内皮祖细胞与噪音性耳聋耳蜗损伤的关系

发布时间：2018-07-16 22:08

【摘要】：目的： 1、建立噪声导致的内耳损伤的动物模型,在噪声性耳聋的动物模型上观察听功能的变化、内耳毛细胞的损伤情况,建立内耳损伤修复的研究平台。 2、观察外周血EPCs的变化和耳蜗血管纹中VEGF、iNOS在噪声暴露后表达变化,阐述噪声性耳聋的耳蜗组织损伤与修复过程中的发病机理。 3、通过干预内源性EPCs、探讨对于噪声性耳聋耳蜗损伤的预防和治疗采取的措施。方法：取健康体重约250g-300g成年SD大鼠应用118±1dBSPL白噪声持续暴露4小时建立噪声性耳聋模型,实验分组为：对照组：未进行噪声暴露；噪声暴露即刻组：噪声暴露后即刻检测：噪声暴露1天组：噪声暴露后1天检测;噪声暴露3天组：噪声暴露后3天检测；噪声暴露7天组：噪声暴露后7天检测,；噪声暴露14天组：噪声暴露后14天检测；各组均为10只；将试验分为两部分,一为单纯噪声暴露对耳蜗损伤修复的影响,二为干预内源性EPCs后噪声暴露对耳蜗损伤修复的影响。SD大鼠根据噪声暴露后即刻组、1天组、3天组、7天组和14天组的分组分别检测各组大鼠的ABR听阈改变、通过Fillo密度梯度离心法获得单个核细胞流式细胞仪鉴定SD大鼠外周血中EPCs数量、通过Western印记和耳蜗冰冻切片免疫组化观察耳蜗血管纹VEGF、iNOS和CD34的表达变化,同时对噪声暴露后的耳蜗行扫描电镜观察。应用EPO皮下注射提高内源性EPCs数量、半胱氨酸饲料喂养制备内源性EPCs数量降低的模型和NS皮下注射,分别观察SD大鼠噪声暴露后即刻组、1天组、3天组、7天组、和14天组ABR听阈改变。结果： 1、噪声暴露后即刻组ABR阈值为最高,其听阈为86.67±6.85dBSPL,听力损伤最为明显,属于TTS期；噪声暴露后7天和14天ABR阈值部分恢复,阈值降低,属于PTS期,其听阈分别为为56.18±5.29dBSPL和57.92±6.20dBSPL; 2、对各组外周血中的EPCs进行测定,观察到EPCs于噪声暴露后1天时最高,数值为91.40±8.13/20万个单核细胞；噪声暴露后14天时EPCs恢复接近正常水平,数值为29.00±14.56/20万个单核细胞； 3、免疫组化及Western印记表明噪声暴露后1天VEGF和iNOS出现高峰,噪声暴露后即刻组表达最低,CD34于噪声暴露后3天和7天达到高峰；4、通过干预内源性EPCs,EPCs升高组对于听力的保护作用明显高于EPCs抑制组。 4、EPCs在内耳损伤后的修复过程中,有促进血管生成,改善内耳微环境的作用,降低EPCs可以导致听力较为严重的下降,提高内源性EPCs的释放,可以保护耳蜗减轻噪声对耳蜗的损伤加快耳蜗损伤后的恢复。结论： 1、噪声性耳聋最终可导致耳蜗损伤,随着时间改变外周血中EPCs的数量也发生改变； 2、VEGF、iNOS参与噪声性耳聋损伤与修复的各个阶段,VEGF和EPCs在噪声损伤的耳蜗血管修复的不同阶段均发挥作用； 3、EPCs在噪声性耳聋耳蜗损伤与修复过程中起着关键作用。
[Abstract]:Objective: 1. To establish an animal model of inner ear injury induced by noise and observe the changes of auditory function and the damage of hair cells of inner ear on the animal model of noise induced deafness. To observe the changes of EPCs in peripheral blood and the expression of VEGF iNOS in stria vascularis of cochlea after noise exposure. The pathogenesis of cochlear tissue injury and repair in noise-induced deafness was described. 3. The prevention and treatment of noise-induced deafness were discussed through the intervention of endogenous EPCs. Methods: healthy 250g-300g adult SD rats were exposed to white noise for 4 hours to establish the model of noise induced deafness. The rats were divided into two groups: control group: no noise exposure; Immediately after noise exposure: 1 day after noise exposure, 3 days after noise exposure, 7 days after noise exposure, 3 days after noise exposure, 7 days after noise exposure. Noise exposure for 14 days: 14 days after noise exposure; 10 rats in each group. The experiment was divided into two parts, one was the effect of pure noise exposure on cochlear injury and repair, Second, the effect of noise exposure to endogenous EPCs on cochlear injury and repair. The ABR hearing threshold of rats in each group was detected according to the group of 1 day after noise exposure and the group of 3 days group and 14 day group, respectively. The number of EPCs in peripheral blood of SD rats was identified by flow cytometry with Fillo density gradient centrifugation. The expression of VEGFN iNOS and CD34 in stria vascularis of cochlea was observed by Western blot and immunohistochemistry. At the same time, the cochlea after noise exposure were observed by scanning electron microscope. The number of endogenous EPCs was increased by subcutaneous injection of EPO, the model of decreasing the number of endogenous EPCs was made by cysteine feed and the rats were injected subcutaneously with NS. ABR hearing threshold changes in 14 days and 14 days group. Results: 1, the ABR threshold was the highest in the group immediately after noise exposure, the hearing threshold was 86.67 卤6.85 d BSPLs, the hearing loss was the most obvious, belonging to the TTS period, the ABR threshold was partially recovered at 7 and 14 days after noise exposure, and the threshold value decreased, belonging to the PTS phase. The hearing thresholds were 56.18 卤5.29dBSPL and 57.92 卤6.20dBSPL2.The EPCs in peripheral blood of each group were measured. The highest EPCs were observed on the 1st day after noise exposure, the values were 91.40 卤8.13200 monocytes, and the EPCs returned to the normal level 14 days after noise exposure. The number of monocytes was 29.00 卤14.56 / 2000.3.Immunohistochemistry and Western blotting showed that VEGF and iNOS appeared peak on the first day after noise exposure, and the lowest expression of CD34 reached the peak at 3 and 7 days after noise exposure. 4. The protective effect of EPCs on hearing was significantly higher in EPCs group than in EPCs inhibition group. 4EPCs could promote angiogenesis and improve the microenvironment of inner ear during the repair of inner ear injury. Reducing EPCs can lead to a serious decline in hearing, improve the release of endogenous EPCs, can protect the cochlea from noise damage and accelerate the recovery of cochlear injury. Conclusion: 1.Noise-induced deafness can eventually lead to cochlear injury and the number of EPCs in peripheral blood changes with time. (2) VEGF and EPCs were involved in various stages of noise-induced deafness injury and repair. 3 EPCs play a key role in the process of cochlear injury and repair in noise-induced deafness.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R764.3

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