盐酸椒苯酮胺对豚鼠耳蜗缺血再灌注损伤后细胞凋亡的影响

发布时间：2018-05-14 02:01

本文选题：耳蜗 + 缺血/再灌注损伤　；参考：《南方医科大学》2015年硕士论文

【摘要】：研究背景听力障碍是常见的耳鼻喉科疾病,世界卫生组织于2013年发布的报告显示,全球约约5.3%人口患有听力障碍,也就是说约有3.6亿的人群受困于残疾性听力,其中约67%的听力障碍患者在发展中国家。作为全球最大的发展中国家,全国约有2057万听力障碍患者,占总人口的16.79‰,患者人数居各类残疾之首,是影响居民生活质量和身体健康的重要疾病之一。因此,探索和发现听力障碍的预防和治疗方法是当前医学界的热点课题,也是我们面临的巨大挑战。听力障碍主要包括感音神经性耳聋、传导性耳聋和混合性耳聋,其中感音神经性耳聋患者数量最多。感音神经性耳聋的致病因素包括老年性、缺血、病毒感染、听神经病、药物耳毒性、遗传、中枢疾病、自身免疫疾病、噪音性以及肿瘤等。它是指内耳和其神经传导通路病变引起的各种听力障碍,通常涵盖螺旋神经节、毛细胞、耳蜗神经、突触复合体以及听中枢等组织和器官的病理性改变。内耳中,感受器细胞包括外毛细胞(outer hair cell,OHC)和内毛细胞内毛细胞(inner hair cell,IHC)两类,它们都可将机械能转变为生物电能,即产生感受器地位电位。螺旋神经节属于传入神经元,可将接收感受器发出的电位信号,并将信号传入中枢,进而产生位置觉或听觉。内、外毛细胞分别可同螺旋神经节的Ⅰ型、Ⅱ型传入神经元树突构成传入突触。听觉的形成过程是内毛细胞感应外界声音信号,并将强度频率不同的机械信号转变为电信号,形成感应定位,进而通过传入突触将信号传递给螺旋神经元,螺旋神经元再将信号传导至中枢系统,中枢系统产生听觉。外毛细胞可放大外界声音信号,提高耳蜗对声音频率的选择性和敏感性,是内皮细胞的重要功能辅助细胞。内毛细胞传入突触的形态、功能、结果和数量的异常改变是引发感音神经性耳聋的重要因素。事实上,在声音信号传入通路中,毛细胞、螺旋神经节等结构元件的损伤都会导致听觉障碍。耳蜗是人听觉系统的关键结构,属于高耗能组织,缺氧缺血状况下会导致传入神经肿胀、毛细胞非自然性死亡进而产生听力损伤；噪音和药物中毒会一定程度上损伤螺旋神经元和毛细胞,也是感音神经性耳聋的常见致病因素。然而有研究显示缺血5min后引起传入神经肿胀可通过再灌注逐渐恢复。感音神经性耳聋的病理现象涵盖了螺旋神经节、毛细胞、神经末梢以及支持细胞的器质性改变,其常见的致病因素包括病毒感染、缺血、老年性退行性变以及耳毒性药物中毒等,其中缺血是导致感音神经性耳聋的最普遍因素,然而血流障碍普遍表现为缺血后再灌注损伤,极少数为纯粹的缺血损伤。比如突发性耳聋普遍认为是内耳微循环障碍引起的听觉障碍。有数据显示单纯缺血引起的损失显著小于缺血后再灌注引起的损失,可见研究内耳缺血/再灌注损伤(I/RI)的相关机理对于预防和治疗内耳疾病十分关键。如今在哺乳动物当中,认为毛细胞发生损伤,则很难再生,目前仍在研究治疗感音神经性耳聋的方式,例如再生毛细胞或是移植耳蜗干细胞进行治疗。出现感音神经性聋的一个关键因素传入神经系统损伤这一病理改变,神经末梢、螺旋神经节细胞以及突触复合体等都属于传入神经系统,突触结构在外界环境的影响会损害其功能,从而出现异常,但是受损螺旋神经节细胞很难进行修复。曾经有实验对其进行研究,损伤后的传入神经可以再生神经纤维,同时毛细胞可以关联再生的神经纤维；与此同时,施万细胞会在螺旋神经节细胞死亡后进行增殖,但是却不能向正常功能神经元细胞进行分化,因此也不能发挥应有的效果。当前,为了使得感音神经性聋患者能够听到外界的声音一般进行助听器的佩戴或是移植人工耳蜗,然而,因为人工耳蜗价格较高,人们也不能适应佩戴助听器,所以没有在大范围应用,因此药物是大部分患者主要的治疗方式。所以,感应神经性聋的探索的方式应放在药物治疗上。盐酸椒苯酮胺(peperphentonamine hydrochloride, PPTA),是我国自主创新研发的钙增敏剂类强心药及心肌保护剂,已获得3个发明专利,11类化学药品临床试验批件,且已经完成了127例Ⅰ期临床试验。临床前研究表明其不仅具有保护受损心肌、增强心功能并能降低心肌耗氧量的双重作用；还可通过增加SOD活性及GSH含量,减少NO含量,发挥神经保护作用,并且可降低缺血脑组织Caspase-1 mRNA的表达,表现出良好的抗凋亡作用。与心脑组织损伤类似。钙超载、自由基、细胞凋亡也是耳蜗损伤的常见原因,PPTA对心脑的保护机制为本研究提供了方向。本实验分为两个部分,第一部分成功建立豚鼠耳蜗缺血/再灌注损伤模型。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。可以有效的减少内耳血流灌注,成功的建立了缺血再灌注模型,该模型手术创伤小,术中动物死亡率低,术后存活率高,适合大量造模,为下一步实验提供了良好的基础。第二部分旨在从细胞凋亡(利用TUNEL技术检测)和与凋亡有关的caspase-1的mRNA表达来探讨椒苯酮胺对内耳缺血/再灌注损伤的保护作用及可能机制,以扩大椒苯酮胺的临床适应症,为椒苯酮胺用于缺血性内耳疾病的治疗提供药理学依据,可望开发出对内耳缺血再灌注损伤具有保护作用的新药。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。方法随机挑选24只3组豚鼠,这些豚鼠的标准必须是200-250g的体重、健康、有着灵敏耳廓反射,三组是空白对照组、正常组以及缺血再灌注组。采用暂时性微动脉夹对双侧椎动脉进行夹闭、用活结对右侧颈总动脉进行结扎可以模拟暂时缺血缺血,制造再灌注模型,实验中对耳蜗血流的检测使用激光多普勒进行检测(CoBF)。结果成功进行缺血模型的建立：双侧椎动脉及右侧颈总动脉通过无创微动脉夹进行夹闭,夹闭5-10分钟后再进行观察,发现各组动物的耳蜗血流只有30%的原来水平,如果在此水平保持不变,说明成功建立了缺血模型。成功建立再灌注模型：1小时缺血模型完成后,不再夹闭右颈总动脉微动脉,将双侧椎动脉微动脉夹取下,避免椎动脉出现断离,对各组动物的CoBF进行检测,灌注10分钟后血量恢复到70%的缺血前水平,该稳定能够进行一段时间的维持,表示成功建立了再灌注模型。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。这可以有效的减少内耳血流灌注,成功的建立了缺血再灌注模型,该模型手术创伤小,术中动物死亡率低,术后存活率高,适合大量造模,为下一步实验提供了良好的基础。第二部分PPTA对豚鼠耳蜗缺血再灌注损伤后细胞凋亡及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。每组中6只用TUNEL法观察细胞凋亡的情况,计算凋亡指数(Apoptotic index, AI),组间比较检测结果；每组中另外6只用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,P0.05为有统计学意义。结果：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,I/RI组与正常组、假手术组、PPTA组比较,凋亡指数明显升高,P0.001。缺血再灌注组耳蜗组织Caspase-1 mRNA表达量显著高于正常组、假手术组和缺血再灌注PPTA组(P0.001)；缺血再灌注PPTA组耳蜗组织Caspase-1 mRNA表达量显著高于正常组(P0.001)；正常组和假手术组耳蜗组织Caspase-1 mRNA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无或罕有凋亡细胞。缺血再灌注组凋亡细胞增多,进行干预后使凋亡细胞显著减少。缺血再灌注耳蜗组织Caspase-1 mRNA的表达显著增高,PPTA可部分但有效地降低Caspase-1 mRNA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Caspase-1 mRNA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。
