急性药物性内耳损伤中CaV1.3钙通道及三种细胞凋亡因子的检测及变化

发布时间：2018-04-05 11:34

本文选题：C57BL/6J小鼠　切入点：氨基糖甙类抗生素　出处：《华中科技大学》2011年硕士论文

【摘要】：目的研究卡那霉素和速尿诱导的耳毒性发生后CaV1.3钙离子通道蛋白在内耳含量的改变,以及三种细胞凋亡因子cyt-c、caspase-3、p53在内耳中的表达改变,并分析其与卡那霉素联合速尿快速诱导耳蜗损伤导致细胞凋亡之间的关系。方法选取4周龄C75BL/6J小鼠为实验对象,将小鼠随机分为实验组和对照组,实验组采用硫酸卡那霉素颈部皮下注射(1g/kg),30分钟后速尿腹腔注射(0.4g/kg);对照组给与等量生理盐水。在注射药物前以ABR检查小鼠听觉功能,注射1周后再检测ABR反应阈值。应用RT-PCR(逆转录聚合酶链反应)检测CaV1.3 mRNA在实验组和对照组中的表达水平。采用免疫组织化学方法确定cyt-c、caspase-3、p53蛋白在耳蜗内的定位,并比较其在实验组和对照组之间的差别。结果给药后实验组ABR的反应阈值较对照组明显上升,两组之间的差异具有统计学意义(P0.001)。实验组CaV1.3 mRNA的表达量较对照组有所减少。免疫组织化学检测发现CaV1.3钙通道蛋白主要分布在血管纹、螺旋韧带、螺旋凸、毛细胞等处,在耳毒性发生后,其表达明显减弱。cyt-c、caspase-3、p53主要分布在血管纹、螺旋韧带、螺旋凸、毛细胞等部位,在实验组小鼠的耳蜗其表达较对照组的表达显著增强。结论应用卡那霉素联合速尿序贯注射能引起小鼠的急性耳蜗损伤和听力的减退。这种药物引起的耳毒性与内耳CaV1.3钙离子通道蛋白的损害呈现相关性,并且与内耳毛细胞、血管纹、螺旋神经元的凋亡有关。
[Abstract]:Objective to study the changes of CaV1.3 calcium channel protein in the inner ear after ototoxicity induced by kanamycin and furosemide, and the expression of three apoptosis factors, cyt-caspase-3 and p53, in the inner ear.The relationship between apoptosis induced by kanamycin combined with furosemide and cochlear injury was analyzed.Methods 4-week-old C75BL/6J mice were randomly divided into experimental group and control group. The experimental group was injected with kanamycin sulfate subcutaneously for 30 minutes and the control group was given the same amount of normal saline.The auditory function of mice was examined by ABR before injection, and the threshold of ABR reaction was measured 1 week after injection.RT-PCR was used to detect the expression of CaV1.3 mRNA in experimental group and control group.The localization of cyt-caspase-3 p53protein in cochlea was determined by immunohistochemical method, and the difference between experimental group and control group was compared.Results the response threshold of ABR in the experimental group was significantly higher than that in the control group, and the difference between the two groups was statistically significant (P 0.001).The expression of CaV1.3 mRNA in the experimental group was lower than that in the control group.Immunohistochemical examination showed that CaV1.3 calcium channel protein was mainly distributed in stria vascularis, helical ligament, helical convex, hair cell, etc. After ototoxicity, the expression of Caspase-3 p53 was significantly decreased in stria vascularis, helical ligament, helical convexity, etc.The expression of hair cells in the cochlea of the experimental group was significantly higher than that in the control group.Conclusion kanamycin combined with sequential furosemide injection can induce acute cochlear damage and hearing loss in mice.The ototoxicity induced by the drug is related to the damage of CaV1.3 calcium channel protein in the inner ear and apoptosis of hair cells vascular striae and spiral neurons in the inner ear.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764

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