【摘要】：目的： 1、建立噪聲導(dǎo)致的內(nèi)耳損傷的動(dòng)物模型,在噪聲性耳聾的動(dòng)物模型上觀察聽功能的變化、內(nèi)耳毛細(xì)胞的損傷情況,建立內(nèi)耳損傷修復(fù)的研究平臺(tái)。 2、觀察外周血EPCs的變化和耳蝸血管紋中VEGF、iNOS在噪聲暴露后表達(dá)變化,闡述噪聲性耳聾的耳蝸組織損傷與修復(fù)過(guò)程中的發(fā)病機(jī)理。 3、通過(guò)干預(yù)內(nèi)源性EPCs、探討對(duì)于噪聲性耳聾耳蝸損傷的預(yù)防和治療采取的措施。 方法： 取健康體重約250g-300g成年SD大鼠應(yīng)用118±1dBSPL白噪聲持續(xù)暴露4小時(shí)建立噪聲性耳聾模型,實(shí)驗(yàn)分組為：對(duì)照組：未進(jìn)行噪聲暴露；噪聲暴露即刻組：噪聲暴露后即刻檢測(cè)：噪聲暴露1天組：噪聲暴露后1天檢測(cè);噪聲暴露3天組：噪聲暴露后3天檢測(cè)；噪聲暴露7天組：噪聲暴露后7天檢測(cè),；噪聲暴露14天組：噪聲暴露后14天檢測(cè)；各組均為10只；將試驗(yàn)分為兩部分,一為單純?cè)肼暠┞秾?duì)耳蝸損傷修復(fù)的影響,二為干預(yù)內(nèi)源性EPCs后噪聲暴露對(duì)耳蝸損傷修復(fù)的影響。SD大鼠根據(jù)噪聲暴露后即刻組、1天組、3天組、7天組和14天組的分組分別檢測(cè)各組大鼠的ABR聽閾改變、通過(guò)Fillo密度梯度離心法獲得單個(gè)核細(xì)胞流式細(xì)胞儀鑒定SD大鼠外周血中EPCs數(shù)量、通過(guò)Western印記和耳蝸冰凍切片免疫組化觀察耳蝸血管紋VEGF、iNOS和CD34的表達(dá)變化,同時(shí)對(duì)噪聲暴露后的耳蝸行掃描電鏡觀察。應(yīng)用EPO皮下注射提高內(nèi)源性EPCs數(shù)量、半胱氨酸飼料喂養(yǎng)制備內(nèi)源性EPCs數(shù)量降低的模型和NS皮下注射,分別觀察SD大鼠噪聲暴露后即刻組、1天組、3天組、7天組、和14天組ABR聽閾改變。 結(jié)果： 1、噪聲暴露后即刻組ABR閾值為最高,其聽閾為86.67±6.85dBSPL,聽力損傷最為明顯,屬于TTS期；噪聲暴露后7天和14天ABR閾值部分恢復(fù),閾值降低,屬于PTS期,其聽閾分別為為56.18±5.29dBSPL和57.92±6.20dBSPL; 2、對(duì)各組外周血中的EPCs進(jìn)行測(cè)定,觀察到EPCs于噪聲暴露后1天時(shí)最高,數(shù)值為91.40±8.13/20萬(wàn)個(gè)單核細(xì)胞；噪聲暴露后14天時(shí)EPCs恢復(fù)接近正常水平,數(shù)值為29.00±14.56/20萬(wàn)個(gè)單核細(xì)胞； 3、免疫組化及Western印記表明噪聲暴露后1天VEGF和iNOS出現(xiàn)高峰,噪聲暴露后即刻組表達(dá)最低,CD34于噪聲暴露后3天和7天達(dá)到高峰；4、通過(guò)干預(yù)內(nèi)源性EPCs,EPCs升高組對(duì)于聽力的保護(hù)作用明顯高于EPCs抑制組。 4、EPCs在內(nèi)耳損傷后的修復(fù)過(guò)程中,有促進(jìn)血管生成,改善內(nèi)耳微環(huán)境的作用,降低EPCs可以導(dǎo)致聽力較為嚴(yán)重的下降,提高內(nèi)源性EPCs的釋放,可以保護(hù)耳蝸減輕噪聲對(duì)耳蝸的損傷加快耳蝸損傷后的恢復(fù)。 結(jié)論： 1、噪聲性耳聾最終可導(dǎo)致耳蝸損傷,隨著時(shí)間改變外周血中EPCs的數(shù)量也發(fā)生改變； 2、VEGF、iNOS參與噪聲性耳聾損傷與修復(fù)的各個(gè)階段,VEGF和EPCs在噪聲損傷的耳蝸血管修復(fù)的不同階段均發(fā)揮作用； 3、EPCs在噪聲性耳聾耳蝸損傷與修復(fù)過(guò)程中起著關(guān)鍵作用。
[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 鹵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 鹵5.29dBSPL and 57.92 鹵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 鹵8.13200 monocytes, and the EPCs returned to the normal level 14 days after noise exposure. The number of monocytes was 29.00 鹵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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R764.3

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