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基质细胞衍生因子-1α/CXCR4/CXCR7轴对骨髓间充质干细胞迁移的影响
王玉兰 何晓梅* 唐薇 顾怡萍 张世春 汤曼 徐铁军 高殿帅
解剖学报 ›› 2014, Vol. 45 ›› Issue (5) : 639-645.
基质细胞衍生因子-1α/CXCR4/CXCR7轴对骨髓间充质干细胞迁移的影响
Effect of stromal cell derived factor-1α/CXCR4/CXCR7 axis on migration of the bone marrow mesenchymal stem cells
目的 探讨基质细胞衍生因子-1α（SDF-1α）受体CXCR4、CXCR7在骨髓间充质干细胞（BMSCs）中蛋白和mRNA的表达；及SDF-1α/CXCR4/CXCR7轴对BMSCs迁移作用的可能机制。方法 体外培养大鼠BMSCs，流式细胞术鉴定细胞表面抗原CD29、CD44和CD34。应用CXCR4特异性拮抗剂AMD3100及CXCR7中和抗体分别阻断CXCR4及CXCR7，通过 Western blotting和RT-PCR分别检测BMSCs蛋白和mRNA的表达变化；Transwell法检测细胞迁移能力。本次实验分为单纯BMSCs组（A）、AMD3100预处理BMSCs组(B)、CXCR7中和抗体预处理BMSCs组(C)及AMD3100+CXCR7中和抗体预处理BMSCs组(D)。结果 经鉴定第3代大鼠BMSCs中CD29和CD44均呈阳性表达，而CD34表达阴性。BMSCs中CXCR4、CXCR7蛋白和mRNA均有表达。与A组相比，B组及D组CXCR4及CXCR7蛋白表达明显受到抑制（P<0.05），C组只有CXCR7蛋白表达降低（P<0.05）；各组CXCR4 mRNA和CXCR7 mRNA的表达差异均无显著性。SDF-1α可以诱导BMSCs迁移，与0μg/L组相比，10μg/L组和100μg/L组穿膜细胞数均显著增多（P<0.01），与10μg/L组相比，100μg/L组穿膜细胞数亦明显增多（P<0.01）；AMD3100和CXCR7中和抗体均能抑制BMSCs的迁移作用（P<0.05），当两者同时作用时，抑制效应更为显著（P<0.05）。结论 BMSCs共表达CXCR4、CXCR7蛋白及mRNA；BMSCs的迁移具有SDF-1α浓度依赖性; SDF-1α/CXCR4 /CXCR7轴介导BMSCs的迁移作用，CXCR4受体和CXCR7受体对BMSCs的迁移可能具有协同促进作用。
Objective To investigate expression of CXCR4 and CXCR7 protein and mRNA, which are the receptors of stromal cell derived factor-1α(SDF-1α), in the bone marrow mesenchymal stem cells(BMSCs); to explore the role of SDF-1α/CXCR4/CXCR7 axis in migration of BMSCs in vitro and the possible mechanism. Method BMSCs were isolated from rats and cultured in vitro. CD29, CD44 and CD34 of the cells were identified by flow cytometry. CXCR4-selective antagonist AMD3100 and CXCR7-specific neutralizing antibody were applied to block CXCR4 and CXCR7 respectively. The expressions of CXCR4 and CXCR7 mRNA and protein on BMSCs were detected with RT-PCR and Western blotting. Transwells chamber test was used to observe the migration of BMSCs. The BMSCs were divided into the BMSCs group(A), the AMD3100 pretreated BMSCs group(B), the CXCR7-specific neutralizing antibody pretreated BMSCs group(C), the AMD3100+CXCR7-specific neutralizing antibody pretreated BMSCs group(D). Result Flow cytometry showed that the expressions of CD44 and CD29 were positive, while the expression of CD34 was negative in the third passage of BMSCs(P3-BMSCs). CXCR4 and CXCR7 protein and mRNA were both expressed in P3-BMSCs. Compared with the A group, the expression of CXCR4 and CXCR7 protein declined significantly in the B group and the D group; the protein expression of CXCR7 in the C group was lower compared with the A group(P<0.05). However, the expression of CXCR4 mRNA and CXCR7 mRNA had no significant difference between groups. SDF-1α factor promoted migration of BMSCs(P<0.05). Compared with the 0μg/L group, the numbers of migrated cells were increased significantly in both of the 10μg/L group and the 100μg/L group(P<0.01). The number of migration of BMSCs was significantly higher in the 100μg/L group than that of the 10μg/L group(P<0.01). AMD3100 and CXCR7-specific neutralizing antibody both inhibited significantly the migration of BMSCs(P<0.05), and the attenuate effect was more significant when they worked together(P<0.05). Conclusion CXCR4 and CXCR7 receptors are coexpressed in P3-BMSCs; the SDF-1α factor can promote the migration of BMSCs in the concentration dependent manner; SDF-1α/CXCR4/CXCR7 axis is involved in the migration of BMSCs, and both of the CXCR4 and CXCR7 receptors have a synergistic promoting effect to the BMSCs migration.
基质细胞衍生因子-1α / CXCR4 / CXCR7 / 骨髓间充质干细胞 / 免疫印迹法 / 大鼠
Stromal cell derived factor-1α / / CXCR4 / CXCR7 / / Bone marrow mesenchymal stem cell / / Western blotting / / Rat
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