本文選題：JNK通路 + 自噬　； 參考：《安徽醫(yī)科大學(xué)》2014年博士論文

【摘要】：目的研究表明細(xì)胞凋亡和自噬之間存在著聯(lián)系,JNK信號(hào)通路在神經(jīng)元變性和穩(wěn)態(tài)方面發(fā)揮重要作用。異氟醚作為一種常用的吸入麻醉藥能對(duì)在體或離體模型誘發(fā)腦損傷,產(chǎn)生神經(jīng)毒性,抑制細(xì)胞增殖,甚至細(xì)胞凋亡。本實(shí)驗(yàn)通過研究自噬調(diào)節(jié)JNK通路在乳化異氟醚(異氟醚的靜脈制劑)誘導(dǎo)神經(jīng)干細(xì)胞凋亡的機(jī)制,來明確怎樣調(diào)節(jié)自噬水平和JNK通路來降低藥物毒性。本研究的結(jié)果將為臨床上圍產(chǎn)期手術(shù)藥物使用及發(fā)育中神經(jīng)保護(hù)與損傷修復(fù)提供理論依據(jù)。方法1.細(xì)胞模型建立購(gòu)買Invitrogen公司Gibco#174;Rat Fetal Neural Stem Cell Kit,加入NSCs培養(yǎng)液(DMEM/F12+2%B27添加劑+l%N2添加劑+20 ng/ml EGF+20 ng/mlb FGF,其中含青霉素鈉200 u/L及鏈霉素100 U/L,p H 7.4)。將細(xì)胞以1×105接種于培養(yǎng)瓶中,每瓶5ml,置37°C,體積分?jǐn)?shù)為5%CO2和95%空氣條件下進(jìn)行培養(yǎng)。傳代至第六代,實(shí)驗(yàn)前一天接種于96孔培養(yǎng)板。調(diào)整濃度為5×104個(gè)/ml。2.實(shí)驗(yàn)分組神經(jīng)細(xì)胞毒性實(shí)驗(yàn)共設(shè)5組:control、脂肪乳劑對(duì)照、7.56,9.52和11.48 mmol/l乳化異氟醚處理胚胎神經(jīng)干細(xì)胞,分別處理6h,12h和24 h,測(cè)量MTT assay的OD值。分別在6h,12h,24h觀察細(xì)胞凋亡情況檢測(cè)技術(shù)A,MTT檢測(cè);B,流式細(xì)胞儀及Annexin V-FITC/PI雙標(biāo)可定量分析早期凋亡細(xì)胞(AV+PI-)、中晚期凋亡細(xì)胞(AV+PI+)、壞死細(xì)胞(AV-PI+)及正常細(xì)胞(AV-PI-)所占的比例。Western blotting檢測(cè)乳化異氟醚處理后凋亡相關(guān)蛋白的變化.6h,12h,24 h乳化異氟醚處理后western blot示胚胎神經(jīng)干細(xì)胞JNK1,JNK2,Bcl-2,聚腺苷酸二磷酸核糖轉(zhuǎn)移酶(PARP),裂解的caspase 3,caspase 3和IRE1蛋白等表達(dá);Lipofectamine試劑轉(zhuǎn)染,連續(xù)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差表示。結(jié)果乳化異氟醚的濃度7.56mmol/L,9.52mmol/L和11.48mmol/L跟脂肪乳對(duì)照組比較,作用神經(jīng)干細(xì)胞24小時(shí),乳化異氟醚顯著地減少細(xì)胞生存,呈劑量依賴性,用6h,12h和24h.三組間細(xì)胞生存和抑制有明顯不同;流式細(xì)胞術(shù)檢測(cè)不同濃度乳化異氟醚24h誘導(dǎo)胚胎神經(jīng)干細(xì)胞的細(xì)胞生存和凋亡(Annexin V陽(yáng)性細(xì)胞比率)。7.56,9.52和11.48 mmol/l乳化異氟醚處理神經(jīng)干細(xì)胞24 h,P0.05.**P0.01vs.脂肪乳對(duì)照組,數(shù)據(jù)記錄采用均數(shù)(±標(biāo)準(zhǔn)差),包括3次獨(dú)立實(shí)驗(yàn)。6h Caspase3*P0.05 vs.脂肪乳和未用乳化異氟醚對(duì)照,12h Caspase3**P0.01 vs.脂肪乳和未用乳化異氟醚對(duì)照,24h乳化異氟醚組,脂肪乳對(duì)照組,未用乳化異氟醚組三組比較,無明顯差異;6h和24 h乳化異氟醚處理PARP**P0.01 vs.未用乳化異氟醚對(duì)照,12h乳化異氟醚處理PARP*P0.05 vs.未用乳化異氟醚對(duì)照,PARP(聚腺苷酸二磷酸核糖轉(zhuǎn)移酶)蛋白;6h,12h乳化異氟醚處理JNK*P0.05 vs.未用乳化異氟醚對(duì)照,24h乳化異氟醚處理JNK*P0.05vs.未用乳化異氟醚對(duì)照,24 h乳化異氟醚處理JNKP0.05 vs.脂肪乳對(duì)照;*P0.05 vs.未用乳化異氟醚組對(duì)照#P0.05 vs.12h脂肪乳對(duì)照P0.05 vs.24h脂肪乳對(duì)照Western blot分析bcl-2蛋白提示內(nèi)質(zhì)網(wǎng)內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡,結(jié)果表明乳化異氟醚接觸Bcl-2表達(dá)下降;LC3B蛋白在各個(gè)時(shí)間點(diǎn)明顯上調(diào)*P0.05,**P0.01 vs.相應(yīng)的脂肪乳對(duì)照組(包括6h,12h和24h處理組),24h乳化異氟醚處理組明顯上調(diào)P0.05 vs.24h未用乳化異氟醚處理組;p62蛋白每種蛋白帶的密度測(cè)量,GAPDH作為內(nèi)參6h和12h處理組*P0.05 vs.相應(yīng)的脂肪乳對(duì)照組,#P0.05vs.12h乳化異氟醚處理,P0.05 vs.24h乳化異氟醚處理;Atg5蛋白每種蛋白帶的密度測(cè)量,GAPDH作為內(nèi)參6h乳化異氟醚處理組Atg5明顯上調(diào),*P0.05 vs.相應(yīng)的脂肪乳對(duì)照組;共焦顯微鏡觀察GFP-LC3標(biāo)記的斑點(diǎn)結(jié)構(gòu)9.52mmol/l乳化異氟醚,脂肪乳劑作為對(duì)照,6h,12h和24h作用于胚胎神經(jīng)干細(xì)胞,GFP-LC3質(zhì)粒轉(zhuǎn)染.流式細(xì)胞術(shù)分析GFP-LC3標(biāo)記的斑點(diǎn)數(shù),*P0.05,**P0.01 vs.相應(yīng)的脂肪乳對(duì)照組,##P0.01 vs.6h乳化異氟醚處理組,P0.01 vs.12h乳化異氟醚處理組。通過電鏡掃描觀察藥物處理后細(xì)胞的自噬形態(tài),不同作用時(shí)間9.52mmol/l乳化異氟醚誘導(dǎo)的神經(jīng)干細(xì)胞自體吞噬體形成。乳化異氟醚處理12 h MTT的OD值比較3-MA組較乳化異氟醚處理組明顯上升***P0.001,Rap組較乳化異氟醚處理組明顯下降**P0.01乳化異氟醚處理組較對(duì)照組明顯下降***P0.001**P0.01,***P0.001 vs.相應(yīng)的對(duì)照組。結(jié)論抑制JNK通路和下調(diào)自噬水平能減少乳化異氟醚誘導(dǎo)的神經(jīng)細(xì)胞凋亡。
