kg-1.d-1)10天。C组(卡那霉素组,12只)前8周用生理盐水代替半乳糖,后10天卡那霉素注射同B组。D组(空白对照组,10只)仅给予生理盐水注射。听性脑干反应(auditory brainstem respones , ABR)检测各组大鼠反应阈,比色法检测各组大鼠膜迷路谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)活力。利用巢氏聚合酶链反应技术检测内耳组织线粒体DNA 4834 bp缺失突变的发生情况。结果用药后A组大鼠线粒体DNA 4834bp缺失突变发生率为100%(28耳/28耳),B组为92.86%(26耳/28耳),C组和D组均无线粒体DNA 4834 bp缺失突变检出。A组GSH-PX活性为59.07±8.70U,B组63.29±12.40U,C组136.67±9.53U,D组142.10±7.02U。A组和D组之间差异具有显著统计学意义(pAD=0.000);A组和B组,C组和D组之间差异没有显著统计学意义(pAB=0.307,pCD=0.151)。A组ABR反应阈平均提高5.36±3.08 dB peSPL,B组为61.79±11.20 dB peSPL ,C组为34.17±4.69 dB peSPL,D组为6.50±3.37 dB peSPL。经校正t′检验,A组和D组之间差异无显著性意义(pAD=0.398),B组和C组分别与D组之间差异有显著性意义(pBD=0.000,pCD=0.000),B组和C组之间差异也有显著性意义(pBC=0.000)。结论D-半乳糖可以诱导大鼠内耳组织拟老化,此模型大鼠内耳组织mtDNA4834bp缺失突变率极高,同时对氨基糖甙类抗生素耳毒性的易感性增强。
实验五核因子与mtDNA4834bp缺失突变相互作用在大鼠内耳对氨基糖甙类药物耳毒性易感中的作用
【摘要】目的研究氨基糖甙类抗生素耳聋易感模型中的大鼠内耳膜迷路中线粒体相对拷贝数,核基因编码的核呼吸因子-1(NRF-1)、过氧化物酶增值子激活受体-γ亚单位共激活因子(PGC-1)及线粒体DNA编码的细胞色素C氧化酶亚单位III(COXIII)的mRNA水平变化,以及COXIII蛋白水平的变化和mtDNA4834bp缺失突变,探讨核因子与mtDNA4834bp缺失突变相互作用在大鼠内耳对氨基糖甙类药物易感中的作用。方法Wistar大鼠16只,随机分为2组,A组(半乳糖加卡那霉素组,10只)皮下注射半乳糖同A组,8周后,腹腔注射硫酸卡那霉素(500mg.kg-1.d-1)10天。B组(空白对照组,6只)第一阶段用生理盐水代替半乳糖注射,第二阶段用药同A组。听性脑干反应(auditory brainstem respones ,ABR)检测各组大鼠反应阈,聚合酶链式反应技术(polymerase chain reaction,PCR)检测内耳线粒体拷贝数(copy number,CN),巢氏聚合酶链反应技术(nest PCR)检测内耳组织线粒体DNA 4834 bp缺失突变的发生情况,半定量的逆转录聚合酶链反应技术(reverse transcription PCR,RT-PCR)检测内耳NRF-1、PGC-1和COXIII的mRNA水平,蛋白免疫印迹法(western-blot)检测内耳COXIII蛋白水平。结果用药后A组大鼠线粒体DNA 4834bp缺失突变发生率为100%,B组无线粒体DNA 4834 bp缺失突变检出。A组ABR反应阈平均提高66.79±7.75 dB peSPL, B组为8.33±6.83 dB peSPL。经校正t′检验,A组和B组之间差异有显著性意义(P<0.01)。A组CN为2.75±1.38,B组5.76±1.04,A组和B组之间差异具有显著统计学意义(P<0.01);NRF-1和PGC-1的mRNA水平均较对照组下降(0.51±0.10 vs 0.94±0.06,2.93±0.98 vs 7.92±1.53, P<0.01)。COXIII的mRNA水平无明显改变(2.83±0.65 vs 3.15±0.53,P>0.05),但是蛋白质水平明显下降(P<0.01)。结论核因子与线粒体基因相互调节,其协同作用可能与大鼠内耳对氨基糖甙类药物易感有关。
Part one: Establishing model of mimetic aging of inner ear with mtDNA mutation in rat
Object To explore the establishment of the mimetic aging effect in the inner ear of the rat. Methods Twenty-four wistar rats were randomly divided into two groups: group A (D-galactose group, n=14), were treated with hypodermic 5% D-galactose (150mg.kg-1.d-1) for 8 weeks and then with intraperitoneal saline for 10days; group B (control group, n=10) were given saline only. Auditory brainstem response(ABR) was used to detect the hearing threshold of rats, and colorimetry was used to analyze the activity of the glutathione peroxidase(GSH-PX). The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results The incidence of mitochondrial DNA 4834bp deletion of group A was100 %(28/28) , while group B was 0. The activity of the GSH-PX in group A(59.07±2.32U) is lower than in group B(142.10±2.21U), the difference between group A and group B is significant(t-test,p=0.000<0.01). 5.36±3.08 dB peSPL variation in ABR threshold was observed in group A, and 6.50±3.37 dB peSPL in group B. The difference in shift of ABR threshold between group A and group B is not significant (t-test,p=0.508>0.05). Conclusion The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats of model present high incidence of mtDNA4834 deletion. But no hearing loss was found in this model.
