發(fā)布時間：2018-05-14 02:01

  本文選題：耳蝸 + 缺血/再灌注損傷�。� 參考：《南方醫(yī)科大學》2015年碩士論文

【摘要】：研究背景聽力障礙是常見的耳鼻喉科疾病,世界衛(wèi)生組織于2013年發(fā)布的報告顯示,全球約約5.3%人口患有聽力障礙,也就是說約有3.6億的人群受困于殘疾性聽力,其中約67%的聽力障礙患者在發(fā)展中國家。作為全球最大的發(fā)展中國家,全國約有2057萬聽力障礙患者,占總人口的16.79‰,患者人數居各類殘疾之首,是影響居民生活質量和身體健康的重要疾病之一。因此,探索和發(fā)現聽力障礙的預防和治療方法是當前醫(yī)學界的熱點課題,也是我們面臨的巨大挑戰(zhàn)。聽力障礙主要包括感音神經性耳聾、傳導性耳聾和混合性耳聾,其中感音神經性耳聾患者數量最多。感音神經性耳聾的致病因素包括老年性、缺血、病毒感染、聽神經病、藥物耳毒性、遺傳、中樞疾病、自身免疫疾病、噪音性以及腫瘤等。它是指內耳和其神經傳導通路病變引起的各種聽力障礙,通常涵蓋螺旋神經節(jié)、毛細胞、耳蝸神經、突觸復合體以及聽中樞等組織和器官的病理性改變。內耳中,感受器細胞包括外毛細胞(outer hair cell,OHC)和內毛細胞內毛細胞(inner hair cell,IHC)兩類,它們都可將機械能轉變?yōu)樯镫娔?即產生感受器地位電位。螺旋神經節(jié)屬于傳入神經元,可將接收感受器發(fā)出的電位信號,并將信號傳入中樞,進而產生位置覺或聽覺。內、外毛細胞分別可同螺旋神經節(jié)的Ⅰ型、Ⅱ型傳入神經元樹突構成傳入突觸。聽覺的形成過程是內毛細胞感應外界聲音信號,并將強度頻率不同的機械信號轉變?yōu)殡娦盘?形成感應定位,進而通過傳入突觸將信號傳遞給螺旋神經元,螺旋神經元再將信號傳導至中樞系統,中樞系統產生聽覺。外毛細胞可放大外界聲音信號,提高耳蝸對聲音頻率的選擇性和敏感性,是內皮細胞的重要功能輔助細胞。內毛細胞傳入突觸的形態(tài)、功能、結果和數量的異常改變是引發(fā)感音神經性耳聾的重要因素。事實上,在聲音信號傳入通路中,毛細胞、螺旋神經節(jié)等結構元件的損傷都會導致聽覺障礙。耳蝸是人聽覺系統的關鍵結構,屬于高耗能組織,缺氧缺血狀況下會導致傳入神經腫脹、毛細胞非自然性死亡進而產生聽力損傷；噪音和藥物中毒會一定程度上損傷螺旋神經元和毛細胞,也是感音神經性耳聾的常見致病因素。然而有研究顯示缺血5min后引起傳入神經腫脹可通過再灌注逐漸恢復。感音神經性耳聾的病理現象涵蓋了螺旋神經節(jié)、毛細胞、神經末梢以及支持細胞的器質性改變,其常見的致病因素包括病毒感染、缺血、老年性退行性變以及耳毒性藥物中毒等,其中缺血是導致感音神經性耳聾的最普遍因素,然而血流障礙普遍表現為缺血后再灌注損傷,極少數為純粹的缺血損傷。比如突發(fā)性耳聾普遍認為是內耳微循環(huán)障礙引起的聽覺障礙。有數據顯示單純缺血引起的損失顯著小于缺血后再灌注引起的損失,可見研究內耳缺血/再灌注損傷(I/RI)的相關機理對于預防和治療內耳疾病十分關鍵。如今在哺乳動物當中,認為毛細胞發(fā)生損傷,則很難再生,目前仍在研究治療感音神經性耳聾的方式,例如再生毛細胞或是移植耳蝸干細胞進行治療。出現感音神經性聾的一個關鍵因素傳入神經系統損傷這一病理改變,神經末梢、螺旋神經節(jié)細胞以及突觸復合體等都屬于傳入神經系統,突觸結構在外界環(huán)境的影響會損害其功能,從而出現異常,但是受損螺旋神經節(jié)細胞很難進行修復。曾經有實驗對其進行研究,損傷后的傳入神經可以再生神經纖維,同時毛細胞可以關聯再生的神經纖維；與此同時,施萬細胞會在螺旋神經節(jié)細胞死亡后進行增殖,但是卻不能向正常功能神經元細胞進行分化,因此也不能發(fā)揮應有的效果。當前,為了使得感音神經性聾患者能夠聽到外界的聲音一般進行助聽器的佩戴或是移植人工耳蝸,然而,因為人工耳蝸價格較高,人們也不能適應佩戴助聽器,所以沒有在大范圍應用,因此藥物是大部分患者主要的治療方式。所以,感應神經性聾的探索的方式應放在藥物治療上。鹽酸椒苯酮胺(peperphentonamine hydrochloride, PPTA),是我國自主創(chuàng)新研發(fā)的鈣增敏劑類強心藥及心肌保護劑,已獲得3個發(fā)明專利,11類化學藥品臨床試驗批件,且已經完成了127例Ⅰ期臨床試驗。臨床前研究表明其不僅具有保護受損心肌、增強心功能并能降低心肌耗氧量的雙重作用；還可通過增加SOD活性及GSH含量,減少NO含量,發(fā)揮神經保護作用,并且可降低缺血腦組織Caspase-1 mRNA的表達,表現出良好的抗凋亡作用。與心腦組織損傷類似。