大鼠高胆固醇血症与听觉器官线粒体DNA4834缺失的关系

目的　探讨大鼠高胆固醇血症对听觉器官线粒体DNA483 4 缺失以及听力下降的影响。方法　 6 0只大鼠分为实验组 (38只 )和对照组 (2 2只 ) ,实验组大鼠以高胆固醇饲料喂养 ,时间为 6个月 ,建立高胆固醇血症大鼠模型 ;对照组则在相同条件下以普通饲料喂养。通过听性脑干电反应(auditorybrainstemresponses,ABR)检查实验前后ABR阈上升程度。取左侧耳蜗 (实验组 2 1个 ,对照组10个 )和左侧蜗神经核 (实验组 2 7个 ,对照组 13个 ) ,提取DNA ,通过套式聚合酶链反应 (nestpolymerasechainreaction ,nestPCR)扩增检测听觉器官线粒体DNA483 4 (mitochondrialDNA ,mtDNA483 4 )缺失 ,比较两组动物的ABR阈上升程度和听觉器官的mtDNA483 4 缺失率并进行统计分析。结果　①高胆固醇饲料喂养可造成实验组大鼠血清胆固醇水平明显升高 (t检验 ,P <0 0 5 ) ;②两组大鼠随年龄增加ABR阈均上升 ,高胆固醇血症大鼠的ABR阈上升更加明显 ,和对照组的差异具有显著性 (t检验 ,P<0 0 5 ) ;③所有标本都扩增到正常mtDNA编码tRNA和NADH基因 ,实验组听觉器官mtDNA483 4 缺失发生率较对照组明显增高 ,其差异具有显著性 (χ2 检验 ,P <0 0 5 )。结论　长期高胆固醇血症可以加剧大鼠的听力下降 ,听觉器官mtDNA483 4 缺失可能是其作用     

人听觉器官线粒体DNA4977缺失与老年聋的关系

目的　探讨mtDNA4 977缺失与老年聋的关系。方法　采用聚合酶链反应 (polymerasechainreaction ,PCR)及套式PCR技术 ,扩增正常及缺失区mtDNA片段 ,pGEM TEasy载体连接扩增产物 ,重组、克隆目的DNA进行序列分析。检测 6 7耳 (4 1例 )颞骨切片、2 1例蜗核 (双侧 )、2 0例颞叶脑组织、2 0例心肌和 2 2例肝脏组织中mtDNA4 977缺失的发生率 ,根据患者生前的临床资料分为老年组与非老年组、老年聋组与老年听力正常组进行比较分析。结果　被检所有标本均成功扩增到正常mtDNA编码tRNA和ND1片段的NADH基因。在老年人多种组织中检测到了mtDNA4 977缺失 ,老年组不同器官组织中mtDNA4 977缺失发生率均明显高于非老年组 ,两组之间的差异具有显著性意义 (χ2 检验 ,P <0 0 5 )。老年聋患者颞骨切片和蜗核组织中mtDNA4 977缺失的发生率明显高于老年听力正常组 ,其差异具有显著性意义 (χ2 检验 ,P <0 .0 5 ) ;而与听觉无关的心肌和肝脏组织中mtDNA4 977缺失发生率在两组之间差异无显著性。结论　mtDNA4 977缺失的发生与老化有关 ;内耳和蜗核组织中mtDNA4 977缺失与老年聋的发生有关。     

大鼠高胆固醇血症与听觉器官线粒体DNA4834缺失的关系

目的探讨大鼠高胆固醇血症对听觉器官线粒体DNA4834缺失以及听力下降的影响.方法 60只大鼠分为实验组(38只)和对照组(22只),实验组大鼠以高胆固醇饲料喂养,时间为6个月,建立高胆固醇血症大鼠模型;对照组则在相同条件下以普通饲料喂养.通过听性脑干电反应(auditory brainstem responses, ABR)检查实验前后ABR阈上升程度.取左侧耳蜗(实验组21个,对照组10个)和左侧蜗神经核(实验组27个,对照组13个),提取DNA,通过套式聚合酶链反应(nest polymerase chain reaction,nest PCR)扩增检测听觉器官线粒体DNA4834(mitochondrial DNA, mtDNA4834)缺失,比较两组动物的ABR阈上升程度和听觉器官的mtDNA4834缺失率并进行统计分析.结果①高胆固醇饲料喂养可造成实验组大鼠血清胆固醇水平明显升高(t检验,P<0.05);②两组大鼠随年龄增加ABR阈均上升,高胆固醇血症大鼠的ABR阈上升更加明显,和对照组的差异具有显著性(t检验,P<0.05);③所有标本都扩增到正常mtDNA编码tRNA和NADH基因,实验组听觉器官mtDNA4834缺失发生率较对照组明显增高,其差异具有显著性(χ2检验,P<0.05).结论长期高胆固醇血症可以加剧大鼠的听力下降,听觉器官mtDNA4834缺失可能是其作用机制之一.     

