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1.
经鼓阶胚胎干细胞内耳导入的初步观察 总被引:2,自引:2,他引:0
目的观察未经体外诱导的胚胎十细胞(embyonic stem cells,ESC)导入听力正常大鼠内耳的可行性以及导入后的存活和分布情况,为ESC内耳移植治疗由毛细胞缺失导致的感音神经性耳聋提供实验基础和理论依据。方法5—6周龄Wistar大鼠,10只,右耳为实验耳:经鼓阶打孔法植入带有绿色荧光蛋白(EGFP)的ESCs;左耳为对照组,不实施手术。术前1周与术后即刻行听性脑干反应(ABR)检查,取双侧耳蜗做冰冻切片,观察ESCs植入内耳后存活和分布情况。结果术后动物存活8只,麻醉效果好无干扰完整测完ABR动物5只。鼓阶打孔途径导入耳蜗的ESCs大部分于鼓阶聚集悬浮,少数可在鼓阶基底膜嵴和鼓阶外侧壁处贴壁;未在柯替器等中阶部位巾发现有ESCs的分布。ABR检测结果显示鼓阶打孔途径导入方法对大鼠听力影响较小。结论胚胎干细胞可经耳蜗底转鼓阶打孔途径导入耳蜗。干细胞在内耳成功存活,并且对内耳损伤小,因此。它可以作为内耳细胞移植的重要方式。 相似文献
2.
目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性,为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠,术前及术后分别行听性脑干反应(ABR)检查。实验组(12只)以重组腺病毒携带的增强型绿色荧光蛋白基因(enhancedgreenfluorescentprotein,EGFP),对照组(8只)以人工外淋巴液注入豚鼠圆窗龛内。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片,耳蜗冰冻切片观察。结果于圆窗龛内注入腺病毒携带目的基因的转导方法对听力无明显影响。转染耳蜗及对侧耳蜗内目的基因呈广泛表达。5天组表达产物最高,14天组逐渐降低。对照组耳蜗未见EPFP表达。结论于圆窗龛内注入腺病毒携带目的基因转导的方法对耳蜗无明显毒害作用,且能够将目的基因成功转导至双侧耳蜗组织并广泛表达。 相似文献
3.
目的:观察地塞米松(dexamethasone ,DEX)加氨茶碱(amionophylline ,AMI)治疗脂多糖(lipopo‐lysaccharide ,LPS)诱导的豚鼠急性听力损失的疗效及耳蜗组蛋白去乙酰化酶2(histone deacetylase 2,HDAC2)的表达,探讨治疗中 HDAC2对糖皮质激素(glucocorticoid ,GC)抵抗的影响。方法选取50只健康白色豚鼠,其中10只为对照组,仅双耳鼓阶注入5μl人工外淋巴液(artificial perilymph ,AP)(AP组);其余40只鼓阶注入5μl(5 mg/ml)的LPS后再随机分为4组,每组10只:模型组(LPS组),无其他处理;LPS+DEX组:术前30 min及术后24小时腹腔注射DEX 1 mg/kg ;LPS+AMI组:术前30 min及术后24小时腹腔注射AMI 20 mg/kg ;LPS+DEX+AMI组:术前30 min及术后24小时腹腔注射AMI 20 mg/kg及DEX 1 mg/kg。所有动物给药前及鼓阶注射术后48小时行ABR检测后取耳蜗,采用免疫荧光法,观察耳蜗基底膜铺片及耳蜗组织冰冻切片中HDAC2的分布及空间定位,用ELISA法测定耳蜗组织中HDAC2的含量。结果 LPS组鼓阶注射LPS后48小时全频听力损失,由低频向高频逐渐加重;LPS+DEX组及LPS+ AMI组16、32 kHz ABR阈移较LPS组改善明显(<0.05),LPS+DEX+AMI组各频率ABR阈移均低于LPS+DEX组(P<0.05),以16 kHz处最显著(P<0.01)。耳蜗基底膜铺片及耳蜗冰冻切片免疫染色结果表明HDAC2广泛分布于耳蜗中,且主要存在于胞核内。8、16、32 kHz ABR阈移与耳蜗内HDAC2的含量呈负相关(P<0.05)。结论 AMI可提高豚鼠耳蜗HDAC2的表达,HDAC2可改善治疗中GC抵抗,对提高GC治疗L PS导致的急性听力损失的敏感性有一定的作用。 相似文献
4.