鈣超載、自由基、細胞凋亡也是耳蝸損傷的常見原因,PPTA對心腦的保護機制為本研究提供了方向。本實驗分為兩個部分,第一部分成功建立豚鼠耳蝸缺血/再灌注損傷模型。通過暫時性微動脈夾夾閉雙側椎動脈及右側頸總動脈,夾閉1小時制造缺血模型,松開三條動脈制造再灌注模型。可以有效的減少內耳血流灌注,成功的建立了缺血再灌注模型,該模型手術創(chuàng)傷小,術中動物死亡率低,術后存活率高,適合大量造模,為下一步實驗提供了良好的基礎。第二部分旨在從細胞凋亡(利用TUNEL技術檢測)和與凋亡有關的caspase-1的mRNA表達來探討椒苯酮胺對內耳缺血/再灌注損傷的保護作用及可能機制,以擴大椒苯酮胺的臨床適應癥,為椒苯酮胺用于缺血性內耳疾病的治療提供藥理學依據,可望開發(fā)出對內耳缺血再灌注損傷具有保護作用的新藥。第一部分耳蝸缺血/再灌注(I/R)豚鼠模型的建立目的建立豚鼠耳蝸缺血/再灌注損傷模型。方法隨機挑選24只3組豚鼠,這些豚鼠的標準必須是200-250g的體重、健康、有著靈敏耳廓反射,三組是空白對照組、正常組以及缺血再灌注組。采用暫時性微動脈夾對雙側椎動脈進行夾閉、用活結對右側頸總動脈進行結扎可以模擬暫時缺血缺血,制造再灌注模型,實驗中對耳蝸血流的檢測使用激光多普勒進行檢測(CoBF)。結果成功進行缺血模型的建立：雙側椎動脈及右側頸總動脈通過無創(chuàng)微動脈夾進行夾閉,夾閉5-10分鐘后再進行觀察,發(fā)現各組動物的耳蝸血流只有30%的原來水平,如果在此水平保持不變,說明成功建立了缺血模型。成功建立再灌注模型：1小時缺血模型完成后,不再夾閉右頸總動脈微動脈,將雙側椎動脈微動脈夾取下,避免椎動脈出現斷離,對各組動物的CoBF進行檢測,灌注10分鐘后血量恢復到70%的缺血前水平,該穩(wěn)定能夠進行一段時間的維持,表示成功建立了再灌注模型。結論通過暫時性微動脈夾夾閉雙側椎動脈及右側頸總動脈,夾閉1小時制造缺血模型,松開三條動脈制造再灌注模型。這可以有效的減少內耳血流灌注,成功的建立了缺血再灌注模型,該模型手術創(chuàng)傷小,術中動物死亡率低,術后存活率高,適合大量造模,為下一步實驗提供了良好的基礎。第二部分PPTA對豚鼠耳蝸缺血再灌注損傷后細胞凋亡及caspase-1的]mRNA表達的影響目的：1.觀察缺血再灌注損傷后豚鼠耳蝸細胞凋亡的情況。2.對缺血再灌注損傷后豚鼠耳蝸caspase-1的mRNA的表達與細胞凋亡間的關系進行分析。3.探討PPTA對豚鼠缺血再灌注損傷后凋亡細胞的保護作用。方法：采用前述的缺血再灌注模型,將48只豚鼠隨機分為4組,每組12只,分別為正常組、假手術組、缺血再灌注組對照組、缺血再灌注PPTA組(缺血60min行再灌注后立即經股靜脈注射10mg/kg鹽酸椒苯酮胺),缺血再灌注對照組以等量生理鹽水代替椒苯酮胺注射,24小時后取標本。每組中6只用TUNEL法觀察細胞凋亡的情況,計算凋亡指數(Apoptotic index, AI),組間比較檢測結果；每組中另外6只用RT-PCR法檢測caspase-1的mRNA的表達,并比較PPTA干預前后的變化。運用SPSS13.0統計軟件對實驗數據進行方差分析,用LSD檢驗比較組間差異,P0.05為有統計學意義。結果：TUNEL法顯示正常組、假手術組和缺血再灌注PPTA組耳蝸各部位沒有或僅有個別部位出現細胞凋亡,缺血及再灌注對照組耳蝸凋亡細胞明顯增多,PPTA干預后凋亡細胞顯著減少,I/RI組與正常組、假手術組、PPTA組比較,凋亡指數明顯升高,P0.001。缺血再灌注組耳蝸組織Caspase-1 mRNA表達量顯著高于正常組、假手術組和缺血再灌注PPTA組(P0.001)；缺血再灌注PPTA組耳蝸組織Caspase-1 mRNA表達量顯著高于正常組(P0.001)；正常組和假手術組耳蝸組織Caspase-1 mRNA表達量差異無統計學意義(P=0.825)。結論：通過豚鼠耳蝸缺血再灌注損傷模型PPTA及對照試驗。正常組豚鼠耳蝸各部位無或罕有凋亡細胞。缺血再灌注組凋亡細胞增多,進行干預后使凋亡細胞顯著減少。缺血再灌注耳蝸組織Caspase-1 mRNA的表達顯著增高,PPTA可部分但有效地降低Caspase-1 mRNA的表達。提示細胞凋亡是耳蝸缺血再灌組損傷的一種細胞損傷形式,PPTA可能通過抑制Caspase-1 mRNA的表達,通過減少細胞凋亡而對缺血再灌注損傷后的耳蝸起保護作用。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R764

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相關期刊論文 前1條

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