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1.
Objective To study expression of adenovira1-mediated Hathl-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane (RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with AdHath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and fro zensections were examined. Results ABR tests showed no significant change of hearing after the surgery.Strong fluorescence staining in the cochleae was seen in Ad-Hathl-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with tittle decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.  相似文献   

2.
目的 探讨Hathl(human atonal homolog 1)基因对大鼠耳蜗大上皮嵴(greater epithelialridge,GER)细胞的诱导分化作用.方法 取出牛后1 d大鼠耳蜗,利用酶消化和机械分离相结合的方法分离出GER细胞,并行体外培养.以腺病毒为载体,对GER细胞培养物进行Hath1基因和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因(ad-Hath1-EGFP)的转染,单纯转染EGFP(ad-EGFP)作为对照组,对感染后不同灭数的标本行毛细胞特异性标记物myosin Ⅶa免疫组化染色鉴定.结果 ad-Hathl-EGFP组中的GER细胞中出现了myosin Ⅶa和EGFP双标记细胞,并且从感染后第5天开始出现myosin Ⅶa阳性细胞,随后阳性细胞的数量有所增加,但增加不明显;而ad-EGFP组感染后3~12 d的GER标本均未见myosin Ⅶa阳性细胞.结论 GER细胞可能是耳蜗毛细胞的前体细胞,Hath1基冈过表达可以诱导GER细胞向毛细胞样细胞(myosin Ⅶa阳性)分化.  相似文献   

3.
Math1基因内耳导入后噪声性聋豚鼠听功能改变观察   总被引:2,自引:1,他引:1  
目的观察Math1基因内耳导入对噪声性聋豚鼠听功能的影响,探讨Math1基因过表达对噪声损伤耳蜗的生物学效应,为内耳基因治疗提供实验基础和理论依据。方法经脉冲噪声致聋的豚鼠45只(各频率ABR阈值均≥95dB SPL),雌雄不限,实验开始时体草250~300g。随机分为3组:Ad—Math1-EGFP组(30只);Ad—EGFP组(5只);空白组(10只)。各组豚鼠在基因转导后4周、8周分别测试双耳ABR。测试完毕后处死动物,观察听泡及耳蜗尤炎性病变者记录听阈结果。结果Math1导入后4周,导入耳各频率ABR阈值低于对照耳(右耳),也低于Ad—EGFP组及空内组,平均达到85dBSPL。Math1导入后8周,导入耳各频率ABR阂值低于对照耳(右耳),也低于Ad—EGFP组及空白组,与4周时比较,进一步好转,平均达到75dB SPL。结论Math1基因内耳导入可使噪声导致全聋的豚鼠听功能部分恢复,为噪声性聋的治疗打开了新的思路和手段。  相似文献   

4.
Cross-links between stereocilia in the guinea pig cochlea   总被引:4,自引:0,他引:4  
  相似文献   

5.
Effects of exposure to noise on ion movement in guinea pig cochlea   总被引:1,自引:0,他引:1  
Healthy guinea pigs were exposed to broad band noise at levels between 95 and 115 dBA for 7 days. A significant decrease of the sound-induced cochlear responses, together with a substantial increase of the endocochlear potential, was observed in guinea pigs exposed to noise at 105 or 115 dBA. Microsamples of the endolymph obtained from these guinea pigs showed a significant increase of K+ and Cl- concentrations and a decrease of Na+ concentration, when compared with those from control animals. The K+, Na+ and Cl- concentrations in the perilymph were not markedly affected by noise exposure. When the perilymphatic space was perfused with artificial perilymph containing 43K, 22Na or 36 Cl, the uptake of radiotracers into the endolymph showed a single exponential function of the perfusion time. When compared with rate constants in normal animals, the value of rate constant for K+ was significantly decreased in animals exposed to noise. These results indicate that ionic permeability changes of the endolymph-perilymph barrier are a significant factor in the physiological mechanisms underlying noise-induced hearing loss.  相似文献   

6.
Guinea pigs, which were either anesthetized (A) or conscious (U), were exposed to four 2 h sessions of broad-band noise of 96 dB SPL. Cochlear microphonics and N1 thresholds were measured prior to killing from 1 to 13 days later. The cochleas were examined by SEM and by section.The U series suffered less N1 threshold loss and recovered within 10–13 days, while the large initial loss in the A series did not completely reverse within the period of study. Initially, the IHC stereocilia in the basal half of the cochlea showed marked bending, the affected area being somewhat more extensive in the A group. These hairs gradually recovered, although not progressively. On the other hand, the disturbance to OHC stereocilia, which appeared to be less pronounced early on and was more apically centered, developed over time into marked permanent damage. The outermost row consistently showed the greatest effect with hairs becoming elongated or fused, and occasionally lost altogether. Susceptibility to noise varied between individuals of both groups.The recovery of N1 threshold was concomitant with the recovery of the erectness of the IHC hairs.  相似文献   

