【作者】 王斌；

【导师】 迟放鲁； 高文元； 朱晓燕；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 噪声性聋及药物性聋是常见的感音神经性聋,严重影响人们的生活质量。长期以来,对声损伤和庆大霉素耳毒性的防治一直是广大耳科工作者致力研究的课题。地塞米松作为合成的糖皮质激素,具有多种生理功能。已有研究发现,地塞米松能减小噪声及氨基甙类药物的耳毒性,但对这一现象机制的研究少有报道。地塞米松作为糖皮质激素,通过基因组途径及非基因组途径发挥作用。在基因组途径中,地塞米松通过与位于胞浆的糖皮质激素受体（Glucocorticoidreceptor,GR）结合,进而影响某些基因的转录与表达,因此了解GR在耳蜗的的分布很有必要。既往的研究应用酶联免疫吸附试验（ELISA）、原位杂交、免疫组织化学方法研究糖皮质激素在内耳的分布,而应用直观的免疫荧光技术分析GR在耳蜗的分布及声损伤对GR表达的影响还未见报道。Notch信号系统几乎涉及所有细胞的增殖、分化活动,Hes1（hairy andenhancer of split 1）为Notch的基本效应因子。Hes1 mRNA表达水平增高提示Notch信号活化,导致CDK（Cyclin-dependent kinases,周期性蛋白依赖激酶）抑制物p21^{Wafl/ Cip}的聚集,而后者与细胞凋亡密切相关。噪声、地塞米松与hes1mRNA表达的关系可能是地塞米松拮抗声损伤的机制之一,对这一现象未见相关报道。地塞米松拮抗氨基甙类抗生素耳毒性的在体研究已有报道,离体实验避免了全身因素的干扰和影响,使研究途径更加便捷可靠。观察地塞米松是否拮抗庆大霉素对体外培养的Corti器的耳毒性,尚未见报道。抑制外毛细胞Ca²⁺敏感的外向K⁺电流可能是氨基甙类抗生素耳毒性的机制之一,地塞米松是否通过非基因组途径影响该电流,从而拮抗庆大霉素耳毒性,尚未见报道。因此,本研究的主要目的是探讨地塞米松对声损伤及庆大霉素耳毒性的影响及相关机制。以免疫荧光技术定位,荧光强度半定量分析为指标,探讨了糖皮质激素受体在豚鼠耳蜗的分布,声损伤后对其分布及各区域表达强度的影响。以听觉脑干诱发电位（ABR）,耳蜗形态学为指标探讨地塞米松减轻耳蜗声损伤的作用;用RT-PCR及荧光实时定量PCR技术,探讨噪声及地塞米松对豚鼠耳蜗hes1mRNA表达水平的影响。以免疫荧光技术定位,耳蜗形态学毛细胞计数为指标,探讨地塞米松减轻庆大霉素对离体培养的耳蜗Corti器的毒性作用。以听觉脑干诱发电位（ABR）为指标探讨地塞米松减轻庆大霉素耳毒性的作用;应用全细胞膜片钳记录技术探讨庆大霉素耳毒性及地塞米松拮抗庆大霉素耳毒性的离子通道机制。第一部分地塞米松拮抗内耳声损伤的实验研究一、糖皮质激素受体在豚鼠耳蜗的分布及声损伤对其表达的影响目的:研究GR在豚鼠耳蜗的分布及声损伤对豚鼠耳蜗GR表达的影响。方法:豚鼠随机分为二组:实验组予噪声暴露,噪声为白噪声,强度115dBSPL,暴露时间3h。暴露结束后2h断头剥取耳蜗。正常对照组不做处理,与实验组同时剥取耳蜗。制备耳蜗冰冻切片,免疫荧光技术观察豚鼠耳蜗GR表达情况,并进行荧光强度半定量分析。结果:（1）豚鼠耳蜗GR主要的阳性部位在螺旋韧带、血管纹、骨螺旋唇、Corti器以及螺旋神经节。螺旋韧带、血管纹、骨螺旋唇以及螺旋神经节等区域GR荧光强度无明显统计学意义（P＞0.05）,而上述各区域与Corti器的GR荧光强度相比,差异有显著意义（P＜0.05）。（2）声损伤后豚鼠耳蜗GR荧光强度在骨螺旋唇、corti器以及螺旋神经节较正常对照组无统计学意义（P＞0.05）,而螺旋韧带、血管纹区域荧光强度较正常对照组明显减低,有显著统计学意义（P＜0.01）。结论:GR在豚鼠耳蜗分布于螺旋韧带、血管纹、corti器和螺旋神经节,其中螺旋韧带、血管纹区域表达最强,corti器区域表达最弱;声损伤降低耳蜗各区域GR的表达,以血管纹、螺旋韧带最显著。二、地塞米松拮抗耳蜗声损伤及对豚鼠耳蜗hes1表达水平的影响目的:探讨地塞米松拮抗声损伤及与耳蜗hes1 mRNA的表达的关系。方法:在预实验的基础上,豚鼠随机分为5组,空白对照组,NS ip qd+植物油im qd;噪声组（N）,NS ip qd+植物油im qd+噪声暴露;地塞米松+噪声组（Dex+N）,Dex ip qd+植物油im qd+噪声暴露;RU486+噪声组（RU+N）,NS ip qd+RU486 im qd+噪声暴露;地塞米松+RU486+噪声组（Dex+RU+N）,Dex ip qd+RU486 im qd+噪声暴露。其中地塞米松剂量1mg/kg,浓度0.5mg/ml;RU486剂量20mg/kg,浓度25mg/ml。药物作用时间均为5d。噪声暴露于白噪声3小时,强度115dB SPL。噪声暴露结束即刻处死。取下颞骨,冰盒中取出耳蜗,用荧光实时定量PCR（real-time-PCR）检测耳蜗组织hes1 mRNA的表达。同样分组条件下,在噪声暴露前、噪声暴露结束后24小时测定Click和短纯音（2、4、6、8kHz）ABR反应阈值,空白对照组在实验开始和结束各测定一次ABR。各组动物进行耳蜗NBT染色及基底膜铺片。结果:（1）白噪声115dB SPL,暴露3h,促进豚鼠hes1 mRNA表达增加约3倍;地塞米松预处理5d,可以使hes1 mRNA表达水平接近于对照组。Dex-RU486-noise组hes1 mRNA表达水平接近于noise组,说明RU486基本上阻断了地塞米松下调豚鼠耳蜗hes1 mRNA表达水平的作用。（2）各组ABR平均阈移:空白对照组阈移为0.5～1.40dB;噪声组click阈移为29.56dB,短纯音（2、4、6、8kHz）阈移为32.75～34.78dB;地塞米松+噪声组click阈移为11.46dB,短纯音（2、4、6、8kHz）阈移为12.75～15.85dB;RU486+噪声组click阈移为26.93dB,短纯音（2、4、6、8kHz）阈移为30.15～33.26dB;地塞米松+RU486+噪声组click平均阈移为27.38dB,短纯音2、4、6、8kHz阈移为30.26～33.52dB。噪声组、地塞米松+RU486+噪声组分别与地塞米松+噪声组相比,全频的ABR阈移均有显著性差异（p＜0.05）。（3）耳蜗基底膜NBT染色显示噪声组底回损伤较重,地塞米松+噪声组损伤轻微。结论:地塞米松对耳蜗声损伤具有拮抗作用;下调耳蜗hes1 mRNA的表达水平,减少细胞凋亡,可能是地塞米松对声损伤发挥保护作用的机制之一。第二部分地塞米松拮抗庆大霉素耳毒性的实验研究一、地塞米松拮抗庆大霉素耳毒性作用的离体实验研究目的:采用Corti器体外培养的方法,观察地塞米松是否对庆大霉素的耳蜗毒性具有拮抗作用。