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...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(enhanced green fluorescent protein,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。展开更多
目的研究腺病毒携带目的基因经小鼠耳后入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(en...目的研究腺病毒携带目的基因经小鼠耳后入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。展开更多
Objective To study expression of adenoviral-mediated Hath1-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 guine...Objective To study expression of adenoviral-mediated Hath1-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 Ad-Hath1-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 frozen sections were examined. Results ABR tests showed no significant change of hearing after the surgery. Strong fluorescence staining in the cochleae was seen in Ad-Hath1-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with little 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.展开更多
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...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。展开更多
Background In recent years the interest of sustained drug delivery into inner ear is promising, at the same time a great deal of novel oral drugs using biodegradable vehicles have been produced to achieve sustained dr...Background In recent years the interest of sustained drug delivery into inner ear is promising, at the same time a great deal of novel oral drugs using biodegradable vehicles have been produced to achieve sustained drug release. The aim of this study was to use biodegradable vehicles to release dexamethasone in the round window membrane application. Methods Dexamethasone gels composed of alginate and chitin were prepared and the release-permeating profiles were studied using a reproducible in vitro apparatus. A longer-period time course was simulated using the parameters acquired in this study. The data obtained in this study was compared with those of other studies in intratympanic drug delivery, and an appropriate initial dosage was extrapolated. Results The combination of alginate and chitin could efficiently restrict dexamethasone diffusion and the time course suggested a sustained drug concentration within 24 hours. A higher initial dosage was estimated to achieve a stable therapeutic concentration in vivo. Conclusion The combination of alginate and chitin could be used as vehicle for sustained release of dexamethasone in intratympanic application.展开更多
目的探讨人工耳蜗植入术中不同刺激位置对电诱发听觉脑干反应(EABR)波形的影响。方法选取CT,MRI影像耳蜗形态正常的双侧感音神经性耳聋患30例,均为单侧植入。植入术中先后电刺激术耳圆窗龛与圆窗膜,比较分析刺激强度在1.0-3.0mA之间时2...目的探讨人工耳蜗植入术中不同刺激位置对电诱发听觉脑干反应(EABR)波形的影响。方法选取CT,MRI影像耳蜗形态正常的双侧感音神经性耳聋患30例,均为单侧植入。植入术中先后电刺激术耳圆窗龛与圆窗膜,比较分析刺激强度在1.0-3.0mA之间时2种不同位置处EABR阈值以及Ⅲ波、Ⅴ波引出率、潜伏期和波间期的差别。结果①EABR阈值在圆窗龛刺激中为1.745±0.09692 m A,圆窗膜刺激中为1.275±0.1160 m A(P<0.05)。②EABRⅢ波检出率圆窗龛刺激为66.667%,圆窗膜刺激为93.333%(P<0.05)。③Ⅲ波潜伏期圆窗龛刺激为2.215±0.04600ms,圆窗膜刺激为2.180±0.03566ms。④EABRⅤ波圆窗龛刺激检出率为50%,圆窗膜刺激检出率为93.333%(P<0.05)。⑤EABRⅤ波潜伏期圆窗龛刺激为4.059±0.08122ms,圆窗膜刺激为3.951±0.06329 ms。⑥EABRⅢ-Ⅴ间期圆窗龛刺激为1.880±0.06285 ms,圆窗膜刺激为1.807±0.04218 ms。结论圆窗膜刺激EABR波形稳定,可重复性强,受肌电反应干扰小,引出率高,以较小的刺激强度能取得较好的波形,对听觉传导通路的评估更为准确。展开更多
We investigated the pharmacokinetics of caroverine in the perilymph,cerebrospinal fluid and plasma after systemic and local administrations in guinea pigs by using high-performance liquid chromatography.Auditory brain...We investigated the pharmacokinetics of caroverine in the perilymph,cerebrospinal fluid and plasma after systemic and local administrations in guinea pigs by using high-performance liquid chromatography.Auditory brainstem responses were measured to evaluate auditory functional effect.The results showed that local application was a both safe and efficient method.We further reviewed literature and pinpointed that the round window is effectively local drug delivery means for future inner ear treatment.展开更多
文摘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(enhanced green fluorescent protein,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。
文摘目的研究腺病毒携带目的基因经小鼠耳后入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。
基金National NaturalScience Foundation grants No.30730040 and No.30628030.
文摘Objective To study expression of adenoviral-mediated Hath1-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 Ad-Hath1-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 frozen sections were examined. Results ABR tests showed no significant change of hearing after the surgery. Strong fluorescence staining in the cochleae was seen in Ad-Hath1-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with little 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.
文摘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。
基金This work was supported by a grant from Beijing Medical Development Foundation(No.2002-2012)
文摘Background In recent years the interest of sustained drug delivery into inner ear is promising, at the same time a great deal of novel oral drugs using biodegradable vehicles have been produced to achieve sustained drug release. The aim of this study was to use biodegradable vehicles to release dexamethasone in the round window membrane application. Methods Dexamethasone gels composed of alginate and chitin were prepared and the release-permeating profiles were studied using a reproducible in vitro apparatus. A longer-period time course was simulated using the parameters acquired in this study. The data obtained in this study was compared with those of other studies in intratympanic drug delivery, and an appropriate initial dosage was extrapolated. Results The combination of alginate and chitin could efficiently restrict dexamethasone diffusion and the time course suggested a sustained drug concentration within 24 hours. A higher initial dosage was estimated to achieve a stable therapeutic concentration in vivo. Conclusion The combination of alginate and chitin could be used as vehicle for sustained release of dexamethasone in intratympanic application.
文摘目的探讨人工耳蜗植入术中不同刺激位置对电诱发听觉脑干反应(EABR)波形的影响。方法选取CT,MRI影像耳蜗形态正常的双侧感音神经性耳聋患30例,均为单侧植入。植入术中先后电刺激术耳圆窗龛与圆窗膜,比较分析刺激强度在1.0-3.0mA之间时2种不同位置处EABR阈值以及Ⅲ波、Ⅴ波引出率、潜伏期和波间期的差别。结果①EABR阈值在圆窗龛刺激中为1.745±0.09692 m A,圆窗膜刺激中为1.275±0.1160 m A(P<0.05)。②EABRⅢ波检出率圆窗龛刺激为66.667%,圆窗膜刺激为93.333%(P<0.05)。③Ⅲ波潜伏期圆窗龛刺激为2.215±0.04600ms,圆窗膜刺激为2.180±0.03566ms。④EABRⅤ波圆窗龛刺激检出率为50%,圆窗膜刺激检出率为93.333%(P<0.05)。⑤EABRⅤ波潜伏期圆窗龛刺激为4.059±0.08122ms,圆窗膜刺激为3.951±0.06329 ms。⑥EABRⅢ-Ⅴ间期圆窗龛刺激为1.880±0.06285 ms,圆窗膜刺激为1.807±0.04218 ms。结论圆窗膜刺激EABR波形稳定,可重复性强,受肌电反应干扰小,引出率高,以较小的刺激强度能取得较好的波形,对听觉传导通路的评估更为准确。
文摘We investigated the pharmacokinetics of caroverine in the perilymph,cerebrospinal fluid and plasma after systemic and local administrations in guinea pigs by using high-performance liquid chromatography.Auditory brainstem responses were measured to evaluate auditory functional effect.The results showed that local application was a both safe and efficient method.We further reviewed literature and pinpointed that the round window is effectively local drug delivery means for future inner ear treatment.
