摘要
目的:利用Boyden chamber体外迁移体系初步探索基质细胞衍生因子-1(SDF-1)在骨髓源间充质干细胞(MSC)迁移中的作用及其信号转导机制.方法: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仪分析其表面标记(CD133,CD34,CD90,CD105)等鉴定MSC特征;以P3-MSC为实验材料,利用Boyden chamber体外迁移体系,观察不同浓度的hSDF-1对MSC迁移的影响,以50nmol渥曼青霉素,10mmol LY294002,50mmol PD98059,10mmol U73122,0.1g/L AMD3100等不同处理P3-MSC,观察最适宜浓度的hSDF-1对MSC迁移影响的信号转导机制.结果:培养的MSC呈现出CD90,CD105强阳性,具有成骨、成脂肪等多向分化能力;MSC体外迁移能力随着hSDF-1α浓度(1,10,100,500μg/L)的递增而逐渐增强,并且hSDF-1α浓度在100μg/L时,MSC迁移到滤膜上的细胞数接近于峰值;渥曼青霉素,LY294002,PD98059,U70312,AMD3100,维拉帕米对MSC迁移均有影响,其中U73122,AMD3100对MSC迁移阻断的效应最显著.结论:SDF-1/CXCR4所介导的MSC迁移与丝裂原活化蛋白激酶(MAPK),磷脂酰肌醇特异性磷脂酶C和蛋白激酶C(PKC)等信号途径有关,且PKC途径可能处于中心环节.
AIM: To explore the role of stromal cell-derived factor-1 (SDF-1) in the migration of mesenchymal stem cell (MSC) and its signal transduction mechanism. METHODS: MSC culture was performed with the classical whole bone marrow adhering method; characteristics of MSC were identified through the induction of multiple-differentiation into osteoblasts or lipoblasts, and surface marker assay ( CD133, CD34, CD105) ; by using Boyden chamber in vitro migration assay system, the effects of SDF-1 concentrations on P3 MSC migration were observed and the signal transduction pathways related to P3-MSC migration were analyzed. RESULTS: Cultured MSC had the potential of menbranedifferentiation and highly expressed CD105 and CD90; the efficiency of MSC migration into the filter membrane increased gradually with the increase of hSDF-lconcentration (1, 10, 100, 500 μg/L), and MSC number in the filter membrane was approximately maximum when hSDF-1 was 100 μg/L. 50 nmol wortmannin, 10 mmol LY294002, 50 mmol PD98059, 10 mmol U73122 and 0. 1 g/L AMD3100 were all able to decrease the number of MSC in the filter membrane, and what's more, U73122 and AMD3100 treatments were most effective in blocking the MSC migration. CONCLUSION: SDF-1/CXCR4-mediated MSC migration is related to mitogen activated protein kinase ( MAPK), phosphatidylinositol phospholipase C ( PI-PLC ) and protein kinase (PKC) signal pathways, but PKC signal pathway may be the central role in MSC migration.
出处
《第四军医大学学报》
北大核心
2007年第19期1753-1756,共4页
Journal of the Fourth Military Medical University
基金
湖北省自然科学基金(2005ABA079)
湖北省卫生厅资助项目(JX3B29)