Part two: The comparison of three ways for extracting nucleic acid from rat’s hair
Objective To discuss the method of extracting nucleic acid from rat’s hair. Methods The method with PCR buffer、SDS-proteinase K or Chelex-100 were used to extract the nucleic acid from rat’s hair separately, and the isolated nucleic acid was analyzed by PCR and electrophoresis. Results The success rate of extracting mtDNA from the hair papilla, we found the method with SDS-proteinase K is lower than the other methods (χ1=42.421,χ2=28.800,P<0.001). This is same as the result of extracting nuclear DNA (χ1=49.091,χ2=30.767,P<0.001). While no difference has been found between the method with PCR buffer and with chelex-100(mtDNA:χ=0.296;nuclear DNA:χ=0.048,P >0.05).The method with PCR buffer can’t extract nucleic acid from hair shafts, the method with SDS-proteinase K can’t extract mtDNA from one or two hairs, the method with Chelex-100 can extract mtDNA and nuclear DNA from single rat’s hair or hair shafts. Conclusion The method with Chelex-100 is suitable for extracting nucleic acid from hair.
Part three:The incidence of mitochondrial DNA 4977bp deletion mutation among the different tissues
Object To explore incidence of mtDNA4834bp deletion in the inner ear,kidney,brain,blood,skeletal muscle,spleen,heart,liver,lung and hair of model rat and elucidate its potential pathological significance.Meanwhile, to determine possibility of hair instead of blood preparation used in clinical examination of mtDNA deletion. Methods Twenty-four Wistar rats were randomly divided into two groups: group A (D-galactose group , n = 14) ,which were treated with hypodermic 5% D-galactose (150 mg·kg - 1·d - 1 ) for 8 weeks and then with intraperitoneal saline for 10 days,and group B (control group , n = 10) , which were given saline only. The various tissues were harvested and mitochondrial DNA was amplified to identify the 4834bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results CD can be examined in various tissue of group A.incidence of CD in inner ear,temporal muscle and liver are highest(100%),kidney and brain are second(92.86%),spleen is 85.71%,heart、lung and hair are 71.43%,blood is the lowest(35.71%).The difference of incidence of CD between blood and inner ear was significant(p=0.001<0.01,two-tailed test,Fisher’s test),but it is not significant between other tissue and inner ear(pa=1.000,pb=0.481, pc=0.098,pall>0.05,two-tailed test,Fisher’s test)。in group B, CD can be investigated only in liver(20%),brain and kidney(10%),the other tissue was negative. Conclusion Result indicates that D-galactose can induce mtDNA4834bp deletion not only in inner ear, but also in many other tissue.The difference of incidence of CD may concern with degree of caryocinesia and activity of oxidative phosphorylation. Meanwhile, the result indicated that incidence of CD in hair can represent in inner ear more suitably.Hair can be used in clinical examination of mtDNA deletion instead of blood.
Part four: The sensitivity to the ototoxicity of aminoglycoside antibiotic of the animal model with mimetic aging in the inner ear
Object To research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of aminoglycoside antibiotic. Methods fifty wistar rats were randomly divided into four groups: group A (D-galactose group, n=14),were treated with hypodermic 5% D-galactose (150mg.kg-1.d-1) for 8 weeks and then with intraperitoneal saline for 10days; group B (D-galactose and kanamycin group, n=14),were given the same dose of D-galactose but kanamycin (500mg.kg-1.d-1.) instead of saline; group C (kanamycin group, n=12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n=10) were given saline only. Auditory brainstem response(ABR) was used to detecte the hearing threshold of rats, and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results The incidence of mitochondrial DNA4834bp deletion mutation of group A was 100 % (28/ 28), group B was 92.86%(26/ 28),and group C or group D was 0. The activity of GSH-PX in group A was 59.07±8.70U,in group B was 63.29±12.40U,in group C was 136.67±9.53U,in group D was 142.10±7.02U. The difference between group A and D is significant (pAD=0.000).while the difference between group A and B is not significant (pAB=0.307), similarly as between group C and group D(PCD=0.151). 5.36±3.08 dB peSPL variation in ABR threshold was observed in group A, 61.79±11.20 dB peSPL in group B, 34.17±4.69 dB peSPL in group C,and 6.50±3.37 dB peSPL in group D.No difference was found between group A and D(pAD=0.398), while the difference in shift of ABR threshold between group B and group C(or group D) is significant(pBD=0.000, pCD=0.000). Conclusion The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rat model present high incidence of mtDNA4834 deletion. The mutation can greatly enhance the sensitivity of the inner ear to the aminoglycoside antibiotic.
