【作者】 臧洪瑞；

【导师】 姜学钧；

【作者基本信息】 中国医科大学 ， 耳鼻咽喉学， 2004， 硕士

【摘要】 前言 JNK是丝裂原活化蛋白激酶系统的重要成员，是细胞内信号传导的重要通路，与细胞凋亡有密切关系。氨基糖苷类药物的耳毒性已经公认，已有研究表明氨基糖苷类药物可在耳蜗通过凋亡诱导毛细胞死亡。丁胺卡那霉素作为一种新型的半合成广谱抗生素，疗效显著，但听觉障碍率为15.7％。本实验结合畸变产物耳声发射，全耳蜗铺片，及免疫组化方法，研究丁胺卡那霉素耳毒性的病变特点及活化JNK在毛细胞的表达，初步探讨耳毒性机制。 材料和方法 材料：耳廓反射灵敏的健康白色红目豚鼠12只，体重250～300克，雄性。随机分成实验组和对照组二组，每组6只，实验组：丁胺卡那霉素300mg·kg^-1·d^-1，肌肉注射；对照组：生理盐水等量肌肉注射，连续7天。方法：（1）DPOAE记录方法：在隔声室内，豚鼠处于麻醉状态进行双耳DPOAE检测。采用LB563-EA型耳声发射分析测试系统（IHC公司，美国）。选择初始纯音f₁和f₂，f₂／f₁=1.22，强度差L₁-L₂=10dBSPL，取DPOAE幅值超过本底噪声3dB为DPOAE检出标准。测试频率f₀[f₀=（f₁×f2）^1/2]为1、2、4、6、8kHz处的幅值。（2）免疫组织化学检查：各组豚鼠麻醉状态下行第二次DPOAE检测后，麻醉下开胸，4℃生理盐水经心脏灌流，冲出全部红细胞后4％多聚甲醛灌流固定。断头，快速取出右侧听泡，分离出耳蜗，蜗尖打孔，开放圆窗、卵圆窗，将耳蜗置于相同固定液中24小时。甲酸-甲酸钠溶液脱钙一周，每日更换脱钙液。再移入30％蔗糖溶液4℃下过夜。OCT包埋，沿蜗轴平行方向行10μm厚冰冻切片准备作免疫组化染色（SABC法）。待切片干燥后，入3％过氧化氢溶液10分钟去除内源性过氧化物酶，正常山羊血清室温20分钟封闭，加一抗（兔抗鼠P-JNK多克隆抗体），4℃过夜，再加二抗（生物素化羊抗兔IgG），室温孵育20分钟后加链霉亲和素-生物素-过氧化物酶复合物（streptavidin-biotin-peroxidasecomplex，SABC）37℃反应20分钟，二氨基联苯胺显色，光镜下控制反应时间，蒸馏水终止显色，苏木素复染，梯度酒精脱水，二甲苯透明，中性树胶封片，光镜下观察。空白对照用PBS代替一抗，其余步骤不变。乳腺癌细胞作为阳性对照。（3）耳蜗铺片标本制备:各组豚鼠麻醉状态下行第二次DPOAE检测后，快速断头处死，取左侧听泡，从蜗尖灌注新鲜配制的0 .5%硝酸银溶液2一3nil，用4%多聚甲醛溶液灌注并固定，解剖显微镜下剥离基底膜，分回封片，自然光下曝光2一3小时，光镜下观察，摄片。实验数据采用x土s表示，采用SPSSI 1 .0统计软件行t检验。结果 1.DPOAE幅值检测实验组在用药后DPOAE幅值较用药前除IkHz外，其余均出现下降，其2、4、6、skHz幅值差值同对照组比较有显著性差异（P<0 .01）。 2.耳蜗铺片实验组内毛细胞个别肿胀，外毛细胞纤毛部分消失，结构不清，排列不整齐，有散在空位。外毛细胞损伤主要在底回，第二回，且多为OHCI和oHcZ。对照组内外毛细胞基本正常，排列整齐，细胞间隔均匀，边界清楚。 3.P一JNK在耳蜗毛细胞表达实验组Corti器中外毛细胞有棕黄色颗粒免疫表达，信号反应呈核型，而在对照组Corti器及空白对照均呈阴性反应，实验组中内毛细胞及支持细胞也无免疫反应。讨论 氨基糖昔抗生素的耳毒性已经公认。绝大多数听力损害与毛细胞损害有关，毛细胞是一种机械一电能转换器，毛细胞通过其静纤毛感受基底膜振动的刺激，导致静纤毛机械变行，而诱发一系列的换能过程，最后产生引起听神经兴奋性电活动信号。丁胺卡那霉素主要损害外毛细胞，探讨其耳毒性机制具有临床意义。 DPOAE是由两个具有一定频比关系的纯音同时刺激所诱发的声音能量，反映了耳蜗外毛细胞的活动，常用于评估外毛细胞的功能状态，实验结果显示实验组在用药后DPOAE幅值在2、4、6、skHz处明显下降，同对照组相比有显著差异（P<0.01），频率越高，其幅度下降越大，表明丁胺卡那霉素对豚鼠的外毛细胞损害越重，听力损害有高频损失严重的趋势。耳蜗铺片中发现外毛细胞损害主要表现在底回和第二回，也提示高频损失严重。Chiocfo等认为:氨基糖贰类药物对末梢听器感觉毛细胞损害是主要病理变化;首先受损的是外毛细胞（OHC），OHC受损始于底回，渐向顶回扩展，各回OHC中，以OHCI最易损坏，OHCZ次之，OHC3损坏最轻;内毛细胞（IHc）受损晚于OHC，在我们试验中发现，外毛细胞比内毛细胞损害严重，主要表现在底回和第二回。在同一回外毛细胞中，靠近隧道的外毛细胞较内侧毛细胞易受损害。观察结果与Chiocfo等描述所相似。 研究发现，用紫外线照射细胞后，一种蛋白激酶能够使c一Jun氨基末端活性区Se巧3和Ser73发生磷酸化，据此把该蛋白激酶称为C一Jun氨基末端激酶（c一J皿N一te丽n沮kinase JNK），是丝裂素活化蛋白激酶（MAPK）家族的重要成员之一。研究表明许多外界应激刺激引起JNK活性增高，进而活化转录因子，影响基因表达，作为应激信号传递的关键激酶，其与凋亡发生密切有关。 已有研究表明氨基糖贰类药物可以在耳蜗中、前庭中通过凋亡诱导毛细胞死亡，并且有更多还原

