本文關(guān)鍵詞： 內(nèi)質(zhì)網(wǎng)應(yīng)激 螺旋神經(jīng)節(jié) 退變過程 caspase12 凋亡 自噬 活性氧 可逆的 神經(jīng)毒性作用 LC3　出處：《華中科技大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分硫酸卡那霉素慢性致聾大鼠螺旋神經(jīng)節(jié)細(xì)胞變性的時(shí)序性及機(jī)制研究 目的：建立硫酸卡那霉素慢性致聾大鼠模型。研究硫酸卡那霉素慢性致聾大鼠螺旋神經(jīng)節(jié)細(xì)胞的形態(tài)學(xué)動(dòng)態(tài)變化及其退變時(shí)程,探討內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)是否參與螺旋神經(jīng)節(jié)退變過程。 方法：選取耳廓反射正常,聽力正常的成年大鼠為研究對(duì)象,大腿內(nèi)側(cè)皮下每天注射硫酸卡那霉素500mg/kg,連續(xù)注射10天。在給藥后第1,7,14,28,56,70和140天觀察螺旋神經(jīng)節(jié)神經(jīng)元細(xì)胞形態(tài)學(xué)變化和定量分析螺旋神經(jīng)節(jié)細(xì)胞密度變化,以及在給藥后的各個(gè)時(shí)間點(diǎn)檢測(cè)Bip, IRE1α, caspase-12, Phospho-eIF2-alpha (Ser51), Phospho-PERK (Thr980), GADD153和ATF-6α的表達(dá),同時(shí)行TUNEL染色。 結(jié)果：1、聽功能：正常成年大鼠在4K,8K,16K,24K和32K Hz頻率的ABR平均閾值分別為24.00±3.16,20.50±4.38,25.00±5.77,30.50±5.50和34.00±3.94dB SPL。在卡那霉素治療1,7,14,28,56,70和140天后,4KHz頻率ABR平均閾值分別為37.00±10.33,37.50±13.79,57.00±8.24,60.00±11.06,72.00±8.56,48.00±9.78和50.00±11.55dB SPL；8KHz頻率ABR平均閾值分別為42.00±12.51,40.50±11.17,52.00±11.35,63.00±9.19,81.00±8.43,56.50±5.80和57.00±14.18dB SPL；16KHz頻率ABR平均閾值分別為是59.00±11.50,58.00±11.83,64.50±6.85,67.00±11.35,83.50±5.30,66.00±12.43和61.50±19.59dB SPL；24KHz頻率ABR平均閾值分別是61.50±12.26,61.00±9.66,73.50±9.14,80.50±5.99,86.00±5.68,76.50±11.80和68.50±15.64dB SPL。在32KHz頻率平均閾值分別為71.50±6.69,67.50±11.37,80.50±8.96,81.00±7.75,86.50±6.26,75.50±9.56和75.00±13.33dB SPL。卡那霉素給藥后,除4KHz在1和7天外,各個(gè)頻率ABR平均閾值在每個(gè)時(shí)間點(diǎn)與正常對(duì)照組相比均有顯著升高(P0.001)。2、螺旋神經(jīng)節(jié)細(xì)胞計(jì)數(shù)：對(duì)照組和實(shí)驗(yàn)組(給藥后1,7,14,28,56,70和140天)底回SGN平均細(xì)胞密度分別為(/mm2)2334.06±169.62,2100.00±237.01,1664.47±139.45,1400.03±279.79,1334.66±85.96,1314.85±164.18,1166.43±41.69和1140.27±96.09。除卡那霉素給藥后1天外,對(duì)照組和各實(shí)驗(yàn)組之間的SGC計(jì)數(shù)有顯著性差異(P0.001)。3、SGC超微結(jié)構(gòu)變化：(I型)SGCs數(shù)量大幅減少,導(dǎo)致單個(gè)細(xì)胞之間的空間擴(kuò)大。大部分SGCs變?yōu)闃渫粯有螤罴捌浼?xì)胞器和細(xì)胞內(nèi)容分布更加緊湊,表現(xiàn)出電子致密的外觀。同時(shí),核周體髓鞘變得更薄、更松弛。亞細(xì)胞水平形態(tài)結(jié)構(gòu)上,在卡那霉素給藥后早期,內(nèi)質(zhì)網(wǎng)擴(kuò)張明顯及線粒體進(jìn)行性腫脹,隨后從給藥后28天至140天逐漸恢復(fù)。線粒體在卡那霉素給藥后出現(xiàn)局灶性空泡化。4、TUNEL染色：在卡那霉素給藥后7,14和28天,TUNEL和caspase-12染色雙陽性的SGCs細(xì)胞數(shù)明顯增多,并于給藥后第7天,TUNEL和caspase-12染色雙陽性的SGCs細(xì)胞數(shù)升高最明顯。5、western blot分析：IRE1α的表達(dá)在給藥后7天(P0.05),14天(P0.01)和56天(P0.05)顯著升高(P0.05)。與對(duì)照組相比,BIP表達(dá)在給藥后1天到56天上調(diào)(P0.05),而在140天下調(diào)(P0.01)。GADD153蛋白水平在給藥后1天顯著上調(diào)(P0.05),持續(xù)至70天(P0.05)并且在卡那霉素治療后70天達(dá)到最高水平(P0.01)。其次,在與對(duì)照組相比,P-PERK蛋白水平從給藥后7天至140天上調(diào)(P0.05),P-eIF2α蛋白水平從卡那霉素治療后1天至56天上調(diào)(P0.05)。第三,ATF-6a表達(dá)在對(duì)照組和實(shí)驗(yàn)組中幾乎檢測(cè)不到。最后,Caspase-12的蛋白水平在給藥后1(P0.01),7(P0.01),14(P0.05)和56天(P0.01)顯著上調(diào),而在140天下調(diào)(P0.05) 結(jié)論：1、硫酸卡那霉素引起螺旋神經(jīng)節(jié)細(xì)胞退變是持續(xù)性的。