低氧诱导小鼠胎肝间质细胞表达红细胞生成素

Expression of erythropoietin in fetal liver stromal cells of mice induced by hypoxia

目的:观察低氧培养条件下小鼠胎肝间质细胞中红细胞生成素的表达。方法:实验于2004-10/2005-01在暨南大学医学院血液病研究所完成。取孕14.5d小鼠胚胎肝细胞进行贴壁培养及传代,生长至次融合状态的第5代细胞在体积分数为0.95的N2和体积分数为0.05的CO2混合气条件下培养,分为培养0(对照组),2,4,8,16,32h组,在相应时间下应用半定量反转录-聚合酶链反应和Westernblot技术检测其红细胞生成素基因表达。结果:①在常氧压下培养的胎肝间质细胞中,红细胞生成素mRNA及其蛋白水平均较低。②低氧培养则可显著提高胎肝间质细胞红细胞生成素基因的mRNA及其蛋白水平。红细胞生成素mRNA在低氧培养4h时即显著增高,8h时达到高峰水平,聚合酶链反应产物红细胞生成素与β-actin信号比从对照组(4.92±2.31)%增加到(26.69±7.94)%,差异有显著性意义(P<0.01)。红细胞生成素蛋白在低氧培养4h时显著增高,16h时达到高峰水平,Western检测红细胞生成素与β-actin信号从对照组(3.57±2.34)%增加到(19.81±6.56)%,差异有显著性意义(P<0.01)。结论:低氧预处理可诱导胎肝间质细胞表达红细胞生成素,提示低氧预处理小鼠胎肝间质细胞在组织缺血损伤中的潜在治疗作用,如治疗脑缺血和心肌缺血等疾病。

AIM： To observe the expression of erythropoietin （EPO） in fetal liver stromal cells under hypoxia condition in mice. METHODS： The experiment was performed in Institute of Hematology of Medical College of Jinan University from October 2004 to January 2005. Liver cells from 14.5 days old mouse fetuses were cultured, and passaged. Subconfluent cells of the fifth passage were cultured in hypoxia condition （95% N2 ＋ 5% CO2）. It was divided into 0, 2, 4, 8, 16, and 32 hours group （control group）. The expression of erythropoietin was detected by semiquantitative reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. RESULTS： ① EPO mRNA and EPO protein were very low level in normal cultured fetal liver stromal cells, ② Levels of both EPO mRNA and EPO protein increased significantly under hypoxia condition. Expression of EPO mRNA increased at 4 hours of hypoxia, and reached peak at 8 hours.It was significant difference of EPO of PCR and β-actin compared with control group, （26.69±7.94）% vs （4.92±2.31）%,（P 〈 0.01）. EPO protein increased at 4 hours, and reached peak at 16 hours, It was significant difference of EPO of Western and β-actin compared with control group, （ 19.81±6.56）% vs （3.57±2.34）%,（P 〈 0.01 ）. CONCLUSION： The expression of EPO mRNA and EPO increases in foetus liver interstitial cells under hypoxia condition, which indicates that foetus liver interstitial cells may play an important role in treatment of ischemic injury such as cerebral ischemia and myocardial ischemia etc.