The Effect Of Alveolar Echinococcosis On The Differentiation Of BMSC Into Fibroblasts

Objective:1.To establish in vitro mouse bone marrow mesenchymal stem cells in mice in vitro respectively with Alveolar echinococcosis protoscolices and echinococcus granulosus protoscolices co-culture system,testing different time points between groups BMSCs ? type collagen,collagen type ? expression situation.To investigate whether the two types of echinococcosis former head of larva to fibroblast differentiation of BMSCs have different impact.2.To establish in vitro mouse bone marrow mesenchymal stem cells in mice in vitro respectively with alveolar echinococcosis protoscolices and echinococcus granulosus protoscolices co-culture system,The expression of TGF-bate 1,Smad7 and phosphorylated smad2/3 in BMSCs at different time points were detected.To investigate whether two types of echinococcosis procariae affect mouse bone marrow mesenchymal stem cell fibrosis through TGF-bate /Smad signaling pathway.3.It is expected to elucidate the mechanism by the inability of alveolar echinococcosis to formation of fibrocystic wall in the same way as echinococcus granulosus.It provides a new theoretical basis for the treatment of invasive growth of alveolar echinococcosis.Methods:1.Bone of lower limb of C57BL/6 mice was extracted for 3-5 weeks,and bone marrow mesenchymal stem cells were isolated and cultured by adherent culture.Purified BMSCs by culture passage in vitro,and induce differentiation and identification.2.The procariae of alveolar echinococcosis protoscolices were extracted from gerbils infected with echinococcus alveolar respectively.The protuberous segment of echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus.3.The protoscolices and BMSCs of mice were co-cultured with six-well plates,Grouping according to experimental design: the simple control group(BMSCs cultured alone);alveolar echinococcosis group(alveolar echinococcosis protoscolices and BMSCs co-cultured);echinococcus granulosus group(echinococcus granulosus protoscolices and BMSCs co-cultured).At 1,3,5 and 7 days after culture,cell RNA,protein and supernatant were collected.By real-time fluorescent quantitative PCR detection between groups BMSCs collagen ?,collagen ? mRNA expression level;Western blot test groups of BMSCs collagen ?,collagen ? expression level;ELISA detection of each cell in the supernatant collagen ?,collagen ? concentration.4.Alveolar echinococcosis protoscolices and echinococcus granulosus protoscolices were co-cultured with BMSCs of mice.The simple control group(BMSCs cultured alone);alveolar echinococcosis group(alveolar echinococcosis protoscolices and BMSCs co-cultured);echinococcus granulosus group(echinococcus granulosus protoscolices and BMSCs co-cultured).At 1,3,5 and 7 days after culture,cell RNA,protein and supernatant were collected.By real-time fluorescent quantitative PCR detection between groups BMSCs TGF-beta 1 and Smad7 mRNA expression level;the expression levels of BMSCs Smad7 and phosphorylated smad2/3 protein in each group were detected by Western blot.Results:1.BMSCs were successfully isolated and cultured from mice and identified by induction experiments.2.Alveolar echinococcosis protoscolices and echinococcus granulosus protoscolices were extracted respectively.The activity was found to be good.3.Cultivation of 1,3,5,7 day,real-time fluorescence quantitative PCR detection showed that,The alveolar echinococcosis group collagen ?,collagen ? mRNA relative expression were significantly lower than the echinococcus granulosus group and simple control group;echinococcus granulosus group collagen ?,collagen ? mRNA expression of relative amount were significantly higher than other two groups.Western blot test,the alveolar echinococcosis group collagen ?,collagen ? protein relative expression were significantly lower than the echinococcus granulosus group and simple control group;echinococcus granulosus group collagen ?,collagen ? protein relative expression were significantly higher than other two groups.ELISA method,the alveolar echinococcosis group in the supernatant collagen ?,collagen ? content were significantly lower than the echinococcus granulosus group and simple control group;echinococcus granulosus group in the supernatant collagen ? and collagen ? content were significantly higher than other two groups.The differences among the three groups were statistically significant(P<0.05).4.Cultivation of 1,3,5,7 day,real-time fluorescence quantitative PCR detection showed that,The alveolar echinococcosis group TGF-beta 1 mRNA relative expression were significantly lower than the echinococcus granulosus group and simple control group;the relative expression of Smad7 mRNA was higher than that of other groups.Echinococcus granulosus group TGF-beta 1 mRNA expression of relative amount were significantly higher than other two groups,the relative expression of Smad7 mRNA was lower than other two groups.Western blot test,the alveolar echinococcosis group phosphorylation Smad2/3 protein relative expression were significantly lower than the echinococcus granulosus group and simple control group;the relative expression of Smad7 protein was higher than other twogroups.Echinococcus granulosus group phosphorylation Smad2/3 protein relative expression were significantly higher than other two groups.The relative expression of Smad7 protein was lower than that of the control group.The differences among the three groups were statistically significant(P<0.05).Conclusion:1.Alveolar echinococcosis protoscolices may be through the TGF-beta /smad signaling pathway,Promoted the mesenchymal stem cells secrete Smad7,inhibit secrete the collagen type ?,collagen type ? and TGF-beta 1;inhibit fibrosis of mesenchymal stem cells.It may be related to the inability of alveolar echinococcosis to formation of fibrocystic wall in the same way as echinococcus granulosus.2.Echinococcus granulosus protoscolices may be through the TGF-beta /smad signaling pathway.Promoted the mesenchymal stem cells secrete collagen type ?,collagen type ? and TGF-beta 1;Promoted fibrosis of mesenchymal stem cells.It may be related to the formation of fibrocystic wall in echinococcosis.