维甲酸对高氧暴露下原代培养的胎鼠肺泡II型上皮细胞和成纤维细胞增殖与凋亡的影响  

Effect of Retinoic Acid on the Primary Rat Embryonic Type II Alveolar Epithelial Cell and Lung Fibroblasts Proliferation and Apoptosis Exposed to Hyperoxia

作　　者：李文斌[1] 常立文[1] 容志惠[1] 王华[1] 卢红艳[1] 汪鸿[1] 刘伟[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030

出　　处：《细胞生物学杂志》2007年第1期115-121,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(No.30471824);国家"十五"科技攻关计划(No.2004BA720A11)资助项目~~

摘　　要：探讨高氧暴露对原代培养的胎鼠肺泡II型上皮细胞(AECII)、成纤维细胞(LFs)增殖和凋亡的影响以及维甲酸(RA)的保护作用机制。通过建立高氧暴露原代培养的胎鼠AECII和LFs模型,以RA作为干预方式,采用流式细胞术(膜联蛋白V-PI双标记)检测AECII和LFs凋亡,West-ern印迹检测AECII增殖细胞核抗原(PCNA)、p53及caspase-3表达和LFsPCNA表达。结果发现:(1)与空气对照比较,高氧暴露12h,膜联蛋白V(+)PI(-)和膜联蛋白V(+)PI(+)标记AECII数均显著升高(14.41±1.15vs2.80±0.19,P<0.01;61.07±3.06vs1.49±0.11,P<0.01);RA对空气暴露下AECII坏死、凋亡无明显影响,但明显下调高氧暴露下膜联蛋白V(+)PI(-)和膜联蛋白V(+)PI(+)标记AECII数(8.04±0.79vs14.41±1.15,P<0.01;27.57±2.32vs61.07±3.06,P<0.01)。(2)高氧、RA对LFs坏死、凋亡无明显影响。(3)高氧暴露12h,明显降低AECIIPCNA表达(P<0.01),显著提高其p53(P<0.01)和caspase-3活性片段(P<0.01)表达;RA显著上调高氧暴露下AECIIPCNA表达(P<0.01),下调其p53和caspase-3活化片段表达(P<0.01)。(4)高氧、RA对LFsPCNA表达无明显影响。由此提示,高氧暴露,导致AECII大量凋亡、坏死,增殖受到抑制,同时,LFs所受影响较小,两种细胞对高氧暴露的差异性行为可能是导致未成熟肺组织异常重构的重要原因;RA通过降低AECII凋亡、坏死从而对高氧肺损伤具有保护作用。To explore the effect of hyperoxia exposure on proliferation and apoptosis of the primary rat embryonic type Ⅱ alveolar epithelial cells （AECⅡ） and lung fibroblasts （LFs）, and the protective effect of retinoic acid （RA） on hyperoxia lung injury. The primary rat embryonic AECⅡ and LFs （gestation 19-20 d） were cultured in vitro. For the study of RA effects, AECⅡ and LFs were exposed to hyperoxia in the presence or absence of RA for 12 h. Their apoptosis were analyzed by annexin V/propidium iodide （PI） double Staining and flow cytometry. The expression of PCNA, p53 and caspase-3 in AECⅡ and that of PCNA in LFs were determined by Western blot. Results showed that： （1） Quantitative data from flow cytometry analyses （PI/annexin V double staining） demonstrated that there was a significant increase in positive cells of both annexin V （＋） PI （-） and annexin V （＋） PI （＋） after 12 h of hyperoxia in AECⅡ （14.41±1.15 vs 2.80±0.19, P〈0.01; 61.07±3.06 vs 1.49±0.11, P〈0.01）. RAhad no effect on apoptosis and necrosis of AECII exposed in room air, but markedly decreased hyperoxia-induced AECⅡ apoptosis and necrosis（8.04±0.79 vs 14.41 ± 1.15, P〈0.01; 27.57± 2.32 vs 61.07± 3.06, P〈0.01 ）. （2） The apoptosis and necrosis of LFs were not changed by hyperoxia exposure and/or RA treatment. （3） Western blot analyses showed that, after 12 h of hyperoxia, PCNA was reduced markedly （P〈0.01）, p53 and active fragment of caspase-3 （17 kDa） were increased in AECⅡ （P〈0.01）. In hyperoxia exposure, RA decreased the expression of p53 and active fragment of caspase-3 markedly （P〈0.01）, improved the expression of PCNA （P〈0.01）. （4） Hyperoxia exposure and/or RA treatment had no effect on the expression of PCNA in LFs. It was concluded that, hyperoxia exposure lead to numerous AECⅡ apoptosis and necrosis and inhibited AECⅡ proliferation, but did not change LFs survival, both of which were involved in abnormal lung remod

关 键 词：高氧 维甲酸 肺泡Ⅱ型上皮细胞 肺成纤维细胞 凋亡 增殖 

分 类 号：Q813.1[生物学—生物工程]