左归丸靶向C-X-C趋化因子受体4对人脐带间充质干细胞体外迁移的影响

Effect of Zuogui Pill Targeting CXCR4 on the Migration of Human Umbilical Cord Mesenchymal Stem Cells In Vitro

目的:探讨左归丸通过靶向调控C-X-C趋化因子受体4(CXCR4)表达对人脐带间充质干细胞(hUC-MSCs)体外迁移的影响。方法:采用组织块法分离培养hUC-MSCs并进行流式鉴定。利用CCK-8检测不同浓度左归丸对hUC-MSCs增殖的影响,细胞划痕和Transwell实验分析左归丸对hUC-MSCs体外迁移的影响,实时聚合酶链反应(PCR)、蛋白印迹法和流式细胞术分析hUC-MSCs中CXCR4表达变化。结果:流式细胞术结果显示,hUC-MSCs表面高表达CD73和CD90,极低表达CD34和CD45。CCK-8结果显示,0.1、0.2 mg/mL左归丸均能明显促进hUC-MSCs增殖(P<0.05)。细胞划痕和Transwell实验结果显示,0.1、0.2 mg/mL左归丸能明显促进hUC-MSCs迁移,最佳浓度为0.2 mg/mL,CXCR4拮抗剂普乐沙福则能抑制其促迁移作用(P<0.05)。实时PCR、蛋白印迹法和流式细胞术结果显示,左归丸能上调hUC-MSCs中CXCR4 mRNA和蛋白表达水平,并促进CXCR4蛋白在细胞膜表达(P<0.05)。结论:左归丸通过上调CXCR4表达促进hUC-MSCs的体外迁移。

Objective:To investigate the effect of Zuogui Pill on the migration of human umbilical cord mesenchymal stem cells(hUC-MSCs)in vitro by regulating the expression of C-X-C chemokine receptor 4(CXCR4).Methods:The hUC-MSCs were isolated through tissue block method and identified by flow cytometry.CCK-8 method was used to analyze the proliferation of hUC-MSCs treated with different concentrations of Zuogui Pill.Cell scratch and Transwell assays were performed to detect the effect of Zuogui Pill on the migration of hUC-MSCs.The expression of CXCR4 in hUC-MSCs was detected by real-time PCR,Western blot,and flow cytometry.Results:The flow cytometry showed that hUC-MSCs highly expressed CD73 and CD90,while extremely lowly expressed CD34 and CD45 on their surfaces.CCK-8 assay indicated that 0.1 and 0.2 mg/mL Zuogui Pill significantly promoted the proliferation of hUC-MSCs(P<0.05).By cell scratch and Transwell assays,it was found that 0.1 and 0.2 mg/mL(optimal concentration)Zuogui Pill enhanced the migration of hUC-MSCs,and CXCR4 antagonist AMD3100 attenuated its migration-promoting effect(P<0.05).Real-time PCR,Western blot,and flow cytometry demonstrated that Zuogui Pill up-regulated the CXCR4 mRNA and protein expression of hUC-MSCs,and promoted the expression of CXCR4 on cell surface(P<0.05).Conclusion:Zuogui Pill facilitates the migration of hUC-MSCs in vitro by up-regulating the expression of CXCR4.