方法:成功建立耳蜗Corti器体外培养系统后,选取出生3d的SD大鼠,随机分为3组,正常对照（Control）组,进行Corti器体外培养,72h后中止培养;庆大霉素（GM）组,培养48h后加入庆大霉素液（浓度0.5mmol/L）,作用24h后中止培养;地塞米松+庆大霉素（Dex+GM）组,培养24h后加入地塞米松溶液（浓度100ng/mL）,培养48h后加入庆大霉素液（浓度0.5mmol/L）,培养72h后中止培养。各组中止培养后,Rhodamine-phalloidin染色,免疫荧光技术观察Corti器毛细胞生长状况。结果:正常对照（Control）组毛细胞无明显缺失;庆大霉素（GM）组外毛细胞有明显缺失,而内毛细胞基本无明显改变,三排外毛细胞中以内两排损害较重;地塞米松+庆大霉素（Dex+GM）组可见外毛细胞的缺失程度明显减轻,内毛细胞基本无明显改变。结论:地塞米松预处理对离体耳蜗Corti器的庆大霉素耳毒性具有拮抗作用。二、地塞米松拮抗庆大霉素耳毒性作用的电生理学研究目的:探讨地塞米松拮抗庆大霉素耳毒性的离子通道机制。方法:（1）豚鼠随机分为6组,生理盐水（control）组,生理盐水0.5ml,ip;庆大霉素（GM）组,庆大霉素80mg/kg,im;地塞米松低浓度（Dex-1）组,地塞米松0.2mg/kg ip;地塞米松高浓度（Dex-h）组,地塞米松1mg/kg ip;地塞米松低浓度+庆大霉素（Dex-1+GM）组,地塞米松0.2mg/kg ip,庆大霉素80mg/kg,im;地塞米松高浓度+庆大霉素（Dex-h+GM）组,地塞米松1mg/kg ip,庆大霉素80mg/kg,im。给药前及给药结束后,测定ABR反应阈,计算ABR阈移。（2）全细胞膜片钳技术记录单离外毛细胞钙敏感外向钾电流。分别观察0.1、1、10、100、1000umol/L庆大霉素对钙敏感外向钾电流的影响;全细胞膜片钳技术记录单离外毛细胞钙敏感外向钾电流。分别观察0.1、1、10、100、1000umol/L地塞米松对钙敏感外向钾电流的影响。结果:（1）生理盐水（control）组、Dex-1组和Dex-h的ABR平均阈移很小,为1.0～1.6dB;GM组click平均阈移为10.6dB,短纯音在5.0～21.0dB之间,并以6kHz及8kHz的阈移最为明显;Dex-1+GM组Dex-h+GM组click平均阈移为5.8～7.2dB,短纯音在2.5～5.2dB之间,与GM组相比有显著性差异（p＜0.05）。（2）庆大霉素以浓度依赖性方式抑制外毛细胞Ca²⁺敏感的外向K⁺电流;地塞米松以浓度依赖方式增大外毛细胞Ca²⁺敏感的外向K⁺电流。结论:地塞米松能够显著降低庆大霉素对豚鼠的耳毒性,并且对豚鼠听功能没有明显影响;庆大霉素抑制外毛细胞Ca²⁺敏感的外向K⁺电流,可能是急性耳中毒的机制之一;地塞米松拮抗庆大霉素耳毒性的作用可能是通过激活外毛细胞Ca²⁺敏感的外向K⁺通道的非基因组效应实现的。更多还原

【Abstract】 Noise and drugs induced heating loss as neurosensory deafness influence our quality of life severely.It is a long-term topic for many otologists to devote themrselves to investigate the prevention and cure of acoustic trauma and ototoxicity of gentamicin.Dexamethasone,as a synthetical glucocorticoid,has lots of physiological functions.It is reported that dexamethasone attenuates ototoxicity of noise and aminoglycosides drugs,however,its mechanisms are known little.Dexamethasone,as a kind of glucocorticoid,produces a marked effect through binding with glucocorticoid receptors located in the kytoplasm.It is essential to comprehend the distribution of glucocorticoid receptor（GR） in cochlea.Investigative methods including ELISA,in-situ hybridization,immunohistochernistry in previous studies.Few literatures can be fiound on the distribution of GR in cochlea and effects due to the acoustic trauma.Notch signaling is involved in proliferation differentiation of almost all cells.Hes1（hairy and enhancer of split 1） is its effective agent.The elevation of Hes1 mRNA level suggests the activation of Notch signaling,leading to gathering of CDKI p21^Wafl/Cip,which is closely related to the cell apoptosis.The relationship among the acoustic trauma,dexamethasone and Hes1 mRNA level can be one of mechanisms of dexamethasone antagonizing the acoustic trauma.However,little is known about it.It is reported dexamethasone antagonizes AmAn ototoxicity in vivo.Experiments in vitro avoid the systemic interferences,which make the