经完整圆窗膜途径EGFP基因在大鼠耳蜗中的表达

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。     

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

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。     

经大鼠圆窗膜bFGF基因转导防治爆震性聋的实验研究

目的以阳离子脂质体介导bFGF(basic fibroblast growth factor,碱性成纤维细胞生长因子)基因,经完整圆窗膜途径转染爆震后的大鼠耳蜗,观察bFGF的表达及其对爆震性聋的防治作用。方法 SD大鼠30只,随机分为四组:空白对照组(n=6),仅接受155dB SPL脉冲噪声暴露20次;EGFP(enhanced green fluorescent protein,增强型绿色荧光蛋白)对照组(n=8),噪声暴露后即刻导入EGFP/阳离子脂质体复合物;bFGF治疗组(n=8),噪声暴露后即刻导入EGFP-bFGF/阳离子脂质体复合物;bFGF保护组(n=8),噪声暴露前3天导入EGFP-bFGF/阳离子脂质体复合物。各组动物分别于噪声暴露前及噪声暴露后1天、3天、7天、14天行ABR阈值测试。噪声暴露后14天耳蜗取材做基底膜铺片,荧光显微镜观察,并做bFGF免疫荧光染色验证bFGF的表达。结果爆震后1天,bFGF保护组ABR阈值低于空白对照组及EGFP对照组,且差异均有显著性意义(P<0.05)。爆震后3天、7天和14天,bFGF治疗组及bFGF保护组ABR阈值均低于两个对照组,且差异有显著性意义(P<0.05)。而爆震前及爆震后bFGF治疗组和bFGF保护组间的ABR阈值差异均无显著性意义(P>0.05)。术后14天,荧光显微镜直接观察及bFGF免疫荧光染色均检测到毛细胞内有bFGF的定位表达。结论将阳离子脂质体介导的bFGF基因经圆窗膜导入大鼠耳蜗能够表达,表达产生的bFGF对爆震所致的耳蜗损害有一定的保护作用,它能够减轻爆震后听。     

小鼠内耳圆窗基因导入的实验研究

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&#177;9.94dBSPL,人工外淋巴液组ABR反应阈为60.83&#177;9.70dBSPL,均较术前（37.86&#177;8.59和34.16&#177;8.04dBSPL）及空白对照组（40.83&#177;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。     

氯化钠等物质豚鼠圆窗膜上对前庭功能行为影响的观察

在豚鼠的单侧或双侧圆窗膜上旋转ＮａＣｌ，ＫＣｌ或蔗糖晶体，观察其前庭功能行为受影响情况。结果：置ＮａＣｌ或ＫＣｌ的动物中，单侧置药组和双侧不同时置药组的部分动物出现刺激性和麻痹性双相眼震或其中一相眼震，而双侧同时置药组动物均未出现类似现象。各组的部分动物均可诱发变向性位置性眼震。放置蔗糖对前庭功能行为无影响。提示圆窗外单纯高渗透压对前庭无明显影响，放置ＮａＣｌ或ＫＣｌ后Ｎａ＾＋，Ｋ＾＋进入内耳可能     

携带c-myc基因重组腺病毒的构建和鉴定及其在豚鼠耳蜗中的表达与分布

目的 构建携带c-myc基因的重组腺病毒表达载体,并观察其在豚鼠耳蜗内的表达分布,为探讨该基因的功能奠定实验基础.方法 利用细菌内同源重组的方法.构建携带c-myc基因的重组腺病毒表达载体(Ad.c-myc-EGFP).并分别应用酶切、逆转录聚合酶链反应(RT-PCR)和测序方法 鉴定腺病毒的构建情况.利用豚鼠耳蜗内显微注射术、Western blot及荧光显微镜观察注射后该蛋白在耳蜗中的表达及分布特性.结果 经酶切、RT-PCR和测序鉴定,质粒构建正确,重组腺病毒Ad.c-myc-EGFP构建成功.Ad.c-myc-EGFP腺病毒豚鼠耳蜗内注射后第三天便可通过荧光显微镜观察到绿色荧光蛋白的广泛分布,且WesternBlot方法 证实它可以上调耳蜗内c-Myc蛋白的表达.结论 本实验成功构建了携带c-myc基因的重组腺病毒表达载体.它可以有效地感染豚鼠耳蜗内各种细胞.为深入研究C-MYC基因在耳蜗中的作用奠定了实验基础.     

