【摘要】：背景與目的:阻塞性睡眠呼吸暫停(OSA)引起并發(fā)癥可涉及多個(gè)系統(tǒng),對(duì)于中樞神經(jīng)系統(tǒng)(CNS)來說成人和兒童均引起認(rèn)知功能障礙,包括嗜睡、注意力下降、記憶力減退、情緒和表達(dá)障礙。間歇低氧(IH)是OSA的重要病理生理特征之一,主要通過海馬CA1區(qū)域神經(jīng)元的凋亡引起認(rèn)知功能障礙。CNS IH可引發(fā)活性氧簇過度產(chǎn)生,導(dǎo)致炎性物質(zhì)大量產(chǎn)生致神經(jīng)元凋亡和壞死并加重OSA相關(guān)認(rèn)知功能障礙。因此本研究通過原代培養(yǎng)海馬神經(jīng)元5-7 d,并進(jìn)行分組,經(jīng)低氧暴露后測(cè)定不同分組炎癥因子的表達(dá)水平,從而探討神經(jīng)炎癥在間歇低氧引起的神經(jīng)認(rèn)知障礙中的作用。實(shí)驗(yàn)方法:采用孕16-18d SD孕鼠的胎鼠分離其海馬神經(jīng)元細(xì)胞,原代培養(yǎng)5-7天后,進(jìn)行實(shí)驗(yàn)。首先進(jìn)行細(xì)胞形態(tài)學(xué)觀察,分別于不同時(shí)間采用倒置相差顯微鏡觀察細(xì)胞形態(tài),采集細(xì)胞形態(tài)圖像;采用結(jié)構(gòu)性微管相關(guān)蛋白-2(MAP-2)標(biāo)記神經(jīng)元胞體和樹突,DAPI染核進(jìn)行免疫熒光染色鑒定,在熒光顯微鏡下觀察細(xì)胞、采集圖像。挑選細(xì)胞長(zhǎng)勢(shì)良好、無污染的細(xì)胞,隨機(jī)將細(xì)胞分為常氧對(duì)照組和間歇低氧組。采用天津醫(yī)科大學(xué)總醫(yī)院呼吸科仿真系統(tǒng)1.0設(shè)置低氧/再氧合循環(huán)模式,低氧混合氣含1.5%O2、93.5%N2、5%CO2,正常氧混合氣含21%O2、74%N2、5%CO2,暴露頻率6次/h,暴露時(shí)間為4h。再根據(jù)暴露后繼續(xù)常氧培養(yǎng)不同時(shí)間將間歇低氧組細(xì)胞分為2h組,4h組,6h組,8h組。間歇低氧暴露結(jié)束后提取對(duì)照組和間歇低氧組海馬神經(jīng)元細(xì)胞總mRNA,采用Real Time PCR測(cè)定兩組細(xì)胞高遷移率族蛋白1(HMGB1)mRNA相對(duì)表達(dá)水平。待常氧培養(yǎng)結(jié)束后提取細(xì)胞總mRNA和細(xì)胞總蛋白水平,利用Real Time PCR和Western blot方法檢測(cè)對(duì)照組和各培養(yǎng)組HMGB1和N-甲基-D-天冬氨酸(NMDA)相對(duì)表達(dá)水平。采用SPSS和GraphPad Prism進(jìn)行統(tǒng)計(jì)分析。結(jié)果:(1)培養(yǎng)的海馬神經(jīng)元細(xì)胞4h后貼壁明顯,少數(shù)細(xì)胞開始長(zhǎng)出1-2個(gè)突起,細(xì)胞折光性比較強(qiáng),未見明顯細(xì)胞核。神經(jīng)元細(xì)胞接種24h后大多數(shù)細(xì)胞已貼壁,細(xì)胞呈圓形或橢圓形且周圍有光暈,單個(gè)均勻分布,可見有3個(gè)突起或多突起的神經(jīng)元。培養(yǎng)3d后神經(jīng)元突起增多并變長(zhǎng),胞體變大,細(xì)胞聚集成團(tuán),可見突起連接成稀疏的網(wǎng)狀結(jié)構(gòu)。5d后神經(jīng)元胞體成三角形、圓形或多邊形,突起形成較密集的網(wǎng)絡(luò)。7d后神經(jīng)元生長(zhǎng)良好,胞體清晰,突起增粗形成致密的神經(jīng)網(wǎng)絡(luò)。對(duì)培養(yǎng)5-7d的海馬神經(jīng)元細(xì)胞采用神經(jīng)元特異性標(biāo)志物Map-2和細(xì)胞核DAP I染色后在倒置熒光顯微鏡采用不同激發(fā)波長(zhǎng)觀察,可見綠色神經(jīng)元突起和胞體以及藍(lán)色熒光的細(xì)胞核,兩圖重疊后可計(jì)算神經(jīng)元所占比例,計(jì)算陽性細(xì)胞比例在90%以上。(2)IH組和對(duì)照組相比較,IH組HMGB1 mRNA相對(duì)表達(dá)量是對(duì)照組的3.203倍,IH組HMGB1 mRNA表達(dá)水平明顯增高(P0.05)。IH組暴露后繼續(xù)培養(yǎng)不同時(shí)間各組之間HMGB1mRNA相對(duì)表達(dá)水平差異明顯(P0.05),其中4H mRNA相對(duì)表達(dá)量水平最高。約為對(duì)照組的8.142倍(P0.0001),2h HMGB1表達(dá)約為對(duì)照組的2.836倍(P0.05),6h約為對(duì)照組的0.669倍,與對(duì)照組相比表達(dá)量減少(P0.01),8h約為對(duì)照組的0.338倍,與對(duì)照組相比表達(dá)量減少(P0.01)。與2H相對(duì)表達(dá)水平相比,4H表達(dá)量明顯增多(P0.05);6H與2H相比,6H相對(duì)表達(dá)水平增加(P0.05);8H與2H相比,8H相對(duì)表達(dá)水平增高(P0.05);6H與4H相比,6H相對(duì)表達(dá)水平明顯增高(P0.05);8H與4H相比,8H相對(duì)表達(dá)水平增高(P0.05)。(3)IH組暴露后繼續(xù)培養(yǎng)不同時(shí)間各組之間NMDA mRNA相對(duì)表達(dá)水平差異明顯(P0.05),隨著正常氧培養(yǎng)時(shí)間的延長(zhǎng),各組NMDA mRNA相對(duì)表達(dá)水平逐漸增高(P0.05)。2h與對(duì)照組相比相對(duì)表達(dá)水平明顯升高(P0.01),4h與對(duì)照組相比相對(duì)表達(dá)量增加(P0.001),6h與對(duì)照組相比表達(dá)水平明顯增加,8h與對(duì)照組相比表達(dá)量增加(P0.001)。(4)IH組暴露后繼續(xù)培養(yǎng)不同時(shí)間海馬神經(jīng)元細(xì)胞內(nèi)HMGB1蛋白表達(dá)水平差異明顯(P0.05),與對(duì)照組相比4h蛋白表達(dá)水平最高,2h蛋白表達(dá)與對(duì)照組相比增加,6h蛋白表達(dá)水平較對(duì)照組相比明顯減少,8h蛋白表達(dá)水平與對(duì)照組相比明顯降低。IH組暴露后繼續(xù)培養(yǎng)不同時(shí)間海馬神經(jīng)元細(xì)胞內(nèi)NMDA蛋白表達(dá)水平存在明顯差異(P0.05),隨著暴露后常氧培養(yǎng)時(shí)間延長(zhǎng),海馬神經(jīng)元細(xì)胞內(nèi)NMDA蛋白表達(dá)明顯增多。8h海馬神經(jīng)元細(xì)胞表達(dá)NMDA蛋白量最多。結(jié)論:神經(jīng)炎癥在OSA IH引起的神經(jīng)認(rèn)知功能障礙中發(fā)揮重要作用,IH可引起神經(jīng)炎癥因子HMGB1表達(dá)。說明神經(jīng)炎癥和損傷的神經(jīng)元之間形成惡性循環(huán),對(duì)神經(jīng)元造成“二次打擊”。即使IH暴露撤除神經(jīng)元的損傷也持續(xù)存在。瀕臨死亡的神經(jīng)元持續(xù)釋放HMGB1對(duì)于形成和維持這個(gè)惡性循環(huán)有重要意義。即使始動(dòng)因素IH停止后,炎性損傷還能繼續(xù)。IH可以引起神經(jīng)炎癥,但I(xiàn)H不是維持神經(jīng)炎癥的必須條件。隨著炎癥持續(xù)時(shí)間延長(zhǎng),因子HMGB1分泌減少,但NMDA明顯增多,表明可能HMGB1和NMDA可能發(fā)揮協(xié)同作用,引起神經(jīng)元凋亡和功能障礙。