【Abstract】 INTRODUCTIONThe JNKs are a member of the mitogen activated group of protein kinases that are important signal transduct enzymes involved in many facts of cellular regulation including apoptosis. The ototoxic potential of aminoglycoside antibiotics is well known, an increasing body of evidence suggests that HC death after aminoglycoside ototoxicity can occur through apoptosis. Amikacin, a new type of semi - synthetic wide spectrum antibiotics, gains satisfying result in clinical use, but the ratio of hearing loss is 15. 7%. Our research try to figure out the ototoxic character of amikacin and the expression of p - jnk in hair cells ( HCs) , discussing the ototoxic mechanism through DPOAE, cochlea preparation and im-munohistochemistric method.MATERIALS AND METHODSMaterials: 12healthy, otomicroscopically normal guinea pigs weighing 250 - 300g and with a normal Preyer reflex were used. Animals were divided into 2 groups with 6 in each group. Animals in experimental and control group received amikacin (300mg kg-1 d-1) and saline respectively in im. After successive 7days ,all animals were sacrificed. All instillations into the middle ear cavity were performed in the posterior - superior quadrant of the tympanic membrane under a Ziss operating microscope. Methods: (1) Assessment with DPOAE: In sound - proof room, we assess the HC function of guinea pigs under deep anesthesia with DPOAE (Intelligent Hearing Systems, USA). The two applied stimulus pure tones is characterized by its frequency and sound intensty f2/f1 =1.22 , L1 - L2 = 10dBSPL, and we apply SNR beyond 3dB as detectory standard. Wecollect the data of amplitude when frequenry f0[f0 = (fj x f2)1/2] is 1N2N4N6N 8kHz. (2) Immunohistochemistry. All animals from each group were sacrificed after the 2nd DPOAE measurement and fixed via cardiac perfusion with 4% paraformaldehyde after flushing out the red blood cells with 0. 1 m PBS. Both temporal bones of each animal were removed. The bone near the apex, the round and oval window - membrane were opened for better penetration of the fixative. The tissues were immersed in the same fixative solution for 24 h. Decalci-fication of the cochlear was performed in 10% formate - sodium formate for a week. Before embedded in OCT for immunohistochemical analysis, the tissues were dehydrated in 30% saccharose for 24h. The specimens were reduced to sectional series of 10 jxm thickness with a microtome and were mounted on APES. After immersed in 3% H202 and normal goat serum, the sections were incubated with antibody to P - JNK at 37 ^C overnight. A biotinylated anti - rabbit antibody was used as the second AB. For negative controls the primary AB was omitted on one of the mounted sections on all slides. Processing was ultimately performed with a SABC and DAB. The reaction was observed under a light - microscope and was finally stopped by application of PBS. The specimens were dehydrated in baths of ascending ethanol concentration. The primary antibody was replaced with PBS as a control for non - specific binding. ( 3) Cochlear preparation: all the animals were sacrificed after second DPOAE measurement, and the left temporal bones of each animals were removed. Cochlea were perfused with AgN03and fixed with 4% paraformaldehyde from the apex. After exposed under sunlight 2-3 hour, the basal membrane was taken out and observed under microscope. The experimental data were analyzed by SPSS using t test.RESOULTSAssessment with DPOAE: the DPOAE amplitude of experimental animals decreased refer to pre - injection except 1kHz; the decrease amplitude on 2, 4, 6, 8kHz has a significant difference between the experimental group and the control one.Cochlea preparation: The individual inner HCs of experimental group swollen , the cilia of outer HCs miss partially, and there are spaces between the outer HCs which should have arranged uniformly. The damage of outer HCs mainly located in the basal turn and the second turn. The outer hair cell of control group shows no anomaly: had a clear structure and arranged uniformly.更多还原