7.
Distortion product otoacoustic emissions (DPOAE) are a sensitive detector of outer hair cell (OHC) function and were monitored in awake guinea pigs before and after impulse noise damaging the cochlea (peak intensity 153 dB SPL, rise time < 0.1 ms). Animals had stable DPOAE levels before noise exposure. In the first hours after noise exposure DPOAE levels were reduced significantly. Three different patterns of recovery of DPOAE were seen in the post-exposure period: restitution exceeding controls, partial recovery and no recovery. In general, DPOAE levels declined and types of recovery closely corresponded to changes in amplitudes of cochlear microphonics after noise exposure. These data suggest that the monitoring of DPOAE is a suitable method for diagnosing impaired OHC function. Received: 25 February 1999 / Accepted: 1 July 1999  相似文献   

8.
Summary After kanamycin induced degeneration of outer hair cells from guinea pigs the tuning properties of primary auditory nerve fibres are compared with those of normal untreated guinea pigs. The existence of fibres with no alteration of the tuning properties leads to the conclusion that there is no neural interaction between inner and outer hair cells needed to enhance the frequency selectivity.This study was supported by the Deutsche Forschungsgemeinschaft (Bo 421/1-3). Kanamycin was kindly provided by Chemie Grünenthal.  相似文献   

9.
Intracellular recordings were made from inner hair cells in the basal turn of the guinea pig cochlea during transient asphyxia. During these periods the endocochlear potential was reversibly reduced, the hair cell resting membrane potential was slightly hyperpolarized and the cochlear microphonic potential was decreased, all with similar time courses. The eighth nerve compound action potential and DC and AC components of the inner hair cell receptor potential were strongly attenuated and with similar slow time courses. The asymmetrical low frequency receptor potentials became symmetrical, and this change was attributed to alterations in the mechanical properties of the organ of Corti. The desensitization of the cochlea during transient asphyxia was associated with the loss of asymmetry of the inner hair cell receptor potential.  相似文献   

10.
We investigated the effects of noise and salicylate on the guinea pig cochlea by monitoring the acoustic nerve compound action potential (CAP) as well as the 2f1-f2 distortion product in the cochlear microphonics (DP-CM). The alterations of DP-CMs by noise and salicylate revealed an apparent difference, even when the noise-induced and salicylate-induced CAP threshold shifts were equal. This result indicated that noise and salicylate might have an effect on different sites in the cochlea. Combined effects were altered by changing the order in which the two agents were applied. The existing noise-induced damage attenuated the additional ototoxicity of salicylate. In contrast, attenuation did not occur when salicylate administration preceded noise exposure. Considering known effects of noise and/or salicylate altering mechano-electrical transduction (MET) and electro-mechanical transduction of the cochlear outer hair cells, our results seem to suggest that the MET plays a key role in the transduction mechanism in the cochlea.  相似文献   

11.
豚鼠耳蜗单离外毛细胞的外向整流钾电流   总被引:1,自引:0,他引:1  
目的 :观察豚鼠耳蜗单离外毛细胞 (OHC)的电生理特性 ,记录不同长度OHC的外向整流钾电流 ,分析区分外向整流钾电流所包含的通道电流成分 ,研究外向整流钾电流的动力学特征。方法 :采用酶消化法及机械分离OHC。运用全细胞膜片钳技术 ,在电压钳下记录K+ 通道电流。结果 :OHC的全细胞膜电容为 (30 .96±2 .79) pF(n =2 9) ,零电流电位 (30± 2 .1)mV(n =16 ) ,反转电位为 (- 5 1.6 7± 1.84 )mV(n =9)。不同长度OHC的外向整流钾电流存在系统差异 ,短OHC表现出大的钾电导 ,长OHC则相反。 10 0 μmol/L的氯化镉 (Cd Cl2 )抑制了OHC外向整流钾电流的最大电流幅度的 6 0 % ,且改变了电流的动力学特征 ,对峰电流的影响明显大于稳态电流 (P<0 .0 1,n =5 ) ;1mmol/L的四氨基吡啶 (4 AP)抑制了最大电流幅度的 4 3% ,没有改变电流的动力学特征。外向整流钾电流的激活符合Boltzmann方程 ,V1/ 2 =(- 11.0 7± 0 .2 6 )mV ,S =(6 .6 2± 1.74 )mV(n=13)。结论 :外向整流钾电流包含有钙离子激活的钾离子电流、外向延迟整流钾电流和A型电流  相似文献   

