【作者】 袁伟；

【导师】 张学渊；

【作者基本信息】 第三军医大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 一、研究背景和目的听力损失作为名列第十五位的世界疾病,在发达国家,约有一亿人遭受听力损失,且90%为感音神经性聋。据我国残疾人抽样调查统计,听觉残疾呈逐年上升态势。毛细胞病变所导致的听力丧失是耳聋的主要原因,因此如何保护毛细胞已成为科研工作者日益关注的课题。耳蜗毛细胞是将声刺激转化电化学能的末梢感受器,其中内毛细胞是感受器细胞,负责将机械振动转化为与之相连的听神经纤维的动作电位;外毛细胞与增强听神经的高度频率选择性、耳蜗自我的调节功能有关。内、外毛细胞的损伤都可引起不可逆的感音神经性耳聋。耳蜗位于蜗壳内,被其包裹和保护,同时存在血迷路屏障,这种独特的生理结构导致耳蜗在解剖和功能上相对独立,一旦受到损伤很难通过全身或局部循环与外界交流,主要靠自身代谢来排除损伤因素,因此如何提高毛细胞自身的保护功能,保护正常细胞不被进一步破坏是研究这一问题的关键。因此急切寻找一条途径对毛细胞进行持久的保护。机体遇到外界有害因子刺激时会产生防御性反射,而很多细胞在遇到有害因素刺激时会产生一些保护性蛋白,锌指蛋白A20就是其中之一。锌指蛋白A20首先是在人的内皮细胞中作为肿瘤坏死因子(TNF)刺激的应答产物被发现的。通过基因测序发现其可读框编码一个新型锌指蛋白,命名为锌指蛋白A20,简称A20。A20是一种存在于胞液的NF-κB(nuclear factor-κB)活性负反馈调控蛋白,在许多细胞中都发现有A20的表达,A20 mRNA中的3’端非翻译区内有4个规范重复序列ATTTA,正是该序列决定了A20在细胞表达的不稳定性。A20已在多种细胞如内皮细胞、脑神经细胞中显示有抗炎、抗凋亡、坏死作用。研究表明,毛细胞的损伤会导致NF-κB和Caspase-3信号通路的改变,这一点和A20的抗损伤通路之间有共同交叉作用点,前者上调NF-κB和Caspase-3的表达,后者抑制NF-κB和Caspase-3的表达,因此A20可用于毛细胞的防护研究。A20在体内的表达是快速而短暂的,因此需要体外构建载体来持续表达A20,从而发挥其抗损伤作用。腺病毒(Ad)载体具有高效率、低致病性、高滴度及不整合入宿主细胞染色体等优点,已被广泛应用于内耳基因治疗研究。造成毛细胞损伤有各种各样的原因,其中药物致耳毒性是日常生活中很常见的原因;同时现代社会、现代战争中电磁辐射尤其是高频电磁辐射也是威胁人类健康的重要原因,因此本实验选用这两个损伤模型来进行研究。通过研究初步明确锌指蛋白A20对内耳毛细胞的保护作用及其分子机制,为改善和修复受损的听功能提供了理论依据。基于以上分析,本研究首先建立了庆大霉素、高功率微波(HPM)致毛细胞损伤模型,观察了A20在毛细胞正常、损伤条件下的表达规律,然后制备纯化A20重组腺病毒表达载体pAdEasy-1/A20,观察其在庆大霉素、HPM两种损伤条件下对毛细胞的保护作用,并对其可能的机制进行了探讨。二、研究方法和结果1. HPM损伤毛细胞模型的建立:将豚鼠置于反射系数近似为零的微波暗室内进行辐照,辐照剂量分别为30 W/cm2、65 W/cm2、90 W/cm2,辐照时间20min,辐照后3 h、6 h、12 h三个时相点观察。结果发现不同功率的辐照对豚鼠的神经行为、体温产生程度不一的影响,说明HPM可致豚鼠热效应损伤;ABR阈值测定显示功率65 W/cm2辐照6 h后出现显著性差异;光镜、电镜观察65 W/cm2 6 h后出现形态学改变,毛细胞损伤数目较辐照前有差异,尤其是内毛细胞损伤较大,细胞间出现许多球形物质。这部分实验说明HPM可以造成毛细胞损伤,65 W/cm2 6 h可以作为损伤观察的研究剂量。2.庆大霉素损伤毛细胞模型的建立:给予庆大霉素120㎎/㎏腹腔注射,连续10天,通过ABR、光镜、电镜观察发现庆大霉素功能上可以造成听力下降,形态上可以造成毛细胞损伤,其损伤主要表现为外毛细胞,第一排外毛细胞的损伤较第二、三排重。3.锌指蛋白A20在豚鼠耳蜗毛细胞的表达研究:通过耳蜗冰冻切片、A20原位杂交显示正常情况下A20在体内沉默表达,HPM、庆大霉素损伤后3 h即出现表达,但6 h后表达即减弱,12 h后表达基本消失。说明体内毛细胞存在A20基因,其在损伤条件下出现短暂表达。4. pAdEasy-1/20腺病毒的制备及其在体对毛细胞作用的表达:通过酶切连接的方式将pCAGGS-FLAGmA20质粒的表达框连入pAdtrack-CMV穿梭载体,后者和Adeasy-1在大肠杆菌中进行同源重组,随后转染293细胞进行病毒颗粒包装。经单、双酶切及PCR鉴定正确,大量扩增病毒后,通过GFP报告基因确定病毒滴度为5×109 pfu /ml。5. pAdEeay-1/A20重组腺病毒对毛细胞的保护作用研究:通过耳蜗冰冻切片及免疫组化观察,pAdEeay-1/A20可以成功感染毛细胞;我们把研究对象按毛细胞损伤模型分为庆大霉素和HPM两个大组,每组又分为5个实验组,分别为正常对照组、pAdEeay-1组、pAdEeay-1/庆大霉素注射(或HPM)组、pAdEeay-1/A20/庆大霉素注射(或HPM)组和人工外淋巴液/庆大霉素注射(或HPM)组。结果发现正常对照组处死前后ABR听阈无明显变化,P>0.05;pAdEeay-1组耳蜗给药前、后动物ABR反应阈变化无统计学意义(P>0.05),表明耳蜗给药操作及重组腺病毒对动物ABR反应阈无明显影响,未导致内耳损伤;pAdEeay-1/庆大霉素(或HPM)组处死前后听反应阈值变化明显,P<0.01,提示不含A20的pAdEeay-1未对庆大霉素(或HPM)造成的听功能下降起到保护作用;pAdEeay-1/A20/庆大霉素(或HPM)组及人工外淋巴液/庆大霉素(或HPM)组听反应阈均提高,两组与正常对照组比较差异有统计学意义(P<0.05),但pAdEeay-1/A20/庆大霉素(或HPM)组听阈增高程度较人工外淋巴液/庆大霉素(或HPM)组低,两者统计学比较有差异,两组之间P<0.05,提示A20能够对庆大霉素(或HPM)所造成的听功能下降起到一定的保护作用,但这种保护作用是不完全的。毛细胞形态学观察同样发现pAdEeay-1/A20/庆大霉素(或HPM)组与正常对照组比较毛细胞损害数目有差异(P<0.05),但同时pAdEeay-1/A20/庆大霉素组(或HPM)与pAdEeay-1/庆大霉素组(或HPM)、人工外淋巴液/庆大霉素(或HPM)组比较毛细胞损伤数目又有有明显差异(P<0.01)。说明A20可以在形态上对内、外毛细胞起到一定的保护作用。6.锌指蛋白A20保护毛细胞的作用机制:理论上A20抗损伤和毛细胞的损伤通路上都有一个共同作用点就是caspase-3,但A20保护毛细胞是否是作用于caspase-3这一通路还不得知,本研究采用免疫组化,通过对损伤组(庆大霉素、HPM)、pAdEeay-1/A20组比较发现免疫标记在正常对照组和空载体组中都呈阴性反应;损伤组和pAdEeay-1/A20组中冰冻切片标本均显示有大量含棕黄色颗粒细胞出现在耳蜗Corti器内,其中毛细胞和支持细胞均有阳性细胞出现,但pAdEeay-1/A20组的阳性颗粒细胞数明显少于损伤组,损伤组与pAdEeay-1/A20组阳性细胞数经IOD统计,结果具有显著差异性,P<0.05。三、结论1.本实验成功制作电磁辐射(HPM)损伤毛细胞模型,发现HPM可以造成毛细胞损伤,HPM对毛细胞的损伤效应是随剂量和时间的变化而变化的,同时发现HPM主要对内毛细胞造成明显的损伤,而且细胞间有不规则的球状物出现。2.锌指蛋白A20在正常毛细胞中沉默表达,当受到外界损伤因素刺激后出现一过性表达,其表达时效仅数小时。3.通过构建重组pAdEeay-1/A20腺病毒载体以及在毛细胞的有效感染,证实了A20可以从功能和形态上对庆大霉素和HPM造成的毛细胞损伤起到一定的保护作用。4.本研究表明锌指蛋白A20保护毛细胞的作用机制之一可能是通过抑制caspase-3的表达来实现的。更多还原