investigative route more convenient and reliable.However,few literatures can be found on whether dexamethasone antagonizes ototoxicity of gentamicin in vitro.One of possible mechanisms for AmAn ototoxicity is the inhibition of calcium-sensitive outward potassium current of the outer hair cells.Few literatures can be found on whether the ion channel mechanism is involved in dexamethasone antagoning ototoxicity of gentamicin.The present study is designsd to explore the effects of dexamethasone on the acoustic trauma and the ototoxicity of gentamicin,and the possible underlying mechanisms.Immunofluorescent technique fixing,and fluorescence intensity semiquantitative analysis are detected for studying the distribution of GR in cochlea of guinea pig and the effects due to the acoustic trauma.Auditory brainstem response（ABR） and changes in cochlear morphology are detected for studying the effect of dexamethasone attenuate the acoustic trauma in cochlea.RT-PCR and real time PCR technique are used to discuss the effects of the acoustic trauma and dexamethasone on hes1 mRNA level in cochlea of guinea pig.Immunofluorescent technique fixing and changes in cochlear morphology are detected for studying the effect of dexamethasone attenuates the ototoxicity of gentamicin in vitro.Auditory brainstem response（ABR） is detected for studying the effect of dexamethasone attenuating the ototoxicity of gentamicin in vivo.The Whole cell patch clamp technique is utilized to investigate ion channel mechanism of ototoxicity of gentamicin and dexamethasone antagoning it.PartⅠThe Experimental Investigation on Dexamethasone Antagonizes the Acoustic Trauma1.The distribution of GR in cochlea of guinea pig and effects due to the acoustic trauma.Purpose To explore the distribution of GR in cochlea of guinea pig and effects due to the acoustic trauma.Methods White guinea pigs are randomly divided into 2 groups:experimental group and control group.Noise exposure（white noise,115dB SPL,3h） is exerted in experimental group.Decapitation and cochlear separation are done after 2h from the noise exposure termination.Empty treatments are done in the control group. Then,frozen sections are prepare.Immunofluorescent technique fixing,and fluorescence intensity semiquantitative analysis are detected for studying the distribution of GR in cochlea of guinea pig and the effects due to the acoustic trauma.Results（1） The fundamental positive positions of GR are located in spiral ligament,stria vascularis,spiral limbus,organ of Corti and spiral ganglions.For fluorescence intensity of GR,there are no significant difference in spiral ligament, stria vascularis,spiral limbus iand spiral ganglions（P＞0.05）.Hower,there are significant difference between these positions and organ of Corti（P＜0.05）.