本文關(guān)鍵詞： 聽泡注射 慶大霉素 耳蝸 椒苯酮胺 聽力損傷 耳蝸 自噬 Na+-K+-2Cl-聯(lián)合轉(zhuǎn)運(yùn)體 內(nèi)皮素　出處：《南方醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景聽力障礙是主要的致殘疾病,據(jù)2013年WHO報(bào)道,全球約5.3%的人口患有聽力類疾病,其中大部分為發(fā)展中國(guó)家公民。中國(guó)作為最大的發(fā)展中國(guó)家,聽力殘疾人有2700萬,居各類殘疾首位,其中聾啞人有200多萬,并以每年3萬人的數(shù)量增長(zhǎng),造成個(gè)人、家庭和社會(huì)的巨大痛苦和沉重負(fù)擔(dān)。因此,降低人群中聽力障礙疾病的發(fā)病率對(duì)提高人口素質(zhì)、改善生活質(zhì)量有著重大意義,能否找到防聾治聾的有效方法,是我們目前面臨的巨大挑戰(zhàn),也是全社會(huì)的共同責(zé)任。臨床上將耳聾分為傳導(dǎo)性聾、感音神經(jīng)性聾及混合性聾三類,其中以感音神經(jīng)性聾的發(fā)病率最高且治療難度最大,為耳科最大難癥之一。感音神經(jīng)性聾(sensorineural hearing loss, SNHL)又可細(xì)分為感音性聾(sensory hearing loss),其病變部位在耳蝸,主要為螺旋器毛細(xì)胞損傷致聲音的感受功能異常,也稱耳蝸性聾(cochlear hearing loss);以及神經(jīng)性聾(nervous hearing loss),為聽神經(jīng)、聽覺傳導(dǎo)徑路及其各級(jí)神經(jīng)元受損害,致聽覺神經(jīng)沖動(dòng)傳導(dǎo)障礙以及聽皮層功能減退者,也稱蝸后聾(retrocochlear hearing loss)。感音神經(jīng)性聾是臨床常見、多發(fā)、疑難之疾病,包括藥物性聾、突發(fā)性聾、噪聲性聾、老年性聾等。其機(jī)制可能為通過氧自由基、機(jī)械力、鈣超載等致使聽覺功能區(qū)損害,病理改變主要分布于耳蝸和聽神經(jīng)。目前,感音神經(jīng)性聾最有效的治療方法為配戴助聽器或植入人工耳蝸,但是這兩種方法對(duì)聽力損害有所改善的病人使用是受限的,人工耳蝸植入術(shù)雖能獲得很好的效果,但受經(jīng)濟(jì)條件、手術(shù)適應(yīng)癥的限制；佩戴助聽器雖然有助于改善耳聾患者的聽力功能,但其本質(zhì)上并未對(duì)疾病本身進(jìn)行有效的治療。因此,藥物仍然是治療感音神經(jīng)性聾的重要手段。雖然感音性耳聾發(fā)生在急性期,藥物有時(shí)可以改善聽力下降,但傳統(tǒng)藥物治療有一定的限制,因此人們開始尋找新的藥物和治療方法。目前臨床用藥仍以全身給藥方式為主,然而,由于耳蝸血-迷路屏障對(duì)藥物通透性的影響,全身給藥后耳蝸中藥物濃度較低,甚至存在不能通過該屏障的問題,導(dǎo)致感音神經(jīng)性聾選擇全身給藥治療時(shí)效果不理想。而采用耳內(nèi)局部用藥可有效解決耳蝸血-迷路屏障對(duì)藥物有效濃度的影響。慶大霉素(Gentamicin,GM)是常見的殺菌力強(qiáng)抗菌譜廣的氨基糖苷類抗生素(Aminoglycoside antibiotics, AmAn), I臨床應(yīng)用廣泛,且因其特有藥理作用,很多臨床應(yīng)用難以替代。此外,GM全身毒性較小,故本實(shí)驗(yàn)采用GM建立感音神經(jīng)性聾的動(dòng)物模型。鹽酸椒苯酮胺(Piperphentonamine hydrochloride, PPTA)是一類治療心肌缺血再灌注保護(hù)藥,可降低心肌耗氧量,增加心肌細(xì)胞對(duì)鈣離子的敏感性,同時(shí)不引發(fā)鈣超載,目前已順利完成I期藥物試驗(yàn),正在進(jìn)行II期臨床藥藥理實(shí)驗(yàn)中。目前對(duì)于缺血再灌注損傷發(fā)生的機(jī)制研究多圍繞著細(xì)胞線粒體功能障礙、胞內(nèi)鈣超載等離子紊亂、氧自由基等方面進(jìn)行,這些損傷機(jī)制與氨基糖苷類抗生素(AmAn)耳毒性損傷的作用方面十分相似,提示PPTA亦可用于對(duì)GM耳蝸損傷的保護(hù)研究。當(dāng)外部刺激因素持續(xù)存在時(shí),細(xì)胞將發(fā)生死亡。目前大多數(shù)學(xué)者將細(xì)胞的死亡分為3種形式,即細(xì)胞凋亡、細(xì)胞自噬和細(xì)胞壞死。眾多研究已表明感音性耳聾存在著內(nèi)耳毛細(xì)胞的凋亡,但是否存在自噬尚無文獻(xiàn)報(bào)道。先期研究表明PPTA可改善豚鼠耳蝸缺血再灌注損傷,并抑制缺血再灌注后IL-1β、TNF-α,Caspase-3及Fas蛋白的表達(dá)。本次試驗(yàn)通過觀察豚鼠聽覺功能、耳蝸內(nèi)環(huán)境的影響因素、細(xì)胞自噬等方面對(duì)豚鼠耳蝸慶大霉素?fù)p傷及椒苯酮胺的保護(hù)作用和局部用藥情況進(jìn)行研究,探討椒苯酮胺對(duì)耳蝸慶大霉素?fù)p傷保護(hù)過程中細(xì)胞自噬相關(guān)蛋白LC3和Beclin1、NKCC1 mRNA、ET-1表達(dá)的影響,以期為椒苯酮胺用于感音神經(jīng)性聾的治療提供藥理學(xué)依據(jù)。