Part five:The role of nuclear factor in inner ear of rat which sensitivity to the aminoglycoside antibiotic
Object To investigate mitochondrial copy number in membranous labyrinth of inner ear of aminoglycoside antibiotics-induced hearing loss model of rat,the change of mRNA level of nuclear gene encoding NRF-1,PGC-1,mtDNA encoding cytochrome C oxidase COXIII, and the change of protein level of COXIII and mtDNA4834bp deletion and explore interaction between nuclear factor and mtDNA4834bp deletion contribute to sensitivity of the inner ear of rat to aminoglycoside antibiotics. Methods Sixteen Wistar rats were randomly divided into two groups: group A(D-galactose and kanamycin group,n=10) were treated with hypodermic D-galactose (150 mg/kg/d)for 8 weeks and then with intraperitoneal kanamycin (500 mg/kg/d) for 10 days.group B(control group, n = 6) was administrated with saline instead of D-galactose. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats,polymerase chain reaction(PCR) was used to detect inner ear mitochondrial copy number,nest PCR was used to detect the incidence of inner ear mtDNA4834bp deletion,semiquantitative RT-PCR was used to detect the mRNA level of NRF-1、PGC-1 and COXIII and western-blot was used to detect protein level of COXIII in inner ear. Results The incidence of mitochondrial DNA 4834 bp deletion of group A was 100%,while the deletion of group B was 0 . 66.79±7.75 dB peSPL increased in ABR threshold was observed in group A, and 8.33±6.83 dB peSPL in group B. The CN level of group A is 2.75±1.38, group B is 5.76±1.04. The difference between A and B was significant(p<0.01). The mRNA level of NRF-1 and PGC-1 decreased(0.51±0.10 vs 0.94±0.06,2.93±0.98 vs 7.92±1.53, P<0.01). The mRNA level of COXIII didn’t change(2.83±0.65 vs 3.15±0.53,P>0.05 ), but protein level decreased(P<0.01). Conclusion There is mutual regulation between nuclear factor and mtDNA, their cooperation effect may be related with sensitivity of the inner ear of rat to aminoglycoside antibiotics.
【摘要】目的探讨建立大鼠内耳拟老化伴线粒体突变模型,为进一步揭示老年性聋发病机制及其防治策略奠定基础。方法Wistar大鼠24只,随机分为2组,A组(D-半乳糖组,14只),5%D-半乳糖颈部皮下注射(150mg*kg-1*d-1),共8周,继以生理盐水腹腔注射10天;B组(空白对照组,10只)全程仅给予生理盐水注射。听性脑干反应(auditory brainstem respones , ABR)检测两组大鼠反应阈,比色法检测两组大鼠膜迷路谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)活力。利用巢氏聚合酶链反应技术检测内耳组织线粒体DNA 4834 bp缺失突变的发生情况。结果用药后A组大鼠线粒体DNA 4834bp缺失突变发生率为100 %(28耳/28耳) ,B组无线粒体DNA 4834 bp缺失突变检出。A组GSH-PX活力(59.07±2.32U)明显低于B组(142.10±2.21U),其差异具有显著统计学意义(t检验,双侧检验,P=0.000<0.01)。而A组ABR反应阈平均提高5.36±3.08 dB peSPL,B组为6.50±3.37 dB peSPL。经t检验,A组和B组之间差异无显著性意义(t检验,双侧检验,pAD=0.508)。结论D-半乳糖可以诱导大鼠内耳组织拟老化,此模型大鼠内耳组织线粒体DNA4834bp缺失突变率极高,但是ABR反应阈无明显提高。比较,差异无统计学意义(χ=0.296,P >0.05)。提取核DNA成功率,SDS-蛋白酶K法(60.00%)分别与PCR缓冲液法(90.00%)和Chelex-100法(90.83%)比较,差异均有统计学意义(χ1=49.091,χ2=30.767,均P<0.001);PCR缓冲液法与Chelex-100法比较,差异无统计学意义(χ=0.048,P >0.05)。PCR缓冲液法不能从毛干中提取核酸,SDS-蛋白酶K法不能从1、2根鼠毛中提取mtDNA,而Chelex-100法可从毛干和单根鼠毛中提取mtDNA和DNA。结论Chelex-100法对从毛发中提取核酸较为合适。
实验三大鼠线粒体DNA4834bp缺失突变在不同组织器官中的差异表现
【摘要】目的探讨在模型大鼠内耳、肾脏、大脑、血、骨骼肌、脾脏、心脏、肝脏、肺和毛发等不同的组织中,线粒体DNA4834bp缺失突变的发生情况及其可能的病理意义。同时探讨毛发代替血标本用于临床检测线粒体缺失突变的可行性。方法Wistar大鼠24只,随机分为2组,A组(D-半乳糖组,14只),5%D-半乳糖颈部皮下注射(150mg*kg-1*d-1),共8周,继以生理盐水腹腔注射10天;B组(空白对照组,10只)仅给予生理盐水注射。利用巢氏聚合酶链反应技术检测各组织线粒体DNA 4834 bp缺失突变的发生情况。结果用药后A组大鼠各器官均可检出CD,其中内耳、颞肌、肝脏CD检出率最高,为100%,肾脏和大脑次之,为92.86%(a),脾脏85.71%(b),心脏、肺脏和毛发组织为71.43%,血液中检出率最低,仅为35.71%。其中除血液CD检出率与内耳CD检出率比较,差异具有显著统计学意义外(p=0.001<0.01,双尾检验,Fisher’s精确概率检验),其他组织和内耳组织CD检出率之间的差异无统计学意义(pa=1.000,pb=0.481,pc=0.098,p均>0.05,双尾检验,Fisher’s精确概率检验)。而在对照组中,仅肝脏(20%),大脑和肾脏(10%)检出了CD突变,其他组织均未检出CD的存在。结论提示D-半乳糖除了诱导大鼠内耳4834bp缺失突变以外,可以诱导模型多器官CD突变。CD缺失率高低可能与组织的有丝分裂程度和氧化磷酸化活跃程度有关。同时提示毛发CD检出率更能代表内耳CD缺失情况,可以代替血标本用于临床检测线粒体缺失突变。