12.
目的:探讨缝隙连接在维持正常的听功能方面可能发挥的作用。方法:通过向豚鼠外淋巴中注入缝隙连接阻断剂1-庚醇(1-heptanol),然后检测给药前后豚鼠听性脑干诱发反应(ABR)阈值和潜伏期的改变,并借助透射电镜观察药物对豚鼠耳蜗中的缝隙连接结构的影响。结果:注射1-heptanol后,豚鼠ABR阈值显著提高,潜伏期延长(P〈0.01),并随时间推移呈动态升高趋势。同时,通过电镜可以观察到在豚鼠耳蜗中存在着大量的缝隙连接结构,其主要分布在毛细胞和其下的支持细胞间。1-heptanol可以使缝隙连接结构破坏,毛细胞水肿、变性。结论:缝隙连接阻断剂1-heptanol能明显影响豚鼠的听功能,缝隙连接结构在听觉形成过程中起着重要的作用。  相似文献   

13.
目的:探讨豚鼠耳蜗单离外毛细胞(OHC)钾电流(Ik)的正常值及其特性。方法:应用膜片钳全细胞记录技术及多种辅助方法,测试在不同细胞内、外液和电压刺激条件下的Ik、钾尾电流(Iktail)和反转电位。结果:Ik具有明显的电压依赖性和时间依赖性,在20ms内达峰值,平均激活电压约为-32.7mV,从激活电压至0电压时Ik增长最快,在40mV时接近饱和。正常条件下Ik无明显“run-down现象”。细  相似文献   

14.
Isolated outer hair cells (OHC) of the guinea pig cochlea were exposed to external alternating electric fields parallel to the longitudinal axis of the cells. This resulted in oscillations of the cells' length that were measured photoelectrically using a ratiometric light amplifier. At 5 Hz and elongations up to 300 nm, amplitude of the cell length during oscillation was a linear function of the amplitude of the sinusoidal electric field. When increasing the stimulus frequency up to 32kHz, OHC length changes followed the stimulus cycle-by-cycle. Oscillations at frequencies above 32 kHz escaped the experimental approach by their small amplitudes and could not be excluded. The frequency dependence of the motile response measured at 5–12 000 Hz had low-pass filter characteristics in cells of the second, third and fourth turns of the cochlea. However, frequency tuning of the motile response was absent in each OHC and systematic differences between different turns were not observed.  相似文献   

15.
Math1基因内耳导入径路的探索研究   总被引:1,自引:1,他引:0  
目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250—300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组(24只)导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP),对照组(16只)导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应(ABR)检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异(P〉0.05);鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异(P〉0.05),8kHz较术前增高(P〈0.05),16kHz、20kHz较术前明显增高(P〈0.01),术后14天在16kHz、20kHz较术后5天时明显好转(P〈0.01),但较术前仍有增高(P〈0.05)。转导成功率鼓阶打孔组为91.6%,优于完整圆窗膜组的50%。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。  相似文献   

16.
Summary Guinea pigs have been exposed to 20 kHz at 120 dB SPL for exposure durations of 7.5, 6.5, 5.0, and 3.25-min and killed either 3 or 12 weeks post-exposure. One series of guinea pigs exposed for 30-min had cochlear potentials recorded 3 weeks post-exposure. The damage was assessed by surface preparations and quantified as percentage hair cell loss per segment in every row, total number of outer, and inner hair cells missing and the area of total outer hair cell damage. A significantly smaller number of ears were found to be damaged after the shortest exposures, but no significant differences could be detected in the amounts of damage when all the series were compared. Myelinated nerve fibre degeneration had increased after the longer post-exposure interval, but no such differences were observed in the sensory hair cell degeneration.Part of this work was presented at the XXth Workshop on Inner Ear Biology at Geilo, Norway, 4–7 September 1983  相似文献   

