CAJ | 학술논문

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、50 IU/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10 IU/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10 IU/ml EPO刺激4 d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-STAT-5信号途径有关.
목적 관찰중조인촉홍세포생성소(rhEPO)대인골수간충질간세포(MSCs)증식적영향,병탐토기가능궤제.방법 추취건강지원자적골수액,채용첩벽배양법획취제3대MSCs(P3-MSCs),통과세포형태특정관찰、세포면역표형검측이급유도분화적방법 감정MSCs.취경과감정적P3-MSCs,분별여불동농도(0.5、1、5、10、50 IU/ml)rhEPO공배양,응용사갑기우담서염(MTT)법검측세포증식;이과량특이성항체봉폐EPO수체(EPOR),재용MTT법관찰P3-MSCs증식정황;채용면역형광세포화학염색법검측P3-MSCs적EPOR표체,용Western인적검측증식신호통로단백표체.결과 첩벽배양획취적P3-MSCs동시고표체CD105화CD90,저표체CD34화CD45,병가피유도분화위지방、성골급연골세포.MTT결과 현시,급여EPO후,MSCs적증식명현증강,이농도위10 IU/ml자적작용최위현저;동시가입항EPOR항체봉폐EPOR후,MSCs적증식솔명현강저(P<0.01).P3-MSCs적EPOR표체양성;용10 IU/ml EPO자격4 d후,EPOR、린산화단백락안산격매2(pJAK2)급린산화신호전도화전록인자-5(pSTAT-5)적표체균명현상조.결론 EPO능촉진체외배양적골수MSCs증식,해효응경EPOR개도,병여JAK2-STAT-5신호도경유관.
Objective To investigate the effect of recombinant human erythropoietin (rhEPO) on proliferation of human bone marrow-derived mesenchymal stem 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 cytochemical 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.