【Abstract】 1 Background and aimsHearing loss is the fifteenth common disease in the world. Even in developed countries, there are about 100 million people suffering from it, and 90% among them are sensorineural hearing loss. According to the data collected by a sampling survey on the handicapped in our country, auditory deformity is in a trend of rising year by year. Hearing loss due to hair cell damage is the main reason for deafness, so how to protect hair cells becomes the focus of scientific researchers. Cochlear hair cells, the terminal receptor which transforms the acoustic stimulus into chemical energy, consists two kinds of cells, inner and outer hair cells. The former are the receptor cells which transform the mechanical vibration into the action potential of the connected auditory nerves, and the later are involved in the enhancement of the auditory nerves on height-frequency selectivity, and regulation and self-protection of the cochlea. The lesion of either outer or inner hair cells can lead to irreversible sensorineural hearing loss. Although being enveloped and protected in a snail-shaped bony structure and with the help of blood-labyrinth barrier, the cochlea is relatively independent in anatomical and functional aspects. Once it is impaired, it will be difficult to communicate with outside via systemic or local circulation, and has to depend on its own metabolism to eliminate the injury factors. In this way, the key point of this issue is to improve the self-protective ability of hair cells so as to prevent the normal cells from damage. We need to find a factor which can exert consistent protection on hair cells.When there are exogenous deleterious factors, our body will generate defensive reflection to response them, while for cells, they will produce some protective proteins. Zinc finger protein A20 is one of them. This protein was firstly found as the product when TNF was used to treat human endothelial cells. Gene sequencing revealed that it encodes a new zinc protein, so was named as zinc finger protein A20. The zinc finger protein A20 exists in the cytoplasma, which negatively regulates the activity of nuclear factor-kappa B (NF-κB), and is expressed in many cells. There are four standard repeats ATTTA in the 3’ untranslated region of the A20 mRNA, which indicating the unstable expression of A20. A20 exerts anti-inflammation, anti-apoptosis, and anti-necrosis effects in various cells, such as endothelial cells, and brain nerve cells. It has reported that hair cell injury results in the alteration of NF-κB and Caspase-3 signal pathways, leading to up-regulation of these two proteins. While A20 can inhibit the expressions of NF-κB and caspase-3. Thus, we infer A20 can protect hair cells through caspase-3. Since A20 is expressed rapidly and transiently, recombinant expression vector is need to obtain stable expression to perform its anti-injury