豚鼠老化模型听反应阈检测及基因多态性标记分析

目的 建立D-半乳糖致豚鼠老化模型,应用扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分析检测与听力损失相关的分子生物学标记,寻求老年性聋发病的分子机制.方法 51只豚鼠随机分为三组:A组(模型实验组)21只,5%D-半乳糖腹腔注射(200 mg·kd-1·d-1),共6周;B组(模型对照组)15只,仅给予生理盐水注射.A、B两组注射前后分别进行听性脑干反应(ABR)检测,将两组豚鼠置于噪声环境中7 d,每天噪声暴露8 h,结束后再次检测ABR阈值.C组(空白对照组)15只,不加任何处理,仅予以同步检测ABR阈值.用比色法检测三组肝和脑组织的超氧化物歧化酶(superoxide dismutase,SOD)及丙二醛(maleic dialdehyde,MDA)含量.提取三组豚鼠内耳组织DNA,应用AFLP技术筛选差异位点.结果 注射后A组豚鼠ABR阈值较B组提高,但两组阈移之间差异无统计学意义(t=1.14,P>0.05),噪声暴露后,A组ABR阈值(峰等效声压级)平均提高(22.97±10.56)dB,B组提高(14.16±7.36)dB,组间差异具有统计学意义(t=2.78,P<0.05).A组肝和脑组织中SOD活性明显低于B组,MDA含最则明显高于B组,其差异具有统计学意义(P值均<0.01).AFLP分析发现有与耳聋一致的多态性标记.结论 D-半乳糖可以诱导豚鼠衰老,此模型豚鼠听反应阈虽无明显提高,但对噪声的敏感性增加,AFLP检测到的差异位点可能与其对噪声敏感有关.

Detection of hearing threshold and polymorphic molecular marker analysis of guinea pigs of mimetic aging

Objective To explore the establishment of the mimetic aging effect in guinea pigs induced by D-galactose, and to detect the biological indicatrix associated with hearing loss and provide a new tool for molecular pathogenesis of hearing loss.Methods Total of 51 guinea pigs were randomly divided into three groups: group A ( model aging group , n = 25) , which were injected with D-galactose (200 mg ·kg-1·d-1 ) by intra peritoneum for 6 weeks, group B ( model control group , n = 18), which were given the same amount of saline only, and group C(vacant group, n = 15) were not treated.Then, The guinea pigs in group A and B were exposed in noise for 8 days, 8 hours once a day.Auditory brainstem response (ABR) was used to test the hearing threshold of guinea pigs thrice, first before the drug administered, then after 6 weeks the drug used, third after noise exposure.And colorimetry was used to analyze the activity of superoxide dismutase (SOD) and malon dialdehyde (MDA) in brain and liver tissue.The DNA of inner ear tissue was harvested and amplified fragment length polymorphism (AFLP) was used to detect the differential polymorphic markers.Results After injection, there was no significant difference in elevation of ABR threshold between the group A and group B ( t = 1.14, P>0.05 ).However, exposure of noise later, elevation in ABR threshold of ( 22.97±10.56) dB PeSPL was observed in group A, and ( 14.16±7.36) dB peSPL in group B.The was significant difference in variation of hearing threshold between group A and group B ( t = 2.78 ,P<0.05 ).The activity of SOD in brain and liver tissue in group A was lower than that in group B.the level of MDA was opposite between group A and group B.The difference between group A and group B was significant ( P<0.01 ) .A differential polymorphic marker was observed by AFLP. Conclusions The mimetic aging effect of the guinea pigs can be induced by D-galactose, and this model can not directly induce the hearing loss.The differential polymorphic marker possibly act as a predisposing factor which can greatly enhance the sensitivity of the ear to the noise.