骨髓间充质干细胞缺氧后促红细胞生成素信号通路的改变

目的研究骨髓间充质干细胞缺氧后细胞死亡情况及促红细胞生成素信号通路成分的表达改变。方法从Wistar大鼠股骨提取骨髓分离培养骨髓间充质干细胞并在体外扩增。取第4至6代接近融合的骨髓间充质干细胞置于氧浓度为0．5％的缺氧箱内培养24、48、72、96h后行台盼蓝染色计数阳性细胞并提取蛋白行Western blot检测促红细胞生成素、促红细胞生成素受体、HIF-1α、ERK、磷酸化ERK表达改变，另取0．5％缺氧培养48h的细胞免疫荧光染色观察EPO表达改变，Hoechst 33342染细胞核。结果常氧浓度培养对照组骨髓间充质细胞组台盼蓝染色阳性率为3．5％±0．4％，缺氧培养24、48、72、96h组分别为3．9％±0．2％、5．0％±0．4％、5．9％±0．5％、7．1％±0．5％。Western blot和免疫荧光染色发现EPO表达在缺氧48h后开始明显上调。F20R表达在缺氧后24h即开始显著上调且倍数更高。总ERK在对照组和不同缺氧时间组表达改变不明显，但HIF-1α和磷酸化ERK缺氧24h后即上调，72h达高峰。结论骨髓间充质干细胞对单纯缺氧损害较耐受，缺氧后促红细胞生成素信号通路的主要成分均显著上调，提示促红细胞生成素信号通路在骨髓间充质干细胞耐缺氧和旁分泌保护能力中起着重要作用。

Erythropoietin signal pathway is upregulated in mesenchymal stem cell during hypoxia

The multi-potent differentiation capability of mesenchymal stem cells (MSCs) implies potential to achieve patient-specific regenerative therapy for myocardial infarction. However, it is evident that transplanted stem cells do not survive well in the harsh ischemic microenvironment. Erythropoietin (EPO) has been shown to be a potent inhibitor of neuronal and myocardial apoptosis. Here, we investigate the cell viability and the change of erythropoietin signal pathway of MSCs challenged by hypoxia treatment.Methods MSCs were derived from the bone marrow of 2-week-old Wistar rats and expanded. Hypoxia treatment of cells was performed using a ProOx-C-chamber with the oxygen concentration set to 0.5% for the times required.Results The rate of trypan blue staining was 3.5% ± 0.4% in control group, and 3.9% ± 0.2%, 5.0% ±0.5%, 7.1% ± 0.5% in the groups treated with hypoxia for 24, 48, 72, 96 hours. Western blot analysis suggested the expression of EPO in MSCs was significantly upregulated after 48-hour treatment with hypoxia, which was further confirmed by immunofluorescence staining. There was higher and earlier expression of EPOR. And the expression level of total ERK remained constant during the hypoxic treatment. However, the expression of HIF-1α phosphorylated ERK was significantly upregulated after 24-hour treatment with hypoxia and peaked at 72 hours.Conclusions MSCs is not sensitive to hypoxia insult alone. The components in EPO signal pathway (e. g.EPO, EPOR and P-ERK) is upregulated in MSCs after hypoxic treatment, which suggests that EPO signal pathway plays an important role in the hypoxia-tolerance and paracrine protecting capability of MSCs.