effect. Adenovirus vector is well adopted in the treatment targeting inner ear genes because of its high-efficiency, low pathogenicity and high-titer.There are various causes for hair cell injury, and drug poisoning is the most common one. What’s more, in this modern society and during modern war, electromagnetic radiation, especially that of high-frequency, becomes a main reason threatening human’s health,. Base on this, we employed these two kinds of injury models of hearing loss. We aim to found theoretical basis for A20 improving and repairing injured auditory function by exploring its protective effect and possible molecular mechanism on inner hair cells.Based on above mentioned analyses, we established the hair cell injury models induced by gentamicin ototoxicity and high power microwave, observed A20 expression in normal and injured hair cells, then explore the protective effect of A20 by constructing recombinant adenoviral expression vector pAdEasy-1/A20 and the possible mechanism.2 Methods and results2.1 Establishment of HPM-induced hair cell injury modelGuinea pigs was placed in a microwave anechoic chamber with a reflection coefficient of approximately zero, and exposed to radiation of 30, 65 or 90 W/cm2 for 20 min. Then the animals were observed 3, 6 and 12 h after radiation. Changes of neurobehavior function and body temperature were observed in guinea pigs exposure to different power radiation, indicating that HPM leads to thermal effect injury. Significant difference was found in the evoked potentials of auditory brainstem response (ABR) in guinea pigs 6 h after being exposed to 65 W/cm2 radiation. Light and electron microscopy observed that there were more injured hair cells, especially inner hair cells, and many globular objects in the group 6 h after 65 W/cm2 radiation. These results suggested that HPM resulted in hair cell injury and 6 h after 65 W/cm2 radiation could be used as the dose for damage study. 2.2. Establishment of gentamicin -induced hair cell injury modelThis model was established by i.p. injecting 120㎎/㎏ gentamicin for 10 d. ABR, light and electron microscopy discovered that gentamicin caused hearing loss, made the damage of hair cells morphologically, especially those inner cells. The damage of first row hair cells was more severe than those at second and third rows.2.3 Expression of A20 in the cochlear hair cells of guinea pigsIn situ hybridization indicated that A20 was not expressed in normal cochlea, but begun to express 3 h after gentamicin ototoxicity or HPM radiation, declined in 6 h and almost disappeared in 12 h. These data indicated that A20 was transiently expressed only under damage condition in cochlea inner hair cells .2.4 Construction, purification and expression of adenoviral vector pAdEasy-1/20 in hair cells in vivoAfter the expression cassette of plasmid pCAGGS-FLAGmA20 was cloned into shuttle vector pAdtrack-CMV by enzyme ligation, the recombinant was amplified in E. coli Adeasy-1 and then packaged in 293 cells. After identified by single and double enzyme digestion and PCR, the virus was expanded and purified, and yield a titer of 5×109 pfu /ml with the aid of