摘要
目的观察重组人促红细胞生成素（rhEPO）对人骨髓间充质干细胞（MSCs）增殖的影响，并探讨其可能机制。方法抽取健康志愿者的骨髓液，采用贴壁培养法获取第3代MSCs（P3-MSCs），通过细胞形态特征观察、细胞免疫表型检测以及诱导分化的方法鉴定MSCs。取经过鉴定的P3-MSCs，分别与不同浓度（0．5、1、5、10、50IU／ml）rhEPO共培养，应用四甲基偶氮唑盐（MTT）法检测细胞增殖；以过量特异性抗体封闭EPO受体（EPOR），再用MTT法观察P3-MSCs增殖情况；采用免疫荧光细胞化学染色法检测P3-MSCs的EPOR表达，用Western印迹检测增殖信号通路蛋白表达。结果贴壁培养获取的P3-MSCs同时高表达CD105和CD90，低表达CD34和CD45，并可被诱导分化为脂肪、成骨及软骨细胞。MTT结果显示，给予EPO后，MSCs的增殖明显增强，以浓度为10IU／ml者的作用最为显著；同时加入抗EPOR抗体封闭EPOR后，MsCs的增殖率明显降低（P〈0．01）。P3-MSCs的EPOR表达阳性；用10IU／ml EPO刺激4d后，EPOR、磷酸化蛋白酪氨酸激酶2（pJAK2）及磷酸化信号转导和转录因子-5（pSTAT-5）的表达均明显上调。结论EPO能促进体外培养的骨髓MSCs增殖，该效应经EPOR介导，并与JAK2-STAT5信号途径有关。
Objective To investigate the effect of recombinant human erythropoietin （rhEPO） on proliferation of human bone marrow-derived mesenchymal stern cells （MSCs） in culture and its probable molecular mechanism. Methods The aspirates of the bone marrow from healthy volunteers were seeded in culture medium. Then MSCs were isolated by its character adhering to the plastic. After three passages in culture, MSCs were identified by morphological features, cell surface antigens and differentiation into multi lineages. Then P3-MSCs were treated with different concentrations of rhEPO （0. 5, 1, 5, 10, or 50 U/ml）. And proliferation of MSCs was measured by MTT assay. Subsequently, EPO receptor （EPOR） was blocked and MTT was performed for another time. What＇s more, immuofluresence cytoehemical staining was used to detect the expression of EPOR. And the proteins of signal pathway were examined by Western blot. Results The P3 bone morrow-derived adherent cells were positive for CD90 and CD105, while negative for CD34 and CD45, and could differentiate into adipocytes, osteocytes and chondrocytes in induction media. MTT assays showed that the absorbance （A value） in EPO-treated groups was significantly higher than in control group, and 10 U/ml EPO-treated group achieved the most predominant effects. However, the A value was reduced dramatically when the specific anti-EPOR antibody （anti EPOR Ab） was added with rhEPO. Compared with the control group, all those differences were statistically significant （P〈0. 01）. In addition, P3-MSCs were positive for EPOR. And the expression of EPOR, phospho Janus kinase2 （pJAK2） and phospho-signal transducer and activator of transcription （pSTAT-5） was significantly higher in 10 U/ml EPO-treated group than in the control group. Conclusion Erythropoietin can promote proliferation of human bone marrow MSCs in vitro, which is mediated by EOPOR, and related with JAK2-STAT-5 signal pathway.
出处
《中华器官移植杂志》
CAS
CSCD
北大核心
2009年第1期21-25,共5页
Chinese Journal of Organ Transplantation
基金
湖北省自然科学基金（2005ABA081）
湖北省教育厅重点项目（D200724004）
湖北省科技攻关项目（2007AA301C141）
湖北省卫生厅科研基金（QJX200518）
关键词
红细胞生成素
重组
骨髓
间质干细胞
细胞增殖
Erythropoietin, recombinant
Bone marrow
Mesenchymal stem cells
Cell proliferation
