【作者】 王云峰；

【导师】 李华伟； 林曦；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 第一部分:Cx26条件基因敲除小鼠的鉴定背景耳聋是临床上最为常见的感觉障碍之一,新生儿的耳聋将会严重的影响儿童的言语学习和言语能力获得。耳聋可分为先天性耳聋和后天性耳聋。先天性耳聋是新生儿中是最常见的出生缺陷之一,大约每500个出生的婴儿就会有一个被诊断为先天性耳聋,另外,在剩下的儿童中大约每一千个儿童中就会有一个儿童,在成年之前发展为重度感音神经性耳聋。先天性耳聋分为遗传性耳聋和非遗传性耳聋。非综合症型耳聋占遗传性耳聋的80%以上。Cx26基因的突变引起的耳聋人群占遗传性非综合症型语前聋病人总数的的50%以上。近年来,对于Cx26突变导致耳聋的研究进展迅速。但是对于Cx26突变导致耳聋的发病机理和随后的病理变化仍然并不清楚。由于小鼠与人类具有很大的基因同源性,基因敲除小鼠为此项研究提供了很好的方法。本实验室建立了两种新的Cx26条件基因敲除小鼠模型即Foxg1-Cre cCx26ko和Pax2-Cre cCx26ko条件敲除小鼠。为进一步研究Cx26基因敲除对耳蜗的影响,首先需要验证两种小鼠符合条件基因敲除的条件。方法通过Foxg1-Cre and Pax2-Cre小鼠与ROSA26 reporter小鼠进行杂交证明两种小鼠模型在胚胎期,cre已经产生有活性的表达,随后通过PCR的方法来验证所产生小鼠的基因型,挑选出符合特定基因型的Cx26条件敲除小鼠。并用western blot来验证所选小鼠耳蜗中各部位的Cx26蛋白的表达是否有所变化。最后通过对冰冻切片和耳蜗铺片的免疫组化的方法进一步证实在Cx26条件基因敲除小鼠的耳蜗Cx26表达发生的变化。结果在E11.5,E12.5天时Cre已开始在内耳表达,在P1天和P30天的报告小鼠的耳蜗切片显示,Cre表达在小鼠耳蜗的感觉上皮和螺旋神经节。PCR鉴定出符合基因敲除条件的小鼠基因型为Cx26^loxP/loxP;pax2^cre/+和Cx26^loxP/loxP;Foxg1^Cre/+。western blot的结果证明:Cx26蛋白水平在两种基因敲除小鼠的耳蜗corti器表达降低,但在螺旋韧带和血管纹中表达并未降低。cCx26ko小鼠在的耳蜗的冰冻切片和免疫组化显示,Cx26蛋白的表达部位位于骨螺旋板缘和螺旋韧带,而在corti氏器中的表达是缺失的。结论由PCR,western blot和免疫组化得到的结论为,本实验所用的两种小鼠从基因水平和蛋白水平,均符合Cx26基因敲除的条件,两种小鼠的基因型为Cx26^loxP/loxP;pax2^cre/+和Cx26^loxP/loxP;Foxg1^Cre/+。可以用于进一步的研究。第二部分:Cx26条件基因敲除小鼠出生后病理组织学和听觉功能的变化背景Cx26突变占人类非综合症型语前聋患者的一半以上,在不同的人群分布中略有不同。在中国人中,由cx26突变导致的先天性耳聋大约占到总人数的12.2%。其中235delC是最为常见的突变,虽然对于Cx26导致耳聋的报道较多,但多集中在遗传学分析和家系研究,对于Cx26突变的致聋机理仍然不清楚。基因敲除动物模型的出现为我们研究特定基因突变导致的疾病,提供了有力的方法和途径。因此在研究Cx26的过程中,通过研究Cx26条件基因敲除小鼠的出生后的耳蜗的病理学变化,可以为间接地去理解人类Cx26突变导致的病理学改变提供帮助,通过对于Cx26条件基因敲除小鼠的听觉功能研究去探讨其发病机制方法通过免疫组化和树脂切片方法验证与对照小鼠的形态学差异。通过听觉脑干诱发电位和内淋巴电位的测定,了解Cx26条件基因敲除小鼠的听觉改变。通过独立样本t检验,对听力结果进行分析。结果免疫组化的结果发现P2时Prox1和P75及mysinⅥ的染色Cx26条件基因敲除小鼠与对照野生小鼠比较没有差异,Cx26ko基因敲除小鼠模型的树脂切片观察发现,在Cx26ko基因敲除小鼠出生的早期,在P4天与WT小鼠在耳蜗的顶圈和中圈的的形态学比较无显著的变化,P8天时Corti隧道和Nuel间隙未打开,在出生后14天,cCx26ko小鼠的基底膜中有明显的毛细胞死亡和丢失,其主要位置位于中圈,P21天时,耳蜗中圈的感觉上皮全部丢失,仅留下一层类上皮细胞。耳蜗的顶圈没有观察到细胞死亡,但是Corti器形态发育异常,耳蜗中Corti器隧道,和Nuel间隙未打开。在P18天开始出现螺旋神经纤维的退缩,在P21天可以观察到螺旋神经节的部分丢失,在P1m中圈的螺旋神经节出现了更多的丢失和死亡。P2m时仅留下零星的皱缩的螺旋神经节的胞体。测定P15,P21和P1m的的内淋巴电位:在P15到P1m,对照的杂合子野生小鼠的内淋巴电位在92±1毫伏左右,而在P15天的cCx26ko小鼠的内淋巴电位分别为35±1毫伏和66.5±1.5毫伏。P21天cCx26ko小鼠的内淋巴电位分别为33.5±1.5毫伏和71±3毫伏。P1m cCx26ko小鼠的内淋巴电位分别为平均35.5±1.3毫伏和69±1毫伏。cCx26ko小鼠动物模型有严重的听力损失,两组间的t检验发现从4K 8K12K 18K 24K 32K频率与杂子对照小鼠来比较,各个频率均有显著地差异p＜0.01。结论Cx26蛋白在耳蜗中表达水平的降低导致了。cCx26ko小鼠模型的耳蜗在出生后的结构发育异常,听力严重障碍,内淋巴电位有不同程度的降低。第三部分:Cx26条件基因敲除小鼠治疗的初步探索背景对于治疗由Cx26导致的中重度感音神经性耳聋的,最有效的方法就是人工电子耳蜗的植入。但是对于人工耳蜗而言,电信号需要有效地传入大脑的听觉中枢,需要幸存有较为完好的螺旋神经节,以利于电刺激的传入。因此对于cCx26ko基因敲除小鼠而言,要达到拯救听力的目的,拯救螺旋神经节的存活是一种具有可行性的途径之一。另外一种可行的治疗方案就是,根据cCx26ko基因敲除小鼠的病理学变化,从病因学上给予治疗。在内耳细胞死亡之前就进行有效地干预,从而延缓甚至逆转细胞死亡。达到改善或提高听力的目的。本实验室Ahmad等人通过遗传学过表达的方法使Cx30基因敲除小鼠的听力得到了恢复使人们看到了治疗遗传性耳聋的新的方法。即通过过表达一种与之相关联的蛋白水平,拯救其功能。但是此种方法只能通过基因工程的方法来实现,应用腺病毒携带相关的基因进入耳蜗,表达缺失的蛋白,从而提高Cx26的蛋白表达量。就有可能恢复耳蜗的正常形态和功能。方法运用7,8-Dihydroxyflavone对Cx26条件基因敲除小鼠治疗,每日每克体重5ug,P4开始注射直到P64,进行ABR的测定然后进行耳蜗组织学的观察。基因治疗的方法,P14天C57小鼠的耳蜗中阶和圆窗膜注入1ul五型腺病毒,三天后断头取听泡,观察耳蜗铺片和切片的转染情况。结果78DH治疗组的Foxg1-cre cCx26ko小鼠在注射药物78DH每日每克体重5ug的情况下,整个耳蜗连续切片观察来看,从顶圈开始到底圈为止,没有看到明显的细胞崩解的迹象,但是内隧道和Nuel间隙依然没有打开。螺旋神经节的形态基本正常,没有明显的死亡和崩解。78DH药物治疗组与对照组的听力学比较:听觉脑干诱发电位测定的结果在各频率的比较t检验显示没有明显区别（P＞0.05）在P14天的小鼠耳蜗注射Ad5-EGFP,三天后可见在基底膜的支持细胞层有绿色荧光蛋白表达。结论78DH初步证明可以有效的抑制和延缓,Cx26条件基因敲除小鼠中的螺旋神经节丢失和死亡。5型腺病毒可以转染耳蜗的支持细胞。可用于今后的Cx26条件基因小鼠的治疗研究。更多还原

