【作者】 李永贺；

【导师】 谢民强；

【作者基本信息】 南方医科大学 ， 耳鼻咽喉科， 2013， 博士

【摘要】 听力障碍是影响公众身体健康和生活质量的重要因素,数以亿计的人受到威胁。世界卫生组织2013年2月27日在日内瓦表示,全世界有3.6亿人患有耳聋或听力障碍,占全球人口的5%,其中三分之二在发展中国家。中国是世界上最大的发展中国家,有13亿人口,听力障碍残疾人有2057万,占全国人口的16.79%o,绝大部分为感音神经性聋。感音神经性聋的病理改变主要是毛细胞损伤、螺旋神经节、支持细胞及神经末梢的器质性改变,发病原因公认有缺血、病毒感染、耳毒性药物中毒及老年性退行性变等,尤其缺血因素最为常见。而血流障碍中只有少数为单纯的缺血损伤,大部分是缺血后的再灌注损伤。例如突发性耳聋,目前多认为是内耳微循环障碍所致的感音神经性聋。有研究指出缺血后再灌注造成的损伤大于单纯缺血造成的损伤。所以探讨内耳缺血／再灌注损伤(I/RI)的机制将非常有利于内耳疾病的研究。盐酸椒苯酮胺(piperphentonamine hydrochloride,简称椒苯酮胺,PPTA)是由中国医学科学院药物研究所合成筛选出的化学创新药。相关研究表明椒苯酮胺对缺血再灌注损伤的心肌具有保护作用,是钙增敏剂类强心药及心肌保护剂。另外有研究表明其对缺血再灌注损伤脑组织也有保护作用,但国内外尚无椒苯酮胺抗内耳缺血再灌注损伤作用的研究。本实验从听觉功能、细胞形态、炎性因子及细胞凋亡四个方面对豚鼠耳蜗的(I/RI)进行研究,旨在探讨椒苯酮胺对内耳(I/RI)的保护作用及可能机制,为椒苯酮胺用于缺血性内耳疾病的治疗提供药理学依据。本研究分为四个部分：第一部分耳蜗缺血/再灌注(I/R)豚鼠模型的建立目的建立豚鼠耳蜗缺血/再灌注损伤模型,探讨豚鼠耳蜗缺血/再灌注损伤后听功能改变。方法将24只健康、耳廓反射灵敏,体重200-250g豚鼠随机分成3组,分别为正常组、空白对照组、缺血再灌注组。缺血再灌注模型通过暂时性微动脉夹夹闭双侧椎动脉、结扎线活结结扎右侧颈总动脉,实验中激光多普勒检测耳蜗血流(CoBF)。各组动物分别于手术前、缺血再灌注6h、24h、48h行听性脑干反应(auditory brainstem response,ABR)测定,观察ABR各波潜伏期、Ⅰ—Ⅲ波间期和Ⅲ波阈值。结果正常组、假手术组术前、术后ABR阈值无显著变化;缺血再灌注组再灌注6小时、24小时后ABR闽值显著升高(P<0.05),24小时达到高峰,48小时ABR阈值有所恢复,但未恢复正常。结论通过暂时性微动脉夹夹闭双侧椎动脉及右侧颈总动脉,夹闭1小时制造缺血模型,松开三条动脉制造再灌注模型。可致耳蜗损伤,表现为听功能下降,在再灌注后6h、24h、48h,以24h听阈最高,即耳蜗损伤最为严重。第二部分PPTA对耳蜗缺血再灌注损伤的听力保护作用及耳蜗组织扫描电镜和透射电镜观察目的探讨盐酸椒苯酮胺(PPTA)对豚鼠耳蜗缺血再灌注损伤后听功能及耳蜗形态的影响。方法采用前述的缺血再灌注模型,将32只豚鼠随机分为4组,每组8只,每组4只用于扫描电镜观察,剩余4只用于透射电镜观察。分为正常组,假手术组,缺血再灌注对照组,缺血再灌注+盐酸椒苯酮胺组。缺血再灌注+盐酸椒苯酮胺组再灌注后立即经股静脉注射盐酸椒苯酮胺,缺血再灌注对照组以等量生理盐水代替椒苯酮胺注射。测量实验前后各组的听性脑干反应(ABR)波Ⅲ反应阈的变化。24小时迅速断头取听泡,,在扫描电镜、透射电镜下观察耳蜗结构。结果1、正常组、假手术组实验前后ABR波Ⅲ反应阈值无显著变化(P>0.05),缺血再灌注对照组,造模成功后ABR波Ⅲ显著升高(P<0.05),缺血再灌注+盐酸椒苯酮胺组,造模成功后ABR波Ⅲ升高,但低于缺血再灌注对照组(P<0.05)。2、扫描电镜下观察耳蜗组织的形态学变化：正常组及空白对照组豚鼠耳蜗基底膜居外侧3排外毛细胞,1排内毛细胞呈刷状,排列整齐,内、外毛细胞纤毛排列整齐,无缺失、倒伏。缺血再灌注对照组外毛细胞肿胀、缺失,纤毛排列紊乱、倒伏,部分缺失。缺血再灌注+盐酸椒苯酮胺组少量外毛细胞有纤毛倒伏。3、透射电镜下观察：正常组及空白对照组耳蜗毛细胞细胞核圆、核染色质分布均匀,线粒体形态正常,分布均匀,毛细胞突触结构清晰。螺旋神经节神经元未见明显脱髓鞘改变,血管纹内皮细胞核呈梭形；IR组耳蜗毛细胞核边界不清,可见固缩、边集现象,突触结构消失。螺旋神经节可见大量脱髓鞘改变。血管纹内皮细胞核可见固缩、边集现象,呈现充血改变；缺血再灌注+盐酸椒苯酮胺组毛细胞核边界清楚,核染色质分布均匀,线粒体形态分布均匀,无明显肿胀,突触结构尚清晰。螺旋神经节可见少量脱髓鞘变。血管纹内皮细胞核膜完整,无固缩、边集现象。结论PPTA对豚鼠耳蜗的缺血再灌注听力损伤有保护作用,并且可以防止耳蜗缺血再灌注损伤后毛细胞的损伤缺失。第三部分盐酸椒苯酮胺对缺血再灌注后IL-1β和TNF-α的mRNA表达的影响目的1.探讨耳蜗炎性反应与缺血再灌注损伤的关系。2.探讨缺血再灌注后豚鼠耳蜗IL-1β和TNF-α的mRNA的表达与炎性反应的关系。3.探讨PPTA对豚鼠耳蜗缺血再灌注损伤的保护作用机制。方法采用前述的缺血再灌注模型,将24只豚鼠随机分为4组,每组6只,分别为正常组、假手术组、缺血再灌注对照组和缺血再灌注PPTA组。缺血再灌注PPTA组于缺血1小时再灌注后立即经股静脉注射盐酸椒苯酮胺(10mg/kg),缺血再灌注对照组以等量生理盐水代替椒苯酮胺注射,24小时后取标本。用RT-PCR法检测IL-1β和TNF-α的mRNA的表达。用SPSS13.0软件对实验数据进行方差分析,若满足方差齐性,用LSD检验比较组间差异,若不满足方差齐性,用Dunnett’sT3比较组间差异,P<0.05为有统计学意义。结果缺血再灌注对照组耳蜗组织IL-1βmRNA表达量显著高于正常组和假手术组(P<0.001)；缺血再灌注PPTA保护组耳蜗组织IL-1βmRNA表达量显著低于缺血再灌注对照组(P<0.001)；缺血再灌注PPTA保护组耳蜗组织IL-1βmRNA表达量与正常组差异无统计学意义(P=0.056>0.05)。缺血再灌注对照组耳蜗组织TNF-α mRNA表达量显著高于正常组和假手术组(P<0.001)；缺血再灌注PPTA保护组耳蜗组织TNF-α mRNA表达量显著低于缺血再灌注对照组(P<0.001)；缺血再灌注PPTA保护组耳蜗组织TNF-α mRNA表达量高于正常组(P=0.009<0.05)。结论1. PPTA具有拮抗耳蜗组织缺血再灌注损伤作用2. PPTA可能通过抑制炎性反应实现对耳蜗缺血再灌注损伤的拮抗作用第四部分PPTA对豚鼠耳蜗缺血再灌注损伤后细胞凋亡和Fas蛋白、caspase-1的mRNA表达的影响目的：1.观察缺血再灌注损伤后豚鼠耳蜗细胞凋亡的情况。2.对缺血再灌注损伤后豚鼠耳蜗Fas蛋白、caspase-1的mRNA的表达与细胞凋亡间的关系进行分析。3.探讨PPTA对豚鼠缺血再灌注损伤后凋亡细胞的保护作用。方法：采用前述的缺血再灌注模型,将48只豚鼠随机分为4组,每组12只,分别为正常组、假手术组、缺血再灌注组对照组、缺血再灌注PPTA组(缺血60min行再灌注后立即经股静脉注射10mg/kg盐酸椒苯酮胺),缺血再灌注对照组以等量生理盐水代替椒苯酮胺注射,24小时后取标本。用免疫组织化学法(SP)观察Fas蛋白的表达和分布,并用Motic图象分析系统分析单位面积下阳性细胞的积分光密度(IOD)；用TUNEL法观察细胞凋亡的情况；用RT-PCR法检测caspase-1的mRNA的表达,并比较PPTA干预前后的变化。运用SPSS13.0统计软件对实验数据进行方差分析,用LSD检验比较组间差异,P<0.05为有统计学意义。结果：免疫组织化学染色见正常组、假手术组和缺血再灌注PPTA组耳蜗血管纹、螺旋神经节、Corti器Fas表达为弱阳性,缺血再灌注组表达显著增强。IOD值显示正常组、假手术组、缺血再灌注PPTA组之间差异无统计学意义(P>0.05),缺血再灌注组较其他三组显著增强(P<0.05)。TUNEL法显示正常组、假手术组和缺血再灌注PPTA组耳蜗各部位没有或仅有个别部位出现细胞凋亡,缺血及再灌注对照组耳蜗凋亡细胞明显增多,PPTA干预后凋亡细胞显著减少。缺血再灌注组耳蜗组织Caspase-1mRNA表达量显著高于正常组、假手术组和缺血再灌注PPTA组(P<0.001)；缺血再灌注PPTA组耳蜗组织Caspase-1mRNA表达量显著高于正常组(P<0.001)；正常组和假手术组耳蜗组织Caspase-1mRNA表达量差异无统计学意义(P=0.825)。结论：通过豚鼠耳蜗缺血再灌注损伤模型PPTA及对照试验。正常组豚鼠耳蜗各部位Fas为弱阳性表达,无或罕有凋亡细胞。缺血再灌注组Fas表达增强,凋亡细胞增多,进行干预后使凋亡细胞显著减少。缺血再灌注耳蜗组织Caspase-1mRNA的表达显著增高,PPTA可部分但有效地降低Caspase-1mRNA的表达。提示细胞凋亡是耳蜗缺血再灌组损伤的一种细胞损伤形式,PPTA可能通过抑制Fas蛋白及Caspase-1mRNA的表达,通过减少细胞凋亡而对缺血再灌注损伤后的耳蜗起保护作用。更多还原

