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本文旨在探讨粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)促进骨髓间充质干细胞(mesenchymal stem cells,MSCs)活性的机制。采用经典的全骨髓贴壁法培养MSCs,通过成骨、成脂肪等多向诱导分化以及流式细胞仪分析其表面标记(CD133、CD34、CD90、CD105)等鉴定MSCs特征;以第三代MSCs为实验材料,采用MTT法分析G-CSF(20μg/mL)对MSCs活性的影响。随后以50nmol/L wortmannin(磷酰肌醇-3激酶阻断剂)、50μmol/LPD98059(细胞外信号调节激酶阻断剂)、30μmol/LSB203580(丝裂原活化蛋白激酶阻断剂)、10μmol/LH89[蛋白激酶A(protein kinase A,PKA)抑制剂]、20μmol/LY27632(Rho激酶特异抑制剂)、1μmol/Lrapamycin[雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)特异性阻断剂]、10mmol/L straurosporine[蛋白激酶C(protein kinase C,PKC)阻断剂]、6nmol/LG0697(PKCα抑制剂)、50μmol/L Pseudo Z(PKCζ抑制剂)等分别处理MSCs,观察G-CSF影响MSCs活性的信号机制。结果显示,培养的第三代MSCs呈现出CD90、CD105强阳性,具有成骨、成脂肪等多向分化能力;G-CSF促进MSCs活性,H89、Y27632、PseudoZ不能抑制G-CSF对MSCs的激活作用,wortmannin、rapamycin、PD98059、SB203580、G0697部分抑制G-CSF对MSCs的激活作用,straurosporine可完全抑制G-CSF对MSCs的激活作用。以上结果提示,G-CSF激活MSCs效应与AKT-mTOR-PKC途径密切相关,且PKC处于中心环节。
This article aims to investigate the mechanism of granulocyte colony-stimulating factor (G-CSF) promoting mesenchymal stem cells (MSCs) activity. MSCs were cultured by classical whole bone marrow adherent method. The characteristics of MSCs were identified by multi-directional induction of osteogenic and adipogenic differentiation and surface marker (CD133, CD34, CD90, CD105) by flow cytometry. For the experimental material, the effect of G-CSF (20μg / mL) on the activity of MSCs was analyzed by MTT method. Followed by 50 nmol / L wortmannin (phosphoinositide-3 kinase inhibitor), 50 μmol / LPD98059 (extracellular signal-regulated kinase inhibitor), 30 μmol / LSB203580 (mitogen activated protein kinase blocker), 10 μmol / LH89 [inhibitor of protein kinase A (PKA)], 20 μmol / L Y27632 (Rho kinase specific inhibitor), 1 μmol / Lrapamycin [mammalian target of rapamycin (mTOR) specific blocker ], 10mmol / L straurosporine [protein kinase C (PKC) blocker], 6nmol / LG0697 (PKCα inhibitor) and 50μmol / L Pseudo Z (PKCζ inhibitor) Signaling mechanism affecting MSCs activity. The results showed that the third generation of MSCs showed strong positive of CD90 and CD105, and had multi-directional differentiation ability of osteoblast and adipocyte. G-CSF promoted the activity of MSCs. H89, Y27632 and PseudoZ could not inhibit the activation of MSCs by G-CSF Effect of wortmannin, rapamycin, PD98059, SB203580 and G0697 on the activation of MSCs partially inhibited by G-CSF, and straurosporine could completely inhibit the activation of MSCs by G-CSF. These results suggest that the activation of MSCs by G-CSF is closely related to the AKT-mTOR-PKC pathway, and PKC is in the central part.