目的沙鼠耳蜗圆窗应用毒毛旋花甙G(Ouabain),观察沙鼠听觉电生理变化。方法显微镜下将浸有100μl毒毛旋花甙G(Ouabain)1mM的明胶海绵置于沙鼠耳蜗圆窗膜表面,观察应用Ouabain5。12h后沙鼠听性脑干反应(auditorybrainstemresponse,ABR)、耳蜗微音电位(cochlearmicrophoniC,CM)、复合动作电位(compoundactionpotential,CAP)的变化。结果应用Ouabain5h:ABR阈值明显升高(P〈0.05),且波形不典型;CM阈值无明显变化(P〉0.05);CAP阈值明显升高(P〈0.05):应用Ouabain12h:ABR消失;CM阈值无明显变化(P〉0.05);CAP波形消失(P〈0.05)。结论沙鼠圆窗膜表面应用Ouabain(5、12h)导致沙鼠CAP与ABR阈值升高甚至波形消失,而CM阈值不变。为建立听神经病动物模型提供了较好的研究方法。 相似文献
5.
目的 研究经完整圆窗膜途径进行耳蜗基因转导的可行性及安全性,为内耳基因治疗提供实验基础和理论依据。方法 24只SD大鼠术前及术后分别行听性脑干反应(ABR)检查。实验组(18只)用阳离子脂质体携带的增强型绿色荧光蛋白(enhanced green fluoresent protein,EGFP)基因,对照组(6只)用生理盐水,注入置于圆窗龛处的明胶海绵内。分别于术后3、7、14天取双侧耳蜗标本做基底膜铺片观察。结果 于圆窗龛处放置明胶海绵的转导方法对听力无明显影响。转染耳蜗呈明显的绿色荧光。3天组表达产物最高,7天组逐渐降低,14天组更弱。对侧及对照组耳蜗均未见荧光表达。结论 于圆窗龛处放置明胶海绵进行基因转导的方法对听力没有影响,且能够成功转染耳蜗组织,是一种行之有效的方法。 相似文献
6.
目的研究携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)腺病毒(Ad-EG-FP)经由小鼠耳后圆窗径路导入内耳的可行性,分析EGFP在耳蜗内的表达特点。方法 19只健康的8~9周龄C57BL/6J雄性小鼠随机分为三组:Ad-EGFP组7只,人工外淋巴液组6只,两组通过耳后切口圆窗径路注射,分别导入Ad-EGFP和人工外淋巴液;空白对照组6只,未予处理。各组均于术前3日和术后7日行听性脑干反应(ABR)检查;术后7日取出耳蜗于荧光显微镜下观察EGFP在内耳的分布并行免疫组化观察EGFP在基底膜的表达。结果 3组动物术前ABR反应阈差异无统计学意义(P〉0.05),术后7日,Ad-EGFP组ABR反应阈为62.86±9.94dBSPL,人工外淋巴液组ABR反应阈为60.83±9.70dBSPL,均较术前(37.86±8.59和34.16±8.04dBSPL)及空白对照组(40.83±8.61dBSPL)高(P值均〈0.05);空白对照组实验前后ABR反应阈无变化,Ad-EGFP组与人工外淋巴液组术后7天ABR反应阈差异无统计学意义(P值均〈0.05)。Ad-EGFP组Ad-EGFP导入后在基底膜上可见EGFP呈广泛表达,人工外淋巴液组和空白对照组基底膜未见荧光表达。结论外源性基因可经内耳圆窗导入并在耳蜗基底膜上广泛表达。 相似文献
7.
耳后入路圆窗膜显微注射小鼠耳蜗基因转染新途径的研究 总被引:3,自引:0,他引:3
目的研究腺病毒携带目的基因经小鼠耳后人路圆窗膜显微注射途径耳蜗转导的可行性,为以小鼠作为动物模型的内耳基因治疗提供实验基础和解剖学依据。方法12只C57BL/6J小鼠分为2组,实验组(8只)以重组腺病毒携带的增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)、对照组(4只)以人工外淋巴液经耳后入路圆窗膜显微注射注入耳蜗内。分别于术后5、14天取双侧耳蜗标本做基底膜铺片,在激光共聚焦显微镜下观察GFP表达。结果术后动物存活10只(每组死亡1只)。实验组转染后耳蜗底回基底膜及螺旋神经节上目的基因有表达,14天组强于5天组。对照组耳蜗未见荧光表达。结论耳后入路操作简单、损伤小、易于暴露圆窗龛。耳后入路圆窗膜显微注射腺病毒携带目的基因转导的方法能够将目的基因成功转导至耳蜗组织并表达。 相似文献
8.
