目的 探讨高氧对胎鼠肺成纤维细胞（LFs）p53 和增殖细胞核抗原（PCNA）表达的影响。方法 原代培养胎鼠肺 LFs，待生长至亚汇合状态时，随机分为：空气组和高氧组（95% O2/5% CO2）。于培养 12 h 和 24 h 时，采用噻唑蓝（MTT）实验测定细胞增殖状况，半定量 RT-PCR 方法检测 p53 mRNA 表达，Western blot 技术检测 p53 和 PCNA 蛋白的表达。结果（1）与空气组比较，高氧组 12 h 和 24 h 的 LFs 生长抑制率分别为 8% 和 23%；（2）高氧组在 12 h 和 24 h 时 p53 mRNA 表达明显高于空气组（P<0.01）；（3）高氧组在 12 h 和 24 h 时 p53 蛋白表达明显高于空气组（P<0.01），而 PCNA 蛋白表达水平在 24 h 明显低于空气组（P<0.01）。结论 高氧暴露抑制 PCNA 表达、促进 p53 表达，从而抑制 LFs 增殖和 DNA 复制，是导致肺发育异常的重要因素。
Abstract
Objective To study the effect of hyperoxia exposure on the expression of p53 and proliferating cell nuclear antigen（PCNA) in fetal rat lung fibroblasts（LFs). Methods Primary rat embryonic LFs were cultured in vitro.LFs grew to subconfluence and then were randomly divided into air and hyperoxia exposure（95% O2, 5% CO2) groups.After LFs were cultured for 12 and 24 hours, the proliferation was analyzed by MTT. p53 mRNA level was detected by semi-quantitative reverse transcription polymerase chain reaction（RT-PCR). p53 and PCNA protein levels were determined by Western blot. Results After 12 and 24 hours of culture the growth inhibition rate of LFs was 8% and 23% respectively in the hyperoxia exposure group. p53 mRNA and protein levels increased significantly（P<0.01) in the hyperoxia exposure group after 12 and 24 hours of culture compared with the air exposure group. Hyperoxia exposure decreased PCNA expression after 24 hours of culture（P<0.01). Conclusion Hyperoxia exposure increases p53 level and decreases PCNA expression, resulting in inhibitions of LFs proliferation and DNA repair.
关键词
高氧 /
肺成纤维细胞 /
p53 /
增殖细胞核抗原 /
大鼠
Key words
Hyperoxia /
Lung fibroblast /
p53 /
Proliferating cell nuclear antigen /
Rats
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Giordani VM, Debenedictus CM, Wang Y, et al.Epidermal growth factor receptor（EGFR) contributes to fetal lung fibroblast injury induced by mechanical stretch[J].J Recept Signal Transduct Res, 2014, 34(1): 58-63.
[2] Londhe VA, Sundar IK, Lopez B, et al.Hyperoxia impairs alveolar formation and induces senescence through decreased histone deacetylase activity and up-regulation of p21 in neonatal mouse lung[J].Pediatr Res, 2011, 69(5 Pt 1): 371-377.
[3] Jean JC, George E, Kaestner KH, et al.Transcription factor Klf4, induced in the lung by oxygen at birth, regulates perinatal fibroblast and myofibroblast differentiation[J].PLoS One, 2013,8(1): e54806.
[4] Wright CJ, Dennery PA.Manipulation of gene expression by oxygen: a primer from bedside to bench[J].Pediatr Res, 2009,66(1): 3-10.
[5] 李文斌, 常立文, 祝华平, 等.高氧、维甲酸对胎鼠肺成纤维细胞 c-Jun、c-Fos 表达的影响 [J].华中科技大学学报(医学版), 2003, 32(2): 216-219.
[6] Klimova TA, Bell EL, Shroff EH, et al.Hyperoxia-induced premature senescence requires p53 and pRb, but not mitochondrial matrix ROS[J].FASEB J, 2009, 23(3): 783-794.
[7] Maniscalco WM, Watkins RH, Roper JM, et al.Hyperoxic ventilated premature baboons have increased p53, oxidant DNA damage and decreased VEGF expression[J].Pediatr Res, 2005,58(3): 549-556.
[8] Albertine KH, Dahl MJ, Gonzales LW, et al.Chronic lung disease in preterm lambs: effect of daily vitamin A treatment on alveolarization[J].Am J Physiol Lung Cell Mol Physiol, 2010,299(1): L59-L72.
[9] 李文斌, 常立文, 容志惠, 等.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 [J].细胞生物学杂志, 2007, 29(1): 115-121.
[10] McGrath-Morrow SA, Lauer T, Collaco JM, et al.Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status[J].Cytokine, 2014, 65(1): 4-9.
[11] Das KC, Ravi D, Holland W.Increased apoptosis and expression of p21 and p53 in premature infant baboon model of bronchopulmonary dysplasia[J].Antioxid Redox Signal, 2004,6(1): 109-116.
[12] 祝华平, 常立文, 李文斌.CyclinD1 和 p21CIP1在 大鼠肺发育过程中的动态表达 [J].中国当代儿科杂志, 2011, 13(5): 396-400.
基金
国家自然科学基金资助项目（81370099）；湖北省自然科学基金资助项目（2008CDB139、2013CFB087）；华中科技大学自主创新研究基金（国际科技合作专项 2014XJGH008）。
PDF(1419 KB)