本研究分為如下兩個(gè)部分：第一部分豚鼠GM耳蝸損傷模型的建立及PPTA給藥途徑和耳毒性的評(píng)估目的建立確切的由慶大霉素?fù)p傷所致的豚鼠感音神經(jīng)性聾模型,尋找PPTA合適的給藥途徑,并評(píng)估PPTA局部給藥治療內(nèi)耳疾病的一些基本情況,以便進(jìn)一步研究PPTA對(duì)豚鼠耳蝸GM損傷的治療情況。研究?jī)?nèi)容及方法健康成年純白紅目雄性豚鼠32只,體質(zhì)量300-350g,隨機(jī)分為4組,每組8只：對(duì)照組(control group,A組)、手術(shù)組(surgery group,B組)、PPTA組(PPTA group,C組)慶大霉素組(GM group,D組)。對(duì)照組不做處理；手術(shù)組行聽泡外側(cè)壁置管、聽泡內(nèi)注入人工外淋巴液0.1ml/次、3次/d、連續(xù)7d;PPTA組行聽泡外側(cè)壁置管、聽泡內(nèi)注入0.2%PPTA液0.1ml/次、3次/d、連續(xù)7d；GM組腹腔注射慶大霉素(GM),160mg/kg/d,連續(xù)7d。各組完成給藥后進(jìn)行聽性腦干反應(yīng)(ABR)測(cè)定,觀察ABR各波形的閾值及形態(tài)變化。ABR測(cè)定2-4小時(shí)后,手術(shù)組與PPTA組各隨機(jī)挑選2只豚鼠斷頭處死,剩余各6只老鼠再予聽泡內(nèi)注入0.2%PPTA液0.1ml,2組分別于給藥后15min、30min、60min各斷頭處死2只豚鼠,所有斷頭處死豚鼠均迅速取出聽泡,即各時(shí)間點(diǎn)8個(gè)聽泡,快速提取耳蝸外淋巴液,并采用超高效液相色譜-質(zhì)譜技術(shù)(UPLC(?))檢測(cè)其PPTA的藥物濃度。實(shí)驗(yàn)數(shù)據(jù)均以均數(shù)土標(biāo)準(zhǔn)差(賈士S)表示,采用SPSS19.0軟件進(jìn)行one-way AVONA方差分析,一組與多組之間比較時(shí)采用LSD法,P0.05時(shí)差異有統(tǒng)計(jì)學(xué)意義。結(jié)果ABR檢測(cè)反應(yīng)閾：手術(shù)組、PPTA組ABR反應(yīng)閾與對(duì)照組無明顯差異(P0.05)；GM組豚鼠ABR閾值明高于對(duì)照組(P0.001)。未給藥豚鼠耳蝸外淋巴液中為檢測(cè)出PPTA,給藥后15min、30min、60min的豚鼠耳蝸外淋巴液PPTA藥物濃度分別為(ug/ml):29.83±3.11、37.65±3.36、12.25±3.77。結(jié)論慶大霉素致豚鼠聽力損失作用明確,表明其可用于建立豚鼠損傷的動(dòng)物模型；聽泡注射簡(jiǎn)單方便,經(jīng)聽泡注射PPTA后豚鼠耳蝸外淋巴液中藥物濃度較高,同時(shí)豚鼠聽力未見損失,說明PPTA對(duì)豚鼠園窗膜的通透性良好,且其本身不具有耳毒性,可采用局部給藥應(yīng)用于豚鼠耳蝸損傷保護(hù)的研究。第二部分PPTA對(duì)豚鼠耳蝸慶大霉素?fù)p傷細(xì)胞自噬相關(guān)蛋白LC3和Beclin1、NKCC1 mRNA、ET-1表達(dá)的影響目的研究PPTA對(duì)慶大霉素致耳蝸損傷的保護(hù)作用及其與自噬蛋白LC3、beclin1、內(nèi)皮素(Endothelin 1 ET-1)、Na+-K+-2C1-共同轉(zhuǎn)運(yùn)體(Na+-K+-2Cl-cotransporter NKCC1) mRNA表達(dá)的關(guān)系。方法健康成年純白紅目雄性豚鼠60只,體質(zhì)量300-350g,隨機(jī)分為5組、每組12只：對(duì)照組(control group,A組)、模型組(model group,B組)、同期治療組(concurrent treatment group,C組)、造模后對(duì)照組(model control group,D組)、后期治療組(lately treatment group, E組)。所有豚鼠行聽泡置管手術(shù)處理后進(jìn)行給藥：對(duì)照組行腹腔注射生理鹽水十聽泡注入人工外淋巴液連續(xù)7d；模型組腹腔注射GM (160mg·kg-1·d-1)+聽泡注入人工外淋巴液連續(xù)7d；治療組腹腔注射硫酸慶大霉素注射液(160mg·kg-1·d-1)+聽泡注射PPTA連續(xù)7d；造模后對(duì)照組腹腔注射GM連續(xù)7d后,再行人工外淋巴液聽泡內(nèi)連續(xù)注入7d；后期治療組腹腔注射硫酸慶大霉素注射液(160mg·kg-1·d-1)連續(xù)7d后,再行PPTA聽泡內(nèi)連續(xù)注入7d。各組豚鼠完成給藥后進(jìn)行聽性腦干反應(yīng)測(cè)定(auditory brainstem response, ABR),分析各組ABR閾值變化及相關(guān)關(guān)系。所有豚鼠完成ABR結(jié)果測(cè)定后斷頭處死,迅速取出雙側(cè)聽泡并分離耳蝸,免疫印跡法檢測(cè)beclinl和LC3表達(dá)、PCR檢測(cè)NKCC1 mRNA的表達(dá)、免疫組織化學(xué)染色法檢測(cè)ET-1的表達(dá)情況。