【Abstract】 PARTⅠValidation of the Cx26 Conditional Knockout Mice ModelBackground Deafness is one of the most prevalent sensory disturbances in human and neonatal deafness affects severely their verbal learning and communication ability. The deafness can be divided into congenital deafness and acquired deafness. Congenital deafness is a very frequent disorder occurring in approximately 1 in 500 live births and the remaining children inherit the tendency to develop hearing loss 1 in 1000 live births before the adult.Among those deafness cases,non-syndrome hereditary deafness accounted for more than 80%in hereditary deafness.The non-syndrome hereditary deafness caused by Cx26 gene mutations contribute to about 50%of inherited prelingual deafness cases.In the past several years,researches of the deafness caused by Cx26 mutation were numerous.However the molecular mechanismes of this kind of the deafness are still unknown and the corresponding mouse animal models are appropriate for studying.To investigate molecular mechanisms of deafness caused by Cx26 mutations,we generated two types of conditional Cx26（cCx26） null mice by cross mating.The genotypes of these mice are Foxgl-Cre cCx26ko and Pax2-Cre cCx26ko.Methods Cre reporter mice（Rosa26R mice） were used to monitor tissue specific Cre activation.Then the genotype was validated by regular PCR.Western blot was used to test the cx26 protein expression in cochlear of these mice.The immunohistochemisty of Cx26 and Cx30 was observed in order to locate the distribution of Cx26 and Cx30 in the cochlear of the mouse model.Results The expression of Cre started at as early as E11.5.The cochlear section showed that the Cre was expressed at sensory epithelial cell layer in the organ of Corti and spiral ganglion neurons.The PCR results revealed that the mice genotype as Cx26^loxP/loxP;pax2^cre/+ and Cx26^loxP/loxP;Foxg1^Cre/+.Western Blot results demonstrated that the Cx26 protein level was down-regulated only in the organ of Corti,not in the spiral ligament and Stria Vascularis.The cochlea frozen-section further displayed that the Cx26 was absent in the organ of Corti cell,but was still kept in the spiral ligament and spiral limbus.Conclusions The PCR,Western blot and immune labelling results demonstrated the Cx26 protein was absent in the epithelial cell layer of organ of Corti.In other words,the two conditional Cx26 null mice model were validated and can be further used in our following studiesPARTⅡPostnatal Pathology of the Cochlea in Cx26 Conditional Knockout MiceBackground Mutations in GJB2 gene are a major cause of autosomal recessive congenital heating loss.The general mutation rate of GJB2 is 12.2%（16/131） and the 235delC is the most common type of deafness-causing mutation in Chinese patients with congenital heating loss.Although there were some reports of Cx26 deafness cases,most of them focused on genetic analysis and family studies.The mechanism underlying Cx26 mutation-induced deafness remains unclear.Gene knockout animal models provide a powerful ways and means.Through studying the pathological changes and investigate the auditory function of the cochlea of the conditional Cx26 gene knockout mice,we can try to understand indirectly the cause of the deafness induced by Cx26 mutations in human being.Methods tmmunohistochemistry and resin sections in cCx26KO mice and control mice were used to determine the morphological differences in the organ of Corti.Through the auditory brainstem evoked potential and the determination of endolymphatic potential,the hearing of cCx26KO mice model were evaluated.Results We found no