实验四大鼠内耳拟老化模型对氨基糖甙类抗生素耳毒性的易感性
【摘要】目的研究大鼠内耳拟老化模型对氨基糖甙类抗生素的易感性。方法Wistar大鼠50只,随机分为4组,A组(半乳糖组,14只)5%D-半乳糖颈部皮下注射(150mg.kg-1.d-1),共8周,继以生理盐水腹腔注射10天;B组(半乳糖加卡那霉素组,14只)皮下注射半乳糖同A组,8周后,腹腔注射硫酸卡那霉素(500mg.kg-1.d-1)10天。C组(卡那霉素组,12只)前8周用生理盐水代替半乳糖,后10天卡那霉素注射同B组。D组(空白对照组,10只)仅给予生理盐水注射。听性脑干反应(auditory brainstem respones , ABR)检测各组大鼠反应阈,比色法检测各组大鼠膜迷路谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)活力。利用巢氏聚合酶链反应技术检测内耳组织线粒体DNA 4834 bp缺失突变的发生情况。结果用药后A组大鼠线粒体DNA 4834bp缺失突变发生率为100%(28耳/28耳),B组为92.86%(26耳/28耳),C组和D组均无线粒体DNA 4834 bp缺失突变检出。A组GSH-PX活性为59.07±8.70U,B组63.29±12.40U,C组136.67±9.53U,D组142.10±7.02U。A组和D组之间差异具有显著统计学意义(pAD=0.000);A组和B组,C组和D组之间差异没有显著统计学意义(pAB=0.307,pCD=0.151)。A组ABR反应阈平均提高5.36±3.08 dB peSPL,B组为61.79±11.20 dB peSPL ,C组为34.17±4.69 dB peSPL,D组为6.50±3.37 dB peSPL。经校正t′检验,A组和D组之间差异无显著性意义(pAD=0.398),B组和C组分别与D组之间差异有显著性意义(pBD=0.000,pCD=0.000),B组和C组之间差异也有显著性意义(pBC=0.000)。结论D-半乳糖可以诱导大鼠内耳组织拟老化,此模型大鼠内耳组织mtDNA4834bp缺失突变率极高,同时对氨基糖甙类抗生素耳毒性的易感性增强。
实验五核因子与mtDNA4834bp缺失突变相互作用在大鼠内耳对氨基糖甙类药物耳毒性易感中的作用
【摘要】目的研究氨基糖甙类抗生素耳聋易感模型中的大鼠内耳膜迷路中线粒体相对拷贝数,核基因编码的核呼吸因子-1(NRF-1)、过氧化物酶增值子激活受体-γ亚单位共激活因子(PGC-1)及线粒体DNA编码的细胞色素C氧化酶亚单位III(COXIII)的mRNA水平变化,以及COXIII蛋白水平的变化和mtDNA4834bp缺失突变,探讨核因子与mtDNA4834bp缺失突变相互作用在大鼠内耳对氨基糖甙类药物易感中的作用。方法Wistar大鼠16只,随机分为2组,A组(半乳糖加卡那霉素组,10只)皮下注射半乳糖同A组,8周后,腹腔注射硫酸卡那霉素(500mg.kg-1.d-1)10天。B组(空白对照组,6只)第一阶段用生理盐水代替半乳糖注射,第二阶段用药同A组。听性脑干反应(auditory brainstem respones ,ABR)检测各组大鼠反应阈,聚合酶链式反应技术(polymerase chain reaction,PCR)检测内耳线粒体拷贝数(copy number,CN),巢氏聚合酶链反应技术(nest PCR)检测内耳组织线粒体DNA 4834 bp缺失突变的发生情况,半定量的逆转录聚合酶链反应技术(reverse transcription PCR,RT-PCR)检测内耳NRF-1、PGC-1和COXIII的mRNA水平,蛋白免疫印迹法(western-blot)检测内耳COXIII蛋白水平。结果用药后A组大鼠线粒体DNA 4834bp缺失突变发生率为100%,B组无线粒体DNA 4834 bp缺失突变检出。A组ABR反应阈平均提高66.79±7.75 dB peSPL, B组为8.33±6.83 dB peSPL。经校正t′检验,A组和B组之间差异有显著性意义(P<0.01)。A组CN为2.75±1.38,B组5.76±1.04,A组和B组之间差异具有显著统计学意义(P<0.01);NRF-1和PGC-1的mRNA水平均较对照组下降(0.51±0.10 vs 0.94±0.06,2.93±0.98 vs 7.92±1.53, P<0.01)。COXIII的mRNA水平无明显改变(2.83±0.65 vs 3.15±0.53,P>0.05),但是蛋白质水平明显下降(P<0.01)。结论核因子与线粒体基因相互调节,其协同作用可能与大鼠内耳对氨基糖甙类药物易感有关。
Part one: Establishing model of mimetic aging of inner ear with mtDNA mutation in rat
Object To explore the establishment of the mimetic aging effect in the inner ear of the rat. Methods Twenty-four wistar rats were randomly divided into two groups: group A (D-galactose group, n=14), were treated with hypodermic 5% D-galactose (150mg.kg-1.d-1) for 8 weeks and then with intraperitoneal saline for 10days; group B (control group, n=10) were given saline only. Auditory brainstem response(ABR) was used to detect the hearing threshold of rats, and colorimetry was used to analyze the activity of the glutathione peroxidase(GSH-PX). The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results The incidence of mitochondrial DNA 4834bp deletion of group A was100 %(28/28) , while group B was 0. The activity of the GSH-PX in group A(59.07±2.32U) is lower than in group B(142.10±2.21U), the difference between group A and group B is significant(t-test,p=0.000<0.01). 5.36±3.08 dB peSPL variation in ABR threshold was observed in group A, and 6.50±3.37 dB peSPL in group B. The difference in shift of ABR threshold between group A and group B is not significant (t-test,p=0.508>0.05). Conclusion The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats of model present high incidence of mtDNA4834 deletion. But no hearing loss was found in this model.