17.
目的:探讨强噪声对豚鼠耳蜗细胞死亡的机制及磷酸化c-Jun氨基末端激酶(JNK)信号通道在强噪声诱导耳蜗细胞凋亡的作用。方法:将实验组豚鼠暴露在4kHz窄带噪声120dBSPL噪声环境中4h,噪声刺激停止后1d、4d、14d组及对照组(每组各8只)在处死前测ABR。取每组4只豚鼠耳蜗作石蜡切片,另外4只豚鼠提取耳蜗总蛋白。脱氧核糖核甘酸末端转移酶介导的缺口末端标记技术(TUNEL)检测耳蜗凋亡细胞,免疫组织化学及WesternBlot方法检测JNK信号途径蛋白质P-JNK、P-c-Jun的表达。结果:实验组耳蜗Corti器毛细胞、血管纹和螺旋神经节细胞存在TUNEL阳性细胞,以1d组最多,逐渐减少,14d组最少,而对照组未见阳性细胞。免疫组织化学观察到实验组P-JNK、P-c-Jun有免疫反应阳性,定位于细胞核,对照组未见阳性细胞。Western Blot检测P-JNK、P-c-Jun含量在噪声刺激后迅速增高并快速活化,1d、4d达到高峰,随后逐渐下降,但在14d仍然维持较高水平。结论:强噪声可以通过诱导凋亡造成耳蜗细胞损伤,同时P-JNK标志着JNK信号途径的激活,提示JNK信号通道可能也是介导强噪声诱导豚鼠耳蜗细胞凋亡信号通道之一。  相似文献   

18.
Objective: To investigate the expression of the miniature pig cochlea after AAV1 transfect into the cochlea via round window membrane (RWM).

Methods: Twenty miniature pigs are equally divided into four experimental groups. Twelve miniature pigs are equally divided into four control groups. Each pig was transfected with the AAV1 in the experimental group via RWM and each pig was transduced with the artificial perilymph in the control group. The expression of green fluorescent protein (GFP) was observed at 2 weeks, 3 weeks and 4 weeks, respectively. Likewise, AAV1 was delivered into the guinea pigs cochleas using the same method, and the results were compared with that of the miniature pigs.

Results: The expression was mainly in the inner hair cells of the miniature pig. The expression of GFP began to appear at 2 weeks, reached the peak at 3 weeks. It also expressed in Hensen’s cells, inner pillar cells, outer pillar cells, spiral limbus, and spiral ligament. In the meanwhile, AAV1 was delivered into guinea pig cochlea via the same method, and AAV1 was also expressed in the inner hair cells. But the expression peaked at 2 weeks, and the efficiency of the inner hair cell transfection was higher than that of the pig.

Conclusion: AAV1 can be transformed into miniature pig cochlea via scala tympani by the RWM method efficiently.  相似文献   


19.
目的:在近成年豚鼠耳蜗内携带atoh1/EGFP基因的腺病毒是否能够将基膜上残留的支持细胞转分化为新的毛细胞。方法:选用体重在200~250g的健康花豚鼠12只,将构建同时表达atoh1和EGFP基因的E1/E3区缺失的人类免疫5型腺病毒5μl,通过耳蜗侧壁打孔灌注导入中阶内淋巴系统。其中6只于灌注2周后处死,6只灌注4周后处死,基膜铺片观察EGFP、毛细胞标记物myosinⅦa和Dapi核染色情况。结果:灌注2周处死的6只豚鼠中有2只在基膜外毛细胞外区域发现有散在的细胞核大、细胞体梭形的、表达EGFP的新生细胞。灌注4周的动物中有3只在基膜原外毛细胞位置和外毛细胞外区域发现有相似的同时表达EGFP和Myo-sinⅦa的新生细胞。结论:atoh1基因可以在近成年豚鼠体内将部分支持细胞转分化为毛细胞样细胞。这部分支持细胞位于外毛细胞位置和外毛细胞外区域的基膜上。  相似文献   

20.
链霉素对豚鼠耳蜗外毛细胞离子电流的影响   总被引:3,自引:0,他引:3  
使用全细胞式记录膜片钳技术,研究链霉素对豚鼠耳蜗外毛细胞离子电流的影响。结果显示:1mmol/L链霉素分别使钾电流和钙电流减少;链霉素对电压依赖性钙电流的半数抑制浓度为56.08μmol/L,最大抑制率为21.7%,表明链霉素以浓度依赖性方式,部分不可逆地阻断豚鼠耳蜗外毛细胞电压依赖性钙电流,对钾电流的阻滞作用部分是阻断Ca^2+内流的结果。  相似文献   

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