腹侧听泡外入路小鼠耳蜗鼓阶基因导入的实验研究 总被引:1,自引:0,他引:1
目的研究腺病毒携带目的基因经腹侧听泡外入路转导耳蜗鼓阶底转的可行性及目的基因的表达特点,为内耳基因治疗提供实验基础和理论依据。方法16只健康5周龄C57BL/6J小鼠,腺病毒组10只,以重组腺病毒携带有Hath-1和增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP),人工外淋巴液组6只以人工外淋巴液,经腹侧听泡外入路导入耳蜗鼓阶底转。分别于术后第7天分别行听性脑干反应(ABR)检查后取双侧耳蜗标本做基底膜铺片、耳蜗冰冻切片观察基因的表达。结果经腹侧听泡外入路转导耳蜗鼓阶底转的转导方法对听力影响较小。腺病毒组耳蜗内目的基因呈广泛表达。对照组耳蜗未见荧光表达。结论经腹侧听泡外入路转导耳蜗鼓阶底转的转导方法对听力影响较小,且能够将目的基因成功转导至耳蜗组织并广泛表达。 相似文献
9.
目的 探讨卡那霉素和速尿联合用药对豚鼠耳蜗听功能的影响。方法实验组50只豚鼠肌肉注射卡那霉素500mg/kg,同时静脉注射速尿40mg/kg,空白对照组4只豚鼠。于药物注射3天(25只)和7天(25只)后行听觉脑干诱发电位(auditory brainstem response,ABR).听神经复合动作电位(compound action potential,CAP)及耳蜗微音电位(cochlear microphonic,CM)检测,观察所有动物的中耳结构,对实验组和对照组豚鼠耳蜗听功能进行统计学分析。结果观察所有实验组豚鼠的中耳结构可见鼓膜完整.无充血或者穿孔.听骨链完整.听骨结构无破坏或者移位。豚鼠用药后听阈的个体差异较大,用药后同一频率CAP阈值低于ABR阈值,有的豚鼠用药后CAP阈值轻度提高,但是CM明显异常;将听阈〉95 dB SPL的豚鼠分为用药3天组和用药7天组,发现用药3天组各频率(2kHz,4kHz,8kHz和16kHz)比正常对照组同一频率的听力阈值明显提高(方差分析P〈0.01,差异有显著性),而用药3天组和用药7天组各频率(2kHz,4kHz,8kHz和16kHz)比较阈值没有差别(方差分析P〉0.05,差异没有显著性),用药后较高频率(8kHz,16kHz)的听力损失大于较低频率(2kHz,4kHz)。结论卡那霉素和速尿联合用药后3天可使豚鼠听功能严重受损,因此,卡那霉素和速尿联合用药是建立耳聋模型的一种快速而有效的方法。 相似文献
10.
内耳疾病基因治疗方法的研究 总被引:1,自引:0,他引:1
目的:探讨一种高效、安全和简便的内耳疾病基因转移和导入方法。方法:将30只健康豚鼠随机分为A、B、C 3组,每组10只。用Ad—GFP报告基因分别由圆窗、耳蜗底回和脑脊液三种途径导入30只豚鼠内耳,在术前和术后测定听性脑干反应(ABR)阈值和畸变产物耳声发射(DPOAE)振幅改变。再行耳蜗铺片,观察AdGFP在内耳组织中的转染表达和耳蜗毛细胞的形态结构。结果:三种途径导入Ad—GFP在耳蜗螺旋神经节、血管纹和Corti器上均有转染和表达。在术后3 d的表达产物最高,7 d后减弱,10 d后更弱。圆窗和耳蜗底回钻孔导入手术可引起蜗内出血,凝血块淤积在耳蜗顶回的中阶,造成顶回毛细胞损害。而脑脊液给药途径无此现象。ABR阈值在各组动物手术前后无明显改变,而圆窗和耳蜗底回给药途径造成DPOAE幅度明显下降,脑脊液给药组无DPOAE改变。结论:应用腺病毒载体进行基因转移通过圆窗和耳蜗底回钻孔途径可引起蜗内出血,影响DPOAE幅度,而应用脑脊液给药途径则相对安全和方便。 相似文献
11.
CHEN Wei HU Yin-yan YANG Shi-ming GUO Wei SUN Jian-he HAN Dong-yi ZHAI Suo-qiang YANG Wei-yan David Z.Z.He 《中华耳科学杂志(英文版)》2008,3(1):18-23
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
12.
