沙鼠耳蜗cDNA文库的构建

为了建立沙鼠耳蜗的cDNA文库 ,先提取沙鼠耳蜗的mRNA并经oligo(dT)引物合成cDNA第一链 ,经LD PCR法扩增合成双链cDNA后 ,克隆到λTriplex2中扩增文库。用耳蜗特异的prestin蛋白基因引物作PCR ,鉴定文库。结果显示 ,文库的库容量为 1.8× 10 6pfu ,重组率为 80 % ,用prestin蛋白基因引物可扩增出 86 3bp的prestin蛋白基因。研究表明 ,构建的文库具有较好的库容量及重组率 ,且插入片段很大 ,为从分子生物学方面研究耳蜗打下了基础     

儿茶素对拟衰老大鼠耳蜗氧自由基生成的影响

【目的】观察儿茶素对衰老大鼠动物模型耳蜗氧自由基生成的影响,初步探讨儿茶素对听觉老化的保护效应及其机制。【方法】通过腹腔注射D-半乳糖（D-gal）建立大鼠衰老动物模型,以腹腔注射生理盐水为阴性对照组,检测其听觉脑干诱发电位（ABR）明确衰老动物听力减退情况,检测大鼠耳蜗组织丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性;并观察胃内灌注儿茶素后衰老动物模型上述指标的变化。【结果】衰老模型组大鼠与对照组大鼠相比较,ABR阈值增高,耳蜗组织中MDA含量明显增高,SOD活性明显降低（P〈0.05）;儿茶素保护组大鼠耳蜗组织中MDA含量和SOD活性水平较对照组无变化（P〉0.05）,而与衰老模型组相比却逆转上述指标变化,并能提高听力（P〈0.05）。【结论】氧自由基及其引发的脂质过氧化反应参与了听觉老化过程,儿茶素可能通过提高SOD活性、减少MDA生成发挥清除自由基抗氧化作用,从而改善听觉功能。     

Prestin蛋白的稳定高表达卵巢细胞株的建立

目的:建立体外稳定高表达耳蜗外毛细胞马达蛋白(prestin)的多克隆中国仓鼠卵巢细胞株(CHO)。方法:采用prestin质粒转染CHO细胞进行表达,高浓度博来霉素400~800mg/L筛选出多克隆细胞株,传代培养,同时长时间维持低浓度抗生素(200~400mg/L)。收集所筛选的多克隆细胞株,提取总RNA,采用针对prestin蛋白不同片段设计的3对引物,进行逆转录-聚合酶链反应(RT-PCR),对扩增产物进行凝胶电泳分析。采用免疫荧光染色方法鉴定所筛选多克隆细胞株的prestin蛋白表达。结果:采用3对不同的prestin引物的RT-PCR结果均为阳性,而对照组则为阴性;利用prestin蛋白的特异性抗体,对所筛选的多克隆细胞株进行免疫荧光染色鉴定结果显示阳性率>90%。结论:成功获得稳定高表达prestin蛋白的多克隆CHO细胞株,转染表达率高。     

CO2激光对豚鼠耳蜗结构影响的实验研究

目的 研究低峰功率超脉冲CO2激光对豚鼠耳蜗结构的影响.方法 将24只豚鼠随机分为激光2W组、激光4 W组及对照组,每组8只,前两组分别以超脉冲CO2激光功率2 W、4 W行左耳蜗底周造孔,对照组只手术暴露耳蜗底周.术后21 d处死动物,应用耳蜗基底膜铺片、扫描电镜技术观察豚鼠耳蜗形态学的改变.结果 实验组与对照组的豚...     

神经生长因子在小鼠耳蜗中的表达分布及年龄相关性变化

目的探讨神经生长因子(NGF)在小鼠耳蜗中的表达分布与年龄的相关性变化。方法选取5、7月龄的快速老化小鼠亚系8(SAMP8)作为实验组,而同龄抗快速老化小鼠亚系1(SAMR1)作为对照组。分别观察其耳蜗中NGF免疫组化染色及增龄性变化情况。结果在小鼠耳蜗中,NGF在螺旋神经节细胞、内外毛细胞、听神经纤维及血管纹中均有表达,且螺旋神经节细胞及内外毛细胞为主要表达部位。耳蜗螺旋神经节细胞及内外毛细胞NGF免疫组化染色平均光密度值:NGF蛋白在不同月龄快速老化小鼠耳蜗组织中螺旋神经节细胞及毛细胞均有表达,第5、7月龄SAMP8小鼠耳蜗螺旋神经节细胞及毛细胞中的NGF蛋白表达较同龄SAMR1小鼠明显降低(P<0.05)。结论 NGF蛋白在小鼠耳蜗中有表达并且表达水平随着小鼠年龄增长而降低,说明NGF蛋白可能与维持耳蜗的功能状态有关。     

