ARC在新霉素诱导的HEI-OC-1细胞凋亡中的作用和机制研究

发布时间：2018-01-15 02:09

本文关键词：ARC在新霉素诱导的HEI-OC-1细胞凋亡中的作用和机制研究　出处：《南京医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:感音神经性耳聋在临床上很常见,原因主要在于耳蜗毛细胞在噪音、耳毒性药物、衰老等因素的诱导下,容易发生凋亡,而成年哺乳动物的毛细胞不能自发再生。带caspase凋亡募集域的凋亡抑制因子(Apoptosis repressor with caspase recruitment domain,ARC)是具有调节内、外源性凋亡通路的关键因子,然而目前还没有关于ARC与内耳毛细胞的相关报道。本研究利用耳蜗毛细胞样细胞,HEI-OC-1 细胞(House Ear Institute Organ of Corti l cell),来研究ARC在新霉素诱导的耳蜗毛细胞凋亡中的作用及机制。方法:实验采用PCR,Western blot,免疫荧光来检测ARC在耳蜗毛细胞和HEI-OC-1细胞内是否表达及新霉素作用后表达含量的变化。接着通过siRNA下调ARC表达,TUNEL染色及流式细胞仪检测Annexin V/PI,来观察ARC对新霉素诱导的HEI-OC-1细胞凋亡的调节作用。最后通过RT-qPCR、免疫荧光、流式细胞仪检测ARC表达下调后,HEI-OC-1细胞内活性氧、线粒体膜电位、凋亡因子变化,来研究ARC调节新霉素诱导的HEI-OC-1细胞凋亡的机制。结果:实验首先发现ARC在耳蜗毛细胞和HEI-OC-1细胞均有表达,但在耳蜗支持细胞没有表达。其次发现ARC在耳蜗毛细胞和HEI-OC-1细胞的表达量在新霉素作用后逐渐下降,并且当我们用siRNA下调ARC表达后,HEI-OC-1细胞对新霉素的敏感度增加,表明ARC参与调节新霉素诱导的HEI-OC-1细胞的凋亡。最后实验发现ARC表达下调将增加HEI-OC-1细胞内活性氧的含量,促使线粒体膜电位下降,增加促凋亡因子的表达,从而调节新霉素诱导的HEI-OC-1细胞凋亡。结论:本研究的成果表明ARC参与调节耳蜗毛细胞凋亡,可作为研究耳蜗毛细胞凋亡的基因靶点。
[Abstract]:Objective: sensorineural hearing loss is common in clinic. The main reason is that the cochlear hair cells in noise, ototoxic drugs, senescence induced by other factors, prone to apoptosis, hair cells and not in adult mammals. The apoptosis of spontaneous regeneration with caspase apoptosis inhibiting factor recruitment domain (Apoptosis repressor with caspase recruitment domain, ARC) is a regulating, the key factor of exogenous apoptosis, however, there is no relevant reports about ARC and the inner hair cells. This study uses the cochlear hair cell like cells, HEI-OC-1 cells (House Ear Institute Organ of Corti L cell), and to study the mechanism of ARC in neomycin induced cochlear hair cells apoptosis effect. Methods: PCR, Western blot, immunofluorescence to detect whether ARC in cochlear hair cells and HEI-OC-1 cells and the expression of neomycin after expression of the content Change. And then using the siRNA expression of ARC, TUNEL staining and detection of Annexin V/PI by flow cytometry, to observe the effect of ARC on apoptosis of HEI-OC-1 cells induced by neomycin. Finally by RT-qPCR, immunofluorescence, detection of ARC expression by flow cytometry, active oxygen HEI-OC-1 cells, mitochondrial membrane potential, apoptosis to study the mechanism of factor changes, ARC regulates HEI-OC-1 cell apoptosis induced by neomycin. Results: the first found expression of ARC in cochlear hair cells and HEI-OC-1 cells are, but in the cochlear supporting cells did not express. Then found the expression of ARC in cochlear hair cells and HEI-OC-1 cells decreased gradually after the effect of neomycin in, and when we use the siRNA expression of ARC, HEI-OC-1 cells increased sensitivity to neomycin, suggesting that ARC is involved in the regulation of apoptosis of HEI-OC-1 cells induced by neomycin. Finally the experimental ARC table To cut will increase the active oxygen content in HEI-OC-1 cells, the decrease of mitochondrial membrane potential, increased expression of Pro apoptotic factors, thereby regulating the apoptosis of HEI-OC-1 cells induced by neomycin. Conclusion: the results of this study indicate that ARC is involved in the regulation of apoptosis of cochlear hair cells, can be used as a target gene on the apoptosis of cochlear hair cells.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R764.43

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