【Abstract】 Hearing impairment is the important factor that affects public health and quality of life, which threatens hundreds of millions of people. On February27,2013, according to World Health Organization in Geneva, there were360million people with deafness or hearing impairment all over the world, accounting for5%of the world’s population and two-thirds of which in developing countries. As the world’s largest developing country, China has20.57million hearing-disabled people, accounting for16.79‰of the population of1.3billion; the vast majority of them are sensorineural deafness.The main pathological changes of sensorineural deafness are the damage to hair cells, spiral ganglion, sertoli cells and physical changes of nerve endings, as we all know, the reasons include ischemia of cochlear,virus infection, ototoxic drugs poisoning and senile degeneration etc, especially ischemia is the most common factor. Blood disorders cause by only a few of isolated ischemic damage, mostly by reperfusion injury after ischemia. Sudden deafness, for example, now thought to be caused by the inner ear microcirculation of sensorineural deafness. So to explore the mechanism of inner ear ischemia reperfusion injury will be very beneficial to the research of inner ear disease.piperphentonamine hydrochloride (PPTA)is chemical innovation drug, synthetized and screened by the Chinese academy of medical sciences institute of drug synthesis. Related studies show that PPTA has the protective effect of myocardial ischemia-reperfusion injury, is Calcium sensitization agent cardiac medicine and myocardial protective agent, there are studies show that protective effect to cerebral ischemic reperfusion injury. At home and abroad, there are not studies about PPTA protect the inner ear research of ischemia-reperfusion injury.The study was to explore PPTA on the protective effects of inner ear ischemia/reperfusion injury and the possible mechanism, and to expand the clinical indications of PPTA by the methods of auditory sense,cell morphology,inflammation factor and cell apoptosis. The indexes include evoked auditory brainstem responses(ABR),scanning electron microscope(SEM),Fas protein,TUNEL method, and mRNA expression of IL-1β,TNF-α and Caspase-1with RT-PCR skill.The research aim to provide pharmacological basis for the treatment of ischemic inner ear disease,and to develop a protective drugs to the inner ear ischemia reperfusion injury. This study consists of four parts:Part Ⅰ Cochlear Ischemia/Reperfusion (I/R) Model in Guinea PigsObjectiveThe purpose of this study was to establish an animal model of cochlea ischemia/reperfusion(I/R) injury through vertebrobasilar artery ischemia reperfusion and to investigate the changes of auditory function in guinea pigs.MethodsTwenty-four healthy guinea pigs with normal Preyer’S reflex weighed from200to250g were used in this study. They were randomly