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粒细胞集落刺激因子促进骨髓源间充质干细胞活性的机制

陈龙, 程范军, 刘岐焕, 唐俊明, 曾琴兵, 孔霞, 郭凌郧, 王家宁

郧阳医学院附属东风医院血液科，十堰 442006；郧阳医学院临床医学研究所，十堰 442000

摘要

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 (20 μg/mL)对MSCs 活性的影响。随后以50 nmol/L wortmannin (磷酰肌醇-3 激酶阻断剂)、50 μmol/L PD98059 (细胞 外信号调节激酶阻断剂)、30 μmol/L SB203580 (丝裂原活化蛋白激酶阻断剂)、10 μmol/L H89 [蛋白激酶A (protein kinase A, PKA)抑制剂]、20 μmol/L Y27632 (Rho 激酶特异抑制剂)、1 μmol/L rapamycin [雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)特异性阻断剂]、10 mmol/L straurosporine [蛋白激酶C (protein kinase C, PKC)阻断剂]、6 nmol/L G0697 (PKCα 抑制 剂)、50 μmol/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、 Pseudo Z 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。

关键词： 粒细胞集落刺激因子; 间充质干细胞; 蛋白激酶C

分类号：R329

[Mechanism of granulocyte colony-stimulating factor for promoting cell viability of bone marrow mesenchymal stem cells.] [Ariticle in Chinese]

Chen Long, CHENG Fan-Jun, LIU Qi-Huan, TANG Jun-Ming, ZENG Qin-Bing, KONG Xia, GUO Ling-Yun, WANG Jia-Ning

1Department of Hematology, Dongfeng Hospital, Yunyang Medical College, Shiyan 442006, China； Institute of Clinical Medicine, Yunyang Medical College, Shiyan 442000, China

Abstract

The present study was aimed to investigate the mechanism of the granulocyte colony-stimulating factor (G-CSF) on the viability of the bone marrow mesenchymal stem cells (MSCs). MSCs were cultured by classical whole bone marrow adhering method, and the MSCs were analyzed for the cell surface differentiation markers CD34, CD133, CD90 and CD105 by flow cytometry (FCM). The ability of the MSCs to differentiate into osteocytes and adipocytes was tested in osteogenic and adipogenic mediums, separately. The effect of G-CSF (20 μg/mL) on the passage 3 MSCs viability was evaluated by MTT method, and the molecular mechanism of the G-CSF mediated effects was assayed through the pretreatment of the signal pathway inhibitors including 50 nmol/L wortmannin (phosphatidylinoesitol 3 kinase inhibitor), 50 μmol/L PD98059 [extracellular signal-regulated-kinase1/2 (ERK1/2) inhibitor], 30 μmol/L SB203580 (p38 mitogen-activated protein kinase inhibitor), 10 μmol/L H89 (protein kinase A inhibitor), 20 μmol/L Y27632 (Rho kinase inhibitor), 1 μmol/L rapamycin [mammalian target of rapamycin (mTOR) inhibitor], 10 mmol/L straurosporine [protein kinase C (PKC) inhibitor], 6 nmol/L G0697 (PKCα inhibitor) and 50 μmol/L Pseudo Z (PKCζ inhibitor). Cultured passage 3 MSCs expressed CD90 and CD105 strongly, and showed the ability of multi-differentiation into osteocytes and adipocytes. G-CSF promoted the viability of MSCs, and the promotion was completely inhibited by PKC inhibitor straurosporine and partially inhibited by wortmannin, rapamycin, PD98059, SB203580 or G0697. However, its effect was not inhibited by H89, Y27632 and Pseudo Z. It is thus suggested that the promoting effect of G-CSF on MSCs viability was closely related to AKT-mTOR-PKC signal pathway, and PKC maybe the central role in the signal pathway.

Key words: granulocyte colony-stimulating factor; mesenchymal stem cell; protein kinase C

收稿日期：2008-06-04　　录用日期：2009-01-15

通讯作者：程范军　　E-mail: chengfanjun001@sina.com

引用本文：

陈龙, 程范军, 刘岐焕, 唐俊明, 曾琴兵, 孔霞, 郭凌郧, 王家宁. 粒细胞集落刺激因子促进骨髓源间充质干细胞活性的机制[J]. 生理学报 2009; 61 (2): 169-174.

Chen Long, CHENG Fan-Jun, LIU Qi-Huan, TANG Jun-Ming, ZENG Qin-Bing, KONG Xia, GUO Ling-Yun, WANG Jia-Ning. [Mechanism of granulocyte colony-stimulating factor for promoting cell viability of bone marrow mesenchymal stem cells.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (2): 169-174 (in Chinese with English abstract).