[Abstract]:BACKGROUND AND OBJECTIVE: Obstructive sleep apnea (OSA) can cause complications involving multiple systems. For the central nervous system (CNS), cognitive impairment occurs in adults and children, including lethargy, decreased attention, memory loss, mood and expression disorders. Intermittent hypoxia (IH) is one of the important pathophysiological characteristics of OSA, mainly through. Neuronal apoptosis in hippocampal CA1 region leads to cognitive impairment. CNS IH can induce excessive production of reactive oxygen species (ROS) clusters, resulting in excessive production of inflammatory substances that induce neuronal apoptosis and necrosis and aggravate OSA-related cognitive impairment. Methods: The hippocampal neurons of SD pregnant rats from 16 to 18 days of gestation were isolated and cultured for 5 to 7 days. Cell morphology was observed under microscope and cell morphology images were collected. Cells were labeled with structural microtubule-associated protein-2 (MAP-2) and identified by immunofluorescence staining with DAPI staining. Cells were observed under fluorescence microscope and collected images. Hypoxia group. Hypoxia/reoxygenation cycle mode was set up in the Simulation System 1.0 of Respiratory Department of Tianjin Medical University General Hospital. Hypoxia mixture contained 1.5% O2, 93.5% N2, 5% CO2, normal oxygen mixture contained 21% O2, 74% N2, 5% CO2. The exposure frequency was 6 times/h and the exposure time was 4 hours. Total mRNA of hippocampal neurons in control group and intermittent hypoxia group was extracted after intermittent hypoxic exposure, and the relative expression levels of high mobility group protein 1 (HMGB1) mRNA in the two groups were determined by Real Time PCR. The relative expression levels of HMGB1 and N-methyl-D-aspartate (NMDA) in the control group and the cultured groups were detected by lot method. SPSS and GraphPad Prism were used for statistical analysis. Most of the cells adhered to the wall 24 hours after inoculation. The cells were round or elliptical with halo around them. There were three or more neurites in the cells. After 3 days of culture, the neurites increased and lengthened, the cell bodies became larger, the cells aggregated into clusters, and the neurite bodies formed triangular and round structures. After 7 days, the neurons grew well, the soma was clear, and the processes were thickened to form a dense neural network. The hippocampal neurons cultured for 5-7 days were stained with Map-2, a neuron-specific marker, and DAP I of the nucleus. Different excitation wavelengths were observed under the inverted fluorescence microscope. Compared with the control group, the relative expression of HMGB1 mRNA in IH group was 3.203 