實(shí)驗(yàn)數(shù)據(jù)以均數(shù)士標(biāo)準(zhǔn)差(X±s)表示,采用SPSS 19.0軟件進(jìn)行one-way AVONA方差分析,一組與多組之間比較時(shí)采用LSD法,驗(yàn)水準(zhǔn)α為0.05。結(jié)果ABR反應(yīng)閾：治療組豚鼠ABR閾值明顯低于于模型組(P0.001),但明顯高于對(duì)照組(P0.001),后期治療組AB值明顯低于模型對(duì)照組(P0.001),但明顯高于治療組(P0.001),模型組、模型后對(duì)照組ABR反應(yīng)閩差異無統(tǒng)計(jì)學(xué)意義(P0.05)。自噬相關(guān)蛋白LC3和Beclinl的表達(dá)：模型組LC3Ⅱ(與actin的比值)及Beclinl的表達(dá)量較其余四組明顯升高,后期治療組LC3 II(與actin的比值)及Beclinl的表達(dá)量較造模后對(duì)照組低。模型組NKCClmRNA表達(dá)較余四組明顯升高(p0.05),后期治療組NKCClmRNA表達(dá)明顯低于造模后對(duì)照組(p0.05),造模后對(duì)照組NKCClmRNA表達(dá)較對(duì)照組明顯升高(p0.05)。ET-1蛋白免疫組化表達(dá)結(jié)果：模型組耳蝸各部位ET-1表達(dá)較其余四組明顯增高,對(duì)照組各部位ET-1表達(dá)低于其余四組,后期治療組各部位ET-1表達(dá)較造模后對(duì)照組降低。結(jié)論P(yáng)PTA可通過抑制耳蝸內(nèi)ET-1表達(dá)和NKCC的激活,維持耳蝸內(nèi)環(huán)境的穩(wěn)態(tài),維持耳蝸的正常生理功能,保護(hù)耳蝸組織免受損傷,保護(hù)聽力；通過調(diào)節(jié)自噬程序的發(fā)生來減輕耳蝸?zhàn)允尚约?xì)胞損傷,進(jìn)而減少聽力損失。PPTA對(duì)豚鼠慶大霉素的耳蝸損傷保護(hù)主要體現(xiàn)為拮抗作用,其抗損傷作用需早期及時(shí)應(yīng)用。
[Abstract]:On the background of hearing disorder is a major cause of disability, according to a 2013 WHO report, about 5.3% of the world's population suffers from hearing diseases, most of which are developing countries Chinese citizens. As the largest developing country, there are 27 million hearing disabilities, among all kinds of disabilities, the deaf people have about 2000000, and growth in the number of 30 thousand people each year, resulting in individual, family and society of great suffering and heavy burden. Therefore, to reduce the incidence of disease prevalence of hearing impairment to improve the quality of the population, is of great significance to improve the quality of life, to find effective methods to prevention and treatment of hearing loss, is a huge challenge facing us, but also the common responsibility of the whole society. Clinically, deafness divided into conductive deafness, sensorineural hearing loss and mixed deafness in three categories, with sensorineural hearing loss and the highest incidence of treatment is the most difficult, most difficult for otology disease A. Sensorineural hearing loss (sensorineural hearing, loss, SNHL) can be subdivided into sensorineural deafness (sensory hearing loss), the lesions in the cochlea, mainly for spiral hair cells injury induced by sound feeling dysfunction, also known as cochlear deafness (cochlear hearing loss); and