基于IGF-1/SDF-1/CXCR4轴研究黑地黄丸含药血清对骨髓间充质干细胞增殖迁移的影响

Study on Effect of Heidihuang Pill Containing Serum on Proliferation and Migration of Bone Marrow Mesenchymal Stem Cells Based on IGF-1/SDF-1/CXCR4 Axis

目的探索黑地黄丸对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、迁移的影响及机制。方法全骨髓细胞贴壁法分离BMSCs并鉴定。MTT检测黑地黄丸对BMSCs增殖的影响并确定最佳干预浓度。将BMSCs分为对照组、黑地黄丸含药血清组、黑地黄丸含药血清+胰岛素样生长因子1受体(insulin-like growth factor 1 receptor,IGF-1R)抑制剂组、黄芪甲苷含药血清组,采用细胞划痕实验检测各组BMSCs的划痕愈合率,采用Transwell实验检测BMSCs的迁移数量。ELISA法检测培养上清中胰岛素样生长因子(insulin-like growth factor,IGF)-1的含量,Western blot检测BMSCs中IGF-1、趋化因子受体(C-X-C chemokine receptor type,CXCR)4、基质细胞衍生因子(stromal cell-derived factor,SDF)-1的表达。结果成功提取大鼠BMSCs,1%~10%的黑地黄丸含药血清可促进BMSCs增殖,且5%黑地黄丸含药血清促增殖作用最佳。与对照组比较,5%黑地黄丸含药血清促进BMSCs的迁移(P<0.05),并可显著增加BMSCs培养上清中IGF-1的水平(P<0.05),上调BMSCs内IGF-1、CXCR4的表达(P<0.05),下调SDF-1的表达(P<0.05)。与5%黑地黄丸含药血清组比较,IGF-1R抑制剂可抑制BMSCs的迁移(P<0.05),显著下调CXCR4的表达(P<0.05),上调SDF-1的表达(P<0.05)。结论黑地黄丸可促进BMSCs增殖,并可通过上调BMSCs内IGF-1的表达,促进IGF-1与IGF-1R的结合,增加表面趋化因子CXCR4的表达,进而调控SDF-1/CXCR4轴,促进BMSCs迁移。

Objective To explore the effect and mechanism of Heidihuang pill,on the proliferation and migration of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated by whole bone marrow cell adherence method,and then identified.The effect of Heidihuang pill,on the proliferation of BMSCs was detected by MTT and the optimal intervention concentration was determined.BMSCs were divided into the control group,Heidihuang pill-containing serum group,Heidihuang pill-containing serum+insulin-like growth factor 1 receptor(IGF-1R)inhibitor group,and astragalosideⅣ-containing serum group.The wound healing rate of BMSCs in each group was detected by cell scratch assay,the migration number of BMSCs was detected by Transwell assay,and insulin-like growth factor(IGF)-1 in the culture supernatant was detected by enzyme-linked immunosorbent assay.Western blot was used to detect the expressions of IGF-1,C-X-C chemokine receptor type(CXCR)4,and stromal cell-derived factor(SDF)-1 in BMSCs.Results BMSCs were successfully extracted from rats.1%-10%Heidihuang pill-containing serum could promote the proliferation of BMSCs,and 5%Heidihuang pill-containing serum had the best effect on promoting proliferation.Compared with the control group,5%Heidihuang pill-containing serum promoted the migration of BMSCs(P<0.01),increased the level of IGF-1 in the culture supernatant of BMSCs(P<0.01),and up-regulated the expressions of IGF-1 and CXCR4 in BMSCs(P<0.05),and down-regulated the expression of SDF-1(P<0.05).Compared with the 5%Heidihuang pill-containing serum group,IGF-1R inhibitor could inhibit the migration of BMSCs(P<0.01),significantly down-regulate the expression of CXCR4(P<0.05),and up-regulate the expression of SDF-1(P<0.05).Conclusion Heidihuang pill can promote the proliferation of BMSCs.And it can promote the migration of BMSCs by up-regulating the expression of IGF-1 in BMSCs,promoting the combination of IGF-1 and IGF-1R,increasing the expression of surface chemokine CXCR4,and regulating the SDF-1/CXCR4 axis.