芍药苷对脂多糖干预骨髓间充质干细胞的作用机制  

Mechanism of paeoniflorin on bone marrow mesenchymal stem cells intervened by lipopolysaccharide

作　　者：李敏 于洋 程基焱 Li Min;Yu Yang;Cheng Jiyan(Department of Histology and Embryology,Basic Medical College,Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学基础医学院组织学与胚胎学教研室,四川省泸州市646000

出　　处：《中国组织工程研究》2022年第13期2000-2005,共6页Chinese Journal of Tissue Engineering Research

基　　金：四川省科技厅项目(2014,LY-05),项目负责人:程基焱;四川省教育厅项目(13ZB0258),项目负责人:程基焱。

摘　　要：背景:骨髓间充质干细胞在组织工程、细胞治疗、基因治疗等方面具有强大的应用前景,有效提升骨髓间充质干细胞移植存活率与治疗效果成为研究热点。目的:探讨芍药苷对大鼠骨髓间充质干细胞增殖、迁移能力以及对脂多糖干预下骨髓间充质干细胞中转化生长因子β1、ERK1/2表达的影响。方法:CCK-8实验检测不同浓度芍药苷对骨髓间充质干细胞的增殖作用,划痕实验检测10μmol/L芍药苷对骨髓间充质干细胞迁移能力的影响,实时荧光定量PCR检测10μmol/L芍药苷干预后骨髓间充质干细胞中Ⅰ型胶原、Ⅲ型胶原、Ki67 mRNA表达量。将骨髓间充质干细胞分为对照组、脂多糖组(1 mg/L)、脂多糖+芍药苷组(1 mg/L脂多糖+80μmol/L芍药苷),干预72 h后荧光定量PCR、蛋白免疫印迹检测细胞中转化生长因子β1、ERK1/2(MAPK3/1)基因与蛋白表达水平,免疫荧光法对骨髓间充质干细胞中转化生长因子β1蛋白进行定位与半定量分析。结果与结论:①一定浓度(6.25-12.5μmol/L)芍药苷能促进骨髓间充质干细胞增殖;②与对照组比较,10μmol/L芍药苷组空白划痕面积缩小,差异有显著性意义(P<0.05);③与对照组比较,10μmol/L芍药苷组Ⅰ型胶原、Ⅲ型胶原、Ki67 mRNA相对表达量上调,差异有显著性意义(P<0.01);④脂多糖刺激后骨髓间充质干细胞中转化生长因子β1、MAPK1、MAPK3 mRNA表达量与转化生长因子β1、p-ERK1/2蛋白表达量上调,差异有显著性意义(P<0.01),芍药苷与脂多糖联合干预后,上述基因与蛋白表达量降低,差异有显著性意义(P<0.01);⑤实验结果表明,芍药苷能促进大鼠骨髓间充质干细胞增殖、迁移以及损伤修复相关基因Ⅰ型胶原、Ⅲ型胶原、Ki67表达,下调脂多糖干预的骨髓间充质干细胞中转化生长因子β1、ERK1/2(MAPK3/1)基因与蛋白表达。BACKGROUND:Bone marrow mesenchymal stem cells have strong potential application prospects in tissue engineering,cell therapy and gene therapy.More and more researches have focus on the effective improvement of survival rate and biological effect of bone marrow mesenchymal stem cell transplantation.OBJECTIVE:To investigate the effects of paeoniflorin on bone marrow mesenchymal stem cell proliferation,migration and the transforming growth factor-β1,ERK1/2 genes and proteins expression in bone marrow mesenchymal stem cells intervened by lipopolysaccharide.METHODS:The effect of paeoniflorin at different concentrations on the proliferation of bone marrow mesenchymal stem cells was detected by cell counting kit-8 assay.Scratch test was used to detect the effect of 10μmol/L paeoniflorin on the migration ability of bone marrow mesenchymal stem cells.Real-time fluorescence quantitative PCR was used to detect the expression of collagen I,collagen III and Ki67 mRNA under the intervention of 10μmol/L paeoniflorin.Bone marrow mesenchymal stem cells were divided into control group,lipopolysaccharide group(1 mg/L)and lipopolysaccharide(1 mg/L)+paeoniflorin(80μmol/L)group.After 72 hours of intervention,real-time fluorescence quantitative PCR and western blot assay were used to detect the transforming growth factor-β1 and ERK1/2(MAPK3/1)expression of genes and proteins in cells.Transforming growth factor-β1 protein location and semi-quantitative analysis in bone marrow mesenchymal stem cells were detected by immunofluorescence.RESULTS AND CONCLUSION:(1)Paeoniflorin increased the proliferation rate of bone marrow mesenchymal stem cells within a certain concentration(6.25-12.5μmol/L).(2)The blank scratch area of the 10μmol/L paeoniflorin group was significantly narrowed compared with the control group(P<0.05).(3)Compared with the control group,the expression levels of collagen I,collagen III and Ki67 mRNA were significantly increased in the 10μmol/L paeoniflorin group(P<0.01).(4)Lipopolysaccharide significantly promoted tr

关 键 词：干细胞 骨髓间充质干细胞 芍药苷 脂多糖 转化生长因子Β1 细胞外调节蛋白激酶 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]