川芎嗪上调MMP-2、MMP-9表达促进骨髓间充质干细胞迁移

Ligustrazine Promoted the Migration of Bone Marrow Mesenchymal Stem Cells by Up-regulating MMP-2 and MMP-9 Expressions

目的体外研究川芎嗪(Ligustrazine)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)迁移及基质金属蛋白酶-2、9(matrix metalloproteinase-2,9,MMP-2、9)表达的影响。方法采用全骨髓贴壁法培养BMSCs,采用流式细胞仪检测BMSCs阳性抗原(CD29和CD90)及阴性抗原(CD34和CD45),鉴定BMSCs纯度;将BMSCs分成对照组,川芎嗪25、50、100μmol/L组和GM6001组(100μmol/L川芎嗪加MMPs抑制剂GM6001);川芎嗪处理24 h,采用Transwell细胞迁移实验和细胞划痕实验检测细胞迁移速度;Western blot检测BMSCs的MMP-2、MMP-9蛋白表达。结果第3代BMSCs生长良好、形态均一,BMSCs阳性抗原CD29、CD90表达率分别为96.9%、97.3%,阴性抗原CD34、CD45表达率分别为0.2%、3.0%。与对照组比较,川芎嗪各剂量组迁移的细胞数量、细胞侵袭相对距离增加,BMSCs的MMP-2和MMP-9蛋白表达升高(P<0.05,P<0.01)。与川芎嗪100μmol/L组比较,川芎嗪25、50μmol/L组和GM6001组迁移的细胞数量、细胞侵袭相对距离减少(P<0.01);川芎嗪25、50μmol/L组MMP-2蛋白表达降低(P<0.01)。结论川芎嗪能在体外促进BMSCs的迁移,作用机制与其上调MMP-2、MMP-9蛋白表达有关。

Objective To explore the effect of ligustrazine on the migration of bone marrow mesen-chymal stem cells（BMSCs） and protein expressions of matrix metalloproteinase-2 and-9（M M P-2 and MMP-9） in vitro.Methods BMSCs were in vitro isolated and cultured using whole bone marrow adherent method,and phenotypes[surface positive antigens（CD2 9 and CD9 0） and negative antigens（CD34 and CD45）]identified using flow cytometry.BMSCs were divided into the blank control group,25,50,1 00 μmol/L ligustrazine group,and the GM6001 group（1 00 μmol/L ligustrazine ＋ MMPs inhibitor GM600 1）.The migration of BMSCs was tested by Transwell chamber test and wound healing assay after treated with ligustrazine for 24 h.The protein expressions ofMMP-2 and MMP-9 were detected by Western blot.Results The third passage BMSCs grew well in uniform morphology.The expression rate of CD29,CD90,CD34,and CD45 was 96.9%,97.3%,0.2%,and 3.0%,respectively.Compared with the blank control group,the number of migrated cells and relative distance of cell invasion increased,and the protein expressions of MMP-2 and MMP-9 were elevated in each ligustrazine group（P0.05,P 〈0.0 1）.Compared with 100 μmol/L ligustrazine group,the number of migrated cells and relative distance of cell invasion decreased in 2 5and 50 μmol/L ligustrazine groups and the GM600 1 group（P 〈0.0 1）.Protein expression of MMP-2 decreased in 25 and 50 μmol/L ligustrazine groups（P 〈0.01）.Conclusion Ligustrazine could promote the migration of BMSCs in vitro,and its mechanism might be related to up-regulating ex-pression levels of MMP-2 and MMP-9 protein.