Lumican Promotes Proliferation Of Hepatic Progenitor Cells Through Interaction With Integrin ?2

Cell fate is determined by cell microenvironment,coordinate each other cell fate decided the life activity of tissues and organs.Adding phases of extracellular matrix protein in vitro,simulating the growth conditions of cells in vivo,is expected to promote cell proliferation and function.Lumican is a member of the small leucine-rich proteoglycan(SLRP)family.AS an important component of the extracellular matrix(ECM),it can regulate cell activities.In part one,the fetal liver cells isolated from 12 to 14days mouse embryos were preliminary screening of 2D liver stem/progenitor cells by Kubota's Medium(KM)and 3D organoid culture in hyaluronan hydrogel.In this part,stem-cell derived,functional mouse hepatic organoids in hyaluronan hydrogels was successfully established.We analyzed lumican protein expression level at two stages of liver development,found lumican expression level was higher in fetal liver,demonstrated that lumican,as an important component of extracellular environmftent,played a significant role in hepatic progenitor cells proliferation and function maintain.In the first part,the gene expression level of Lumican in fetal liver tissue was higher than in adult one through Q-PCR.By tissue immunofluorescence staining,we found that Lumican was widely observed in various subtypes of liver including liver parenchyma cells,endothelial cells and liver stellate cells and the mainly expressed and secreted one is liver parenchyma cells.PCNA is a cell proliferation mark,expressed in proliferative cells and it's higher in fetal liver than adult one.,Compared with adult one,fetal liver tissue expressed higher Lumican,at the same time,the Lumican~+cells raise a higher percentage in PCNA~+cells.It suggests that Lumican~+hepatocytes holds stronger proliferative activity.In second and third parts,we discussed the promoting proliferation effect of Lumican in liver stem/progenitor cell and HepaRG and the influence to function.The fetal liver cells isolated from 12 to 14 days mouse embryos were preli-minary screening of 2D liver stem/progenitor colonies by KM.Adding Lumican to intervenes the growth of liver stem/progenitor colonies,we found the positive percentage of CK19~+Ki67~±cells is significantly higher in experimental group than in control group.At the meantime,the expression level of stem genes such as AFP,CK19 and EpCAM were enhanced and the mature and function genes such as ALB,CPS1,and CYP3A41a were weakened with the concentration of Lumican increasing,which was consistent with the cell immunofluorescence results.It showed that Lumican could promote the proliferation of liver stem/progenitor cell and enhance the expression level of progenitor genes in liver.Culturing 2D HepaRG cells with Lumican and examining the percentage of S phase cells by EdU staining,we discovered that as the concentration raised,the proliferation of cells was synchronous growth with concentration dependent effect and the proliferation cell proportion among groups has statistical signifi-cance(P<0.05).Meanwhile,the Ed U staining results was consistent with the cell proliferation results in single day through CCK8.What's more,the expre-ssion level of proliferation genes and liver stem/progenitor genes were boosted,and the functional genes were contrastly decreased which were agreed with the result of cell immunofluorescence.Adding the Lumican to HepaRG 3D culture system,the proliferation genes and liver stem/progenitor genes expression lever of treatmenat group were facilitated and the mature genes were fallen off com-pare to control group,further more,the activity of CYP3A4 and the synthesis function of ALB in treatment group were significantly lower than control.Therefore,Lumican could block the differention to mature hepatocyte of Hepa-RG cells,maintain the cell stemness and promote the cell proliferation.The inte-grin pathway takes an important part in Lumican action progress.The function of small molecule in cell needs the interaction with ligand to activate the intracellular signaling pathway.The Co-IP results indicated that the ITG?2 interacted with Lumican and it was the ligand of the Lumican molecule.Inhibiting the crucial proteins in cell proliferation signaling pathway,the cell proliferation signaling pathway of Lumican in HepaRG cells was selected asERK and mTOR signaling pathway.Through western blotting,we detected the existence of key protein in the tow pathway downstream,like P21 and CCND1in ERK pathway and P70 S6 kinase?in mTOR pathway,what were Lumican-concentration-dependent raising.The protein p ERK,which is the vital protein in ERK pathway,was transiently appeared at 60 minute after treated by Lumican.Lumican stimulates the cell proliferation by activating ERK and mTOR signal-ing pathways through the interaction with ITG?2.We futher discussed the effec-tion to HepaRG and liver regeneration in vivo with the absence of Lumican.After silent the Lumican by siRNA,the proliferation rate and expression level of Lumican in HepaRG were reduced.The expression level of Lumican in the BALB/C mice liver was silent by injecting siRNA of Lumican through hy-drodynamic method.After 24 hours,2/3 volume of the liver was cut and the rest liver tissue was collected after 24 hours and 48 hours.Both PCNA and Lumican were in low expression in normal mice liver,however,they were universally expressed when cut after 24 hours and 48 hours.Moreover,the si RNA knocked one had the similar expression of PCNA and Lumican before the liver cut,ne-vertheless,they were much less after cut 24 hours and 48 hours.The regenera-tion of liver can be delayed with the absence of Lumican.In conclusion,Lumican activates the ERK and mTOR signaling pathways through the interaction with ITG?2 to promote cell proliferation,maintain cell stemness,block cell mature,and the lack of Lumican could retard or restrain the regeneration progress of liver tissue.