本文關(guān)鍵詞：ARC在新霉素誘導(dǎo)的HEI-OC-1細(xì)胞凋亡中的作用和機(jī)制研究　出處：《南京醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 帶caspase凋亡募集域的凋亡抑制因子(ARC) 耳蝸 毛細(xì)胞 凋亡 HEI-OC-1細(xì)胞 細(xì)胞內(nèi)活性氧 線粒體功能 氨基糖苷類

【摘要】：目的:感音神經(jīng)性耳聾在臨床上很常見,原因主要在于耳蝸毛細(xì)胞在噪音、耳毒性藥物、衰老等因素的誘導(dǎo)下,容易發(fā)生凋亡,而成年哺乳動(dòng)物的毛細(xì)胞不能自發(fā)再生。帶caspase凋亡募集域的凋亡抑制因子(Apoptosis repressor with caspase recruitment domain,ARC)是具有調(diào)節(jié)內(nèi)、外源性凋亡通路的關(guān)鍵因子,然而目前還沒有關(guān)于ARC與內(nèi)耳毛細(xì)胞的相關(guān)報(bào)道。本研究利用耳蝸毛細(xì)胞樣細(xì)胞,HEI-OC-1 細(xì)胞(House Ear Institute Organ of Corti l cell),來研究ARC在新霉素誘導(dǎo)的耳蝸毛細(xì)胞凋亡中的作用及機(jī)制。方法:實(shí)驗(yàn)采用PCR,Western blot,免疫熒光來檢測(cè)ARC在耳蝸毛細(xì)胞和HEI-OC-1細(xì)胞內(nèi)是否表達(dá)及新霉素作用后表達(dá)含量的變化。接著通過siRNA下調(diào)ARC表達(dá),TUNEL染色及流式細(xì)胞儀檢測(cè)Annexin V/PI,來觀察ARC對(duì)新霉素誘導(dǎo)的HEI-OC-1細(xì)胞凋亡的調(diào)節(jié)作用。最后通過RT-qPCR、免疫熒光、流式細(xì)胞儀檢測(cè)ARC表達(dá)下調(diào)后,HEI-OC-1細(xì)胞內(nèi)活性氧、線粒體膜電位、凋亡因子變化,來研究ARC調(diào)節(jié)新霉素誘導(dǎo)的HEI-OC-1細(xì)胞凋亡的機(jī)制。結(jié)果:實(shí)驗(yàn)首先發(fā)現(xiàn)ARC在耳蝸毛細(xì)胞和HEI-OC-1細(xì)胞均有表達(dá),但在耳蝸支持細(xì)胞沒有表達(dá)。其次發(fā)現(xiàn)ARC在耳蝸毛細(xì)胞和HEI-OC-1細(xì)胞的表達(dá)量在新霉素作用后逐漸下降,并且當(dāng)我們用siRNA下調(diào)ARC表達(dá)后,HEI-OC-1細(xì)胞對(duì)新霉素的敏感度增加,表明ARC參與調(diào)節(jié)新霉素誘導(dǎo)的HEI-OC-1細(xì)胞的凋亡。最后實(shí)驗(yàn)發(fā)現(xiàn)ARC表達(dá)下調(diào)將增加HEI-OC-1細(xì)胞內(nèi)活性氧的含量,促使線粒體膜電位下降,增加促凋亡因子的表達(dá),從而調(diào)節(jié)新霉素誘導(dǎo)的HEI-OC-1細(xì)胞凋亡。結(jié)論:本研究的成果表明ARC參與調(diào)節(jié)耳蝸毛細(xì)胞凋亡,可作為研究耳蝸毛細(xì)胞凋亡的基因靶點(diǎn)。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R764.43

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1 管明;ARC在新霉素誘導(dǎo)的HEI-OC-1細(xì)胞凋亡中的作用和機(jī)制研究[D];南京醫(yī)科大學(xué);2017年

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