摘要
目的:探讨Smad7基因质粒转染骨髓间充质干细胞(Smad7-EGFP-BMSCs)在体外抑制肝纤维化的机制。方法:分离、纯化大鼠BMSCs并经Smad7基因腺病毒质粒(Ad-Smad7-EGFP)转染建立Smad7-EGFP-BMSCs。实验将大鼠肝星状细胞HSC-T6分为A组、B组、C组和D组,分别与Smad7-EGFP-BMSCs、BMSCs、Smad7质粒及PBS进行共同培养72 h,采用ELISA测定培养液中Smad7和TGF-β1的表达,采用Western印迹法和RT-PCR法测定细胞Smad7、TGF-β1、ColⅠ、α-SMA蛋白和mRNA的表达,采用流式细胞术测定细胞凋亡情况。结果:(1)ELISA结果显示,B组、C组和D组培养液TGF-β1蛋白水平较A组显著降低(P<0.01),而Smad7蛋白水平较A组显著升高(P<0.01);D组TGF-β1蛋白水平较B组和C组显著降低(P<0.01),而Smad7蛋白水平较B组和C组显著升高(P<0.01);(2)Western印迹法和PCR结果显示,B组、C组和D组TGF-β1、ColⅠ和α-SMA蛋白和mRNA表达水平较A组显著降低(P<0.01),而Smad7蛋白和mRNA表达水平较A组显著升高(P<0.01);D组TGF-β1、ColⅠ、α-SMA蛋白和mRNA表达水平较B组和C组显著降低(P<0.01),而Smad7蛋白和mRNA表达水平较B组和C组显著升高(P<0.01);(3)流式细胞仪检测结果显示,B组、C组和D组HSC-T6细胞凋亡率较A组显著升高(P<0.01),而D组细胞凋亡率较B组和C组显著升高(P<0.01)。结论:Smad7基因质粒转染骨髓间充质干细胞可通过作用肝星状细胞TGF-β1信号转导通路以及促进星状细胞凋亡而具有抗肝纤维化的作用。
Objective： To investigate the mechanism of Smad7 gene modified bone marrow mesenchymal stem cells（ Smad7-BMSCs） to prevent hepatic fibrosis in vitro. Methods： Smad7-EGFP-BMSCs were established by isolating and purifying BMSCs of rats,and transfecting Ad-Smad7-EGFP. HSC-T6 were divided into Group A,Group B,Group C and Group D,which were respectively incubated with Smad7-EGFP-BMSCs,BMSCs,Smad7 plasmid and PBS for 72 hours. The level of Smad7 and TGF-β1protein in the culture solution was determined by ELISA. The expression of mRNA and protein of Smad7,TGF-β1,Col Ⅰ and α-SMA in the hepatic stellate cells were respectively determined by Western blot and RT-PCR. Cellular apoptosis was determined by flow cytometry. Results：（ 1） The results of ELISA showed that the level of TGF-β1 protein decreased（ P〈0. 01） but the level of Smad7 protein increased（ P〈0. 01） in Group B,Group C and Group D compared with Group A; the level of TGF-β1 protein decreased（ P〈0. 01） but the level of Smad7 protein increased（ P〈0. 01） in Group D compared with Group B and Group C.（ 2） The results of Western blot and RT-PCR showed that the level of mRNA and protein of Smad7,TGF-β1,Col Ⅰ and α-SMA decreased（ P 〈0. 01） but the level of mRNA and protein of Smad7 protein increased（ P〈 0. 01） in Group B,Group C and Group D compared with Group A; the level of mRNA and protein of Smad7,TGF-β1,Col Ⅰ and α-SMA decreased（ P〈0. 01） but the level of mRNA and protein of Smad7 protein increased（ P〈0. 01） in Group D compared with Group B and Group C.（ 3） The results of flow cytometry showed that the rate of cellular apoptosis decreased（ P〈0. 01）,but the level of Smad7 protein increased（ P〈 0. 01） in Group B,Group C and Group D compared with Group A; the rate of cellular apoptosis decreased（ P 〈0. 01） in Group D compared with Group B and Group C. Conclusion： Smad7-BMSCs can have the effect of anti-hepatic fibrosis by affecting TGF-β1 signal pathway and promoting cellular apoptosis in hepatic stellate cells.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2016年第10期1441-1445,共5页
Chinese Journal of Immunology
基金
深圳市科创委立项课题(JCYJ20140416122812003)