divided into3groups:the normal control group、blank control group、and ischemia reperfusion groups. The ischemia/reperfusion model by transient blocking bilateral vertebral artery and unilateral cephalic artery for60min,the cochlear blood flow(CoBF)was monitored continuously with Laser Doppler flowmeter. The auditory function was evaluated by evoked auditory brainstem responses(ABR).Each animal was measured before the operation and after ischemia-reperfusion(6h、24h、48h).ResultsThe threshold of ABR of the normal control group and blank control group, which was not significant change before and after every experiment (P>0.05).The changes of ABR during the ischemia-reperfusion group consisted of a prolonged in all wave latencies and interpeak latencies Ⅰ-Ⅲ, an increase of the auditory threshold, especially24hours after I/R, threshold declined when48h,but a statistically significant difference of the auditory threshold shift compared with pre-operation(P<0.05). A statistically significant difference of the auditory threshold shift before and after ischemia/reperfusion(P<0.05).ConclusionWe had successfully established an animal model by blocking bilateral vertebral artery and unilateral cephalic artery in guinea pigs.This model could increase the threshold.The highest auditory threshold of Ischemia reperfusion injury was measured at24h. Part2PPTA Protects Cochlea from Ischemia/Reperfusion Injury and the Change of Histomorphology under Scanning Electron Microscope and Transmission Electron MicroscopeObjectiveTo investigate the effects of PPTA on auditory brainstem responses(ABR) and the change of cochlear histomorphology in cochlea of guinea pigs encounted ischemia/reperfusion injury.MethodsUsed the ischemia reperfusion model, thirty-two healthy guinea pigs with normal Preyer’S reflex weighed from200to250g were used in this study. They were randomly divided into four groups:the normal control group、blank control group、 and ischemia reperfusion control group、ischemia reperfusion group treated with PPTA. Four guinea pigs in each group were randomly assigned to SEM, others were assigned to TEM. Ischemia-reperfusion treated with PPTA group immediately injected PPTA via femoral vein after reperfusion, ischemia reperfusion control group inject same dose of NaCl. Threshold of auditory brainstem response(ABR)was measured before and after every experiment.Killed and taken cochlea quickly when24h after reperfusion, observe the change of histomorphology of cochlear under SEM and TEM.Results1、The threshold of ABR of the normal control group and blank control group,which was not significant change before and after every experiment (P>0.05).The changes of ABR during the ischemia/reperfusion control group consisted an increase of the auditory threshold, especially24hours after ischemia/reperfusion, a statistically significant difference of the auditory threshold shift compared with pre-operation(P<0.05). A statistically significant difference of the auditory threshold shift of ischemia reperfusion group treated with PPTA before and after I/R(P<0.05),but better than ischemia/reperfusion.2、We observed the change of cochlear histomorphology of guinea pigs. Three rows of outer hair cells(OHCs),one row of inner outer hair cells in basical membrane arranged neatly,the stereocilium of hair cells arranged neatly,without loss. Outer hair cell