2、ERS參與卡那霉素所致的SGCs凋亡過程。3、部分SGCs凋亡是通過ERS引發(fā)的caspase-12和GADD153上調(diào)介導(dǎo)。 第二部分硫酸卡那霉素慢性致聾大鼠蝸神經(jīng)背側(cè)核神經(jīng)元退變時(shí)序性及其機(jī)制研究 目的：硫酸卡那霉素慢性致聾大鼠后,檢測(cè)蝸神經(jīng)背核(DCN)梭形細(xì)胞超微結(jié)構(gòu)的動(dòng)態(tài)病理變化,探討凋亡或自噬是否參與硫酸卡那霉素對(duì)DCN的神經(jīng)毒性過程。 方法：選取耳廓反射正常,聽力正常的成年大鼠為研究對(duì)象,大腿內(nèi)側(cè)皮下每天注射硫酸卡那霉素500mg/kg,連續(xù)注射10天。給藥后第1,7,14,28,56,70和140天研究成年大鼠DCN梭形細(xì)胞超微結(jié)構(gòu)的動(dòng)態(tài)病理變化和神經(jīng)元密度變化及神經(jīng)元凋亡情況；同時(shí)在給藥后各個(gè)時(shí)間點(diǎn)檢測(cè)JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B和Beclin-1的表達(dá)變化。 結(jié)果：1、聽功能：正常成年大鼠在4K,8K,16K,24K和32K Hz頻率的ABR平均閾值分別為24.00±3.16,20.50±4.38,25.00±5.77,30.50±5.50和34.00±3.94dB SPL在卡那霉素治療1,7,14,28,56,70和140天后,4KHz頻率ABR平均閾值分別為37.00±10.33,37.50±13.79,57.00±8.24,60.00±11.06,72.00±8.56,48.00±9.78和50.00±11.55dB SPL；8KHz頻率ABR平均閾值分別為42.00±12.51,40.50±11.17,52.00±11.35,63.00±9.19,81.00±8.43,56.50±5.80和57.00±14.18dBS PL;16KHz頻率ABR平均閾值分別為是59.00±11.50,58.00±11.83,64.50±6.85,67.00±11.35,83.50±5.30,66.00±12.43和61.50±19.59dB SPL；24KHz頻率ABR平均閾值分別是61.50±12.26,61.00±9.66,73.50±9.14,80.50±5.99,86.00±5.68,76.50±11.80和68.50±15.64dB SPL。在32KHz頻率平均閾值分別為71.50±6.69,67.50±11.37,80.50±8.96,81.00±7.75,86.50±6.26,75.50±9.56和75.00±13.33dB SPL。卡那霉素給藥后,除4KHz在1和7天外,各個(gè)頻率ABR平均閾值在每個(gè)時(shí)間點(diǎn)與正常對(duì)照組相比均有顯著升高(P0.001)。2、DCN梭形細(xì)胞超微結(jié)構(gòu)：給藥后第1天到28天梭形細(xì)胞線粒體和內(nèi)質(zhì)網(wǎng)腫脹逐漸加重,然后在給藥后第28到140天逐漸恢復(fù)；線粒體局灶性空泡形成；同時(shí),在給藥后第1,7,14,28和56天自噬小體和自噬溶酶體明顯增多,隨后逐步恢復(fù),最后殘留為脂褐素。3、神經(jīng)元密度計(jì)數(shù)和LC3陽性神經(jīng)元細(xì)胞計(jì)數(shù)：給藥后第1,7和14天,實(shí)驗(yàn)組的LC3陽性細(xì)胞數(shù)明顯高于對(duì)照組,而實(shí)驗(yàn)組神經(jīng)元細(xì)胞密度與對(duì)照組相比沒有發(fā)生明顯變化。4、western blot分析：首先,JNK1的表達(dá)水平在給藥后第1天沒有明顯改變,但在第7天有顯著上調(diào)(P0.05),隨后逐漸下調(diào)。第二,與對(duì)照組相比,DAPK2和caspase-3表達(dá)沒有明顯改變。第三,Bcl-2蛋白水平初始未發(fā)生變化,直至給藥后140天出現(xiàn)下調(diào)(P0.05)。p-Bcl2蛋白水平在給藥后1天未發(fā)生明顯上調(diào),但于第7天的時(shí)候達(dá)到最高峰(P0.001),并持續(xù)到給藥后第28天(P0.05)。最后,與對(duì)照組相比,Beclin-1在給藥后第1天顯著升高(P0.05),在第7天達(dá)到最高峰(P0.01),然后從給藥后第14天到140天下降,但這種下降趨勢(shì)并沒有統(tǒng)計(jì)學(xué)意義。同時(shí),LC3在第7天顯著上調(diào)(P0.01),從第14天開始到140天下降到正常水平。5、TUNEL染色：與對(duì)照組相比,任意一個(gè)實(shí)驗(yàn)組TUNEL陽性細(xì)胞數(shù)沒有明顯增高。 結(jié)論：1、硫酸卡那霉素可引起DCN梭形細(xì)胞可逆性損傷。2、自噬參與了卡那霉素對(duì)DCN神經(jīng)元的神經(jīng)毒性過程。3、自噬的增高可能是通過JNK1-p-Bcl-2-Beclin-1信號(hào)通路調(diào)節(jié)的。
[Abstract]:Study on the timing and mechanism of spiral ganglion cell degeneration in the first part of kanamycin sulfate chronic deafness rats
