【作者】 王枫；

【导师】 邱建华； 陈阳；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 目的:听觉剥夺效应是指由于声学信息的减少导致的听觉功能的逐渐下降。人们早已注意到,嗅觉、视觉和听觉系统中感觉输入信号的改变会影响相应系统神经网络在形态和功能上的发育与成熟。以往专家认为,对于先天性耳聋的患儿,人工耳蜗的最佳植入时期是2-3岁语言发育期。美国FDA将人工耳蜗植入的最小年龄定为12月。为防止听障儿童的听觉剥夺,获得更好的语言能力,小龄婴幼儿的人工耳蜗植入受到广泛关注。越来越多的文献报道早期植入人工耳蜗有助于听觉的重建和语言的学习。小儿慢性分泌性中耳炎及双侧先天性小耳畸形作为常见的传导性耳聋,均已有较成熟的治疗方法,但人为干预的时机选择目前尚无定论。现有的研究证实听觉传入信号的减少会造成听觉中枢的功能紊乱。而这些研究均是采用人为破坏耳蜗完整性,以牺牲耳蜗功能为代价的。本实验采用外耳道填塞造成气导听觉剥夺动物模型,在探寻听觉传导通路异常的同时,把耳蜗形态和功能的改变作为另一个研究的对象。近年来,科学家们致力于开发干细胞移植来治疗一些传统方法无法治疗的疾病,并且已经取得一定的成绩。耳科学专家也把目光转向干细胞移植,以期发现治疗感音神经性耳聋的新的治疗手段。移植入体内的干细胞必须携带自己的身份标签,科学家才能利用其身份特征有目的地对其生物学特性进行观察。目前已有多种干细胞标记方法,不同的标记方法均有其各自的优缺点。本实验选择了三种不同的方法对移植的干细胞进行标记,观察了移植NSCs在正常成年大鼠耳蜗核的自然转归情况,为进一步研究移植NSCs在听觉剥夺动物模型中的作用进行了初步探索。方法:1. AD组动物饲养于密闭隔音室,于P12d起用助听器耳模材料行耳道填塞至P42d,填塞结束后分别检测DPOAE、ABR。动物灌注固定,行脑干和耳蜗的冰冻切片备用。2.耳蜗Corti器F-actin定位及听觉剥夺后的表达情况。分别取对照组、AD组耳蜗切片行F-actin免疫组化、免疫荧光及鬼笔环肽荧光染色。激光共聚焦观察毛细胞形态和细胞骨架的改变。Western blot进行耳蜗基底膜F-actin蛋白表达的半定量分析。3. TUNEL试剂盒检测凋亡。透射电镜观察螺旋神经节神经元,NSE染色并进行螺旋神经节神经元计数。4. FJC检测耳蜗核神经元变性。尼氏染色行耳蜗核形态学分析和章鱼样细胞计数。5.嗅球NSCs的体外培养及分化鉴定。分别取SD大鼠、C57BL/6-gfp小鼠孕鼠胚胎嗅球NSCs,进行体外培养并传代,用荧光倒置相差显微镜观察培养NSCs细胞的活力以及生长与分化过程。应用免疫细胞化学及免疫荧光染色方法对NSCs进行鉴定。6. SD大鼠胚胎嗅球NSCs的标记。稳定传代的SD大鼠嗅球NSCs,经由脂质体2000介导质粒pEGFP-N2转染后,经G-418筛选传代1月,收集细胞,流式细胞仪检测转染效率。稳定传代的SD大鼠嗅球NSCs,于培养液中加入核荧光染料Hoechst 33342,37℃孵育1小时。7.三种不同标记方法嗅球NSCs的耳蜗核定位注射。分别收取C57BL/6-gfp小鼠NSCs、绿色荧光蛋白转染的SD大鼠NSCs以及Hoechst 33342标记的SD大鼠NSCs,进行大鼠脑干耳蜗核定位注射。将SD大鼠麻醉固定于小动物立体定位仪上,根据大鼠脑立体定位图谱定位左侧耳蜗核,以尖端接有玻璃微电极的微量注射器吸取准备好的一种NSCs悬液,精确定位缓慢推注,手术后正常环境饲养。分别于2d、10d后行灌注固定,切取40μm层厚的脑片进行免疫荧光染色。结果:1. DPOAE检测结果显示,0.5、1、2、4、8kHz各频率DPOAE引出率与AD组相比,在频率0.5、1、4kHz处两组间差异有统计学意义;对照组及AD组ABR阈值分别为18.6538±1.58785dB SPL和28.4000±1.33775dB SPL,差异有统计学意义;ABR显示Ⅰ-Ⅴ波潜伏期对照组与AD组相比,均不具有统计学差异;Ⅰ-Ⅴ波幅值对照组与AD组相比,在Ⅱ波、Ⅲ波、Ⅴ波幅值AD组与对照组差异显著。AD组与对照组Ⅰ-Ⅴ波间期比较,对照组为3.978±0.435ms,AD组为3.629±0.416ms,P<0.001,差异显著,具有统计学意义。2. F-actin在耳蜗Corti器各类细胞均有表达,主要分布于静纤毛和内、外柱细胞,OHCs胞浆中分布较少。AD组耳蜗Corti器F-actin的表达较对照组均有减弱,在分布集中的细胞和部位减弱得更为明显。Western blot结果显示F-actin在听觉剥夺大鼠耳蜗基底膜表达量减少,其相对表达量与正常成年大鼠耳蜗基底膜F-actin相对表达量做比值,具有显著性差异。3.对照组耳蜗Corti器在血管纹处可查见少量TUNEL阳性细胞,在基底膜处未见TUNEL阳性细胞。AD组螺旋缘处有一定量的TUNEL阳性细胞,且有沿其表面分布的倾向,基底膜处查见支持细胞中有TUNEL阳性细胞,IHCs、OHCs均为TUNEL阴性。4.螺旋神经节神经元分为Ⅰ型和Ⅱ型两种神经元。透射电镜结果显示,AD组螺旋神经节神经元发生退行性变,胞体缩小,细胞间距增大。胞浆膜性结构增多,靠近胞膜,卫星细胞发育差,未形成髓鞘。胞浆内细胞器减少、退化。对照组螺旋神经节神经元NSE染色显示正常神经元NSE主要存在于胞浆,胞体着色明显,核着色略浅淡,胞体排列有一定密度,不能从形态上区分两型神经元。胞体发出的神经纤维着色较胞体略浅。AD组免疫组化显示神经元显色较对照组明显变浅,形态模糊,排列紊乱,神经纤维着色基本正常。神经元计数对照组结果为10.643±1.104,AD组结果为7.883±0.987, P<0.005,两者具有统计学差异。5.脑干切片FJC染色,在耳蜗核部位查见明显的绿色荧光阳性的细胞突起,部分呈纤维断端,部分为细长丝状,并有分支形成,但没有发现阳性的神经元胞体。6.脑干耳蜗核各核团神经元计数AD组小于对照组,具有统计学意义;神经元密度也小于对照组,但无统计学差异。PVCN章鱼样细胞计数和细胞直径AD组小于对照组,具有统计学意义;细胞密度和面积也小于对照组,但无统计学差异。7. C57BL/6-gfp小鼠、SD大鼠嗅球NSCs在培养过程中形成细胞球,表达nestin;诱导分化后可分化为MSE阳性的神经元和GFAP、Galc阳性的神经胶质细胞。8. C57BL/6-gfp小鼠嗅球NSCs在紫外激发光下呈现明亮的绿色荧光;SD大鼠嗅球NSCs EGFP转染后在紫外激发光下部分细胞呈现GFP阳性,呈现明亮的不规则形或环形,较小细胞球绿色荧光强度较弱。Hoechst 33342标记的SD NSCs荧光显微镜下呈现明亮的蓝色荧光。9.三种NSCs脑干耳蜗核定位注射2d后,注射局部均可见到注射针道和荧光阳性的细胞团聚集。定位注射10d后,C57BL/6-gfp小鼠嗅球NSCs的注射局部无绿色荧光阳性细胞;转染EGFP SD大鼠嗅球NSCs的注射局部仅见少量散在绿色荧光阳性细胞,这些细胞发出细长绿色突起,数量不等。经由免疫荧光染色后在绿色荧光阳性细胞中查见NSE免疫反应阳性、GFAP免疫反应阳性细胞而未见GalC免疫反应阳性细胞。Hoechst 33342标记的SD NSCs注射部位及脑干表面均有大量蓝色荧光阳性细胞存在,脑干表面蓝色荧光阳性细胞沿脑膜分布,延伸到较远部位,血管壁细胞也呈蓝色荧光阳性。注射局部蓝色荧光阳性细胞聚集,并有向特定方向延伸的趋势。结论:1.正常成年大鼠耳蜗Corti器F-actin在各类细胞内均有表达,内、外柱细胞着色较强,呈现明亮的红色荧光。