swollen,loss, stereocilium which arranged disorder, collapsed of ischemia reperfusion control group.Ischmia reperfusion group treated with PPTA better than ischemia reperfusion control group.3. In normal group and sham group, membranes of hair cell nuclei were clearly and completely,however,membranes of hair cell were incompletely and typical changes of chromatin condensation and aggression were observed in ischemia reperfusion group.It is much better in PPTA treated group compared with ischemia reperfusion group.The changes of demyelinations in spiral ganglion of ischemia reperfusion group were much more than PPTA treated group. Congestion was observed in stria vascularis in ischemia reperfusion group,but it is not obvious in PPTA treated group,normal group and sham group.ConclusionIntravenous application of PPTA in the early period of cochlear ischemia/reperfusion can protect hearing in guinea pigs, reduced the damage of cochlea. Part3Effect of PPTA on mRNA expression of IL-1β and TNF-α in Guinea Pigs injured by ischemia reperfusionObjective1.To investigate the relationship between inflammatory response and ischemia reperfusion injury of cochlear.2.To investigate the relationship between inflammatory response and mRNA expression of IL-1βand TNF-α of cochlear.3.To investigate the protective effects of piperphentonamine hydrochloride on cochlear of guinea pigs.MethodsThe ischemia reperfusion injury model is the same as mentioned above.24guinea pigs were randomly divided into the normal group, the sham group,the ischemia reperfusion group and the PPTA treated group(10mg/kg). Guinea pigs of PPTA treated group were subjected to ischemia for1hour induced by bilateral vertebral arteries and unilateral carotid artery and then injected piperphentonamine hydrochloride from femoral vein after reperfusion immediately. Ischemia reperfusion group were treated with the same volume of physiological saline instead. Cochlear were removed after24hours of reperfusion.The mRNA expression of IL-1β and TNF-α were measured by Real Time PCR. All data was analyzed by one-way ANOVA using SPSS13.0. LSD test was used if equal variances are assumed. Dunnett’s T3were used if not. A value of P<0.05was considered as statistically significant.ResultsIschemia reperfusion group significantly increased the transcription level of IL-1β mRNA as compared with that of normal group and sham group (P<0.001). In comparison with that in PPTA treated group, the expression levels of IL-1βmRNA were significantly higher (P<0.001). In addition, there is no significant difference between PPTA treated group and normal group(P=0.056>0.05). Compared with normal and sham group, a significantly higher expression of TNF-amRNA was detected in ischemia group(P<0.001). Compared with PPTA treated group, ischemia reperfusion group also had a significantly higher expression of TNF-a mRNA (P<0.001). However, the expression of TNF-amRNA in PPTA treated group is higher than that of normal group (P=0.009<0.05)Conclusions1.Piperphentonamine hydrochloride(PPTA) has an antagonism action toward cochlear ischemia.2.The antagonism action of PPTA towards cochlear ischemia may perform with inhibiting inflammatory reaction. Part4Effect of Fas Protein and mRNA Expression of Caspase-1on Cochlear Ischemia Reperfusion Injury in Guinea Pigs and Protection of PPTAObjective:1. To observe the cell apoptosis in cochlear after ischemia reperfusion in different time.2. To analysis the relationship between expression of Fas protein and transcription of Caspase-1and the cell apoptosis in cochlear after ischemia reperfusion. 