Notch信号在神经肽P物质对高氧暴露肺泡Ⅱ型上皮细胞的保护作用机制  

The effect and molecular mechanisms of Notch signaling in the protection of neuropeptide substance P in hyperoxia-exposed alveolar type Ⅱ epithelial cells

作　　者：董欣鑫[1,2] 姚兰[1] 方芳[1] 许峰[1] 

机构地区：[1]重庆医科大学附属儿童医院PICU,儿童发育疾病研究省部共建教育部重点实验室,重庆400014 [2]太钢总医院,山西太原030003

出　　处：《西安交通大学学报（医学版）》2014年第1期35-39,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30973218);重庆市卫生局2009年度医学科研计划项目(重点项目)(2009-1-40)~~

摘　　要：目的探讨Notch信号在神经肽P物质(substance P,SP)对高氧状态下早产大鼠肺泡Ⅱ型上皮细胞(AECⅡ)的作用机制。方法将原代分离培养的大鼠孕19d胎鼠AECⅡ随机分为4组:空气组和高氧组分别暴露在210mL/L和950mL/L氧气中;高氧+SP组于暴露前加SP;高氧+SP+L703.606组在高氧+SP组基础上加入SP受体拮抗剂L703.606。各组均培养24h后,流式细胞仪检测细胞凋亡率;微孔板检测系统和激光共聚焦显微镜检测超氧化物阴离子水平;四甲基偶氮唑盐(MTT)比色法测定细胞增殖能力;QPCR检测Notch1、Notch3基因的表达;Western blot检测Notch1、Notch3蛋白的表达。结果与空气组比较,高氧组细胞凋亡率及超氧化物阴离子的含量均显著增高,增殖率显著下降(P<0.01);高氧+SP组可显著降低细胞凋亡率及超氧化物阴离子的表达,提高细胞增殖率(P<0.01)。高氧组Notch1基因和蛋白的表达均明显降低(P<0.01),Notch3基因和蛋白水平表达均明显升高(P<0.01),高氧+SP组与单纯高氧组比较,Notch1基因和蛋白水平表达升高,Notch3基因和蛋白水平表达降低,差异具有统计学意义(P<0.01)。高氧+SP+L703.606组与高氧组各指标比较差异均无统计学意义(P>0.05)。结论 SP可通过调控Notch信号通路的表达,抑制高氧诱发的AECⅡ细胞凋亡,减轻氧化损伤,促进细胞增殖的作用。Objective To investigate the effect and molecular mechanisms of Notch signaling in the protection of neuropeptide substance P （SP） in hyperoxia-exposed alveolar type Ⅱ epithelial cells （AECⅡ）. Methods AECⅡwere isolated from the lung of 19-day rat fetus and then cultured in six-well plates. The cells were randomly divided into air group, hyperoxia group, hyperoxia ＋ SP group, and hyperoxia ＋ SP ＋ antagonist group. Air group was exposed to 21% oxygen and hyperoxia group was exposed to 95% oxygen for 24 hours, respectively. SP was added into hyperoxia ＋ SP group before oxygen exposure, while SP and its receptor antagonist （L703. 606） were added into hyperoxia ＋SP ＋ antagonist group simultaneously before oxygen exposure; then both groups were exposed to 95% oxygen for 24 hours. Flow cytometry and MTT colorimetric assay were used to detect the ability of cell proliferation; the intracellular superoxide anion level was detected by SpectraMax MS/M5e and confocal microscopy; the mRNA and protein expressions of Notchl and Notch3 were assessed by QPCR and Western blot analysis, respectively. Results Compared with those in air group, the apoptosis rate and the intracellular superoxide anion concentration were significantly increased, while the proliferation rate of cells was significantly decreased in hyperoxia group （P 〈0.01）. In contrast, the apoptosis rate and the intracellular superoxide anion concentration were lower and the cell proliferation rate were higher in hyperoxia SP group than in hyperoxia group （P〈0.01）. Hyperoxia exposure resulted in significantly decreased expression of Notch1 and increased expression of Notch3 （P〈0.01）, while SP treatment reversed the above results significantly （P〈0.01）. These parameters did not differ significantly between hyperoxia group and hyperoxia q- SP ＋ L703. 606 group. Conclusion SP can reduce the hyperoxia-induced AEC damage and death and promote cell proliferation, which is associated with the activation of Notch signal t

关 键 词：高氧 肺泡Ⅱ型上皮细胞(AECII) 神经肽P物质 NOTCHL NOTCH3 肺损伤 早产大鼠 

分 类 号：R322.35[医药卫生—人体解剖和组织胚胎学] R329.26[医药卫生—基础医学]