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
13.
Math1基因导入成年大鼠前庭有效途径的探索 总被引:1,自引:1,他引:0
目的探索Math1基因导人大鼠前庭简便有效的方法和途径,为前庭功能障碍基因治疗的相关研究提供参考。方法将20只成年Wistar大鼠分为缺失E1、E3基因片段且构建有Math1基因和增强型绿色荧光蛋白报告基凶的复制缺陷型腺病毒(adnovirus—Math1—enhanced green fluorescence protein,Ad—Math1—EGFP)鼓阶导入组和前庭阶导人组.Ad—Math1—EGFP导入组大鼠在右耳通过耳蜗底转鼓阶或前庭阶打孔的方法导人物理滴度为2.1×1011v.p/ml的上述腺病毒5μl。在导入3天、7天后分别将动物处死,进行GFP表达观察。结果导入Ad—Mathl—EGFP3天后,前庭阶导入组大鼠的前庭终末器官及耳蜗均出现明显的GFP阳性表达;而鼓阶导入组的表达则局限于耳蜗,7天后仍未见前庭终末器官的GFP阳性表达。结论耳蜗底转前庭阶打孔可以作为Math1基因导入大鼠前庭简便有效的途径。 相似文献
14.
Darrouzet V Erre JP Dulon D Camicas Van Gout M Aran JM Poutout PA Négrevergne M Dauman R Bébéar JP 《Revue de laryngologie - otologie - rhinologie》1999,120(5):305-316
OBJECTIVES: To establish that susceptance-conductance tympanograms at a probe-tone frequency of 2 kHz reflects the status of the annular ligament (AL) and through it of the cochlea. METHODS: Experimental study in 5 chinchillas and 22 guinea pigs. Six validating experiments were used: blockages of the stapes and of the round window membrane (RWM), fistula of the RWM, fluid removal from the cochlea, injection of saline in the scala tympani (ST) and acoustic trauma (AT). Quantitative data (mean values of Y226, FR, Y2000, G2000 and B2000) and shape of the curves were analyzed before and immediately after lesions were done. RESULTS: Guinea pig was the most convenient provided bulla was vented and the same tip was used along the experiments. Only the shape of the curves are discriminant: 1/a supplementary sharp peak, centered around negative pressures, is observed in Y/G tympanograms in every case of RWM fistulas and in some case of AT. 2/injection of saline into ST induces immediate and reproducible Y2000, G2000, et B2000 curves modifications. 3/RWM and stapes blockages provoke foreseeable stiffening and sharpening of the tympanograms at 2 kHz. 4/on the contrary, fluid removal from the cochlea induces multiple peaks curves. CONCLUSIONS: Experimentally-induced modifications at the AL either direct (stapes blockage) or indirect by AT or decrease/increase of pressure load at the cochlear interface at the footplate result in noticeable, constant, reproducible changes of curves registered at 2 kHz. The stapes behaves both as the plotter of the curves and the interpreter of the inner ear pressure. 相似文献
15.
Drug delivery to the cochlea using PLGA nanoparticles 总被引:4,自引:0,他引:4
Tamura T Kita T Nakagawa T Endo T Kim TS Ishihara T Mizushima Y Higaki M Ito J 《The Laryngoscope》2005,115(11):2000-2005
OBJECTIVES: This study aimed to investigate the efficacy of encapsulating therapeutic molecules in poly lactic/glycolic acid (PLGA) nanoparticles for drug delivery to the cochlea. STUDY DESIGN: An experimental study. METHODS: We examined the distribution of rhodamine, a fluorescent dye, in the cochlea, liver, and kidney of guinea pigs. Intravenous injection of rhodamine or rhodamine-encapsulated PLGA nanoparticles was used to target the fluorescent dye systemically to the liver, kidney, and cochlea, and these molecules were applied locally to the round window membrane (RWM) of the cochlea. The localization of rhodamine fluorescence in each region was quantitatively analyzed. RESULTS: After systemic application of rhodamine nanoparticles, fluorescence was identified in the liver, kidney, and cochlea. The systemic application of nanoparticles had a significant effect on targeted and sustained delivery of rhodamine to the liver but not the kidney or cochlea. Rhodamine nanoparticles placed on the RWM were identified in the scala tympani as nanoparticles, indicating that the PLGA nanoparticles can permeate through the RWM. Furthermore, the local application of rhodamine nanoparticles to the RWM was more effective in targeted delivery to the cochlea than systemic application. CONCLUSIONS: These findings indicate that PLGA nanoparticles can be an useful drug carrier to the cochlea via local application. 相似文献
16.