氯化钠等物质置豚鼠圆窗膜上对前庭功能行为影响的观察

在豚鼠的单侧或双侧圆窗膜上放置ＮａＣｌ、ＫＣｌ或蔗糖晶体，观察其前庭功能行为受影响情况。结果：置ＮａＣｌ或ＫＣｌ的动物中，单侧置药组和双侧不同时置药组的部分动物出现刺激性和麻痹性双相眼震或其中一相眼震，而双侧同时置药组动物均未出现类似现象。各组的部分动物均可诱发变向性位置性眼震。放置蔗糖对前程功能行为无影响。提示圆窗外单纯高渗透压对前庭无明显影响，放置ＮａＣｌ或ＫＣｌ后Ｎａ＋、Ｋ＋进入内耳可能与前庭兴奋性改变有关。     

腺病毒携带的LacZ基因在豚鼠耳蜗中的表达

目的 带有LacZ基因的腺病毒注入豚鼠耳蜗后,观察不同时间段LacZ基因的表达和分布情况及手术操作对听力的影响,为内耳基因治疗提供理论依据。方法 24只白色豚鼠术前及术后行听性脑干反应(ABR)检查。空白对照组经圆窗注入人工外淋巴液,实验组注入带有LacZ基因的腺病毒。分别于2天、l周、2周后取材。耳蜗标本经β-半乳糖苷酶(X-Gal)组织化学染色后做石蜡切片和耳蜗铺片。结果 腺病毒注入耳蜗后对听力影响不大。经X-Gal染色后整个耳蜗被染成蓝色。2天组表达产物最高,l周后逐渐降低。表达产物主要分布于柯蒂器的内外毛细胞、螺旋神经节细胞、基底膜下的间皮细胞。对照组均未着色。结论 通过圆窗注入腺病毒对听力没有影响,单一位点的接种就能使因子通过耳蜗液扩散到整个耳蜗。有效的基因转移在耳蜗是可行的,但表达相对短暂。     

圆窗膜样品的快速制备与环境扫描电镜观察

环境扫描电镜（ESEM）是一种不同于传统扫描电镜（CSEM）的新型表面超微结构观察工具,其主要特点为：快速、简便的样品制备程序和在较低要求的环境条件下,可直接观察到新鲜的生物样品.目前ESEM已用来观察海洋生物、活细胞、肠腔黏膜等新鲜组织样品,并获得良好的效果.为探讨ESEM在耳科形态学研究中的技术方法和应用价值,本实验以豚鼠圆窗膜为对象,应用Philips-XL30型ESEM观察豚鼠圆窗膜组织的双表面超微结构——即鼓室面及鼓阶面的形态特征,并与以往CSEM的圆窗膜图像进行了比较.     

腺病毒通过不同径路导入豚鼠耳蜗后的实验观察

目的：探讨经不同径路将腺病毒导入豚鼠耳蜗后,被感染耳蜗细胞的分布情况。方法：使用构建有EGFP指示基因的腺病毒分别通过圆窗入路进入外淋巴系统,耳蜗侧壁钻孔中阶入路进入内淋巴系统,耳蜗冷冻切片和组织铺片观察腺病毒感染耳蜗细胞的分布情况。结果：由圆窗入路进入外淋巴系统的腺病毒可以感染血管纹的I型、Ⅳ型和V型纤维细胞、螺旋唇上细胞、前庭膜细胞、Ronsensal孔内的螺旋神经元、前庭阶和鼓阶的上皮细胞,内外毛细胞和支持细胞不被感染;而耳蜗侧壁钻孔中阶入路进入内淋巴系统的腺病毒可以感染听器的支持细胞和血管纹缘细胞等。结论：腺病毒是一种有效的豚鼠耳蜗细胞转染载体,导入耳蜗后,可以将目的基因转染到耳蜗细胞内。通过不同径路将腺病毒导入外、内淋巴系统,耳蜗被感染的细胞范围不一致。仅注入外淋巴系统的腺病毒无法感染内淋巴系统内的细胞,只有将病毒导入内淋巴系统,耳蜗支持细胞才被感染。     

adenoviral-mediated hath1-egfp gene transfer into guinea pig cochlea through intact round window membrane

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.     