sensorineural hearing loss (nervous hearing, loss) for the auditory nerve, auditory pathway and neuron damage, induced auditory nerve impulse conduction disorders and auditory cortex dysfunction, also known as retrocochlear deafness (retrocochlear hearing loss). Sensorineural hearing loss is a common clinical disease, multiple problems, including drug sudden deafness. Hearing loss, noise deafness, presbycusis. The possible mechanism is through mechanical force, oxygen free radical, calcium overload resulting in the damage of auditory function, pathological changes were mainly distributed in the cochlea and auditory nerve. At present, the sense of treatment of sensorineural hearing loss is the most effective For hearing aids or cochlear implants, but the use of these two methods to improve the hearing impairment of patients is limited, cochlear implantation can get good results, but because of economic conditions, indications of surgery; while wearing hearing aid helps to improve deaf hearing function, but the essence of the disease itself is not treated effectively. Therefore, the drug is still an important method in the treatment of sensorineural hearing loss. Although sensorineural hearing loss occurred in the acute stage, drugs can sometimes improve hearing loss, but the traditional drugs have certain limitations, so people began to search for new medicines and methods of treatment. At present, the clinical medication still to the systemic administration mode, however, because of the cochlear blood labyrinth barrier effects on drug permeability, after systemic administration of drug concentration in the cochlea is low, even can't pass the screen Avoidance problems lead to sensorineural hearing loss choose systemic drug treatment effect is not ideal. And the use of topical ear can effectively solve the cochlear blood labyrinth barrier effect of effective drug concentration. Gentamicin (Gentamicin, GM) is a common strong bactericidal antibacterial spectrum of aminoglycoside antibiotics widely (Aminoglycoside antibiotics, AmAn, I) is widely used in clinic, and because of its unique pharmacological effects, clinical application of many difficult to replace. In addition, the GM body is less toxic, so we adopt GM to establish the animal model of sensorineural hearing loss. Peperphentonamine