Deiters细胞呈现中等强度的红色荧光,其他支持细胞着色较弱;OHCs胞浆着色浅淡,纤毛及表皮板处着色强度与柱细胞相仿。而IHCs F-actin的表达与内柱细胞相仿。2.发育关键期给予气导听觉剥夺造成实验动物耳蜗Corti器F-actin的表达减少,毛细胞表皮板处及内、外柱细胞分布减少,可能导致细胞骨架分布异常;从而影响基底膜的顺应性,影响机械电能的正常转换,表现为动物DPOAE及ABR异常。3.气导听觉剥夺所致听觉传入信号的减少不能引起各级神经元的变性乃至进一步的凋亡。但是在耳蜗核发生了神经纤维的变性。螺旋神经节神经元NSE表达减少;神经元数量较正常减少,体积缩小,神经元及卫星细胞均发生退行性变。4.气导听觉剥夺造成了耳蜗核神经元数量和密度的下降,PVCN章鱼样细胞体积缩小,数量减少,密度下降。一系列改变说明,发育关键期对动物进行气导听觉剥夺,对于整个听觉传导通路的发育都产生了明显的影响乃至于发生了功能的改变。5. C57BL/6-gfp小鼠、SD大鼠嗅球分离的细胞体外培养后经鉴定nestin阳性,诱导分化后能够分化为MSE阳性的神经元和GFAP、Galc阳性的神经胶质细胞,说明其来源于神经系统,能自我更新并通过不对称分裂产生神经元和神经胶质细胞。6.以转基因动物C57BL/6-gfp小鼠作为NSCs的来源,优点是取材简便,不需要特殊操作步骤,能够稳定表达绿色荧光蛋白,缺点是实验受动物种类限制。本实验中异种动物NSCs注射是导致移植NSCs消失的主要原因。7.以EGFP质粒转染大鼠NSCs,转染过程操作简便,安全,对NSCs的免疫原性、毒性均较低,转染效率较高,能够很好显示标记细胞的形态,可以作为较好的NSCs的标记方法。8.细胞核染料Hoechst 33342标记大鼠NSCs,同样具有操作简便的优点,且细胞毒性小,标记效率高。缺点是染料可能污染注射局部的活细胞,造成假阳性。更多还原

【Abstract】 Objective:Auditory deprivation effect is a significant reduce of acoustic message, which lead to the decrease of hearing ability. People have already noticed that the input signal of olfactory, vision and audition had an important role in the development of its neural pathway. Experts had thought that 2-3year-old was the perfect time for the artificial cochlea implantation, and FDA also suggested 12month-old as the lower limits of implantation. Now, people incline to take the implantation at an early age to reduce the effect of early auditory deprivation and get a better rehabilitation. More and more literatures shew that it was beneficial to our hearing reconstruction and language learning to take the implantation at an early age. Bilateral chronic SOM of chilidren and congenital microtia are the common cause of conductive hearing loss of children. We have found some mature ideas for the treatment of these, but when to interfer is still uncertain. Studies revealed that the reduce of acoustic message might cause the disorder of auditory center. But these studies were performed under the circumstances of destroying the cochlea. In our study, we prepared the auditory deprivation animal models by plugging their ears, which made it possible to study the changes of both auditory pathway and the modality and function of cochlea.In recent years, scientists have been going in for a new therapy with stem cells transplantation for those awkward diseases, and have made some progress. In the same time, otologists also turn to stem cells transplantation in order to find a new therapy for sensorineural hearing loss. However, the transplanted stem cells must carries their own tag of identity, thus the scientists could observe their biological characters through their specific identity. There are a lot of labeling methods for stem cells, all with its advantages and disadvantages. In our experiment, we compared three of those methods to detect the different results of NSEs transplantation in adult rats, and provide support for the different results of NSEs transplantation into the AD animal models.Methods:1. The experiment group underwent an auditory deprivation with a plugging in the external acoustic meatus form 12 days postnatal to 42 days, raised in a acoustic chamber. Then animals were tested for ABR threshold and DPOAE, and perfused by formalin before preparing the frozen sections of cochlea and