实时荧光定量Taqman探针法检测线粒体基因1555 A》G突变

目的 建立以实时荧光定量Taqman探针技术检测线粒体DNA 1555A>G突变的方法,实现更快速、简便、准确筛查这一突变的目标.方法 设计针对线粒体DNA 1555A>G突变的Taqman探针和引物,摸索建立稳定的检测方法,同时进行可靠性验证.从解放军总医院聋病分子诊断中心耳聋DNA库选取标本132例,遵循双盲法原则,分别以实时荧光定量Taqman普通探针法、试剂盒法和直接测序法检测线粒体DNA 1555A>G突变,将三种方法 检测的结果 进行比对.结果 132例耳聋病例经实时荧光定量Taqman普通探针法检测发现线粒体DNA 1555A>G突变阳性患者32例,阴性100例,与试剂盒法和直接测序法检测结果 完全相符,未发现假阳性和假阴性.结论 实时荧光定量Taqman探针技术检测线粒体DNA 1555A>G突变的方法 具有检测结果 准确直观、简单省时(反应时间由原来的最快6 h缩短到1.5 h),特异性强,敏感性高的优点,适用于对母系遗传性耳聋线粒体DNA 1555A>G突变的大规模筛查或氨基糖甙类抗生素应用前预防性检测.     

母系遗传性聋大家系听力学调查及线粒体DNA突变分析

目的　探讨母系遗传性非综合征性耳聋的听力学特征及分子遗传学机制。方法　选择一遗传聋大家系中 4代 41人为研究对象 ,详细询问病史和全身体格检查 ,行系统听力学测试和线粒体DNA(mtDNA)A15 5 5G突变分析。结果　 2 0例母系亲属均存在A15 5 5G点突变 ;纯音测听 17例呈程度不等的感音神经性聋 ,双耳对称 ,发病年龄 1～ 5 0岁 ,5例听力近 11年间呈进行性下降 ;声导抗测试均为A型鼓室曲线 ,12耳可在低感觉级引出镫骨肌反射 ;听性脑干反应 (auditorybrainstemresponse,ABR)和瞬态诱发耳声发射测试无蜗后病变证据。 2 1名父系亲属及配偶无听力障碍 ,mtDNAA15 5 5G突变检测阴性。结论　本家系以迟发性进行性耳蜗性聋为特点 ,导致听力损失的内在因素为mtDNAA15 5 5G突变 ,而环境因素和核基因可能也参与了突变体mtDNA表型表达的调节。     

药物性聋和噪声性聋后听觉中枢可塑性的研究进展

传统的研究方法经常将外周听器和中枢听觉系统分别进行研究 ,在观察内耳损伤的同时往往没有注意到听觉中枢各级神经元核团的变化。然而 ,近年的研究表明 ,当外周听器被噪声或耳毒性药物损伤后 ,在内耳的结构和功能发生变化的同时 ,听觉中枢以一种意料不到的方式改变了其结构和功能 ,即发生了可塑性 (Plasticity)的改变或重组 (re organization)。不同于外周听器的功能下降 ,其表现为自发活动的增强〔1〕,并可产生耳鸣、听觉过敏和响度重振等症状。毋庸置疑 ,外周听器和中枢听觉系统是一有机的整体 ,两者应该是相互影响的。本文试图将药物性…     

职业性噪声性聋与mtDNA缺失关系的探讨

目的 探讨线粒体DNA(mitochondrial DNA,mtDNA)缺失在噪声性聋发病中的可能作用.方法 在265名从事噪声作业的青年工人中,根据纯音听阈测试结果,分为语频听力损失组和语频听力正常组,其中自愿进入研究的语频听力损失组(A组)25人,双耳言语频率(500、1 000、2 000 Hz)听阈均值≥26 dB HL;在语频听力正常人群中随机抽取27人作为语频听力正常组(B组),两组分别抽取外周血,提取白细胞DNA,应用PCR方法检测mtDNA4977缺失情况.结果 A组mtDNA4977缺失检出率20%(5/25),B组mtDNA4977缺失检出率为0(0/27),A组mtDNA4977缺失检出率明显高于B组(P＜0.05).结论 mtDNA4977缺失可能在噪声性聋的发病中起重要作用,     