Part two: The comparison of three ways for extracting nucleic acid from rat’s hair
Objective To discuss the method of extracting nucleic acid from rat’s hair. Methods The method with PCR buffer、SDS-proteinase K or Chelex-100 were used to extract the nucleic acid from rat’s hair separately, and the isolated nucleic acid was analyzed by PCR and electrophoresis. Results The success rate of extracting mtDNA from the hair papilla, we found the method with SDS-proteinase K is lower than the other methods (χ1=42.421,χ2=28.800,P<0.001). This is same as the result of extracting nuclear DNA (χ1=49.091,χ2=30.767,P<0.001). While no difference has been found between the method with PCR buffer and with chelex-100(mtDNA:χ=0.296;nuclear DNA:χ=0.048,P >0.05).The method with PCR buffer can’t extract nucleic acid from hair shafts, the method with SDS-proteinase K can’t extract mtDNA from one or two hairs, the method with Chelex-100 can extract mtDNA and nuclear DNA from single rat’s hair or hair shafts. Conclusion The method with Chelex-100 is suitable for extracting nucleic acid from hair.
Part three:The incidence of mitochondrial DNA 4977bp deletion mutation among the different tissues
Object To explore incidence of mtDNA4834bp deletion in the inner ear,kidney,brain,blood,skeletal muscle,spleen,heart,liver,lung and hair of model rat and elucidate its potential pathological significance.Meanwhile, to determine possibility of hair instead of blood preparation used in clinical examination of mtDNA deletion. Methods Twenty-four Wistar rats were randomly divided into two groups: group A (D-galactose group , n = 14) ,which were treated with hypodermic 5% D-galactose (150 mg·kg - 1·d - 1 ) for 8 weeks and then with intraperitoneal saline for 10 days,and group B (control group , n = 10) , which were given saline only. The various tissues were harvested and mitochondrial DNA was amplified to identify the 4834bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results CD can be examined in various tissue of group A.incidence of CD in inner ear,temporal muscle and liver are highest(100%),kidney and brain are second(92.86%),spleen is 85.71%,heart、lung and hair are 71.43%,blood is the lowest(35.71%).The difference of incidence of CD between blood and inner ear was significant(p=0.001<0.01,two-tailed test,Fisher’s test),but it is not significant between other tissue and inner ear(pa=1.000,pb=0.481, pc=0.098,pall>0.05,two-tailed test,Fisher’s test)。in group B, CD can be investigated only in liver(20%),brain and kidney(10%),the other tissue was negative. Conclusion Result indicates that D-galactose can induce mtDNA4834bp deletion not only in inner ear, but also in many other tissue.The difference of incidence of CD may concern with degree of caryocinesia and activity of oxidative phosphorylation. Meanwhile, the result indicated that incidence of CD in hair can represent in inner ear more suitably.Hair can be used in clinical examination of mtDNA deletion instead of blood.
Part four: The sensitivity to the ototoxicity of aminoglycoside antibiotic of the animal model with mimetic aging in the inner ear
Object To research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of aminoglycoside antibiotic. Methods fifty wistar rats were randomly divided into four groups: group A (D-galactose group, n=14),were treated with hypodermic 5% D-galactose (150mg.kg-1.d-1) for 8 weeks and then with intraperitoneal saline for 10days; group B (D-galactose and kanamycin group, n=14),were given the same dose of D-galactose but kanamycin (500mg.kg-1.d-1.) instead of saline; group C (kanamycin group, n=12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n=10) were given saline only. Auditory brainstem response(ABR) was used to detecte the hearing threshold of rats, and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results The incidence of mitochondrial DNA4834bp deletion mutation of group A was 100 % (28/ 28), group B was 92.86%(26/ 28),and group C or group D was 0. The activity of GSH-PX in group A was 59.07±8.70U,in group B was 63.29±12.40U,in group C was 136.67±9.53U,in group D was 142.10±7.02U. The difference between group A and D is significant (pAD=0.000).while the difference between group A and B is not significant (pAB=0.307), similarly as between group C and group D(PCD=0.151). 5.36±3.08 dB peSPL variation in ABR threshold was observed in group A, 61.79±11.20 dB peSPL in group B, 34.17±4.69 dB peSPL in group C,and 6.50±3.37 dB peSPL in group D.No difference was found between group A and D(pAD=0.398), while the difference in shift of ABR threshold between group B and group C(or group D) is significant(pBD=0.000, pCD=0.000). Conclusion The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rat model present high incidence of mtDNA4834 deletion. The mutation can greatly enhance the sensitivity of the inner ear to the aminoglycoside antibiotic.