brain stem.2. The location and expression of F-actin in the Corti organ. We observed hair cells and Cytoskeleton of both groups with a laser scanning confocal microscope after immunohistochemistry, immuno-fluorescence and Phalloidin stain of F-actin. Western blot was used to the semiquantitative analysis of F-actin.3. TUNEL was used to test the cell apoptosis. Transmission electron microscope was used to observe the neuron of spiral ganglion. Then we took the immunohistochemical staining of spiral ganglion with NSE antibody, and count the number of its neuron.4. FJC was used to test the neuron degeneration of cochlea nucleus, while Nissl stain was used to observe the cochlea modality and count the number of octopus-like cells.5. In vitro culture, differentiation and identification of neural stem cells derived from the olfactory bulb.We derived the neural stem cells from the olfactory bulb of SD rats and C57BL/6-gfp mice embryos, observed the culture, differentiation progress and viability of stem cells with a fluorescence inverted microscope, and then identified the stem cells with immunocytochemistry and immunofluorescence.6. Labeling of the olfactory bulb neural stem cells derived from SD rats embryos.We transfected the olfactory bulb neural stem cells derived from SD rats embryos with liposome2000-mediated PIRES-EGFP-C2 plasmid, and passaged them for 1 month after screening by G-418.Transfection efficiency of the stem cells was evaluated with flow cytometer. Before collecting the cells, we added Hoechst 33342 to the culture medium to label the transplanted cells, then 37℃incubation for 1 hour.7. Stereotaxic injection of labelled olfactory bulb neural stem cells to the cochlea.The C57BL/6-gfp mice NSCs, SD rats NSCs transfected with green fluorescent protein and stained with Hoechst 33342 were collected and injected to the rats cochlear nucleus. We anesthetized SD rats, fixed them on the stereotaxic apparatus, and located their left cochlea nucleus according to the brain atlas solid positioner. NSCs suspension was drew out into a microinjector with a glass microelectrode in the tip, and injected slowly into the cochlea nucleus. Post-operation animals were bred in normal environment and fixed by perfusion 2 and 10 days after operation. 40μm brain sections were made and observed with immunofluorescence staining.Results:1. DPOAE results shew that rate of control group in the frequency of 0.5、1、2、4、8kHz compared with AD group, there was significant difference between two groups in the frequency of 0.5,1,4kHz. There was significant difference between two groups on the ABR threshold: the control group was 18.6538±1.58785 dB SPL and the AD group was 28.4000±1.33775 dB SPL. The latency ABRⅠ-Ⅴwave of control group compared with those of AD group, there was no significant difference between two groups. The ABRⅠ-Ⅴwave amplitude of control group compared with those of AD group, there was significant difference between two groups inⅡ、Ⅲ、Ⅴwave amplitude. And there was significant difference between two groups inⅠ-Ⅴwave interval. TheⅠ-Ⅴwave interval of control group was 3.978±0.435, while that of AD group was 3.629±0.416.2. F-actin can express in alll kinds of cells of Corti organ, mostly