3. To investigate the application of PPTA.Methods:48healthy guinea pigs were devided into the normal group, negative control group, IRI control group after the application of0.9%NaCl,Group with PPTA (10mg/kg) were given by intraperitoneal injection after onset of ischemia-reperfusion(60min after ischemia). Every groups have12guinea pigs and we would take samples24h after reperfusion.The Fas protein immunoactivity in cochlear was studied by immunohistochemisty and labeling ratio of Fas protein expression was studied by image analysis. The cell apoptosis was studied by TUNEL method. Statistical analysis Was performed with one-way ANOVA of SPSS13.0statistical package and statistical significance was defined as P<0.05.Result:Immunohistochemical staining showed Fas protein expression of cochlear stria, spiral ganglion, Corti’s organ for weakly positive in normal group, control group and the ischemia reperfusion group with the PPTA.Fas protein expression in Ischemia reperfusion group significantly enhanced. IOD values of the normal group, control group, ischemia-reperfusion PPTA protection group there was no statistically significant difference between (P>0.05), ischemia reperfusion group was significantly enhanced than other three groups (P<0.05).No or only individual cell apoptosis appeared in the each part of cochlear in the normal group, control group and the ischemia-reperfusion group with the PPTA with TUNEL method.In the ischemia-reperfusion control group the cochlea apoptotic cells increased obviously.PPTA could significantly reduce apoptotic cells.The expression of Caspase-1mRNA of cochlear in ischemia-reperfusion group is significantly higher than normal group, control group and ischemia-reperfusion group with PPTA (P<0.001).In ischemia reperfusion group with the PPTA the expression rate of Caspase-1mRNA of cochlear was significantly higher than it in normal group (P<0.001). The difference of expression of Caspase-1mRNA of cochlear tissue is not significant between the normal group and the control group (P=0.825).Conclusion:There is slight positive expression of Fas protein in the cochleas of normal group and control groups. In the ischemia group and reperfusion groups, labeling ratio of Fas protein expression and transcription level of Caspase-1mRNA increased significantly compared with the normal group and the control groups. Compared with the model groups, the labeling ratio of Fas system expression and transcription level of Caspase-1mRNA in cochlear decreased in drug(PPTA) groups. There were almost no cell apoptosis in the normal group and the control groups. But there were more cell apoptosis in the ischemia group and the reperfusion groups(P<0.05). The numbers of apotosis cells in the drug(PPTA) groups were smaller than the groups without drug(PPTA). All of above indicates that Fas protein may get involved in the cochlear injury caused by the ischemia reperfusion injury. The ischemia reperfusion injury can cause the cell apoptosis in cochlear. The therapy of PPTA may prevent the cochlear from cellapoptosis in the ischemia reperfusion injury through blocking the expression of Fas protein and transcription of Caspase-1.更多还原