PCR技术对大鼠内耳膜迷路线粒体DNA的检测

目的：建立灵敏、可靠的大鼠内耳膜迷路线粒体ＤＮＡ提取和检测方法。方法：结合ＰＣＲ技术扩增ｍｔＤＮＡ编码ＮＤ１－１６ＳｒＲＮＡ基因的６０１ｂｐ片段,检测大听泡内耳膜迷路线粒体ＤＮＡ方法,并对两种ｍｔＤＮＡ获取方法进行比较。结果：采用Ｓｅｉｄｍａｎ的方法,将１只大鼠的以侧听泡内耳膜迷路作为一个样品,检测１０个样品均成功扩增出编码ＮＤ１－１６ＳｒＲＮＡ基因的６０１ｂｐ片段;而采用ＥｄｒｉｓｍｔＤＮＡ提取     

人类颞骨火棉胶切片中线粒体DNA的扩增及重组测序

目的应用分子生物学技术建立人类颞骨火棉胶切片中的ＤＮＡ分析方法。方法采用多聚酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）配合不同的引物、扩增方式及酶切技术检测长期保存的７例（９侧）颞骨火棉胶切片中微量线粒体ＤＮＡ的片段缺失及点突变，ｐＧＥＭＴ载体进行扩增片段的重组与克隆。结果９侧颞骨ＤＮＡ提取液中均可见正常的１３５ｂｐ扩增片段，巢式ＰＣＲ检出其中２例生前患老年聋者（各查１侧）有线粒体ＤＮＡ大片段缺失，测序结果证实扩增的准确性。结论分子生物学技术在颞骨切片研究中的应用对于提高其回顾性研究水平有重要意义，目前可在耳科疾病的研究中发现相关的基因突变或病原体。     

水杨酸钠对噪声性听力损失影响的实验观察

目的　观察水杨酸钠能否减轻噪声引起的听力损失。方法　将 3 6只健康且耳廓反射正常的花色豚鼠随机分为水杨酸钠实验组、生理盐水对照组、水杨酸钠对照组和噪声暴露组。噪声暴露采用 10 5dBSPL的 4kHz窄带噪声下暴露 2h ,连续 5d。水杨酸钠给药为每天 0 5g/kg体重连续10d。由短声诱发听性脑干反应 (auditorybrainstemresponse ,ABR) ,连续测试其阈值 ;而后取动物双侧耳蜗荧光染色后光镜下行毛细胞计数和形态学观察。结果　ABR阈值测试显示 ,实验组动物在噪声暴露结束后 2 4h的听力略好于对照组 ;形态学观察表明 ,实验组细胞核异常数据低于对照组。结论　水杨酸钠可能在一定程度上具有拮抗噪声引起的听力损失及保护耳蜗毛细胞的作用     

碱基淬灭探针技术单管检测线粒体DNA 12S rRNA A1555G及C1494T突变

目的 应用碱基淬灭探针技术建立单管同时检测线粒体DNA 12S rRNA A1555G和C1494T突变的方法.方法 探针的一端标记6-羧基荧光素,同时构建4个载体做为扩增模板,分别代表可能的4种基因型组合.对117例耳聋患者外周血标本进行线粒体DNA 12S rRNA A1555G和C1494T突变的检测,同时选取15例样本进行DNA测序,验证所建立方法的可靠性和准确性.结果 根据熔解曲线可以清晰地对线粒体DNA 12S rRNA 1555位点和1494位点的基因型做出判断.117例耳聋患者中3例携带A1555G突变(2.56％),1494位点未检出突变;基于碱基淬灭探针技术的单管检测方法与DNA测序的结果一致.结论 碱基淬灭探针单管检测技术可用于临床线粒体DNA突变耳聋基因检测.     

Construction of a DNA Chip for Screening of Genetic Hearing Loss

Objectives

Hearing loss is the most common sensory disorder in humans and genetic causes are estimated to cause more than 50% of all incidents of congenital hearing loss. To develop an efficient method for a genetic diagnosis of hearing loss, we have developed and validated a genetic hearing loss DNA chip that allows the simultaneous analysis of 7 different mutations in the GJB2, SLC26A4, and the mtDNA 12S rRNA genes in Koreans.

