实验性自身免疫性内耳病耳蜗热休克蛋白70的表达

目的 探讨自身免疫性内耳病模型动物耳蜗热休克蛋白的表达。方法 利用同种异体内耳抗原免疫豚鼠,以建立自身免疫性内耳病（ａｕｔｏｉｍｍｕｎｅ ｉｎｎｅｒ ｅａｒ ｄｉｓｅａｓｅ,ＡＩＥＤ）动物模型,并应用免疫组织化学及原位杂位技术,研究热休克蛋白（ｈｅａｔ ｓｈｏｃｋ ｐｍｔｅｉｎ,ｈｓｐ）７０在正常对照组及ＡＩＥＤ模型动物实验组耳蜗中的表达情况。结果 对照组豚鼠螺旋神经节细胞中存在ｈｓｐ７０样蛋白基     

卡那霉素耳中毒后豚鼠耳蜗热休克蛋白的表达

目的　观察卡那霉素对热休克蛋白 70 (HSP70 )在豚鼠耳蜗中表达的影响。方法　取听力正常豚鼠随机分为实验组和对照组各 5只 ,分别给予 2 5 0mg·kg-1·d-1卡那霉素和生理盐水肌注 ,10天后处死。左耳铺片 ,右耳石蜡包埋切片。用免疫组织化学方法检测各组石蜡切片HSP70表达情况 ,通过计算机图像分析系统分析HSP70表达强度。结果　对照组中Corti器、血管纹、螺旋韧带、内螺旋缘、螺旋神经节HSP70表达呈弱阳性 ;耳蜗铺片显示内 ,外毛细胞正常。实验组中Corti器、血管纹、螺旋韧带、内螺旋缘HSP70表达呈强阳性 ,而在螺旋神经节呈弱阳性 ;耳蜗铺片显示外毛细胞大部分缺损。结论　卡那霉素能够诱导HSP70在豚鼠耳蜗中表达     

三种内耳抗原与自身免疫性内耳病的相关性研究

目的探讨3种纯化的内耳抗原与自身免疫性内耳病的关系,并确定其在耳蜗的表达.方法以粗制内耳抗原的3种亚组份(31000、42000～45000和60000蛋白)作为抗原,分别免疫动物(B、C和D组),观察听阈、血清IgG水平和内耳形态学的改变,并应用免疫组织化学方法确定其在耳蜗的表达.A组为对照组,以不含抗原的聚丙烯酰胺凝胶匀浆液代替内耳抗原.结果免疫前各组听阈差异无显著性(F=0.07,P>0.05),免疫后对照组和C组听阈和内耳形态学未见明显改变,B组(9/30只)和D组(7/28只)部分动物出现听力损失.组间听阈差异有显著性(F=9.12,P<0.01).实验组动物血清IgG均显著性升高(F=7.46,P<0.01),B、C和D组与对照组相比差异有显著性.31000蛋白完全分布于耳蜗神经纤维,而42000～45000和60000蛋白分布广泛,螺旋神经节、Corti器、血管纹和螺旋韧带均有分布.结论粗制内耳抗原中31000和60000亚组份均能独立诱发自身免疫性内耳病,31000蛋白具有很高的组织特异性,可能作为一种标志性蛋白用于自身免疫性内耳病的临床诊断.     

内耳免疫反应中细胞凋亡的研究

目的　探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl 2和Bax的表达情况。方法　选用雌性白色豚鼠 16只 ,随机分为实验组和对照组各 8只 ,以钥孔血蓝蛋白全身免疫后 ,实验组以相同抗原进行内耳免疫 ,对照组内耳注射等量的磷酸盐缓冲生理盐水 ,在内耳免疫 7d后处死动物 ,取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术 (terminal deoxynucleotidyltransferasemediatednickendlabeling ,TUNEL)检测内耳凋亡细胞 ,免疫组化检测内耳Fas和FasL以及Bcl 2和Bax的表达。结果　透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变 ,而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞 ,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞 ,TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征 ,对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性 ,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达 ,FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl 2蛋白表达阴性 ,对照组的Corti器、侧壁和     