in stereocilium, inner pillar cells and outer pillar cells, little in the outer hair cells. F-actin expressed in Corti organ of AD was less than that of control group, which was more significant in the concentration position. Western blot results shew F-actin express less in the basilar membrane of AD rats cochlea. And there was significant difference between two groups.3. In the Cordi organ of control group rats, we can see TUNEL-positive cells in the stria vascularis, but no in the basilar membrane. In the AD group rats, we can see TUNEL-positive cells along the spiral limbus surface and in supporting cells of the basilar membrane, but not in the IHCs、OHCs.4. There were two kinds of spiral ganglion neurons: typeⅠand typeⅡ. Transmission electron microscope revealed smaller cell body, bigger cell interval and degeneration of AD spiral ganglion neurons. membrane like tissues proliferated along the plasma. Star cells didn’t develop well, and not form myelin sheath. Cell organs degenerated and decreased. The NSEs immunohistochemical staining shew that NSEs primarily located in cytoplasm of normal neuron. Cells bodies painted remarkably and necleus was faint staining. Arrangement of cells bodies was some kind of intensive. We couldn’t tell the two kinds of neuron according to their modality. The neural fiber was faint staining. The NSEs immunohistochemical staining of AD group shew that the neurons were fainter staining with the fuzzy modality and disorder arrangement. There was significant difference between two groups on the results of neurons counting: AD group were 10.643±1.104, while that of the control group were 7.883±0.987.5.We could find green FJC-positive progress in the cochlea nucleus in the immunohistochemical staining of brain stem sections. Some of the progress was thin and branching. But we didn’t find the FJC-positive neural bodies.6. The neuron counting of cochlea nucleus of AD group was significant smaller than that of the control group. And the neuron density was also smaller than that of the control group, but not significant. The counting of octopus like cells and the cells size of AD group was significant smaller than that of the control group. The cells density was also smaller, but not significant.7. Olfactory bulb neural stem cells of C57BL/6-gfp mice and SD rats embryos formed cell spheres in culture and were nestin positive. They could differentiate into MSE positive neurons and GFAP, Galc positive glial cells.8. Olfactory bulb neural stem cells of C57BL/6-gfp mice presented light green fluorescence in the burst of ultraviolel; EGFP transfected rats NSCs presented light green fluorescence with irregular or ring shape, and were GFP positive. Some smaller cell spheres shew darker fluorescence. Rats NSCs labelled with Hoechst 33342 presented light green fluorescence.9. Two days after injection, pin holes and cells gathering were observed in the injection cite of three group animals. 