Objective: to establish a chronic deafness rat model of kanamycin sulfate. To study the morphological changes and the time course of degeneration of spiral ganglion cells in kanamycin sulfate induced chronic deafness rats, and to explore whether endoplasmic reticulum stress (ERS) is involved in the process of spiral ganglion degeneration.
Methods: normal auricle reflex, hearing normal adult rats as the research object, the inner thigh daily subcutaneous injection of kanamycin sulfate injection 500mg/kg, continuous 10 days. Changes of spiral ganglion cell density at days 1,7,14,28,56,70 and 140 observation of spiral ganglion neuron cell morphology and quantity after administration, and to caspase-12 each time point after drug detection Bip, IRE1 alpha, Phospho-eIF2-alpha (Ser51), Phospho-PERK (Thr980), the expression of GADD153 and ATF-6 alpha, and TUNEL staining.
Results: 1, the auditory function in the rats. 4K, 8K, 16K in normal adult, ABR 24K and 32K Hz the average threshold frequency were 24 + 3.16,20.50 + 4.38,25.00 + 5.77,30.50 + 5.50 and 34 + 3.94dB SPL. in Kanamycin treatment of 1,7,14,28,56,70 and 4KHz after 140 days, the average threshold frequency of ABR are 37. 10.33,37.50 + 13.79,57.00 + 8.24,60.00 + 11.06,72.00 + 8.56,48.00 + 9.78 and 50 + 11.55dB SPL; 8KHz frequency ABR average thresholds were 42 + 12.51,40.50 + 11.17,52.00 + 11.35,63.00 + 9.19,81.00 + 8.43,56.50 + 5.80 and 57 + 14.18dB SPL; 16KHz ABR average frequency threshold was 59 + 11.50,58.00 + 11.83,64.50 + 6.85,67.00 + 11.35,83.50 + 5.30,66.00 + 12.43 and 61.50 + 19.59dB SPL; 24KHz ABR average frequency threshold were 61.50 + 12.26,61.00 + 9.66,73.50 + 9.14,80.50 + 5.99,86.00 + 5.68,76.50 + 11.80 and 68.50 + 15.64dB SPL. The average threshold at the frequency of 32KHz was 71.50 + 6.69,67.50 + 11.37,80.50 + 8.96,81.00 + 7.75,86.50 + 6.26,75.50 + 9.56 and 75 + 13.33dB SPL. kanamycin after administration, in addition to 4KHz in the 1 and 7 days, the average frequency of ABR threshold increased at each time point compared with the normal control group were statistically significant (P0.001.2) count, spiral ganglion cells: control group and experimental group (1,7,14,28,56,70 after administration and 140 days) at the end of the average cell density was SGN (/mm2) 2334.06 + 169.622100.00 + 237.011664.47 + 139.451400.03 + 279.791334.66 + 85.961314.85 + 164.181166.43 + 41.69 and 1140.27 + 96.09. in addition to kanamycin for 1 days after the administration, control group and the experimental group SGC count had significant difference (P0.001.3), the ultrastructure of SGC: (I) SGCs significantly reduce the number of individual cells, lead to the space between the parts. To expand SGCs into dendrites 鏍峰艦鐘跺強(qiáng)鍏剁粏鑳?yōu)鍣ㄥ拰缁嗚優(yōu)鍐呭鍒嗗竷鏇村姞绱у嚕?

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