摘要
目的研究重组人红细胞生成素(rhEpo)处理骨髓间充质干细胞(MSCs)后对Epo受体(EpoR)的影响。方法含rhEpo0,5及10kU.L-1的培养基培养MSCs24,48及72h后,MTT法检测细胞增殖;6d后,免疫荧光染色检测EpoR阳性的细胞百分率。rhEpo预处理24h,再缺氧处理48h,Hoechst染色,通过形态变化检测细胞凋亡率;rhEpo预处理6d,再缺氧处理48h,Western蛋白印迹法检测EpoR,磷酸化Janus激酶2(p-Jak2),磷酸化转导及转录活化蛋白(p-Stat5)及缺氧诱导因子-1a(Hif-1a)蛋白表达。结果MTT结果显示,rhEpo处理的MSCs增殖能力增强;免疫荧光结果显示,随rhEpo浓度的增加,EpoR阳性细胞数增加;对照组,rhEpo5及10kU.L-1组EpoR阳性细胞百分率分别为(8.3±4.2)%,(24.7±8.1)%和(30.8±10.5)%。rhEpo预处理能增强MSCs抗缺氧凋亡的能力,缺氧处理后,对照组、rhEpo5及10kU.L-1组细胞核固缩率分别为(42.2±8.7)%,(21.9±8.0)%和(20.1±7.9)%。Western蛋白印迹结果显示,rhEpo预处理及预处理后行低氧培养的MSCs,随rhEpo浓度的增加,EpoR及下游抗细胞凋亡蛋白p-Jak2,p-Stat5和Hif-1a蛋白表达增加。结论rhEpo可以促进MSCEpoR的表达,并增加EpoR下游抗细胞凋亡蛋白的表达。
AIM To study effect of recombinant human erythropoietin（rhEpo） on up-regulation of erythropoietin receptor（EpoR） in bone marrow mesenchymal stem cells（MSCs）.METHODS MSCs were treated with culture medium which contained rhEpo 0,5 and 10 kU·L^-1,the proliferation of cells was obtained by MTT method at 24,48 and 72 h.The EpoR positive ratio was figured by fluorescence immunity after rhEpo treatment for 6 d.The mitotic arrest to reflect apoptosis observed by Hoechst staining after 24 h pretreatment with rhEpo and 48 h hypoxia.Expressions of EpoR,phosphorylation Janus kinase 2（p-Jak2）,phosphorylation signal transducers and activators of transcription 5（p-Stat5） and hypoxia-inducible factor 1a（Hif-1a） were analyzed by Western blot after rhEpo preteament for 6 d or rhEpo pretreatment for 6 d followed by hypoxia 48 h.RESULTS The proliferation of MSCs treated with rhEpo was stronger than that of control group.Pretreatment with rhEpo could prevent MSCs from hypoxia apoptosis.The mitotic arrest ratios of 3 groups were（42.23±8.70）%,（21.94±8.03）% and（20.15±7.91）%,respectively.Following the increasing of rhEpo concentration,there were more EpoR positive cells.The EpoR positive ratios of 3 groups were（8.29±4.21）%,（24.74±8.14）% and（30.78±10.53）%,respectively.In the pretreated cells or the pretreated cells with hypoxia,following the increasing concentration of rhEpo,the expression of EpoR and its downstream anti-apoptotic proteins p-Jak2,p-Stat5 and Hif-1a was much more stronger than that of rhEpo free cells.CONCLUSION Treatment with rhEpo can induce up-regulation of the EpoR and down-stream anti-apoptosis gene expression in MSC.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2009年第2期122-127,共6页
Chinese Journal of Pharmacology and Toxicology
关键词
受体
红细胞生成素
骨髓间充质干细胞
蛋白表达
细胞凋亡
receptor,erythropoietin
bone marrow mesenchymal stem cells
protein expression
apoptosis