TRPV1在正常BALB/c小鼠耳蜗中的定位表达

目的 研究瞬时受体电位通道蛋白V1(TRPV1)在正常小鼠耳蜗中的定位观察.方法 选择10只健康成年BALB/c小鼠,应用免疫荧光染色方法观察TRPV1在耳蜗中的定位与表达,同时结合电生理指标听脑干反应(auditory brainstem response,ABR)测试小鼠的听力.结果 TRPV1在小鼠耳蜗螺旋器的内、外毛细胞、螺旋神经节细胞和血管纹边缘细胞等都有表达,且主要定位于细胞膜;蛋白质印迹检测结果亦显示小鼠耳蜗中有TRPV1的表达.结论 TRPV1在正常BALB/c小鼠耳蜗的多种细胞中均有表达,这为深入TRPV1在内耳听觉生理和病理生理中的作用奠定基础.     

Dynamic patterns of neurotrophin 3 expression in the postnatal mouse inner ear

Recent studies indicate that neurotrophin 3 (NT3) may be important for the maintenance and function of the adult inner ear, but the pattern of postnatal NT3 expression in this organ has not been characterized. We used a reporter mouse in which cells expressing NT3 also express beta-galactosidase, allowing for their histochemical visualization, to determine the pattern of NT3 expression in cochlear and vestibular organs. We analyzed animals from birth (P0) to adult (P135). At P0, NT3 was strongly expressed in supporting cells and hair cells of all vestibular and cochlear sense organs, Reissner's membrane, saccular membrane, and the dark cells adjacent to canal organs. With increasing age, staining disappeared in most cell types but remained relatively high in inner hair cells (IHCs) and to a lesser extent in IHC supporting cells. In the cochlea, by P0 there is a longitudinal gradient (apex > base) that persists into adulthood. In vestibular maculae, staining gradients are: striolar > extrastriolar regions and supporting cells > hair cells. By P135, cochlear staining is restricted to IHCs and their supporting cells, with stronger expression in the apex than the base. By the same age, in the vestibular organs, NT3 expression is weak and restricted to saccular and utricular supporting cells. These results suggest that NT3 might play a long-term role in the maintenance and functioning of the adult auditory and vestibular systems and that supporting cells are the main source of this factor in the adult.     

Delayed neurotrophin treatment following deafness rescues spiral ganglion cells from death and promotes regrowth of auditory nerve peripheral processes: effects of brain-derived neurotrophic factor and fibroblast growth factor

The extent to which neurotrophic factors are able to not only rescue the auditory nerve from deafferentation-induced degeneration but also promote process regrowth is of basic and clinical interest, as regrowth may enhance the therapeutic efficacy of cochlear prostheses. The use of neurotrophic factors is also relevant to interventions to promote regrowth and repair at other sites of nerve trauma. Therefore, auditory nerve survival and peripheral process regrowth were assessed in the guinea pig cochlea following chronic infusion of BDNF + FGF(1) into scala tympani, with treatment initiated 4 days, 3 weeks, or 6 weeks after deafferentation from deafening. Survival of auditory nerve somata (spiral ganglion neurons) was assessed from midmodiolar sections. Peripheral process regrowth was assessed using pan-Trk immunostaining to selectively label afferent fibers. Significantly enhanced survival was seen in each of the treatment groups compared to controls receiving artificial perilymph. A large increase in peripheral processes was found with BDNF + FGF(1) treatment after a 3-week delay compared to the artificial perilymph controls and a smaller enhancement after a 6-week delay. Neurotrophic factor treatment therefore has the potential to improve the benefits of cochlear implants by maintaining a larger excitable population of neurons and inducing neural regrowth.