泡状棘球绦虫原头蚴抑制骨髓间充质干细胞向成纤维细胞的分化

Alveolar echinococcosis protoscolices inhibits the differentiation of bone marrow mesenchymal stem cells into fibroblasts

背景:肝泡状棘球蚴病生物学特征类似于肿瘤样病变,其具有浸润性生长和转移的生物学特性,增加了手术的难度。外囊壁的纤维化可以抑制泡状棘球蚴的生长,使疾病处于静止期。有关间充质干细胞在泡状棘球蚴病外囊壁纤维化过程中的作用尚不明确。目的:探讨泡状棘球绦虫原头蚴对骨髓间充质干细胞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骨髓间充质干细胞;从感染泡状棘球蚴的沙鼠体内提取泡状棘球绦虫原头蚴,从感染细粒棘球蚴病的羊肝中提取细粒棘球绦虫原头蚴。实验分为3组,泡状棘球蚴组:第3代骨髓间充质干细胞和泡状棘球绦虫原头蚴共培养;细粒棘球蚴组:第3代骨髓间充质干细胞和细粒棘球绦虫原头蚴共培养;单纯对照组为第3代骨髓间充质干细胞单独培养。在培养1,3,5,7 d,采用实时荧光定量PCR检测Ⅰ型胶原、Ⅲ型胶原、转化生长因子β1和Smad7基因表达,Western blot检测Ⅰ型胶原、Ⅲ型胶原、Smad7和磷酸化Smad2/3蛋白表达,ELISA检测培养上清中Ⅰ型胶原和Ⅲ型胶原水平。结果与结论:①实时荧光定量PCR检测显示,泡状棘球蚴组骨髓间充质干细胞中Ⅰ型胶原、Ⅲ型胶原、转化生长因子β1 mRNA相对表达量显著低于单纯对照组和细粒棘球蚴组(P<0.05);Smad7 mRNA相对表达量显著高于单纯对照组和细粒棘球蚴组(P<0.05);②Western blot检测显示,泡状棘球蚴组Ⅰ型胶原、Ⅲ型胶原和磷酸化Smad2/3蛋白相对表达量均显著低于单纯对照组和细粒棘球蚴组(P<0.05),Smad7蛋白相对表达量显著高于单纯对照组和细粒棘球蚴组(P<0.05);③ELISA检测显示,泡状棘球蚴组上清中Ⅰ型胶原和Ⅲ型胶原水平均显著低于单纯对照组和细粒棘球蚴组(P<0.05);④结果表明,泡状棘球蚴原头蚴可能通过TGF-β/smad信号通路,诱导骨髓间充质干细胞分泌Smad7,抑制Ⅰ型胶原、Ⅲ型胶原和转化生长因子β1分泌,从而抑制了骨髓间充质干细胞的纤维化。

BACKGROUND:The biological characteristics of hepatic alveolar echinococcosis are similar to cancer lesions.Its biological characteristics of invasive growth and metastasis increase the difficulty of surgery.The fibrosis of the outer capsule wall can inhibit the growth of echinococcus multilocularis and keep the disease in the quiescent stage.The role of mesenchymal stem cells in the fibrosis of the outer capsule wall of echinococcosis remains unclear.OBJECTIVE:To investigate the effect of alveolar echinococcosis protoscolices on the differentiation of mesenchymal stem cells into fibroblasts.METHODS:Bone marrow mesenchymal stem cells were isolated from femur bone marrow of 4-week-old C57BL/6 mice,and cultured by adherent method.Alveolar echinococcosis protoscolices were extracted from gerbils infected with alveolar echinococcus.The experiment was divided into three groups.The alveolar echinococcosis group was co-cultured with the third generation of bone marrow mesenchymal stem cells and the protocephalus of alveolar echinococcosis protoscolices.The Echinococcus granulosus group was co-cultured with the third generation of bone marrow mesenchymal stem cells and the protocephalus of Echinococcus granulosus protoscolices.The simple control group was cultured with the third generation of bone marrow mesenchymal stem cells.At 1,3,5,and 7 days of cultivation,the real-time fluorescent quantitative PCR was used to detect collagen type I,collagen type Ⅲ,transforming growth factor-beta 1 and Smad7 gene expression.Western blot assay was utilized to determine collagen type I,collagen type Ⅲ,Smad7 and phosphorylated Smad2/3 protein expression.ELISA was applied to measure supernatant collagen type I and collagen type Ⅲ contents.RESULTS AND CONCLUSION:(1)Real-time fluorescent quantitative PCR detection displayed that transforming growth factor-beta 1,collagen type Ⅰ,collagen type Ⅲ mRNA relative expression levels were significantly lower in the alveolar echinococcosis group than in the Echinococcus granulosus group and simple control group(P<0.05).Smad7 mRNA relative expression was significantly higher in the alveolar echinococcosis group than in the Echinococcus granulosus group and simple control group(P<0.05).(2)Western blot assay showed that collagen type I,collagen type Ⅲ and phosphorylated Smad2/3 protein relative expression levels were significantly lower in the alveolar echinococcosis group than in the Echinococcus granulosus group and simple control group(P<0.05).Smad7 protein relative expression was significantly higher in the alveolar echinococcosis group than in the Echinococcus granulosus group and simple control group(P<0.05).(3)ELISA exhibited that supernatant collagen type Ⅰ and collagen type Ⅲ contents were significantly lower in the alveolar echinococcosis group than in the Echinococcus granulosus group and simple control group(P<0.05).(4)Alveolar echinococcosis protoscolices may promote bone marrow mesenchymal stem cells to secrete Smad7,inhibit the collagen type Ⅰ,collagen type Ⅲ and transforming growth factor-beta 1 through the transforming growth factor-beta/Smad signaling pathway,thereby inhibiting the fibrosis of bone marrow mesenchymal stem cells.