[Abstract]:Background hearing impairment is a common disease in the Department of ENT. In 2013, the WHO reported that about 5.3% of the world's population had hearing impairment, that is, about 360 million of the population were trapped in the disability hearing, of which about 67% of the hearing impaired were developing in China. As the largest developing country in the world, the country was the largest developing country in the world. There are about 20 million 570 thousand patients with hearing impairment, accounting for 16.79 per thousand of the total population. The number of patients is the first of all kinds of disabled people. It is one of the important diseases that affect the quality of life and health of the residents. Therefore, the exploration and discovery of the prevention and treatment of hearing impairment is a hot topic in the current medical field. It is also a great challenge for us. It includes sensorineural deafness, conductive deafness and mixed deafness, among which the number of sensorineural deafness is the most. The pathogenic factors of sensorineural deafness include geriatric, ischemia, virus infection, auditory neuropathy, drug ototoxicity, heredity, central disease, autoimmune disease, noise, and tumors. All kinds of hearing impairment caused by the conduction pathway, usually covering spiral ganglion, hair cell, cochlear nerve, synaptic complex, and the pathological changes of tissues and organs such as the auditory center. In the inner ear, the receptor cells include the two types of outer hair cell, OHC, and internal hair cells (inner hair cell, IHC), all of which are available. The mechanical energy is transformed into a bioelectric energy, which produces the positional potential of the receptor. The spiral ganglion belongs to the afferent neuron, which can transmit the potential signals from the receptor and afferent the signal to the center, and then produce position sense or hearing. The process of hearing formation is that the inner hair cells induce the external sound signals and transform the mechanical signals with different intensity frequencies into the electrical signals, forming an induction location and passing the signals to the spiral neurons through the incoming synapses. The spiral neurons then transmit the signals to the central system and the central system produce hearing. Outer hair cells can enlarge the outside world. Sound signals, increasing the selectivity and sensitivity of the cochlea to the frequency of the sound, are the important functions of the endothelial cells. The abnormal changes in the morphology, function, results and quantity of the afferent synapses in the inner hair cells are important factors that cause the sensorineural deafness. In fact, in the afferent pathway of the sound signal, the hair cells, spiral ganglion and so on. Damage to the components of the structure can cause hearing impairment. The cochlea is a key structure of the human auditory system. It belongs to high energy dissipation tissue. Hypoxic-ischemic conditions can cause swelling of the afferent nerve, unnatural death of hair cells and hearing damage. Noise and drug poisoning can damage the spiral neurons and hair cells to some