[Abstract]:Objective to study the relationship between apoptosis and autophagy. The JNK signaling pathway plays an important role in neuronal degeneration and homeostasis. Isoflurane, as a common inhaled anesthetic, can induce brain damage in vivo or in vitro model, produce neurotoxicity, inhibit cell proliferation, and even apoptosis. The mechanism of JNK pathway to induce the apoptosis of neural stem cells induced by the emulsified isoflurane (isoflurane intravenous preparation) is used to clarify how to regulate autophagy and JNK pathway to reduce drug toxicity. The results of this study will provide a theoretical basis for the use of drug use and the repair of neural protection and injury in the clinical perinatal operation. Method 1. cells The model builds Invitrogen company Gibco#174, Rat Fetal Neural Stem Cell Kit, adding NSCs culture liquid (DMEM/F12+2%B27 additive +l%N2 additive +20), including penicillin sodium 200 and streptomycin 100, 7.4). 95% air conditions were cultured, passed to sixth generations, and inoculated on 96 hole culture plates a day before the experiment. 5 groups were set up for 5 x 104 /ml.2. experimental groups of neurotoxicity: control, fat emulsion control, 7.56,9.52 and 11.48 mmol/l emulsified isoflurane treated embryonic deity stem cells, respectively, 6h, 12h and 24 h, respectively, to measure MTT ass Ay, A, MTT detection in 6h, 12h, 24h, B, flow cytometry and Annexin V-FITC/PI double standard can be used for quantitative analysis of early apoptotic cells (AV+PI-), middle and late apoptotic cells (AV+PI+), necrotic cells (AV-PI+) and normal cells after emulsified isoflurane Changes in apoptosis related proteins.6h, 12h, 24 h emulsified isoflurane treated Western blot of embryonic neural stem cells JNK1, JNK2, Bcl-2, poly adenylate two phosphate ribose transferase (PARP), cracking caspase 3, caspase 3 and IRE1 protein expression. The concentration 7.56mmol/L, 9.52mmol/L and 11.48mmol/L were compared with the fat milk control group. The emulsified isoflurane significantly reduced the cell survival for 24 hours. The survival and inhibition of cells in the three groups were significantly different from the 6h, 12h and 24h. groups, and the flow cytometry was used to detect different concentrations of emulsified isoflurane 24h to induce the embryonic neural stem fine. Cell survival and apoptosis (ratio of Annexin V positive cells).7.56,9.52 and 11.48 mmol/l emulsified isoflurane treated neural stem cells 24 h, P0.05.