Hepatic Stellate Cells Induce The Conversion Of Hepatocytes Into Liver Progenitor Cells Through Laminin-?v?6 Integrin

Objective:We investigated the role of hepatic stellate cells?HSCs?in the conversion of hepatocytes into liver progenitor cells in cirrhosis or chronic liver injury at the cellular level,and the mechanism of HSCs participating in the conversion from hepatocytes to liver progenitor cells was explored through RNA sequencing,ELISA,transfection,immunohistochemical staining and other technologies.Methods:Firstly,two cirrhosis mouse models of 6WTAA and 12WCCl₄ were established,and the fibrosis was evaluated by Sirius red and HE staining.Secondly,hepatocytes and HSCs were isolated using a modified version of the two-step collagenase perfusion method,the purity of the cells was demonstrated by IF staining of?SMA and albumin,then hepatocytes were co-cultured with activated hepatic stellate cells with transwell co-culture system,48 hours later,we determined the expression of hepatocytes and liver progenitor cells markers using fluorescence technology.Thirdly,hepatocytes of two groups were subjected to RNA sequencing,gene expression differences between the two groups of hepatocytes were compared at the mRNA level using the Poly A enrichment method,and the number of differentially expressed genes in each signal pathway was statistically analyzed by KEGG enrichment analysis,so as to provide possible signal pathway that may influence the transformation of hepatocytes into liver progenitor cells.In the top-20 signal pathways provided,considering that activated hepatic stellate cells secreting extracellular matrix leads to extracellular aggregation in liver tissue,and combining with latest finding,we selected the ECM-receptor interaction signal pathway for the further mechanism research.Additionally,the concentrate of laminin in the culture medium of activated hepatic stellate cells and the control DMEM group were detected by ELISA,with additional apply of laminin in the culture of hepatocytes,we observed the expression of cell markers compared with control group.Finally,siRNA interference was applied to silence the expression of laminin receptor?integrin?6?to see the expression of cell markers.Results:1.Sirius red and HE staining showed that 6WTAA and 12WCCl₄ successfully induced cirrhosis in mouse models;2.Hepatocytes co-culture with activated hepatic stellate cells group?viewed as HSC+group?had a down-regulation of liver cell markers?HNF4??and an up-regulation of liver progenitor cell markers?CK19,SOX9,OPN?compared to the control group?viewed as HSC-group?,which demonstrated the transformation of partial hepatocytes into liver progenitor cells;3.Heatmap of RNA sequencing showed the differentially expressed mRNA in two groups,and KEGG enrichment analysis provided the top 20 signaling pathways that might play a role in this process;4.An increase of laminin concentration was observed in culture medium of activated HSCs with ELISA compared with control group;5.Hepatocytes cultured with laminin?viewed as laminin group?had a down-regulation of liver cell markers?HNF4??and an up-regulation of liver progenitor cell markers?CK19,SOX9,OPN?compared to the control group,which demonstrated the transformation of partial hepatocytes into liver progenitor cells;6.Immunohistochemical staining showed an up-regulation of integrin?v?6 expression in fibrotic liver compared to control group.Immunofluorescent confocal images showed that LPCs?CK19+cells?also expressed integrin?6 in mice models of liver fibrosis.7.SiRNA-mediated inhibition of integrin?6 expression was demonstrated withfluorescence technology,qRT-PCR and WB.The inhibition group?viewed as siRNA group?had experienced an up-regulation of liver cell markers?HNF4??and a down-regulation of liver progenitor cell markers?CK19,SOX9,OPN?,which indicated that the transformation was inhibited.Conclusion:In liver cirrhosis or chronic liver injury,activated hepatic stellate cells promotes the transformation of liver cells into liver progenitor cells by secreting extracellular matrix laminin and acting on the integrin?v?6 receptor of periportal hepatocytes.