骨髓间充质干细胞旁分泌HGF体外调控肝星状细胞

Hepatic stellate cells are regulated by paracrine HGF from bone marrow mesenchymal stem cells in vitro

目的探讨大鼠骨髓间充质干细胞(BMSCs)与肝星状细胞(HSCs)体外共培养体系中,BMSCs旁分泌肝细胞生长因子(HGF)对HSCs增殖、凋亡、活化的影响。方法全骨髓贴壁法分离、培养、纯化大鼠BMSCs,另培养HSCs。6孔板半透膜建立上下两层细胞非直接接触共培养体系,实验设H组(HSCs单独培养)、H-H组(HSCs与HSCs共培养)、M-H组(BMSCs与HSCs共培养)、M-H-C组(BMSCs与HSCs共培养并加c-met抑制剂),各组细胞培养48 h后,流式细胞仪鉴定BMSCs,检测HSCs凋亡率,MTT法检测HSCs的增殖,免疫荧光共聚焦定量检测HSCs中α-肌动蛋白(α-SMA)的表达量,ELISA法检测共培养体系上清液中HGF的浓度。结果 MSCs高表达阳性表面分子CD29、CD90,低表达造血细胞表面标记CD45;BMSCs能明显抑制HSCs的增殖、活化并促进其凋亡,且M-H组上清液中HGF的浓度明显高于其他组。结论 BMSCs与HSCs共培养过程中,BMSCs通过旁分泌HGF促进HSCs的凋亡,抑制HSCs的增殖、活化。

Objective To investigate the effects of paracrine hepatocyte growth factor(HGF)secreted by rat bone marrow mesenchymal stem cells on proliferation,apoptosis and activation of hepatic stellate cells after rat bone marrow mesenchymal stem cells(BMSCs)and hepatic stellate cells(HSCs)were co-cultured in vitro.Methods The whole bone marrow adherent method was used to isolate,culture and purify rat BMSCs,and HSCs were cultured.The semi-permeable membrane of six-well plate was used to establish a two-layer cell co-culture system with indirect contact.The study consisted of group H(HSCs were cultured alone),H-H group(HSCs and HSCs were co-cultured),M-H group(BMSCs and HSCs were co-cultured),M-H-C group(BMSCs and HSCs were co-cultured with c-met inhibitor).Cells in each group were cultured for 48 hours.BMSCs were identified and the apoptosis rate of HSCs was detected by flow cytometry.The proliferation of HSCs was detected by MTT assay,and the expression level of alpha smooth muscle actin(α-SMA)in HSCs was detected quantitatively by immunofluorescence confocal method.The concentration of HGF in the supernate of the coculture system was determined by ELISA.Results MSCs overexpressed positive surface molecules CD29 and CD90,and low expression of hematopoietic cell surface marker CD45.BMSCs could significantly inhibit the proliferation and activation of HSCs,and promote apoptosis of HSCs.The concentration of HGF in the supernate was significantly higher in M-H group than that in the other groups.Conclusion BMSCs can promote the apoptosis of HSCs,inhibit the proliferation and activation of HSCs through paracrine HGF in the co-culture of BMSCs and HSCs.