内耳免疫反应中细胞凋亡的研究

目的探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl-2和Bax的表达情况。方法选用雌性白色豚鼠16只，随机分为实验组和对照组各8只，以钥孔蛾血蓝蛋白全身免疫后，实验组以相同抗原进行内耳免疫，对照组内耳注射等量的磷酸盐缓冲生理盐水，在内耳免疫7d后处死动物，取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling，TUNEL)检测内耳凋亡细胞，免疫组化检测内耳Fas和FasL以及Bcl-2和Bax的表达。结果透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变，而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞，血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞，TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征，对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性，而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达，FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl-2蛋白表达阴性，对照组的Corti器、侧壁和螺旋神经节细胞Bcl-2蛋白表达阳性。实验组Corti器、侧壁和螺旋神经节细胞Bax蛋白表达阳性，对照组只有螺旋神经节细胞Bax蛋白表达弱阳性，Corti器、侧壁表达阴性。结论内耳免疫反应可诱导细胞凋亡发生，Fas-FasL是此过程的信号转导途径之一，Bcl-2和Bax蛋白在其中起了重要调节作用。     

三种内耳抗原与自身免疫性内耳病的相关性研究

目的 探讨3种纯化的内耳抗原与自身免疫性内耳病的关系,并确定其在耳蜗的表达。方法 以粗制内耳抗原的3种亚组分（31000、42000－45000和60000蛋白）作为抗原,分别免疫动物（B、C和D组）,观察听阈、血清IgG水平和内耳形态学的改变,并应用免疫组织化学方法确定其在耳蜗的表达。A组为对照组,以不含抗原的聚丙烯酰凝胶匀浆液代替内耳抗原。结果 免疫前各组听阈差异无显著性（F＝0．07,P＞0．05）,免疫后对照组和C组听阈和内耳形态学未见明显改变,B组（9／30只）和D组（7／28只）部分动物出现听力损失。组间听阈差异有显著性（F＝9．12,P＜0．01）。实验组动物血清IgG均显著性升高（F＝7．46,P＜0．01）,B、C和D组与对照组相比差异有显著性。31000蛋白完全分布于耳蜗神经纤维,而42000－45000和60000蛋白分布广泛,螺旋神经节、Corti器、血管纹和螺旋韧带均有分布。结论 粗制内耳抗原中31000和60000亚组份均能独立诱发自身免疫性内耳病,31000蛋白具有很高的组织特异性,可能作为一种标志性蛋白用于自身免疫性内耳病的临床诊断。     

IL-2、TNF-αmRNA在迷路炎豚鼠耳蜗中的表达

目的探讨迷路炎时,豚鼠耳蜗IL-2、TNF-α mRNA的表达变化及其与迷路炎的关系.方法随机将24只豚鼠分为实验组和对照组,实验组18只、36耳,将1mg.ml-1内毒素(lipopolysaccharide,LPS)0.2ml缓慢注入中耳腔,对照组6只、12耳注入等量生理盐水,观察6h、48h、14d耳蜗的病理变化;用原位杂交技术检测IL-2、TNF-α mRNA在耳蜗的表达,用Tiger 920图像分析软件分析图象.结果(1)耳蜗形态学改变鼓室注药后6h,实验组豚鼠耳蜗的血管纹、螺旋韧带、蜗螺旋轴静脉等即可见充血、水肿、炎细胞浸润,Corti器支持细胞和感觉细胞轻度肿胀、变性;48h时加重;14d时耳蜗各部分结构恢复正常.对照组豚鼠耳蜗各部分结构无改变;(2)IL-2 mRNA的表达对照组耳蜗无IL-2 mRNA表达;鼓室注射LPS后6h,实验组耳蜗的螺旋神经节、螺旋缘呈阳性表达,血管纹、螺旋韧带呈可疑阳性;而48h、14d无表达;(3)TNF-α mRNA的表达对照组耳蜗无TNF-α mRNA表达;鼓室注射LPS后6h,TNF-α mRNA广泛表达于实验组耳蜗的螺旋神经节、Corti器、螺旋缘、血管纹、螺旋韧带等部位,48h表达明显增强(P＜0.01),14d时仅螺旋神经节呈弱阳性染色.结论IL-2、TNF-α可能与急性迷路炎的发生有关,TNF-α则可能起着更为重要的作用.     

