VEGF通过细胞外信号调节激酶途径促进骨髓源间充质干细胞的增殖

本研究旨在探索血管内皮生长因子(VEGF)对间充质干细胞(MSC)增殖的影响及其可能的机制。采用经典的全骨髓贴壁法培养MSC,通过成骨、成脂肪等多向诱导分化以及流式细胞术分析其表面标记(CD45、CD34、CD90、CD29)等鉴定MSC特征;以培养的第3代MSC(P3MSC)为实验材料,采用MTT法分析20ng/ml的VEGF作用12、36、72小时对MSC增殖的影响;随后以细胞外信号调节激酶(ERK1/2)阻断剂50μmol/L PD98059或丝裂原活化蛋白激酶(p38MAPK)阻断剂30μmol/L SB203580等分别处理P3MSC,观察VEGF是否通过p38MAPK或ERK1/2途径影响MSC的增殖。结果表明:培养的P3MSC表达PDGFR-α、PDGFR-β和NRP1,不表达VEGF-R(Flk1和Flt1)。P3MSC呈现CD90(96.7%)和CD29(94.6%)强阳性以及CD34(0.79%)和CD45(0.84%)阴性特征,具有成骨、成脂肪等多向分化能力;20ng/ml VEGF作用于MSC后,随着时间的延长,MSC增殖也逐渐增强,在72小时达峰值。在50μmol/L PD98059或30μmol/L SB203580处理后,VEGF所介导的MSC增殖效应被阻断,且在对照水平以下。PD98059阻断效应明显强于SB203580的阻断效应。结论:VEGF可能主要通过细胞外信号调节激酶途径调节MSC的增殖。

VEGF Promotes the Proliferation of Bone Marrow Derived Mesenchymal Stem Cells through ERK1/2 Signal Pathway

In order to explore the effect of VEGF on mesenchymal stem cell（MSC） proliferation and its possible signal transduction mechanism,MSC culture was performed with the classical bone marrow adhering method;characteristics of passage 3 rat MSC（P3MSC） was identified through multi-differentiation and surface marker assay（CD34,CD45,CD90,CD29）;P3MSC were treated with 20ng/ml VEGF,and the effect of VEGF on the MSC proliferation was measured during 12,36 and 72 hours by MTT assay.Subsequently,P3MSC were treated with extracellular-signal regulated kinase（ERK1/2） inhibitor PD98059（50 μmol/L） or p38 mitogen-activated protein kinase（p38MAPK）inhibitor SB203580（30 μmol/L） for 30 minutes,the culture medium was replaced with new medium including 20 ng/ml VEGF.After 72 hours,the effect of PD98059 or SB203580 on MSC proliferation mediated by VEGF was measured by MTT assay.The result showed that the cultured MSC expressed PDGFR-α,PDGFR-β and NRP1,but did not express VEGF-R（Flk1 and Flt1）.The MSC had the multi-differentiation ability and displayed the characteristics of CD90＋（96.7%）,CD29＋（94.6%）,CD34-（0.79%） and CD45-（0.84%）.The MSC proliferation rate increased gradually with prolonging of the functioning time of 20 ng/ml VEGF,and MSC proliferation rate may reach to maximum value after treating with 20 ng/ml VEGF for 72 hours.The effect of VEGF on MSC proliferation was found to be abolished,even was under level of control group after treating with PD98059 or SB203580 for 30 minutes.Furthermore,the inhibitory effect of PD98059 on MSC proliferation was obviously higher than that of SB203580.It is concluded that the VEGF can promote MSC proliferation,and its possible mechanism may relate to ERK1/2 pathway.