Methods

A genotyping microarray, based on the allele-specific primer extension (ASPE) method, was used and preliminary validation was examined from the five patients and five controls that were already known their genotypes by DNA sequencing analysis.

Results

The cutoff Genotyping index (GI) of genotyping for each mutation was set up and validated to discriminate among the genotypes. The result of the DNA chip assay was identical to those of previous results.

Conclusion

We successfully designed the genetic hearing loss DNA chip for the first time in Korea and it would be useful for a clinical genetic diagnosis of hearing loss. Further consideration will be needed in order to examine the accuracy of this DNA chip with much larger patient sample numbers.     

氨基糖甙类抗生素致聋家系线粒体DNA1555G点突变分析

目的考察１５５５Ｇ点突变与氨基糖甙类抗生素致聋的对应关系，建立相应的基因诊断方法提供依据。方法收集了３个有明确氨基糖甙类抗生素应用史的母系遗传耳聋家系１３人（包括聋人和听力正常者）的外周静脉血标本，聚合酶链反应扩增线粒体ＤＮＡ，Ａｌｗ２６Ｉ限制性内切酶分析、ＤＮＡ斑点杂交和ＤＮＡ序列分析检测１５５５Ｇ点突变。结果家系１和３的７份样品均为１５５５Ｇ点突变阳性，家系２的６份样品为１５５５Ｇ点突变阴性。结论发现１个１５５５Ｇ点突变阴性的氨基糖甙类抗生素致聋家系，说明线粒体ＤＮＡ１５５５Ｇ点突变不是氨基糖甙类抗生素遗传易感性唯一的分子基础。     

Mutations in the mitochondrial 12S rRNA gene in elderly Chinese people

Conclusion: Our data indicate that the mitochondrial 12S rRNA gene, and particularly the A827G mutation, may be associated with susceptibility to age-related hearing loss. Objective: Hearing loss associated with aging is common among elderly persons. In all genetic backgrounds, mitochondrial DNA (mtDNA) mutations may be one of the most important factors contributing to aging and age-related hearing loss. The mitochondrial 12S rRNA is a hot spot for deafness-associated mutations in Chinese populations. The purpose of the present study was to elucidate the relationship of 12S rRNA gene polymorphisms and age-related hearing loss. Methods: The 12S rRNA gene polymorphisms were detected by direct sequencing. Statistical analyses were performed to assess the associations between age-related hearing loss and 12S rRNA gene variants. Results: We report here a systematic mutational screening of the mitochondrial 12S rRNA gene in 662 elderly subjects from the general population with various hearing threshold levels (211 controls and 451 age-related hearing loss subjects). Mutational screening of the mitochondrial 12S rRNA gene identified 55 nucleotide changes, including 4 mutations localized at highly conserved sites and 51 known variants. Of the known deafness-associated mutations in the mitochondrial 12S rRNA gene, the incidence of the A1555G mutation was 0.15%, A827G was 4.38%, T1095C was 0.45%, and T1005C was 3.78%. The incidence of the other known variants was 0.15–99.85%. We found statistically significant differences in the proportions of subjects with the A827G mutation among the various age-related hearing loss groups and normal controls.     

GJB2 and mitochondrial DNA 1555A>G mutations in students with hearing loss in the Hubei Province of China

Objectives

The GJB2 and MTRNR1 1555A > G mutations are the prevalent causes of hearing loss worldwide. However, the mutation profiles of the two genes are dependent on the ethnic or geographic origins. Therefore, this study was to characterize the forms and frequencies of the two genes in 813 students with hearing loss in Hubei province, Central China.

Methods

Blood samples from 813 students were obtained with informed consent. Genomic DNA was extracted from peripheral blood leukocytes. The target fragments were amplified by polymerase chain reaction (PCR). Sequencing (or enzyme digestion) was applied to identify sequence variations.

Results

Ten different mutations were identified in GJB2 in 146 of the 813 (17.96%) patients and 11.81% (96/813) patients had homoplasmic mtDNA 1555A > G mutation.

Conclusions

This study demonstrated the high prevalence of GJB2 and mtDNA 1555A > G mutations in Central Chinese population. Therefore, it will be effective to perform GJB2 and mtDNA 1555A > G mutation analysis for genetic screening for hearing loss in this population.