extent, and also the sensorineural nerve. A common cause of deafness. However, studies have shown that the swelling of the afferent nerve after 5min ischemia can be gradually recovered through reperfusion. The pathological phenomenon of sensorineural hearing loss covers the spiral ganglion, hair cells, nerve endings, and the organic changes of the supporting cells. The common pathogenic factors include virus infection, ischemia, and old age. Degenerative changes and ototoxic drug poisoning, in which ischemia is the most common cause of sensorineural deafness. However, blood flow disorders are generally characterized by post ischemia reperfusion injury, and a few are pure ischemic injuries. For example, sudden deafness is generally considered as a hearing impairment caused by internal ear microcirculation disorder. Data show simple deficiency. The loss of blood caused by ischemia-reperfusion is significantly less than the loss of reperfusion after ischemia. It is found that the mechanism of the internal ear ischemia / reperfusion injury (I/RI) is crucial for the prevention and treatment of internal ear diseases. Now, it is difficult to regenerate the hair cells in mammals. For example, regenerated hair cells or transplantation of cochlear stem cells. A key factor in sensorineural hearing loss is a pathological change in the nervous system, nerve endings, spiral ganglion cells, and synaptic complexes all belong to the afferent nervous system, and the influence of the synaptic structure on the external environment will damage its function. It is unusual, but the damaged spiral ganglion cells are difficult to repair. There have been experiments on it. After the damage, the afferent nerve can regenerate nerve fibers, while the hair cells can be associated with the regenerated nerve fibers; at the same time, Schwann cells proliferate after the death of the spiral ganglion cells, but they can not do normal work. In order to make sensorineural deaf people can hear the sound of the external hearing and the cochlear transplantation, however, because of the high price of the cochlea, people are not able to adapt to the hearing aids, so they are not widely used. Therefore, the drug is the main treatment of most patients. Therefore, the way to explore the induction of neurogenic deafness should be on the drug treatment. Peperphentonamine hydrochloride (PPTA) is a kind of calcium sensitizer and myocardial protection agent in our own innovation and development. It has obtained 3 inventions and 11 kinds of chemicals. 127 cases of phase I clinical trials have been completed in the bed test. Pre clinical study shows that it not only protects the damaged myocardium, strengthens the cardiac function and reduces the oxygen consumption of the myocardium, but also reduces the NO content by increasing the SOD activity and the content of GSH, and plays a neuroprotective effect, and reduces the Caspase-1 mRNA in the ischemic brain tissue. The expression of PPTA shows a good anti apoptosis effect. It is similar to the injury of heart and brain tissue. Calcium overload, free radical, cell apoptosis are also the common causes of cochlear injury. The protective mechanism of the heart and brain provides the direction for this study. This experiment is divided into two parts. The first part has successfully built the model of the guinea