摘要
目的通过蛋白质组学研究探讨高氧暴露后幼鼠Ⅱ型肺泡上皮细胞(AECⅡ)的损伤机制。方法将早产SD大鼠原代AECⅡ细胞分为常氧组和高氧组,分别置于常氧(21%O2)和高氧(95%O2)中培养24h,于倒置相差显微镜下观察细胞形态学改变,用蛋白质免疫印迹试验(Western Blot)检测凋亡相关蛋白Bcl-2、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)表达水平,以保证高氧模型的成功建立;收集AECⅡ细胞总蛋白,采用串联质谱标签(TMT)标记定量蛋白质组技术检测其蛋白谱,以高氧暴露后差异倍数(FC)>1.5、P<0.05双标准筛选差异蛋白,并对其进行生物信息学分析;依据分析结果,将AECⅡ细胞分为常氧组、高氧组及高氧+MW167组〔细胞置于含95%O2的氧舱前30min加入10μmol/L的γ泌肽酶抑制剂MW167〕,培养24h后,用CCK-8细胞增殖及细胞毒性检测试剂盒检测细胞活性,用实时荧光定量反转录-聚合酶链反应(qRT-PCR)检测Notch通路下游蛋白毛状蛋白(Hes1)和促凋亡蛋白Bax的mRNA表达。结果①光镜下观察,常氧组细胞明显增殖、延展,胞质颗粒物质丰富;高氧组细胞核固缩,胞质内颗粒物质明显减少。与常氧组相比,高氧组细胞caspase-3蛋白表达明显增高,Bcl-2蛋白表达明显降低(caspase-3/GAPDH:1.352±0.086比0.769±0.080,Bcl-2/GAPDH:0.614±0.060比1.361±0.078,均P<0.01)。②蛋白质组学鉴定出差异蛋白162个,其中高氧暴露后上调的差异蛋白主要参与各种刺激的应答,定位于胞外区;高氧暴露后下调的差异蛋白主要与物质合成相关,定位于细胞基质区域。对差异蛋白通路富集分析(KEGGPathway分析)结果表明,细胞色素P450代谢、线粒体氧化磷酸化、Notch信号通路等可能与高氧损伤AECⅡ细胞机制相关。③与常氧组比较,高氧组AECⅡ细胞活性显著下降,Hes1、BaxmRNA表达水平明显升高〔细胞活性(A值):0.060±0.003比1.058±0.017,Hes1mRNA(2^-ΔΔCt):2.235±0.606比1.144±0.107,BaxmRNA(2^-ΔΔCt):2.210±0.240比1.084±0.096,均P<0.05〕。与高氧组比较,高氧+MW167组细胞活性明显升高,Hes1、BaxmRNA表达水平明显下降〔细胞活性(A值):0.271±0.025比0.060±0.003,Hes1mRNA(2^-ΔΔCt):0.489±0.046比2.235±0.606,BaxmRNA(2^-ΔΔCt):1.289±0.041比2.210±0.240,均P<0.05〕。结论高氧肺损伤幼鼠AECⅡ细胞损伤机制可能与细胞色素P450代谢、线粒体氧化磷酸化等途径的改变以及Notch信号通路的激活有关。
Objective To investigate the damage mechanism of typeⅡalveolar epithelial cells (AECⅡ) after hyperoxia exposure by proteomics. Methods The primary AECⅡ of preterm Sprague-Dawley (SD) rats were divided into normoxia and hyperoxia groups, and cultured in room air (21% O2) or hyperoxia (95% O2) condition, respectively. The cell morphology change was observed under an inverted contrast microscope;the protein expressions of Bcl-2 and caspase-3 were detected by Western Blot to ensure a successful model. Total protein in AECⅡ was collected, and mass spectrometry-based tandem mass tag (TMT)-labeled quantitative proteomics were used to detect the change of protein profile. Proteins with changes greater than 1.5-fold and P < 0.05 were considered differentially expressed, and bioinformatics analysis was performed. According to the proteomic results, AECⅡ were divided into three groups: normoxia group, hyperoxia group and hyperoxia+MW167 group (γ-secretase inhibitor MW167 was added to culture medium 30 minutes before they were placed into the chamber). The cell viability was detected by the cell proliferation and toxicity kit (CCK-8), and the expressions of Hes1, Bax mRNA were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results ① The cells in the normoxia group proliferated and prolonged significantly, and the cytoplasmic particulate matter was abundant. In the hyperoxia group, nucleus pyknosis and cytoplasmic particulate matter decreased significantly. Compared with the normoxia group, the expression of caspase-3 in the hyperoxia group was significantly increased, and the expression of Bcl-2 was significantly decreased (caspase-3/GAPDH: 1.352±0.086 vs. 0.769±0.080, Bcl-2/GAPDH: 0.614±0.060 vs. 1.361±0.078, both P < 0.01).② A total of 162 differentially expressed proteins were identified between normoxia and hyperoxia groups, the proteins up-regulated by hyperoxia were commonly associated with response processes to various stimuli, and located in the extracellular region;the proteins down-regulated by hyperoxia were commonly associated with synthesis of substances, and located in the cellular matrix. KEGG Pathway analyses suggested that metabolism by cytochrome P450, oxidative phosphorylation, and Notch signaling pathway were associated with the mechanism of hyperoxia injury on AECⅡ.③Compared with the normoxia group, the viability of cells in the hyperoxia group was significantly decreased, and the expressions of Hes1 and Bax mRNA were significantly increased [cell viability (A value): 0.060±0.003 vs. 1.058± 0.017, Hes1 mRNA (2^-ΔΔCt): 2.235±0.606 vs. 1.144±0.107, Bax mRNA (2^-ΔΔCt): 2.210±0.240 vs. 1.084±0.096, all P < 0.05]. Compared with the hyperoxia group, the viability of cells in the hyperoxia+MW167 group was significantly increased, and the expressions of Hes1 and Bax mRNA were significantly decreased [cell viability (A value): 0.271±0.025 vs. 0.060±0.003, Hes1 mRNA (2^-ΔΔCt): 0.489±0.046 vs. 2.235±0.606, Bax mRNA (2^-ΔΔCt): 1.289±0.041 vs. 2.210±0.240, all P < 0.05]. Conclusion The mechanism of hyperoxia injury on AECⅡ may be related to the metabolism by cytochrome P450, oxidative phosphorylation and activation of Notch signaling pathway.
作者
鲁雪
汪超
张超
肖长雪
梁木林
许峰
Lu Xue;Wang Chao;Zhang Chao;Xiao Changxue;Liang Mulin;Xu Feng(Department of Pediatric Intensive Care Unit,Chongqing Medical University Children's Hospital,Ministry of Education Key Laboratory of Child Developmental Disease Research,Chongqing Key Laboratory of Pediatrics,National International Science and Technology Cooperation Base for Children with Major Developmental Diseases,Chongqing 400014,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2019年第4期474-479,共6页
Chinese Critical Care Medicine
基金
国家自然科学基金(81341021)
重庆市渝中区科技计划项目(20170110).
