| Objectives: Liver autotransplantation is the last resort for the treatment of end-stage Alveolar Echinococcosis(AE).However,most patients lose their lives due to insufficient remnant liver for surgery.Stem cell therapy plays a role in repairing and promoting regeneration in end-stage liver disease,however,there are few studies on alveolar echinococcosis.The present study aims to: 1)determine the role of nestin-positive cells in the repair of liver injury following E.multilocularis infection;2)To investigate the mechanism of Wnt/β-catenin/Nestin signaling axis in the repair of liver injury after E.multilocularis infection;3)To evaluate the effect of ADSCs transplantation on liver regeneration induced by E.multilocularis infection through the Wnt/β-catenin/Nestin signaling axis.Methods: Part I: 1)Nestin-Cre/Rosa26-td Tomato normal transgenic C57BL/6 mice were used to analyze the distribution and expression of Nestin in various organs and at different ages.The proportion of Nestin+ cells in the liver of normal mice at different ages was analyzed by flow cytometry;2)Adult Nestin-cre/Rosa26-td Tomato transgenic C57BL/6 mice were infected for 1 month(early stage),3 months(middle stage),and 5 months(late stage).The pathological changes of liver tissue were observed by HE staining and serological detection of ALT and AST.Sirius red staining and α-SMA immunohistochemical staining were used to observe liver fibrosis in the model mice.The relative expression of Nestin m RNA in liver tissue was detected by real-time fluorescence quantitative PCR.Immunofluorescence staining was used to observe the relationship between Nestin expression and ALB.The expression of PCNA in the liver was detected by immunohistochemistry to analyze liver regeneration;3)Nestin+ cells were characterized as stem cells by bioinformatics protein interaction(PPI)analysis and flow cytometry,and their migration ability was evaluated by transwell assay in vitro.Part II: 1)Establishment of primary hepatocytes around the foci infected with hydatid cells,isolation of hepatic stellate cells,culture in vitro and identification;2)separation bubble ball larva infected lesions around the original sequence generation of liver cell protein spectrum,and the differences in protein function annotation and KEGG analysis,screening protein related to liver regeneration;3)C57BL/6 mouse models of January(early),March(middle)and May(late)months were infected with C57BL/6 mice,and the expression levels of Wnt signaling pathway ligand and cascade proteins in liver tissues of mice in each group were detected by q RT-PCR and Western-Blot;4)Lentivirus interferes with Wnt5 a expression in hepatocytes To observe the expression of Wnt/β-catenin/Nestin signal axis in hepatocytes and the effect on hepatocyte proliferation.Part III: 1)ADSCs were extracted from the groin of C57BL/6 mice and injected into Em infected mice through tail vein after culture,identification and DIL labeling.The transplanted ADSCs mice were killed in January and May,respectively.Through gross observation,the size and number of liver lesions in mice were counted,and the repair effect of ADSCs transplantation on liver injury caused by alveolar cyst infection in mice was evaluated;2)The expression of PCNA in liver tissue was detected by immunohistochemistry to observe the effect of ADSCs transplantation on the proliferation of mice infected with hydatid cells,and the expression of α-SMA and Col1 was detected by immunohistochemistry and ceravine scarlet staining to observe the effect of ADSCs transplantation on the progression of fibrosis in mice infected with hydatid cells.The expression changes of Wnt5 a,β-catenin,GSK3β and p-GSK3β were detected by q RT-PCR and Western-Blot method to analyze the regulatory effects of ADSCs transplantation on the Wnt/β-catenin/Nestin signal axis in the liver of mice infected with hydatid cells.Results: Part I: 1)Nestin is expressed in the heart,liver,spleen,lung,kidney and brain of transgenic mouse Nestin with high expression in the heart,kidney and brain tissue,while only a small amount is expressed in the liver.However,the expression of Nestin in all organs gradually declines with age.In particular,the liver showed little expression;2)The lesion area of adult Nestin-cre/Rosa26-td Tomato transgenic C57BL/6 mice was gradually increased after 1 month,3months,and 5 months,and the liver function AST and ALT were increased,but there was no statistical difference.According to the analysis,the area of scarlatina staining,the area of a-SMA positive and the number of PCNA positive cells of liver proliferation index were increased gradually,with statistical difference;3)The expression of Nestin mainly concentrated around the lesion and gradually increased with the aggravation of infection;Colocalization with hepatocyte marker ALB;4)Bioinformatics protein interaction(PPI)analysis showed that Nestin had interaction with the characteristic genes of embryonic stem cells Nanog and Sox2.The positive rate of CD90 on the cell surface of Nestin+ was45%,and that of CD105 was 60%;5)Nestin+ cells have migration ability in vitro.Part II:1)A total of 138 differentially expressed proteins were screened from the primary hepatocytes surrounding the foci of alveolar cyst infection,including 118 up-regulated proteins and 20 down-regulated proteins.GO enrichment analysis and KEGG analysis showed that the differential proteins were mainly concentrated in cytoskeleton regulation,VEGF signaling pathway,MAPK signaling pathway,plasminogen activation cascade,antigen processing and presentation and other related biological activities.Rcsd1 was closely related to Wnt/β-catenin signaling pathway through the analysis of different proteins;2)The expression trend of key proteins of Wnt/β-catenin signaling pathway in mice infected with hydatid cells was consistent with that of Nestin,and increased gradually with the increase of infection,with statistical significance;3)Interference with Wnt5 a expression of hepatocyte line 7702 can significantly inhibit the Wnt/β-catenin signaling pathway,reduce the expression of Nestin,and inhibit the proliferation of hepatocytes.Part III.1)Confocal laser microscopy showed that the Di L-labeled ADSCs could migrate to the liver tissue of EM-infected mice and reside there;2)The total surface area of liver lesions in EM-infected mice decreased significantly at 5 months after ADSCs transplantation,and the difference was statistically significant.The expression of PCNA increased significantly 5 months after ADSCs transplantation,and the difference was statistically significant;3)The area of collagen fibers with scarlet staining decreased significantly at 5 months after ADSCs transplantation,and the difference was statistically significant.Immunohistochemical results showed that the expression of Col1 and α-SMA decreased significantly at 5 months after ADSCs transplantation;4)ADSCs transplantation could activate the Wnt/β-catenin/Nestin signal axis in the liver of mice infected with alveolar cells,and promote the expression of Nestin around the lesion.Conclusions: 1)The high expression of Nestin around liver lesions in mice infected with alveolaris is correlated with hepatocyte proliferation.2)The Wnt/β-catenin/ Nestin signal axis plays an important role in hepatocyte regeneration after hydatid infection;3)adipose mesenchymal stem cell transplantation alleviates liver injury caused by alveolar infection and promotes regeneration through Wnt/β-catenin/Nestin signaling axis. |
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