hydrochloride (Piperphentonamine hydrochloride PPTA) is a kind of treatment of myocardial ischemia-reperfusion injury drugs can reduce myocardial oxygen consumption, increase the sensitivity of myocardial cells to calcium ion, and does not lead to calcium overload, has successfully completed phase I trial, the ongoing phase II clinical pharmacological experiment.. Study on the mechanism for the injury of the ischemia reperfusion around cell mitochondrial dysfunction, intracellular calcium overload in plasma disorders, oxygen free radicals and other aspects, the mechanism of injury and aminoglycoside antibiotics (AmAn) on the ototoxicity of injury is very similar, suggesting that PPTA can be used for the protection of research on GM cochlear injury. When external the incitants persist, cell death occurs. Most scholars will be cell death is divided into 3 forms, namely cell apoptosis, autophagy and cell death. Many studies have shown that sensorineural hearing loss is the apoptosis of hair cells. But whether autophagy has not been reported. Previous studies showed that PPTA can improve the guinea pig cochlea ischemia reperfusion injury after ischemia reperfusion, and inhibition of IL-1 beta, TNF- alpha, Caspase-3 expression and Fas protein. This experiment through the observation of auditory function in guinea pigs, Influence factors of inner environment, autophagy aspects of protective effect on injury of guinea pig cochlea of gentamicin and JBAT and topical, explore JBAT on autophagy of Cochlea Injury in gentamicin related protein LC3 and Beclin1, NKCC1, mRNA, ET-1 expression, in order to provide pharmacological basis. The treatment of sensorineural hearing loss as JBAT used. This study is divided into two parts as follows: the first part is evaluation and establishment of PPTA GM in the guinea pig cochlea injury model of the route of administration and to establish the exact ototoxicity induced by gentamicin injury in guinea pigs with sensorineural hearing loss model, looking for PPTA suitable route of administration the evaluation and some basic PPTA topical treatment of inner ear diseases, in order to further study the treatment of PPTA GM on cochlear damage in guinea pigs. The research contents and methods of health 鎴愬勾綰櫧綰㈢洰闆勬，

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