Part five:The role of nuclear factor in inner ear of rat which sensitivity to the aminoglycoside antibiotic
Object To investigate mitochondrial copy number in membranous labyrinth of inner ear of aminoglycoside antibiotics-induced hearing loss model of rat,the change of mRNA level of nuclear gene encoding NRF-1,PGC-1,mtDNA encoding cytochrome C oxidase COXIII, and the change of protein level of COXIII and mtDNA4834bp deletion and explore interaction between nuclear factor and mtDNA4834bp deletion contribute to sensitivity of the inner ear of rat to aminoglycoside antibiotics. Methods Sixteen Wistar rats were randomly divided into two groups: group A(D-galactose and kanamycin group,n=10) were treated with hypodermic D-galactose (150 mg/kg/d)for 8 weeks and then with intraperitoneal kanamycin (500 mg/kg/d) for 10 days.group B(control group, n = 6) was administrated with saline instead of D-galactose. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats,polymerase chain reaction(PCR) was used to detect inner ear mitochondrial copy number,nest PCR was used to detect the incidence of inner ear mtDNA4834bp deletion,semiquantitative RT-PCR was used to detect the mRNA level of NRF-1、PGC-1 and COXIII and western-blot was used to detect protein level of COXIII in inner ear. Results The incidence of mitochondrial DNA 4834 bp deletion of group A was 100%,while the deletion of group B was 0 . 66.79±7.75 dB peSPL increased in ABR threshold was observed in group A, and 8.33±6.83 dB peSPL in group B. The CN level of group A is 2.75±1.38, group B is 5.76±1.04. The difference between A and B was significant(p<0.01). The mRNA level of NRF-1 and PGC-1 decreased(0.51±0.10 vs 0.94±0.06,2.93±0.98 vs 7.92±1.53, P<0.01). The mRNA level of COXIII didn’t change(2.83±0.65 vs 3.15±0.53,P>0.05 ), but protein level decreased(P<0.01). Conclusion There is mutual regulation between nuclear factor and mtDNA, their cooperation effect may be related with sensitivity of the inner ear of rat to aminoglycoside antibiotics.
引文
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8.孔维佳,韩月臣,王莹等。维生素E、辅酶Q10对大鼠内耳组织线粒体DNA4834bp缺失突变的预防作用。中华耳鼻咽喉科杂志,2004,39(12),707-711。
9. Van Tuyle GC,Gudikote JP,Hurt VR,et al.M ultiple,large deletion in rat mitochondrial DNA:evidence for a major hot spot.Mutat Res,1996,349:95-107.
10. Rotig A, Bourgeron T, Chretien D, et al.Spectrum of mitochondrial DNA rearrangements in the Pearson marrow-pancreas syndrome, Hum. Mol.Genet.,1995,4 :1327-1330.
11. Seidman MD, Bai U,Khan DJ, et al. Mitochondrial DNA deletions associated with aging and presbyacusis. Arch, Otolaryngol Head Neck Surg,1997,123:1039-1045.
12. Bai U, Seidman MD, Hinojosa R ,et al.Mitochondrial DNA deletions associated with aging and possibly presbycusis: a human archival temporal bone study. Am. J. Otol,1997, 18:449–453.
13. Lortholary O,Tod M,Cohen Y,Petitjean O.Aminoglycosides.Med Clin North Am.1995;79:761-98.
14. Sande MA,Mandell GL.Antimicrobial agent.In:Gilman AG,Rall TW,Nics AS,Taylor,editors.Goodman and Golman’s The Pharmacological Basis of Therapeutics,8th edn.Elmsford,NY:Pergamon Press;1990.P.1098-116.
15. Kidwell DT,Mckeown JW,Grider JS,et al.Acute effects of gentamicin on thick ascending limb function in the rat.European journal of Pharmacology,1994,270,97-103
16. Bates DE. Aminoglycoside ototoxicity. Drugs Today ( Barc ) .2003, 39(4):227-85.Review
17. Selimoglu E. Aminoglycoside-induced ototoxicity. Curr Pharm Des.2007; 13(1):119-26. Review
18. Kopke RD,Weisskopf PA,Boone JL,et al.Reduction of noise-induced hearing loss using L-NAC and salicylate in the chinchilla.Hear Res.2000,149(1-2):138-46
19.线粒体DNA突变在感音神经性聋发病中作用的研究。王琼,孔维佳。2002
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6.孔维佳,汪吉宝,钟乃川.豚鼠耳蜗电图及听觉脑干电位同步记录法的探讨.临床耳鼻咽喉科杂志,1989, 3:148-152.