Connor SE Holland NJ Agger A Leong AC Varghese RA Jiang D Fitzgerald O'Connor A 《Acta oto-laryngologica》2012,132(9):932-937
Abstract Conclusions: The round window membrane (RWM)-intentioned approach is superior to the traditional bony cochleostomy (BC) approach in obtaining electrode placement within the scala tympani (ST). Objective: Cochlear implant outcome is influenced by several factors, including optimal placement and retention of the electrode array within the ST. The present study aimed to assess whether the RWM route is superior to a traditional BC for placement and retention of the electrode array in the ST. Methods: This was a prospective consecutive non-randomized comparison study. All patients were implanted with the Advanced Bionics 1J electrode array. The RWM approach (n = 32) was compared with a traditional BC group (n = 33). The outcome measure was the electrode position as judged within the scalar chambers at four points along the basal turn using postoperative computed tomography (CT). Results: When the mean position scores were compared, the RWM-intentioned group had significantly more electrodes directed towards the ST compartment than the BC group (p < 0.001). The RWM electrodes achieved 94% ST retention compared with 64% for the BC group (p < 0.05). All electrodes stayed in the ST in the RWM group, whereas in the BC group 9% crossed from the ST to the scala vestibuli. 相似文献
17.
E. B. King A. N. Salt H. T. Eastwood S. J. O’Leary 《Journal of the Association for Research in Otolaryngology》2011,12(6):741-751
Although intratympanic (IT) administration of drugs has gained wide clinical acceptance, the distribution of drugs in the
inner ear following IT administration is not well established. Gadolinium (Gd) has been previously used as a marker in conjunction
with magnetic resonance imaging (MRI) to visualize distribution in inner ear fluids in a qualitative manner. In the present
study, we applied gadolinium chelated with diethylenetriamine penta-acetic acid (Gd-DTPA) to the round window niche of 12
guinea pigs using SeprapackTM (carboxlmethylcellulose-hyaluronic acid) pledgets which stabilized the fluid volume in the round window niche. Gd-DTPA distribution
was monitored sequentially with time following application. Distribution in normal, unperforated ears was compared with ears
that had undergone a cochleostomy in the basal turn of scala tympani and implantation with a silastic electrode. Results were
quantified using image analysis software. In all animals, Gd-DTPA was seen in the lower basal scala tympani (ST), scala vestibuli
(SV), and throughout the vestibule and semi-circular canals by 1 h after application. Although Gd-DTPA levels in ST were higher
than those in the vestibule in a few ears, the majority showed higher Gd-DTPA levels in the vestibule than ST at both early
and later time points. Quantitative computer simulations of the experiment, taking into account the larger volume of the vestibule
compared to scala tympani, suggest most Gd-DTPA (up to 90%) entered the vestibule directly in the vicinity of the stapes rather
than indirectly through the round window membrane and ST. Gd-DTPA levels were minimally affected by the implantation procedure
after 1 h. Gd-DTPA levels in the basal turn of scala tympani were lower in implanted animals, but the difference compared
to non-implanted ears did not reach statistical significance. 相似文献
18.
Communication between the perilymphatic scalae and spiral ligament visualized by in vivo MRI 总被引:2,自引:0,他引:2
We evaluated the transport of Gadolinium-diethylenetriaminepentaacetate-bismethylamide (Gd-DTPA-BMA) through the round window (RW) membrane into the perilymphatic space with 4.7-T MRI in an animal study and 1.5-T MRI in humans. After administration of Gd-DTPA-BMA onto the intact RW membrane of guinea pig, Gd-DTPA-BMA uptake was observed in the basal turn and part of the second turn within 40 min. The scala tympani, scala vestibuli, the fibrous part of the spiral ligament and semicircular canal all showed uptake of Gd-DTPA-BMA. All turns of the cochlea were filled with Gd within 10 min in the perforated RW membrane administration group and within 30 min in the intravenous administration group. In patients who accepted middle ear injection of Gd-DTPA-BMA, uptake was observed within 2 h in the basal turn and semicircular canal. After 12 h the apex did still not show any uptake. Gd-DTPA-BMA is transported from the RW to the semicircular canal, the scala tympani and scala vestibuli without passing the helicotrema. 相似文献