reporter gene GFP.2.5 Protective effect of recombinant adenovirus pAdEeay-1/A20 on hair cells Immunohistochemical methods indicated that the recombinant adenovirus could infect hair cells in vivo. The guinea pigs in this study were divided into normal control, pAdEeay-1 group, pAdEeay-1 and gentamicin/(or HPM) group, pAdEeay-1/A20/ gentamicin/(or HPM) group, and artificial perilymph solution/gentamicin group. There was no significant difference in hearing threshold of ABR in the animals of normal control group and of pAdEeay-1 group before and after drug administration into the cochlea, indicating that this way of administration and recombinant adenovirus had no obvious effect on the hearing threshold of ABR. Significant change was observed in animals from pAdEeay-1 and gentamicin/(or HPM) group, suggesting that there was no protective effect of pAdEeay-1 on hearing loss. The hearing threshold of ABR was significantly higher in pAdEeay-1/A20/ gentamicin/(or HPM) group and artificial perilymph solution/ gentamicin group in comparison with that in normal control group. That of pAdEeay-1/A20/ gentamicin/(or HPM) group was significantly lower than that of artificial perilymph solution/ gentamicin group. These results implied that A20 exerted protective effect on hearing loss induced by gentamicin ototoxicity or HPM radiation, but the protection was not complete. Morphological study found that there were lesser injured hair cells in pAdEeay-1/A20/ gentamicin/(or HPM) group and artificial perilymph solution/ gentamicin group than in normal control group, with the former better than the later group, indicating that A20 could protect outer and inner hair cells morphologically.2.6 Mechanism of A20 protecting hair cellsTheoretically, the pathway of A20 exerting its anti-injury effect has a crossing point with the damage mechanism of hair cells, that is, caspase-3. We aimed to investigate whether caspase-3 played a role in this protective process. Immunohistochemical method was used to detect its expression in above mentioned group. It was negatively expressed in normal control group and of pAdEeay-1 group, strongly expressed in gentamicin/(or HPM) group and pAdEeay-1/A20/ gentamicin/(or HPM) group. It was distributed in the organ of Corti, hair cells and cells of Deiters. But the caspase-3 expression was significantly milder in pAdEeay-1/A20/ gentamicin/(or HPM) group than in gentamicin/(or HPM) group.3 Conclusion3.1 HPM-induced hair cell injury model is successfully established in guinea pigs. HPM is proved to damage hair cells in a dose- and time- dependent manner, and mainly injures inner hair cells.3.2 Zinc finger protein A20 is not expressed in normal cochlear hair cells, but transiently expressed after the stimulation of external injury factors.It,s expression maintained only several hours.3.3 Zinc finger protein A20 has certain protective effect on the function and morphology of hair cells after gentamicin ototoxicity or HPM radiation via the construction of recombinant adenovirus pAdEeay-1/A20 and its transfection to hair cells.3.4 Zinc finger protein A20 may protect hair cells by inhibiting the expression of caspase-3.更多还原