7. Fischel-Ghodsian N. Mithondrial deafness mutation reviewed. Hum Mut.1999,13:261-270
8. Lee CM, Lopez ME, Weindruch R, Aiken JM. Association of age - related mitochondrial abnormalities with skeletal muscle fiber atrophy. Free Radic Biol Med.1998,25:964-972
9. Barrientos A,Casademont J, Cardellach F, Estivill X, Urbano-Marquez A, Nunes V. Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging. Brain Res Mol Brain Res 1997,52:284-289
10. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle,liver,and heart.J Biol Chem.2000,275:3343-3347
11. Janice AN, Elice MB, Timothy CH, Richard S, Richard FB. Development of aQuantitative PCR(TaqMan)Assay for Relative Mitochondrial DNA Copy Number and the Common Mitochondrial DNA Deletion in the Rat. Environ Mol Mutat. 2004, 44:313-320
12. Puigserver, P. et al. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92, 829–839 (1998).
13. J.M. Weitzel, C. Radtke, H.J. Seitz, Two thyroid hormone-mediated gene expression patterns in vivo identified by cDNA expression arrays in rat, Nucleic Acids Res. 29 (2001) 5148– 5155.
14. Wu Z, Puigserver P, Andersson U,et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivatorPGC-1. Cell 98: 115–124, 1999.
15. L.Huo, R.C.Scarpulla. Mitochondrial DNA instability and periimplantationle- thality associated with targeted disruption ofnuclearrespiratory factor 1 in mice, Mol. Cell. Biol. 21 (2001) 644–654.
16. R. Bergeron, J.M. Ren, K.S. Cadman, et al.Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis, Am. J. Physiol: Endocrinol. Metab. 281 (2001),E1340– E1346.
17. Larsson NG,Wang J,Wilhelmsson H,et al. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.Nat Genet,1998;18:231-236
18. Escriva H,Rodriguze-Pena A,Vallejo CG. Expression of mitochondrial genes and of the transcription factors involved in the biogesis of mitochondria Tfam,NRF-1 and NRF-2,in rat liver,testis and brain. Biochimie,1999;965-971
19. Bykhovskaya Y, Estivill X, Taylor K,et al.Candidate locus for a nuclear modifier gene for maternally inherited deafness. Am J Hum Genet. 2000 Jun;66(6):1905-10. Epub 2000 Apr 27
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2.孔维佳,韩月臣,王莹等。维生素E、辅酶Q10对大鼠内耳组织线粒体DNA4834bp缺失突变的预防作用。中华耳鼻咽喉科杂志,2004,39(12),707-711。
3.孔维佳,汪吉宝,钟乃川.豚鼠耳蜗电图及听觉脑干电位同步记录法的探讨.临床耳鼻咽喉科杂志,1989, 3:148-152.
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1. Nagley P, Mackay IR, Baumer A, et al. Mitochondrial DNA mutation associated with aging and degenerative disease, Ann. N. Y. Acad. Sci, 1992, 673:92-102.
2. Moraes CT, DiMauro S, Zeviani M, et al., Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome, N. Engl. J. Med., 1989,320 :1293-1299.
3. Prezant TR, Agapian JV, Bohlman MC, et al. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness, Nat. Genet,1993, 427: 289-294.
4. Fischel-Ghodsian N, Bykhovskaya Y, Taylor K, et al. Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations. Hearing Res,1997,110:147–154.
5. Gadaleta MN, Rainaldi G, Lezza AM, et al. Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rats. Mutat. Res,1992, 275: 181-193.
6. Wei-Jia Kong, Ying Wang, Qing Wang, et al. The relation between D-galactose injection and mitochondrial DNA 4834bp deletion mutation. Exp. Gerontol, 2006,41:628-634.
7.孔维佳,汪吉宝,钟乃川.豚鼠耳蜗电图及听觉脑干电位同步记录法的探讨.临床耳鼻咽喉科杂志,1989, 3(3):148-152.
8.孔维佳,韩月臣,王莹等。维生素E、辅酶Q10对大鼠内耳组织线粒体DNA4834bp缺失突变的预防作用。中华耳鼻咽喉科杂志,2004,39(12),707-711。
9. Van Tuyle GC,Gudikote JP,Hurt VR,et al.M ultiple,large deletion in rat mitochondrial DNA:evidence for a major hot spot.Mutat Res,1996,349:95-107.
10. Rotig A, Bourgeron T, Chretien D, et al.Spectrum of mitochondrial DNA rearrangements in the Pearson marrow-pancreas syndrome, Hum. Mol.Genet.,1995,4 :1327-1330.
11. Seidman MD, Bai U,Khan DJ, et al. Mitochondrial DNA deletions associated with aging and presbyacusis. Arch, Otolaryngol Head Neck Surg,1997,123:1039-1045.
12. Bai U, Seidman MD, Hinojosa R ,et al.Mitochondrial DNA deletions associated with aging and possibly presbycusis: a human archival temporal bone study. Am. J. Otol,1997, 18:449–453.
13. Lortholary O,Tod M,Cohen Y,Petitjean O.Aminoglycosides.Med Clin North Am.1995;79:761-98.
14. Sande MA,Mandell GL.Antimicrobial agent.In:Gilman AG,Rall TW,Nics AS,Taylor,editors.Goodman and Golman’s The Pharmacological Basis of Therapeutics,8th edn.Elmsford,NY:Pergamon Press;1990.P.1098-116.
