The Role Of Related Signal Pathways To HGF/c-Met In Proliferation Of Hepatic Progenitor Cells

Part I Isolation and identification of hepatic progenitor cells fromnormal rat liverObjective: To explore the techniques of isolation and identification of hepaticprogenitor cells from normal rat liver.Methods: According to expression of surface marker CD90.1, progenitor cells wereisolated by immunomagnetic cell sorting from liver of normal Wistar rats. The markers ofisolated cells were observed by immunohistochemistry (IHC) and polymerase chainreaction (PCR).Results: The percentage of CD90.1positive cells in non-parenchymal cells of normalrat liver was0.32%.98.2%of isolated cells by immunomagnetic cell sorting were CD90.1positive cells in flow cytometric cell sorting. Flesh isolated CD90.1positive cells werepositive for OV-6and strong positive for CK-19and negative for HNF-4a in IHC andPCR.Conclusion: Cells with hepatic progenitor cells exist among liver of normal rats. PartⅡIn vitro cultivation and differentiation of hepatic progenitor cellsfrom normal rat liverObjective: To explore the techniques of culturing hepatic progenitor cells from normalrat liver and inducing them into hepatoeyte-like cells in vitro.Methods: CD90.1positive cells isolated from normal rat liver were cultured in vitrowith different concentration of HGF. Proliferation of hepatic progenitor cells wasevaluated by methyl thiazol tetrazolium (MTT) colorimetry. Differentiation of hepaticprogenitor cells was analyzed using IHC ang realtime RT-PCR detection.Results: At the stage of primary culture, hepatic progenitor cells cultivated with50 ng/ml HGF exhibited good proliferative ability. Cell colony formed at3week. At the stageof subculture, hepatic progenitor cells cultivated with30ng/ml HGF showed good status ofproliferation and differentiation. The cell became the same size as hepatocyte at8week.IHC results showed that fresh isolated cells were negative for HNF-4a, ALB and positivefor CK-19, AFP, while cells at8week became negative for CK-19, AFP and positive forHNF-4a, ALB. Realtime RT-PCR detection showed that fresh isolated cells had highexpression of CK-19mRNA, AFP mRNA and almost no expression of HNF-4a mRNA,ALB mRNA, while cells at8week held high expression of HNF-4a mRNA, ALB mRNAand nearly no expression of CK-19mRNA, AFP mRNA.Conclusion: Hepatic progenitor cells from normal rat liver can proliferate by in vitroculture and be induced into hepatoeyte-like cells successfully. HGF plays an important rolein this process. Part ⅢThe role of related signal pathways to HGF/c-Met in proliferationof hepatic progenitor cellsObjective: To investigate the role of PI3K/AKT, STAT3and ERK1signaltransduction pathway relating to HGF/c-Met in proliferation of hepatic progenitor cells.Methods: when primary cultured in vitro of hepatic progenitor cells was conducted,observation group was added Su11274, an inhibitor of c-Met phosphorylation. MTTcolorimetry was used to test the ability of cell proliferation. Expression of PI3K/AKT,STAT3and ERK1were detected by realtime RT-PCR and western blot.Results: When concentration of Su11274was2μM, proliferation of hepaticprogenitor cells was obviously inhibited. Expression of c-Met, PI3K, AKT, STAT3andERK1mRNA were detected and similar between observation group and control group.Expression level of c-Met protein in control group increased at6d and were1.250.29,1.560.22,1.590.30,1.610.27,1.600.28at6d,8d,10d,12d,14d. Expressionlevel of p-c-Met protein in control group were0.820.25,1.160.27,1.150.24,1.150.29,1.160.22at6d,8d,10d,12d,14d. There was no difference in expression level ofc-Met protein between two groups (P=0.71). Expression level of p-c-Met protein inobservation group were0.450.12,0.360.10,0.220.08,0.130.03at8d,10d,12d, 14d and lower than control group (P=0.026). Expression level of PI3K and AKT proteinwere similar between two groups. But expression level of p-PI3K protein in observationgroup were1.360.14,1.280.15,1.140.14at10d,12d,14d and reduced obviouslycompared to control group (P=0.033). Expression level of p-AKT protein in observationgroup were0.360.11、0.310.09、0.220.11at10d,12d,14d and attenuated sharply(P=0.014).There was no difference in expression level of STAT3protein between twogroups (P=0.73). Expression level of p-STAT3protein at6d,8d,10d,12d,14d were0.220.11,0.180.06,0.130.05,0.130.07,0.130.05in control grop and0.220.08,0.170.05,0.150.05,0.110.07,0.060.02in observation group(P<0.001).Expression level of ERK1protein in control group augmented at6d and were0.670.12,0.730.16,0.760.15,0.780.12,0.820.13at6d,8d,10d,12d,14d.Change of ERK1protein in observation group was similar to it in control group (P=0.57).Expression level of p-ERK1protein increased from6d in both groups and no differencewas observed between two groups (P=0.81).Conclusion: PI3K/AKT and STAT3signal transduction pathway might be relativedownstream pathways of HGF/c-Met in proliferation of hepatic progenitor cells.