热休克蛋白70及其抗体与实验性自身免疫性内耳病

目的　了解抗原免疫对热休克蛋白 70 (heatshockprotein 70 ,HSP70 )在豚鼠耳蜗中表达的影响。方法　以同种异体内耳组织为抗原免疫豚鼠 ,用免疫组织化学方法检测豚鼠耳蜗中HSP70的表达并进行计算机图像处理 ;对豚鼠膜迷路组织进行免疫转印分析以测定HSP71 ;以纯化的人类重组HSP71为抗原 ,用免疫转印技术 (Westernblot)检测动物血浆中HSP71抗体。结果　HSP70在正常豚鼠耳蜗中呈低水平表达。抗原免疫后 ,实验组动物耳蜗HSP70免疫活性较对照组明显增强 ,两组间的吸光度值差异有显著性 (P <0 0 1 ) ;免疫后大多数动物 (1 2 / 2 0 )体内产生了抗HSP71抗体 ,实验组HSP71抗体阳性率显著高于对照组 (P <0 0 1 )。结论　同种膜迷路组织作为抗原免疫豚鼠能够诱导HSP70在耳蜗内合成增加 ;表明HSP70与自身免疫性内耳病有一定联系。     

内耳免疫反应中的细胞凋亡活性及其与FasL表达的相关性

目的 观察内耳免疫反应过程中是否存在细胞凋亡以及细胞凋亡活性与FasL表达相关性。方法16只雌性白色豚鼠。随机分为实验组与对照组，每组各8只。所有动物均先以钥孔虫戚血蓝蛋白全身免疫。然后，实验组再以相同抗原进行单侧内耳局部免疫，对照组则于单侧内耳注射等量磷酸盐缓冲生理盐水。3天后处死动物，取内耳免疫侧（或对照注射侧）耳蜗制备石蜡切片，脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术检测内耳细胞凋亡活性，免疫组化法检测内耳FasL表达水平。结果实验组动物内耳Cord器毛细胞、血管纹的缘细胞和螺旋神经节细胞中存在阳性着色的凋亡细胞，对照组豚鼠内耳则否。实验组动物内耳Corti、蜗管侧壁、螺旋神经节细胞FasL表达阳性。对照组则为阴性。结论 内耳免疫反应可诱导局部细胞凋亡活性增高，FasL在此过程中可能发挥重要作用。     

纯化内耳P0蛋白诱发实验性自身免疫性内耳病动物模型

目的：用从内耳组织中纯化的P0蛋白免疫豚鼠,建立P0蛋白诱发的自身免疫性内耳病动物模型,研究其在自身免疫性内耳病中的作用。方法：采用制备性SDS-PAGE从内耳组织中分离、纯化P0蛋白。以纯化的豚鼠内耳P0蛋白作为抗原免疫豚鼠,观察其听性脑干反应阈,血清中抗体水平和内耳形态学的改变,并用免疫组织化学法确定P0蛋白在耳蜗的分布情况。结果：SDS-PAGE结果显示纯化的蛋白质只在分子量为30 000的位置上出现单一的蛋白染色带,Western blot结果示该蛋白即为P0蛋白。免疫后有22%豚鼠的听性脑干反应阈升高,对照组动物无变化。实验组血清IgG显著升高（F=6.48,P〈0.01）,反应阈提高豚鼠的螺旋神经节细胞均有不同程度的数目减少,蜗轴小血管周围有炎性细胞浸润。P0蛋白在耳蜗仅分布于螺旋神经节、蜗轴神经纤维的髓鞘上。结论：用制备性SDS-PAGE能够成功地从内耳纯化P0蛋白,用于自身免疫性内耳病的研究。P0蛋白在部分豚鼠能够诱发自身免疫性内耳病,可能是自身免疫性内耳病的自身抗原之一。     

Expression of heat shock protein 70 in immune response of the guinea pig inner ear]

OBJECTIVE: To explore whether the immune response of the inner ear could induce heat shock protein (hsp) 70 in guinea pig cochlea. METHODS: A model of autoimmune inner ear disease (AIED) was established by systemically immunizing the guinea pig with the homologous crude inner ear antigen (CIEAg). The immunized cochleae and normal control cochleae were examined for the expression of hsp70 with techniques of immunohistochemistry and in situ hybridization. RESULTS: In the control animals, the expression of the hsp70-like protein appeared only in the spiral ganglion, whereas in the cochleae with CIEAg immunization, strong expression of the hsp70-like protein and its mRNA appeared in the spiral ganglion as well as in the stria vascularis and the spiral ligament. The hearing thresholds were significantly increased in 10 out of 28 cochleae (35.7%) with CIEAg immunization. CONCLUSION: The results suggest that the immune response of the inner ear can induce the expression of hsp70 in the guinea pig cochlea.     

