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 factor β 1 (TGF-β 1), collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescent quantitative PCR, the protein expressions of TGF-β 1, collagen type Ⅰ, collagen type Ⅲ, Smad7, and phosphorylated Smad2/3 were detected by Western blot, and the contents of collagen type Ⅰ and collagen type Ⅲ in 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-β 1, collagen type Ⅰ, and collagen type Ⅲ in the experimental group were significantly higher than those in the control group; the relative protein expressions of TGF-β 1, collagen type Ⅰ, collagen type Ⅲ, 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 type Ⅰ and collagen type Ⅲ in 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 type Ⅰ, collagen type Ⅲ, and TGF-β 1 by TGF-β 1/Smad signal pathway, which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.
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-β 1、Ⅰ 型胶原、Ⅲ 型胶原基因表达，Western blot 检测两组 BMSCs 中 TGF-β 1、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-β 1、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-β 1、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-β 1/Smad 信号通路，促进 BMSCs 分泌 Ⅰ 型胶原、Ⅲ 型胶原和 TGF-β 1，进而促进 BMSCs 纤维化。.
Keywords: Bone marrow mesenchymal stem cells; collagen type Ⅰ; collagen type Ⅲ; echinococcus granulosus; fibrosis; signaling pathway; transforming growth factor β1.