自噬调节JNK通路在乳化异氟醚诱导神经干细胞凋亡的机制研究

发布时间：2018-05-18 00:16

本文选题：JNK通路 + 自噬　；参考：《安徽医科大学》2014年博士论文

【摘要】：目的研究表明细胞凋亡和自噬之间存在着联系,JNK信号通路在神经元变性和稳态方面发挥重要作用。异氟醚作为一种常用的吸入麻醉药能对在体或离体模型诱发脑损伤,产生神经毒性,抑制细胞增殖,甚至细胞凋亡。本实验通过研究自噬调节JNK通路在乳化异氟醚(异氟醚的静脉制剂)诱导神经干细胞凋亡的机制,来明确怎样调节自噬水平和JNK通路来降低药物毒性。本研究的结果将为临床上围产期手术药物使用及发育中神经保护与损伤修复提供理论依据。方法1.细胞模型建立购买Invitrogen公司Gibco#174;Rat Fetal Neural Stem Cell Kit,加入NSCs培养液(DMEM/F12+2%B27添加剂+l%N2添加剂+20 ng/ml EGF+20 ng/mlb FGF,其中含青霉素钠200 u/L及链霉素100 U/L,p H 7.4)。将细胞以1×105接种于培养瓶中,每瓶5ml,置37°C,体积分数为5%CO2和95%空气条件下进行培养。传代至第六代,实验前一天接种于96孔培养板。调整浓度为5×104个/ml。2.实验分组神经细胞毒性实验共设5组:control、脂肪乳剂对照、7.56,9.52和11.48 mmol/l乳化异氟醚处理胚胎神经干细胞,分别处理6h,12h和24 h,测量MTT assay的OD值。分别在6h,12h,24h观察细胞凋亡情况检测技术A,MTT检测;B,流式细胞仪及Annexin V-FITC/PI双标可定量分析早期凋亡细胞(AV+PI-)、中晚期凋亡细胞(AV+PI+)、坏死细胞(AV-PI+)及正常细胞(AV-PI-)所占的比例。Western blotting检测乳化异氟醚处理后凋亡相关蛋白的变化.6h,12h,24 h乳化异氟醚处理后western blot示胚胎神经干细胞JNK1,JNK2,Bcl-2,聚腺苷酸二磷酸核糖转移酶(PARP),裂解的caspase 3,caspase 3和IRE1蛋白等表达;Lipofectamine试剂转染,连续数据用均数±标准差表示。结果乳化异氟醚的浓度7.56mmol/L,9.52mmol/L和11.48mmol/L跟脂肪乳对照组比较,作用神经干细胞24小时,乳化异氟醚显著地减少细胞生存,呈剂量依赖性,用6h,12h和24h.三组间细胞生存和抑制有明显不同;流式细胞术检测不同浓度乳化异氟醚24h诱导胚胎神经干细胞的细胞生存和凋亡(Annexin V阳性细胞比率)。7.56,9.52和11.48 mmol/l乳化异氟醚处理神经干细胞24 h,P0.05.**P0.01vs.脂肪乳对照组,数据记录采用均数(±标准差),包括3次独立实验。6h Caspase3*P0.05 vs.脂肪乳和未用乳化异氟醚对照,12h Caspase3**P0.01 vs.脂肪乳和未用乳化异氟醚对照,24h乳化异氟醚组,脂肪乳对照组,未用乳化异氟醚组三组比较,无明显差异;6h和24 h乳化异氟醚处理PARP**P0.01 vs.未用乳化异氟醚对照,12h乳化异氟醚处理PARP*P0.05 vs.未用乳化异氟醚对照,PARP(聚腺苷酸二磷酸核糖转移酶)蛋白;6h,12h乳化异氟醚处理JNK*P0.05 vs.未用乳化异氟醚对照,24h乳化异氟醚处理JNK*P0.05vs.未用乳化异氟醚对照,24 h乳化异氟醚处理JNKP0.05 vs.脂肪乳对照;*P0.05 vs.未用乳化异氟醚组对照#P0.05 vs.12h脂肪乳对照P0.05 vs.24h脂肪乳对照Western blot分析bcl-2蛋白提示内质网内质网应激介导的凋亡,结果表明乳化异氟醚接触Bcl-2表达下降;LC3B蛋白在各个时间点明显上调*P0.05,**P0.01 vs.相应的脂肪乳对照组(包括6h,12h和24h处理组),24h乳化异氟醚处理组明显上调P0.05 vs.24h未用乳化异氟醚处理组;p62蛋白每种蛋白带的密度测量,GAPDH作为内参6h和12h处理组*P0.05 vs.相应的脂肪乳对照组,#P0.05vs.12h乳化异氟醚处理,P0.05 vs.24h乳化异氟醚处理;Atg5蛋白每种蛋白带的密度测量,GAPDH作为内参6h乳化异氟醚处理组Atg5明显上调,*P0.05 vs.相应的脂肪乳对照组;共焦显微镜观察GFP-LC3标记的斑点结构9.52mmol/l乳化异氟醚,脂肪乳剂作为对照,6h,12h和24h作用于胚胎神经干细胞,GFP-LC3质粒转染.流式细胞术分析GFP-LC3标记的斑点数,*P0.05,**P0.01 vs.相应的脂肪乳对照组,##P0.01 vs.6h乳化异氟醚处理组,P0.01 vs.12h乳化异氟醚处理组。通过电镜扫描观察药物处理后细胞的自噬形态,不同作用时间9.52mmol/l乳化异氟醚诱导的神经干细胞自体吞噬体形成。乳化异氟醚处理12 h MTT的OD值比较3-MA组较乳化异氟醚处理组明显上升***P0.001,Rap组较乳化异氟醚处理组明显下降**P0.01乳化异氟醚处理组较对照组明显下降***P0.001**P0.01,***P0.001 vs.相应的对照组。结论抑制JNK通路和下调自噬水平能减少乳化异氟醚诱导的神经细胞凋亡。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

【参考文献】