Efficacy of gene transfer through the round window membrane: an in vitro model

The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM. The round window including the bony niche of a CD1 mouse was removed under an operating microscope and fixed with adhesive on the base of a petri dish through which a hole had been drilled. Toluidine blue was injected into the niche containing a hyaluronic acid ester sponge against the round window membrane. The niche was closed with a fascia. A plastic tube containing PBS was fixed on the opposite side, from where the samples were collected at different time points. The concentration of toluidine blue was evaluated spectrophotometrically. An adenoviral vector containing green fluorescent protein (GFP) marker gene was injected into the niche. Samples were collected from the opposite side at different time points. The presence of the vector was studied with GFP PCR. We also modulated the permeability of the RWM by treating it with clinically applicable detergents, histamine or silver nitrate. Silver nitrate and trichloracetic acid caused destruction of the surface epithelium of the RWM as shown by light microscopy. Both toluidine blue and adenoviral vectors passed through the RWM in a time-dependent fashion. RWM cells expressed GFP after Ad-GFP treatment. The permeability of the RWM was decreased after treatment with different detergents, histamine or silver nitrate. RWM offers an atraumatic route to the inner ear. Compared with more invasive gene delivery methods, this technique represents a safer and clinically more viable route of cochlear gene delivery.     

Combined rupture of Reissner's membrane and round window: an experimental study in guinea pigs: experimental double-membrane rupture

OBJECTIVE: Hearing loss caused by combined rupture of Reissner's membrane and the round window (RW) membrane (the double-membrane rupture) may differ depending on the site of the lesion on Reissner's membrane. The purpose of this experimental study was to reveal the relationship between the hearing impairment and the site of the lesion on Reissner's membrane. BACKGROUND: According to experimental studies on perilymphatic fistula (PLF), profound hearing loss is not induced by rupture of RW alone, but by the double-membrane rupture. However, the mechanism responsible for hearing loss in the double-membrane rupture remains unclear. METHODS: Compound action potentials (CAPs) of the cochlear nerve in response to tone pip stimuli (1, 2, 4, and 8 kHz) were recorded before the lesion, 90 minutes after the Reissner's membrane rupture, and 90 minutes after subsequent laceration of the RW. Reissner's membrane was ruptured at one of the four turns for comparison. RESULTS: The double-membrane rupture caused a more severe increase in CAP thresholds than seen with separate ruptures, when the Reissner's membrane was ruptured at the second turn. Such pronounced increase in threshold was not seen in ears with the rupture at other turns. CONCLUSIONS: The double-membrane rupture causes varying degrees of hearing loss depending on the site of the lesion of Reissner's membrane. When the Reissner's membrane was ruptured at the second turn, the most severe hearing loss was detected.     

Cochlear gene transfer: round window versus cochleostomy inoculation.

Two possible approaches for cochlear gene transfer have been inoculation via the round window membrane and through a cochleostomy. The aim of this study was to determine which of the two is more effective. Using both approaches, normal-hearing and deafened guinea pigs were inoculated with adenovirus carrying the reporter gene lacZ. After 5 days, the animals were killed and the cochlear tissue was stained with X-gal. The distribution and intensity of staining was estimated by a score system developed to compare gene transfer results between animals. We found that gene transfer via the cochleostomy resulted in a better distribution throughout the cochlea and in higher staining intensity, due to more efficient transfection. Auditory brainstem response (ABR) results showed that neither virus inoculation through a cochleostomy nor through the round window membrane had a significant effect on the click-ABR threshold measured on day 5 following virus injection. Gene transfer via both approaches was also found to be more effective in deafened animals than in hearing animals.     