difference between the cCx26 null mice and the WT mice in the Proxl and P75 cochlear staining,indicating that the supporting cell can differentiate normally in the cCx26 null mice cochlear.The cochlear morphology was then observed in the cochlear resin section.At P4,no difference was observed in the cochlea.From P8,the tunnel of Corti started to open in WT cochlea,however,the cCx26 null mice stalked and never finished opening of tunnel of Corti.Starting at around P14,the out hair cell in the middle turn of Cochlea started to die and all of the epithelial cells in the organ of Corti including HC,supporting cells were totally lost at around P21.In contrast,the apical turn epithelial cells were spared.The neuron fiber degenerate at P18 and some of the SGN started to die at around P21.Most of the SGNs were dead at P1m and only few condensed SGNs were found in the P2m cochlear.The endolymph potential was measured in the WT and Cx26 null mice model respectively.In the WT,the EP is about 90±1 Mv from P15 to adult level.In contrast,the EP in the age corresponding cCx26 null mice displayed two distinct groups,the lower group has a low EP at about 35±1 mv and the higher group has a EP of 66.5±1.3mv.The ABR heating test showed that the two cCx26 null mice models were profoundly deaf and the threshold was significantly Higher than the WT at each tested frequency（4K,8K,12K,18K,24K and 32K）Conclusions The cCx26 null mice were severely deaf and the EP was impaired.The cochlear development was delayed and the organ of Corti was abolished from middle turn to basal turn,the apical turn was spared.PARTⅢExperimental Treaments for Cx26 Conditional knockout miceBackground For the treatment of the severe sensotineural heating loss induced Cx26 mutation,the most effective way is cochlear implantation.Performance with cochlear implants is highly variable and depends on many factors.The existence of the spiral ganglion is one of the important reasons.So increase the survival of spiral ganglion is an effective method.Another possible treatment options is overexpressing the related Cxs by a novel transgenic strategy;however,the translation of the finding into clinical applications is not applicable so far.Based on our finding,there is time window for the cell death in the organ of Corti.Therefore,timely intervene by injection drugs and gene therapy may prevent the organ of Corti cell death.Methods The conditional Cx26 gene knockout mice were treated by the 7,8-Dihydroxyflavone from P4 till P64.Then the ABR is tested and the cochlear of cCx26ko mouse was observed to evaluate the morphological changes.Another method is gene delivery.Ad5-EGFP was injected into the scala media through a cochleostomy in C57 mouse,three days later,the pattern of EGFP in the cochlear was observed.Results After the 78DH injection at a dose of 5ug/g/d,the mice did not show much improvement of hearing threshold（P＞0.05）.However,all of the epithelial cells in the organ of Corti and the SGNs were kept intact after treatment although the tunnel of Corti was still unopened.The expression of GFP was firstly detected after 3 days injection of Ad5-Egfp in the supporting cells of cochlear.Conclusions 78DH can effectively protect the cell death in the organ and SGN in the Cx26 null mice model.Ad5-Egfp can be expressed in the supporting cells and can be a potential method for the treatment for cCx26 null deafness mice model.更多还原