times higher than that in control group, and the expression level of HMGB1 mRNA in IH group was significantly higher than that in control group (P 0.05). The relative expression level of HMGB1 mRNA in each group was significantly different (P 0.05), among which the relative expression level of 4H mRNA was the highest, about 8.142 times (P 0.0001), 2 hours HMGB1 expression was 2.836 times (P 0.05), 6 hours HMGB1 expression was 0.669 times (P 0.01), 8 hours was 0.338 times (P 0.01), and compared with the control group. Compared with 2H, 4H expression increased significantly (P 0.05), 6H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 6H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), 8H relative expression increased (P 0.05), and 8H relative expression increased (P 0.05) compared with 4H relative expression level. Relative expression level of NMDA mRNA was significantly different between groups at different time after exposure (P 0.05). With the prolongation of normal oxygen culture time, the relative expression level of NMDA mRNA gradually increased (P 0.05). The relative expression level of NMDA mRNA in each group was significantly higher than that in the control group at 2 h (P 0.01), 4 h (P 0.001), 6 h and 6 h, respectively. The expression level of HMGB1 protein in hippocampal neurons of IH group was significantly higher than that of control group at 8 hours (P 0.001). (4) The expression level of HMGB1 protein in hippocampal neurons of IH group was significantly different at different time after exposure (P 0.05). Compared with control group, the expression level of HMGB1 protein was the highest at 4 hours, the expression level of 2H protein was higher than that of control group, and the expression level The expression level of NMDA protein in hippocampal neurons of IH group was significantly lower than that of control group at 8 hours after exposure. The expression level of NMDA protein in hippocampal neurons of IH group was significantly different at different time after exposure (P 0.05). The expression of NMDA protein in hippocampal neurons increased significantly with the prolongation of normoxic culture time after exposure. Conclusion: Neuritis plays an important role in neurocognitive impairment induced by OSA-IH, and IH can induce the expression of neuroinflammatory factor HMGB1. This indicates that there is a vicious circle between neurons with neuroinflammation and injury, which can cause a "second blow" to neurons. Continuous release of HMGB1 from dying neurons is important for the formation and maintenance of this malignant cycle. Inflammatory damage can continue even after the initiating factor IH ceases. IH can cause neuroinflammation, but IH is not a necessary condition for maintaining neuroinflammation. As the duration of inflammation prolongs, the secretion of HMGB1 decreases, but NMDA is clear. Significant increase indicates that HMGB1 and NMDA may play a synergistic role in inducing neuronal apoptosis and dysfunction.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R766

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