离体圆窗膜对地塞米松不同制剂的通透性

目的 制备离体圆窗膜试验模型，分析海藻酸钠（alginate sodium，ALG）、羧甲基甲壳胺（carboxymethyled chitosan，CHI）2种缓释剂对地塞米松（dexamethasone，DXM）透膜释放的效力。方法 模拟中耳及外淋巴腔环境，设计圆窗膜离体试验模型；制备不同浓度的海藻酸钠和羧甲基甲壳胺的地塞米松磷酸钠组。利用圆窗膜离体试验模型，于9h内的不同时间点采样，用高效液相色谱（high performance liquid chromatography，HPLC）法测定浓度，计算地塞米松对圆窗膜的累积通透量并进行方程模拟。结果 对照组在给药后3h内迅速通透圆窗膜，3h后接近平衡；25g／L海藻酸钠分别与4g／L或6g／L羧甲基甲壳胺的配方缓释作用最强（P〈0．01）；单独应用25g／L海藻酸钠的缓释作用次之；单独应用4g／L羧甲基甲壳胺仅具有微小促渗作用。结论 离体圆窗膜通透模型允许地塞米松分子自由通透；添加合适剂量的缓释材料——海藻酸钠与羧甲基甲壳胺的地塞米松磷酸钠制剂具有明显缓释作用，有可能成为未来用于内耳局部给药缓释材料的理想选择。     

Diffusion of amikacin at the membrane of the round window. Histologic and electrophysiologic study in newborn guinea pigs]

The aim of our study was to evaluate the permeability of the round window membrane for amikacin during postnatal development. The morphologic and electrophysiologic changes in the cochlea were dependent on the applied concentration of amikacin. When giving high doses a complete degeneration of hair cells and deafness could be found. In these cases an influence on the contralateral cochlea could be proved.     

透明质酸圆窗灌注对豚鼠内耳功能和形态学的影响

为探索透明质酸中耳应用对内耳的影响，以２６只健康豚鼠于左耳用２％透明质酸圆窗灌注前和灌注后５、１４、２８天检测前庭和听觉功能，并行基底膜铺片及颞骨火棉胶包埋连续切片。发现２％透明质酸圆窗灌注后５天，滤波短声０．２５～１０ｋＨｚ诱发的听神经动作电位（ＡＰ）阈值均提高（Ｐ＜０．０５），术后１４天在１、２、４ｋＨｚＡＰ阈值明显改善（Ｐ＜０．０５），术后２８天均恢复至术前水平（Ｐ＞０．０５），灌注前后ＥＮＧ眼震时间无明显变化（Ｐ＞０．０５）。耳蜗Ｃｏｒｔｉ器和前庭终器组织学检查无明显损害。结论：２％透明质酸圆窗灌注对内耳听觉功能有暂时性影响，未见内耳组织学改变，透明质酸具有缓慢渗透脱水作用，可作为药物缓释体用于中耳及内耳手术。     

光化学法诱导豚鼠耳蜗微循环障碍模型

目的 通过光化学法建立豚鼠耳蜗微循环障碍模型以及观察血管纹、耳蜗毛细胞形态学变化.方法 ①将豚鼠随机分成5个组.实验组分成3个组,颈外静脉注入四氯四碘荧光素二钠(rose bengal,RB),用波长为(540±40)nm、光强为(500～600)mW/cm2绿光照射打开的听泡,各组选用不同光照时间诱导耳蜗微循环变化.对照组分2个组,对照Ⅰ组仅颈外静脉注入RB而不行光照;对照Ⅱ组不注入RB而仅行光照耳蜗.②分别对5个组动物行听性脑干反应及形态学检测.结果 ①每个实验组两次ABRⅢ波反应阈差异均有统计学意义(P＜0.05);②形态学观察实验组均出现耳蜗微循环障碍的变化,螺旋器以外毛细胞破坏为主.结论 采用光化学法可致耳蜗微循环障碍及毛细胞破坏,方法简便,易于实施,重复率高.