【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。更多还原

【Abstract】 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.更多还原