Relationship between three inner ear antigens with different molecular weights and autoimmune inner ear disease

Crude inner ear antigen (CIEAg) can induce autoimmune inner ear disease (AIED) although it is not known which subcomponent of CIEAg is involved. In this study, we investigated the relationship between 3 purified inner ear antigens (31, 42-45 and 60 kD proteins) and AIED, and determined their distribution in normal guinea pig cochlea. Three groups of guinea pigs were immunized with the three inner ear antigens and one group served as a control. The hearing thresholds, serum IgG level and morphological changes in the inner ear were observed. The expression of the three antigens in the cochlea was detected using immunohistochemical techniques. No obvious changes in hearing thresholds or inner ear morphology were observed between the control and 42-45 kD groups. Animals immunized with the 31 or 60 kD proteins showed a significant increase in hearing thresholds (p < 0.05 vs control), accompanied by morphological changes in the inner ear. The serum IgG level was increased significantly (p < 0.05) in all immunized animals. The 31 kD protein was distributed in the cochlear nerve and spiral ganglion, while the 42-45 and 60 kD proteins were distributed widely, being found in the spiral ganglion, organ of Corti, stria vascularis and spiral ligament. These results suggest that two subcomponents of CIEAg (the 31 and 60 kD proteins) may induce AIED independently, that several inner ear antigens may contribute to the pathogenesis of AIED and that the 31 kD protein is of high tissue specificity and may be used as a marker protein for the clinical diagnosis of AIED.     

Influence of carbon dioxide laser irradiation on the morphology and function of guinea pig cochlea

Both experimental and clinical studies have demonstrated that carbon dioxide laser is suitable for stapedotomy. The aim of this study was to investigate morphological, electrophysiological and functional changes in the inner ear after irradiation with CO(2) laser set with different energy parameters. A cochleostomy in the basal cochlear turn of guinea pig cochleae was performed with CO(2) laser of 1, 2 and 3 w, respectively. The cochleae were removed three weeks after laser irradiation. The auditory evoked brainstem response (ABR) was measured before and after laser application and immediately before removal of the cochlea. Immunohistochemical methods were used to examine inducible nitric oxide synthase (iNOS/NOSII) and heat-shock protein 70 (Hsp70) concentrations in the cochlea after laser application. The organ of Corti was studied by scanning electron microscopy. Worse hearing loss was observed in animals receiving higher-power CO(2) laser. These findings correlated with more intense injury of the cochlear ultrastructure and with positive expression of iNOS and Hsp70 in spiral ganglion cells, nerve fibres, supporting cells of the organ of Corti and cells of the spiral ligament. The CO(2) laser as a noncontact procedure is shown to be effective and safe if the total amount of energy is kept within the limits applied in this study. Nitric oxide and stress proteins play important roles in the traumatic mechanism of the inner ear, which are related to hearing loss and injury of the ultrastructure of the inner ear.     

同种异体内耳组织免疫后豚鼠内耳形态学变化

目的建立一高阳性率、可供圆窗给药治疗内耳病研究用的自身免疫性内耳病动物模型。方法实验动物为86只豚鼠,其中32只用于制备粗制内耳抗原。其余动物随机分为实验组42只,对照组1、2各6只。将实验组动物腹腔注射环磷酰胺进行预处理,2d后用粗制内耳抗原行皮下多点接种,分别于接种后4、6、8、10、12、14、20d时用光镜观察动物内耳形态学变化,并检测其血清中IgG、IgM、C3、CH50、循环免疫复合物(CIC)水平。对照组1不行任何处理,对照组2不接种内耳抗原,余同试验组。结果接种后豚鼠耳蜗、前庭、内淋巴囊等部位出现以淋巴细胞为主的炎性细胞浸润,尤以前庭阶、鼓阶、螺旋神经节区域及耳蜗底圈等部位为重。接种后4d时,67%动物内耳有炎性细胞浸润;8~12d时,100%动物出现炎性细胞浸润;接种14d后,炎性细胞浸润明显减少。接种6~14d时,内耳尚有血管周围炎、螺旋神经节细胞变性、内淋巴积水等改变。结论将豚鼠采用环磷酰胺预处理及同种异体内耳抗原接种,可建立一高阳性率的自身免疫性内耳病动物模型。     