**P0.01vs. fat milk control group, data recorded using mean number (+ standard deviation), including 3 independent experiment.6h Caspase3*P0.05 vs. fat emulsion and unemulsified isoflurane control, 12h Caspase3**P0.01 Vs. fat emulsion and unemulsified isoflurane control, 24h emulsified isoflurane group, fat milk control group, without emulsified isoflurane group three groups, no significant difference; 6h and 24 h emulsified isoflurane treatment PARP**P0.01 vs. no emulsified isoflurane control, 12h emulsified isoflurane treated PARP* P0.05 vs. without emulsified isoflurane control, PARP (polyadenyl two phosphorus) 6h, 12h emulsified isoflurane treatment JNK*P0.05 vs. did not use emulsified isoflurane control, 24h emulsified isoflurane treated JNK*P0.05vs. did not use emulsified isoflurane control, 24 h emulsified isoflurane treated JNKP0.05 vs. fat milk control, *P0.05 vs. did not use emulsified isoflurane group compared with #P0.05 fat milk control fat milk control fat Milk control Western blot analysis Bcl-2 protein suggests endoplasmic reticulum stress mediated apoptosis. The results showed that the expression of Bcl-2 in the emulsified isoflurane decreased, and the LC3B protein was obviously up-regulated at every point of time *P0.05, and the **P0.01 vs. corresponding fat milk control group (including 6h, 12h and 24h treatment group), and the emulsified isoflurane treatment group up regulated 4h did not use the emulsified isoflurane treatment group; the density of each protein band of p62 protein was measured, GAPDH was used as the control group of *P0.05 vs. in the internal parameter 6h and 12h treatment group, #P0.05vs.12h emulsified isoflurane treatment, P0.05 vs.24h emulsified isoflurane treatment, Atg5 protein per protein band density measurement, GAPDH as the internal parameter of emulsified isoflurane treatment group G5 was obviously up-regulated, *P0.05 vs. corresponding fat milk control group; confocal microscopy was used to observe the dot structure of GFP-LC3 labeled 9.52mmol/l emulsified isoflurane, fat emulsion as control, 6h, 12h and 24h acted on embryonic neural stem cells, GFP-LC3 plasmid transfection. Flow cytometry was used to analyze the number of markings marked by GFP-LC3, *P0.05, **P0.01 accordingly fat emulsion The control group, ##P0.01 vs.6h emulsified isoflurane treatment group, P0.01 vs.12h emulsified isoflurane treatment group. The autophagy morphology of the cells after the drug treatment was observed through the electron microscope, and the different action time 9.52mmol/l emulsified isoflurane induced autophagosus in the neural stem cells. The oemulsification of the emulsified isoflurane treated 12 h MTT was compared with that of the 3-MA group and the emulsification. The treatment group of fluoroether significantly increased ***P0.001, Rap group was significantly lower than the emulsified isoflurane treatment group, **P0.01 emulsified isoflurane treatment group decreased significantly ***P0.001**P0.01, ***P0.001 vs. corresponding control group. Conclusion inhibition of JNK pathway and down regulation of autophagy can reduce the apoptosis induced by emulsified isoflurane.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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相關(guān)期刊論文 前1條

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本文編號(hào)：1903507