pig cochlear ischemia / reperfusion injury. The occlusion of the bilateral vertebral artery and the right common carotid artery was made by clamping the arterioles. The ischemia model was made for 1 hours and three arteries were loosened to make the reperfusion model. The ischemia reperfusion model could be effectively reduced and the model of ischemia reperfusion was successfully established. This model has a small operation injury, low mortality in the operation and a high survival rate after operation. It is suitable for a large number of models. The second part provides a good basis for the next experiment. The second part aims to explore the protective effect and possible mechanism of pepylphenyl ketamine on ischemia / reperfusion injury in the inner ear from apoptosis (using TUNEL Technology) and the expression of apoptosis related caspase-1 to expand the clinical indications of polyamines and the use of zanone amine for ischemia. The treatment of internal ear diseases provides pharmacological basis, and it is expected to develop new drugs that have protective effects on internal ear ischemia reperfusion injury. The first part of the cochlear ischemia / reperfusion (I/R) guinea pig model is established to establish a guinea pig cochlear ischemia / reperfusion injury model. Methods 24 3 groups of Guinea pigs were selected randomly. The standard of these guinea pigs must be 200-25. 0g's weight, health, and sensitive auricular reflex, three groups are blank control group, normal group and ischemia-reperfusion group. Temporary microarterial clamp is used to clamp bilateral vertebral arteries, and ligature of the right cervical artery can be used to simulate temporary ischemia and ischemia, and the model of reperfusion is made, and the test of cochlear blood flow is used in the experiment. Light Doppler was detected (CoBF). Results the ischemic model was successfully established: the bilateral vertebral artery and the right common carotid artery were clamped through the noninvasive Microartery clamp, and then observed after 5-10 minutes. It was found that the cochlear blood flow of each animal was only 30% of the original level. If the level remained unchanged at this level, the ischemia was successfully established. Model. Successfully established the reperfusion model: after 1 hours of ischemia model, the arterioles of the right common carotid artery were no longer clipped and the bilateral vertebral artery microarteries were clipped to avoid the vertebral artery disconnection. The CoBF of the animals in each group was detected, and the blood volume was restored to 70% of the pre blood level after 10 minutes of perfusion, and the stability could be maintained for a period of time. A model of reperfusion was established successfully. Conclusion by clamping the bilateral vertebral artery and the right common carotid artery with temporary Microartery clamp, the ischemia model was created for 1 hours and three arteries were loosened to make the reperfusion model. This could effectively reduce the blood perfusion of the inner ear. The model of ischemia reperfusion was successfully established. The operation was small and the operation was small and the operation was small. The mortality of the animals is low, the survival rate is high after the operation, it is suitable for a large number of models and