同种异体内耳组织免疫后豚鼠听功能与内耳形态学变化的关系

目的 观察豚鼠接受环磷酰胺处理及免疫后其听功能与内耳形态学变化的关系。方法 采用86只白色红目豚鼠作为实验对象，其中32只动物提供粗制内耳抗原。实验组（42只）动物于腹腔注射环磷酰胺2d后用粗制内耳抗原接种，接种后4．6、8、10、12、14、20d时接受ABR检测及内耳形态光镜观察。对照组1（6只）不行任何处理，对照组2（6只）注射环磷酰胺，但不接种抗原。结果 接种后4d时67％动物ABR阈值明显提高，8d时为83％，14d时降为58％，20d时所有动物阈值恢复正常。光镜观察发现：接种后4d时，实验动物听功能受损耳出现炎性细胞浸润；8—12d时，所有实验动物内耳均有类似改变；6—14d时，内耳尚有血管周围炎、螺旋神经节细胞变性等改变，相关形态变化均以听功能受损耳明显为重；接种14d时，内耳炎性细胞浸润明显减轻，20d时，绝大多数动物内耳形态基本恢复正常。结论 豚鼠接受环磷酰胺预处理及同种异体内耳抗原接种后，其听功能与内耳形态学的变化基本相一致。     

Experimental autoimmune inner ear disease

A guinea pig animal model of autoimmune inner ear disease is presented. The functional, anatomical, and immunological inner ear changes were tested electrophysiologically, histologically and by the immunofluorescence test. Using a homologous crude inner ear antigen (CIEAg) we were able to induce endolymphatic hydrops, vasculitis, mild cellular infiltration of the endolymphatic sac, and occasional spiral ganglion degeneration. Threshold shift was seen in 20% of the tested ears. Specific fluorescence was revealed around the modiolar vessels and in the basilar membrane. The endolymphatic sac and duct showed occasional fluorescence in the epithelial and/or subepithelial layers. The findings were discussed in light of the other models immunized with various forms of inner ear antigens. Similarities between the detected specific fluorescence and the fluorescence revealed by sera of patients with cochleovestibular disorders were discussed.     

实验性急性中耳细菌感染诱发的热休克反应

目的 探讨细菌抗原和内耳抗原之间的联系及热休克反应对其抗原的影响。方法 用抗哺乳类热休克蛋白（ｂｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ，ｈｓｐ）７０抗体识别绿脓杆菌、变形杆菌、大肠杆菌、金黄色葡萄球菌和肺炎克雷白杆菌抗原，进行免疫转印分析，测试正常豚鼠膜迷路中ｈｓｐ７０的表达水平。用肺炎克雷白杆菌制作急性中耳感染动物模型，观察细菌的内耳与哺乳类ｈｓｐ７０家族，正常豚鼠膜迷路表达ｈｓｐ７０水平极低。肺炎     

The role of heat shock protein 70 and its autoantibody in experimental autoimmune inner ear disease]

OBJECTIVE: To examine the effect of antigen stimulation on induction of the heat shock protein 70(HSP70) in the guinea pig cochlea and to explore the role of HSP70 and its autoantibody in the pathogenesis and diagnosis of autoimmunize inner ear disease (AIED). METHODS: To establish the animal model of AIED, homologous crude inner ear antigen was used to immunize the guinea pig. The expression of HSP70 was detected by immunocytochemistry and the relative staining densities were quantified with a light microscope image analysis system. The autoantibodies against HSP71 in the plasma of animals were tested by Western blot using purified recombinant human HSP71 as antigen. RESULTS: HSP70 presented in the normal guinea pig cochlea at a lower level. After immunological challenge, the levels of HSP70 immunoreactivity in the immunized animals were significantly increased as compared with control animals. Optical densities of cochleae of immunized animals were significantly greater than those of animals in the control group (P < 0.01). Most of the immunized animals had developed autoantibodies against HSP71. The incidence of autoantibody in the experimental group was significantly different (P < 0.01) from the control group. CONCLUSION: Antigen stimulation could lead to an increase in the synthesis of HSP70 in the guinea pig cochlea. The detection of autoantibody against HSP71 might have significance in the diagnosis of AIED.     

噪声诱导豚鼠耳蜗应激蛋白表达的研究

目的：探讨噪声对耳蜗应激蛋白（ＳＰ,亦即ＨＳＰ）的诱导。方法：用免疫组织化学结合图像分析技术检测噪声刺激后豚鼠耳蜗ＨＳＰ７０的表达。结果：在正常状态下耳蜗表达ＨＳＰ７０的部位（Ｃｏｒｔｉ器,螺旋神经节,血管纹,齿间细胞）经１００ｄＢ声强级的白噪声暴露４５ｍｉｎ后,其ＨＳＰ７０的表达均增强,其中以Ｃｏｒｔｉ器和螺旋神经节更明显,Ｐ值均小于０．０１。结论：白噪声对豚鼠耳蜗ＨＳＰ７０表达具有明显的诱导作用。