（2） The fluorescence intensities of GR located in spiral limbus,organ of Corti and spiral ganglions are similar in noise exposure group and control group（P＞0.05）.The fluorescence intensities of GR in spiral ligament,stria vascularis are much lower in noise exposure group（P＜0.01）.Conclusion The distribution of GR in cochlea of guinea pig are located in spiral ligament,stria vascularis,spiral limbus,organ of Corti and spiral ganglions.The highest and least position are lateral wall and organ of Corti,respectively.Acoustic trauma can decrease GR expression in all positions of cochlea,particularly in spiral ligament and stria vascularis.2.Dexamethasone attenuates the acoustic trauma via down-regulating cochlear hes1 mRNA levelPurpose To explore that dexamethasone attenuates the acoustic trauma and the relationship with hes1 mRNA level in cochlea.Methods Based on the preliminary experiment,guinea pigs are randomly divided into 5 groups:control group（NS ip qd,vegetable oil im qd）,noise group（NS ip qd,vegetable oil im qd and noise exposure）,dex+noise group（Dex ip qd,vegetable oil im qd and noise exposure）,RU486+noise group（NS ip qd,RU486 im qd and noise exposure）,dex+RU486+noise group（Dex ip qd,RU486 im qd and noise exposure）. The dose and concentration of dexamethasone are 1mg/kg,0.5mg/ml,and that of RU486 are 20mg/kg,25mg/ml.All drugs are administered once a day for consecutive 5d.Animals are exposed to white noise（115dB SPL,3h）.Immediately after the animals are sactificed,the cochlea are removed and made into tissue homogenate.The hes1 mRNA expression in cochlea is determined by quantitive realtime-PCR.Hearing thresholds of auditory brainstem responses（ABR_S） of experimental animals are measured before and 24h after noise exposure.For control group,ABR_s are measured in initiation and end of experiment.The NBT staining and basal membrane stretched preparation are practiced in all groups.Results（1） White noise（115dB SPL,3h） makes the cochlear hes1 mRNA level to 3 times compared with the control group.Dexamethasone pretreatment for 5d makes the cochlear hes1 mRNA level close to that of control group.The cochlear hes1 mRNA level of dex+RU486+noise group is similar with that of noise group,which suggest that RU486 almost blocks the effect that dexamethasone down-regulates cochlear hes1 mRNA level.（2） The mean threshold shifts of ABR of control group,noise group,dex+noise group,RU486+noise group and dex+RU486+noise group are 0.5 dB,29.56dB,11.46dB,26.93 dB,27.38dB after click and 0.5～1.40dB,32.75～34.78dB,12.75～15.85dB,30.15～33.26dB 30.26～33.52dB after short tone,respectively.There are significant differences for the mean threshold shifts of ABR in noise group,dex+RU486+noise group compared with those of dex+noise group（p＜0.05）.