10days after injection, no positive cells were found in the injection cite of C57BL/6-gfp mice NSCs. Only some positive cells with slender green processes were found in the injection cite of EGFP transfected rats NSCs. NSE and GFAP positive cells were observed in the green fluorescence positive cells with the immunofluorescence stain, no Galc positive ones. A lot of blue fluorescence shew in the injection cites of Hoechst 33342 labelled SD NSCs and the surface of brainstem, and the latter extended to distant places along the meninges. Cells of blood vessels also shew a blue fluorescence positive. In the pin hole, blue fluorescence positive cells gathered and tended to extend.Conclusion:1. F-actin can express in all kinds of cells of Corti organ, mostly in inner pillar cells and outer pillar cells. They were light red fluorescence. Deiters cells were middle light red fluorescence, while the other supporting cells were faint. The OHCs were faint straining in the cytoplasm. The staining of OHCs cilium, and IHCs were the same as that of pillar cells.2. Early auditory deprivation of air-conduction could lead to the F-actin loss of Corti organ. The loss of F-actin in the cuticular plates and pillar cells could cause the disorder of cytoskeleton, the compliance of basal membrance and its converting function of mechanical energy and electric energy, which resulted in the abnormal ABR and DPOAE.F-actin expressed in Corti organ of AD was less than that of control group, which was more significant in the concentration position. Western blot results shew F-actin express less in the basilar membrane of AD rats cochlea. And there was significant difference between two groups.3. Auditory deprivation of air-conduction which lead to the reduce of the auditory signal didn’t result in the neuron degeneration and apoptosis. But we found the degeneration of neural fibers in the cochlea nucleus. In the AD group, NSE stained faint in the spiral ganglion. The number of neurons decreased and the size smaller. Both neurons and star cells degenerated. 4. Auditory deprivation of air-conduction resulted in the loss of neuron and its density. In the AD group, the PVCN octopus like cells was smaller, less number and density. All of these indicated that the auditory deprivation of air-conduction in the crucial time might cause the great effect on the development of auditory neural pathway and its function.5. Olfactory bulb neural stem cells of C57BL/6-gfp mice and SD rats embryos formed cell spheres in culture and were nestin positive. They could differentiate into MSE positive neurons and GFAP, Galc positive glial cells. These indicated that those cells were from nervous systerm and could self-renewal and generated other cells through asymmetric cell divisions.6. It was easy and convenient to derive the NSCs from C57BL/6-gfp mice. The C57BL/6-gfp mice NSCs could present stable green flurescence. But the animal races were limited. And we estimated that the race difference was the main reason of NSCs death.7. It was simple and safe in the process of transfectting the NSCs with EGFP, which was less immunogenicity or toxicity to NSCs and higher transfection efficiency. It could wonderfully display the modality of tagged cells and could be taken as a perfect way to tag the cells.8. It was also simple, low toxicity and high efficiency to label NSCs with Hoechst 33342. But Hoechst 33342 stain may stain the cells in the injection cite and lead to false positive.更多还原