provides a good basis for the next experiment. Second the effect of PPTA on the apoptosis and the expression of caspase-1 in the cochlear ischemia and reperfusion injury of guinea pigs: 1. the apoptosis of the cochlear cells of the rat after ischemia reperfusion injury was observed by.2. for ischemia reperfusion The relationship between the expression of mRNA in the cochlear caspase-1 and the apoptosis of the cochlea of guinea pigs after injections was analyzed by.3. to explore the protective effect of PPTA on the apoptotic cells after ischemia reperfusion injury in guinea pigs. Methods: 48 guinea pigs were randomly divided into 4 groups, 12 rats in each group, with the foregoing ischemia reperfusion model, respectively, in the normal group, the sham operation group, and the ischemia reperfusion. Group control group, ischemia reperfusion group PPTA (ischemic 60min after reperfusion immediately after intravenous injection of 10mg/kg hydrochloride pepperamine hydrochloride), ischemia reperfusion group in the same amount of saline instead of zanthoxanine injection, 24 hours after the sample. 6 of each group only used TUNEL method to observe the cell withering, and calculate the apoptosis index (Apoptotic index, AI). The other 6 in each group was used to detect the mRNA expression of Caspase-1 only by RT-PCR, and to compare the changes before and after the intervention of PPTA. The experimental data were analyzed by SPSS13.0 statistical software, and the difference between the groups was compared with the LSD test, and the P0.05 was statistically significant. Results: the TUNEL method showed the normal group, the sham operation group and the ischemia reperfusion. There was no or only individual part of apoptosis in the cochlear parts of the PPTA group. The apoptotic cells in the cochlea were significantly increased in the ischemia and reperfusion group, and the apoptotic cells decreased significantly after PPTA. The apoptosis index of the I/RI group was significantly higher than the normal group, the sham operation group and the PPTA group, and the Caspase-1 mRNA expression in the cochlear tissue of the P0.001. ischemia reperfusion group. Significantly higher than normal group, sham operation group and ischemia reperfusion group PPTA (P0.001), Caspase-1 mRNA expression in cochlear tissue of group PPTA of ischemia reperfusion was significantly higher than that of normal group (P0.001), and there was no statistical difference between normal group and sham operation group on the expression of Caspase-1 mRNA in cochlear tissue (P=0.825). Conclusion: through ischemia and reperfusion in cochlea of guinea pigs The damage model PPTA and the control test. There were no or rare apoptotic cells in the normal group of guinea pig cochlea. The number of apoptotic cells increased significantly in the ischemia reperfusion group, and the apoptotic cells decreased significantly. The expression of Caspase-1 mRNA in the cochlear tissue of ischemia reperfusion was significantly increased, and PPTA could partly reduce the expression of Caspase-1 mRNA. Apoptosis is a form of cell damage in the cochlear ischemia reperfusion group, and PPTA may protect the cochlea after ischemia reperfusion injury by inhibiting the expression of Caspase-1 mRNA and reducing the apoptosis.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R764

【参考文献】