Hath1基因和DAPT治疗大鼠耳聋的初步研究

发布时间：2018-04-16 19:39

本文选题：卡那霉素 + 速尿　；参考：《福建医科大学》2011年硕士论文

【摘要】：感音神经性聋严重影响着人类的健康和生存质量,但目前尚无有效治疗方法,毛细胞再生是治疗感音神经性聋的关键。随着分子生物学、分子遗传学、基因工程技术等的不断发展,尤其是近年来基因调控内耳毛细胞再生方面的研究逐步深入,为治疗感音神经性聋开辟了一条崭新的道路。本研究通过建立药物性聋动物模型,并进行Hath1基因和DAPT联合导入内耳诱导毛细胞再生,探索感音神经性聋药物治疗的方法。第一部分速尿和卡那霉素联合用药后大鼠听功能和内耳毛细胞损害观察目的:探讨速尿和硫酸卡那霉素联合用药快速制备大鼠药物性聋动物模型的方法。方法:实验组大鼠静脉注射速尿和/或肌肉注射硫酸卡那霉素,正常对照组不予任何处理。给药3天、7天、2个月后行听觉脑干诱发电位(auditory brainstem response ABR)阈值检测,给药7天后行耳蜗扫描电镜、全耳蜗基底膜铺片及内耳冰冻切片免疫荧光染色和内耳冰冻切片HE染色观察。结果:联合应用不同剂量速尿和硫酸卡那霉素后,大鼠ABR阈值出现不同程度的提高,给药3天、7天、2个月后阈值两两比较均无统计学意义;扫描电镜下耳蜗毛细胞出现由底回到顶回、由外毛细胞到内毛细胞的不同程度损伤。而单用速尿或硫酸卡那霉素的大鼠并不出现阈值提高或毛细胞损伤。耳蜗内、外毛细胞完全损伤的大鼠,支持细胞大部分完好,前庭毛细胞未见损伤。结论:速尿和硫酸卡那霉素具有明显的耳毒性协同作用,联合用药可导致大鼠听功能严重受损并且用药2个月致聋大鼠听力无恢复,但对耳蜗支持细胞和前庭毛细胞损伤较轻;速尿和卡那霉素联合用药是建立大鼠药物性聋动物模型的一种简单、快速且较可靠的方法。第二部分Hath1基因和DAPT联合导入内耳诱导毛细胞再生的初步研究目的:探讨Hath1基因和DAPT联合导入内耳后,Hath1在内耳的表达及诱导毛细胞再生的情况。方法:正常大鼠与致聋大鼠(ABR阈值 110dB SPL)各15只,经背侧入路暴露听泡,去除部分听泡骨暴露鼓阶,于鼓阶近圆窗处打孔微量进液器导入Hath1基因,随后用胶囊渗透压泵经鼓阶持续导入DAPT 4周。行ABR阈值检测,耳蜗扫描电镜观察及全耳蜗基底膜铺片荧光染色观察。结果:所有大鼠非导入耳ABR阈值导入前后无改变,正常大鼠导入耳ABR阈值较导入前提高约10dB SPL,致聋大鼠导入耳ABR阈值导入前后无改变;与导入前相比较,所有正常大鼠与致聋大鼠的导入耳没有发现毛细胞增多或再生。结论:Hath1基因和DAPT联合内耳导入未能诱导药物性聋大鼠耳蜗毛细胞再生,致聋大鼠毛细胞再生和听力改善可能需要多基因联合导入。
[Abstract]:Sensorineural hearing loss seriously affects human health and quality of life, but there is no effective treatment. Hair cell regeneration is the key to the treatment of sensorineural hearing loss.With the development of molecular biology, molecular genetics and genetic engineering technology, especially the research on gene regulation of hair cell regeneration in inner ear, it opens a new way for the treatment of sensorineural hearing loss.In this study, the animal model of drug-induced deafness was established, and the hair cell regeneration was induced by the combination of Hath1 gene and DAPT to induce hair cell regeneration, and the method of drug therapy for sensorineural hearing loss was explored.Part I observation of auditory function and hair cell damage in inner ear of rats after combined therapy of furosemide and kanamycin objective: to study the method of rapid establishment of drug induced deafness model in rats by combination of furosemide and kanamycin sulfate.Methods: rats in the experimental group were given furosemide intravenously and / or kanamycin sulfate intramuscularly.The threshold value of auditory brainstem evoked potential (ABAEP) brainstem response ABR was detected after 3 days and 7 days, and cochlea scanning electron microscope was used after 7 days of administration. Immunofluorescence staining of the whole cochlear basement membrane and frozen sections of the inner ear and HE staining of the frozen sections of the inner ear were observed.Results: after different doses of furosemide and kanamycin sulfate, the threshold value of ABR in rats increased in different degree, after 3 days and 7 days, there was no significant difference between the two groups after 2 months.Under scanning electron microscope, the hair cells of cochlea were damaged from the bottom to the apical gyrus, and from the outer hair cells to the inner hair cells.However, rats treated with furosemide or kanamycin sulfate had no threshold elevation or hair cell injury.In the rat cochlea, most of the Sertoli cells were intact and the vestibular hair cells were not damaged.Conclusion: furosemide and kanamycin sulfate have obvious ototoxic synergistic effects. Combined administration of kanamycin can cause severe impairment of auditory function in rats and no recovery of hearing in deaf rats after 2 months of administration, but the damage to cochlear Sertoli cells and vestibular hair cells is mild.The combination of furosemide and kanamycin is a simple, rapid and reliable method to establish a rat model of drug-induced deafness.Part two: preliminary study on the induction of hair cell regeneration by Hath1 gene and DAPT in inner ear objective: to investigate the expression of Hath1 in inner ear and the induction of hair cell regeneration in inner ear by Hath1 gene and DAPT.Methods: 15 normal rats and 15 deafness rats were exposed to auditory bubble via dorsal approach, and some of the auditory alveolar bone were removed. The Hath1 gene was introduced into the microinjector at the near round window.Then the capsule osmotic pump was continuously introduced into DAPT for 4 weeks.The threshold value of ABR, the scanning electron microscope of cochlea and the fluorescence staining of the whole cochlea basement membrane were observed.Results: there was no change in the threshold of ABR before and after the introduction of ABR in all rats. The threshold of ABR in normal rats was higher than that in the premise of introducing ABR. The threshold of ABR in deaf rats was not changed before and after the introduction of ABR, and compared with that before introduction.Hair cells were not found to increase or regenerate in all normal and deaf rats.Conclusion the combination of DAPT and 1% Hath1 gene can not induce cochlear hair cell regeneration in drug-induced deafness rats. The regeneration of hair cells and hearing improvement in deafness rats may require the combination of multiple gene transduction.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764.35

【引证文献】