细粒棘球绦虫原头节促进BMSCs纤维化的实验研究

Study on effect of echinococcus granulosus protoscolices on fibrosis of bone marrow mesenchymal stem cells

目的探讨细粒棘球绦虫原头节对BMSCs向成纤维细胞分化的影响。方法取4周龄C57BL/6小鼠股骨骨髓,利用贴壁培养法分离培养BMSCs;从感染细粒棘球蚴的羊肝中提取细粒棘球绦虫原头节。实验分为2组,实验组取第3代BMSCs和细粒棘球绦虫原头节共培养,对照组为单纯第3代BMSCs。共培养前后倒置显微镜观察BMSCs形态;培养1、3、5、7 d,采用实时荧光定量PCR检测两组BMSCs中TGF-β1、Ⅰ型胶原、Ⅲ型胶原基因表达,Western blot检测两组BMSCs中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、Smad7和磷酸化Smad2/3蛋白表达,ELISA检测两组上清中Ⅰ型胶原和Ⅲ型胶原蛋白含量。结果细粒棘球绦虫原头节与BMSCs共培养7 d后观察到BMSCs形态发生改变,变成梭形和不规则三角形,更接近单独培养的小鼠成纤维细胞。培养1、3、5、7 d,实时荧光定量PCR检测示,实验组TGF-β1、Ⅰ型胶原、Ⅲ型胶原mRNA相对表达量均显著高于对照组;Western blot检测示,实验组TGF-β1、Ⅰ型胶原、Ⅲ型胶原、磷酸化Smad2/3蛋白相对表达量均显著高于对照组,Smad7蛋白相对表达量显著低于对照组;ELISA检测示,实验组上清中Ⅰ型胶原和Ⅲ型胶原蛋白含量均显著高于对照组。上述指标两组间比较差异均有统计学意义(P<0.05)。结论细粒棘球绦虫原头节可能通过TGF-β1/Smad信号通路,促进BMSCs分泌Ⅰ型胶原、Ⅲ型胶原和TGF-β1,进而促进BMSCs纤维化。

Objective To investigate the effect of echinococcus granulosus protoscolices on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into fibroblasts.Methods Femur bone marrow of 4-week-old C57BL/6 mice was taken and BMSCs were isolated and cultured by adherent culture.Echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus.The experiment was divided into two groups.The experimental group was co-cultured with the 3rd generation BMSCs and the echinococcus granulosus protoscolices,and the control group was the 3rd generation BMSCs.Before and after co-culture,the morphology of BMSCs and the activity of echinococcus granulosus protoscolices were observed by inverted microscope.After cultured for 1,3,5,and 7 days,the mRNA expressions of transforming growth factorβ1(TGF-β1),collagen typeⅠ,and collagen typeⅢwere detected by real-time fluorescent quantitative PCR,the protein expressions of TGF-β1,collagen typeⅠ,collagen typeⅢ,Smad7,and phosphorylated Smad2/3 were detected by Western blot,and the contents of collagen typeⅠand collagen typeⅢin the supernatant of the two groups were detected by ELISA.Results After 7 days of co-culture,the morphology of BMSCs changed into fusiform and irregular triangle,which was closer to the mouse fibroblasts.The relative mRNA expressions of TGF-β1,collagen typeⅠ,and collagen typeⅢin the experimental group were significantly higher than those in the control group;the relative protein expressions of TGF-β1,collagen typeⅠ,collagen typeⅢ,and phosphorylated Smad2/3 in the experimental group were significantly higher than those in the control group,and the relative protein expression of Smad7 in the experimental group was significantly lower than that in the control group;the contents of collagen typeⅠand collagen typeⅢin the supernatant of the experimental group were significantly higher than those in the control group.The differences between the two groups were significant(P<0.05).Conclusion Echinococcus granulosus protoscolices may promote the secretion of collagen typeⅠ,collagen typeⅢ,and TGF-β1 by TGF-β1/Smad signal pathway,which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.