（3） The cochlear surface preparation showed that the damage in noise group is severe but very slight in dex+noise group.Conclusion Dexamethasone has a potential protective role against the acoustic trauma possibly by down-regulating cochlear hes1 mRNA level.PartⅡThe Experimental Investigation on Dexamethasone Antagonizes the Ototoxicity of Gentamicin1.Dexamethasone attenuates the gentamycin-induced ototoxicity in vitroPurpose To explore whether dexamethasone attenuates the gentamycin-induced ototoxicity in vitro.Methods Based on successful establishment of organ Corti’s organ cultural system,the p3 SD rat are randomly divided into 3 groups:Control group,GM group and Dex+GM group.Corti’s organs are cultured for 72h.Rhodamine-phalloidin staining and immunofluorescent technique are used for observing growing status of Corti’s organs.Results No significant hair calls deletion is found in control group.Significant OHC deletion is found in GM group,while no obvious change in IHC.OHC deletion degree is better in Dex+GM group,while no obvious change in IHC.Conclusion Pretreatment of dexamethasone attenuates the gentamycin-induced ototoxicity in vitro.2.Electrophysiologic studies for flexamethasone attenuates the gentamycin-induced ototoxicityPurpose To explore ion channel mechanism of dexamethasone attenuates the gentamycin-induced ototoxicity.Methods（1）Guinea pigs are randomly divided into 6 groups:control group（NS 0.5ml,ip）,GM group（GM 80mg/kg,im）,Dex-1 group（Dex 0.2mg/kg ip）,Dex-h group（Dex 1mg/kg ip）,Dex-l+GM group（Dex 0.2mg/kg ip,GM 80mg/kg,im） and Dex-h+GM group（Dex 1mg/kg ip,GM 80mg/kg,im）.Before and after all drugs are adminietersd,ABRs is measured,and the threshold shifts are calculated afterward.（2） The whole-cell patch clamp techniques are used to study the effects of gentamycin（0.1,1,10,100,1000umol/L） and dexamethasone（0.1,1,10,100, 1000umol/L） on calcium-sensitive outward potassium currents in the isolated outer hair cells of guinea pig cochlea.Results（1）The mean threshold shifts of ABR are 1.0-1.6dB in control group,Dex-l group and Dex-h group,10.6dB of click and 5.0-21.0dB of tone in GM group with the most prominent at 6,8KHz stimuli.For Dex-l+GM and Dex-h+GM groups,the mean threshold shifts are 5.8-7.2dB after click,2.5-5.2dB after short tone with the values of click and 6、8KHz significantly lower than those in GM group （p＜0.05）.（2） Gentamycin reduces calcium-sensitive outward potassium currents in a concentration dependent manner,while dexamethasone increases it as same manner as gentamycin.Conclusion Dexamethasone can remarkably attenuates the gentamycin-induced ototoxicity,and has no obvious effect on auditory function.Gentamycin reduces calcium-sensitive outward potassium currents in OHC,which suggests that is one of the mechanisms of acute ototoxicity.The possible mechanism of Dexamethasone antagonizing the ototoxicity of gentamicin is increased calcium-sensitive outward potassium currents in OHC.更多还原