15. Kidwell DT,Mckeown JW,Grider JS,et al.Acute effects of gentamicin on thick ascending limb function in the rat.European journal of Pharmacology,1994,270,97-103
16. Bates DE. Aminoglycoside ototoxicity. Drugs Today ( Barc ) .2003, 39(4):227-85.Review
17. Selimoglu E. Aminoglycoside-induced ototoxicity. Curr Pharm Des.2007; 13(1):119-26. Review
18. Kopke RD,Weisskopf PA,Boone JL,et al.Reduction of noise-induced hearing loss using L-NAC and salicylate in the chinchilla.Hear Res.2000,149(1-2):138-46
19.线粒体DNA突变在感音神经性聋发病中作用的研究。王琼,孔维佳。2002
1. Clayton, D. A. Replication and transcription of vertebrate mitochondrial DNA. Annu. Rev. Cell. Biol. 1991(7):453-478
2. Dengler R, Wohlfarth K, Zierz S. Muscle fatigue, lactate, and pyruvate in mitochondrial myopathy with progressive external ophthalmoplegia. Muscle Nerve. 1996 Apr;19(4):456-62.
3. Bradfod BL,Bruce MS.Towards a molecular understanding of adaptive thermogenesis. Nature.2000;404(6):652-660
4. Evans MJ, Scarpulla RC. NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells. Genes Dev. 1990 Jun;4(6):1023-34.
5. M.J. Evans, R.C. Scarpulla, NRF-1: a trans-activator of nuclearencoded res- piratory genes in animal cells, Genes Dev. 4 (1990),1023–1034.
6.孔维佳,汪吉宝,钟乃川.豚鼠耳蜗电图及听觉脑干电位同步记录法的探讨.临床耳鼻咽喉科杂志,1989, 3:148-152.
7. Fischel-Ghodsian N. Mithondrial deafness mutation reviewed. Hum Mut.1999,13:261-270
8. Lee CM, Lopez ME, Weindruch R, Aiken JM. Association of age - related mitochondrial abnormalities with skeletal muscle fiber atrophy. Free Radic Biol Med.1998,25:964-972
9. Barrientos A,Casademont J, Cardellach F, Estivill X, Urbano-Marquez A, Nunes V. Reduced steady-state levels of mitochondrial RNA and increased mitochondrial DNA amount in human brain with aging. Brain Res Mol Brain Res 1997,52:284-289
10. Barazzoni R, Short KR, Nair KS. Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle,liver,and heart.J Biol Chem.2000,275:3343-3347
11. Janice AN, Elice MB, Timothy CH, Richard S, Richard FB. Development of aQuantitative PCR(TaqMan)Assay for Relative Mitochondrial DNA Copy Number and the Common Mitochondrial DNA Deletion in the Rat. Environ Mol Mutat. 2004, 44:313-320
12. Puigserver, P. et al. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92, 829–839 (1998).
13. J.M. Weitzel, C. Radtke, H.J. Seitz, Two thyroid hormone-mediated gene expression patterns in vivo identified by cDNA expression arrays in rat, Nucleic Acids Res. 29 (2001) 5148– 5155.
14. Wu Z, Puigserver P, Andersson U,et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivatorPGC-1. Cell 98: 115–124, 1999.
15. L.Huo, R.C.Scarpulla. Mitochondrial DNA instability and periimplantationle- thality associated with targeted disruption ofnuclearrespiratory factor 1 in mice, Mol. Cell. Biol. 21 (2001) 644–654.
16. R. Bergeron, J.M. Ren, K.S. Cadman, et al.Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis, Am. J. Physiol: Endocrinol. Metab. 281 (2001),E1340– E1346.
17. Larsson NG,Wang J,Wilhelmsson H,et al. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.Nat Genet,1998;18:231-236
18. Escriva H,Rodriguze-Pena A,Vallejo CG. Expression of mitochondrial genes and of the transcription factors involved in the biogesis of mitochondria Tfam,NRF-1 and NRF-2,in rat liver,testis and brain. Biochimie,1999;965-971
19. Bykhovskaya Y, Estivill X, Taylor K,et al.Candidate locus for a nuclear modifier gene for maternally inherited deafness. Am J Hum Genet. 2000 Jun;66(6):1905-10. Epub 2000 Apr 27
1. Poyton RO, McEwen JE. Crosstalk between nuclear and mitochondrial genomes. Annu Rev Biochem. 1996;65:563-607.
2. Kung SD, Moscarello MA, Williams JP。Studies with cholroplast and mitochondrial DNA. I. Evidence of sequence homology between chloroplast and nuclear DNA (Broad Bean) and between mitochondrial and nuclear DNA (rat liver). Biophys J. 1972 May;12(5):474-83.
3. DE-XING ZHANG, GODFREY M, HEWITT.Nuclear DNA analyses in genetic studies of populations:practice, problems and prospects. Molecular Ecology 2003; 12,563-584.
4. Hadler HI, Devadas K, Mahalingam R. Selected nuclear LINE elements with mito- chondrial-DNA-like inserts are more plentiful and mobile in tumor than in normal tissue of mouse and rat. J Cell Biochem. 1998 Jan 1;68(1):100-9.
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