| BackgroundThe liver regenerate post partial hepatectomy and recovery to its primary size,the cirrhosis of the liver followed the chronic hepatitis B,and liver cancer occured again after hepatectomy for hepatic cancer,these symptoms indicate that liver has huge ability of regeneration,and growth factors and cyteskin start and mediate hepatic regeneration.Adult liver stem ceils which take part in hepatic regeneration is play key role in the physiologic and pathological course for hepatocytes development,reproduction,differentiation and carcinogenenesis.The recent studies suggest that the critical protein of the wnt signal pathway,β-catenin which attends directly the target gene transcription and cell-cell adhesion is power medicated factor for cell reproduction and differentiation,regulates stem cell self-renewal and differentiation,stem cell maintenance and proliferation,and promotes fate determination in stem cells.Wnt signal pathway plays a crucial role in controlling stem cell development including stem cells proliferation,differentiation,self-renewal,and beginning,development and metastasis of hepatocellular carcinoma and colon cancer.Liver stem cells endowed with the bipotential capacity of differentiating into hepatocytes and bile epidermal cells.There are three ways for hepatic stem cells to go:①Stem cell produces new stem cells by self-renewal;②Stem cells differentiate into mature hepatocytes and bile epidermal cells:③Stem cells carcinogenesis due to disorder of differentiating procedure of stem cell.It is the two facts that there is the same local micro-environment for hepatic stem cell and hepatocellular carcinoma cell and hepatic stem cells were discovered in tumor tissue and close to tumor tissue in liver cancer model and clinical specimen from the patients with liver cancer that indicate hepatocellular carcinoma can originate from the hepatic stem cell.The recent studies indicated that wnt signal pathway not only controlled the proliferation and differentiation of stem cells,but regulates cancer stem cells.However, there is little study about if wnt signal pathway plays role in hepatic stem cells,and the more precise method,such as western blotting was not used to examinedβ-catenin expression in cytoplasm and nucleus of hepatocellular carcinoma.There is little study about difference inβ-catenin expression and inducing differentiation between liver stem cells and liver cancer cells Therefore,the present study examined theβ-catenin expression on the hepatic stem cells and carcinoma cells,and expected to find the difference ofβ-catenin distribution at membrane,in cytoplasm and nucleus,and the changes in cellar morphology,growth curve,albumin and AFP,andβ-catenin distribution after induced differentiation,in order to research the role in regulation and controlling proliferation and differentiation of hepatic stem cells and proliferation of liver cancer cells.Objectives1.The present research developed a model to activate oval cell,and utilizing selective enzymatic digestion and percoll density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population.The marker of liver stem cells,c-kit was examined.We hoped to find more economic and practical methods to isolate,culture and identify liver stem cells,and expected to get more hepatic stem cells for the following research.,and study the pattern of morphologic changes in induced differentiated into mature hepatocytes and bile epidermal cells.2.The difference of wnt signal pathway between hepatic stem cells and cancer cells and relation among cell proliferation,differentiation and wnt signal pathway were conducted by comparing the expression and distribution of key molecular,such asβ-catenin on hepatic stem cells with cancer cells.3.The changes in cellar morphology,growth curve,function of secreting albumin and AFP,and the distributive difference ofβ-catenin in the hepatic stem cells and cancer cells based on the same inducing differentiated medium were examined in order to study the role ofβ-catenin signal in cell proliferation and differentiation.4.This study was expected to examined if there is wnt signal pathway in liver oval cells,and it plays key role in regulating its proliferation and differentiation.MethodsWe have established a new model combining N-2-fluorenylacetamide(AAF) treatment with partial hepatectomy(PH) to activated oval cells,then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population.Freshly isolated cells were cultured in DMEM/F12 medium which was supplemented with 10%FCS,SCF 20ng/ml,HGF 10ng/ml,EGF 10ng/ml,LIF 10 ng/ml for cell proliferation.The marker of stem cells, c-kit was examined by immunfluorescence technique and western blotting analysis to identify hepatic oval cells.RT-PCR analysis was used to identify the expression of albumin and CK19 mRNA of new isolated hepatic stem cells.Immunohistrochemistry techniques and western blotting analysis were used to examined theβ-catenin expression and distribution at membrane,in cytoplasm and nucleus of hepatic stem cells and cancer cells so as to find difference between hepatic stem cells and cancer cells.Hepatic stem cells and cancer cells were induced to differentiate by DMEM/F12 medium with SCF 20ng/ml,HGF 10ng/ml,EGF 10ng/ml,1.5%DMSO and HSS 20ng/ml in order to observe the changes in cellar morphology and the distributive difference ofβ-catenin at the hepatic stem cells and cancer cells.Results1.The activity of the freshly isolated oval cells was more than 90%,the morphologic characterization in color and size is not the same,diameter of the oval cells is 1/4～1/6 diameter of hepatocytes.Large and round hepatocytes displayed firstly in course of inducing to differentiate,and the morphological changes between hepatic oval cells and hepatocytes were observed.Immunfluorescence staining of freshly isolated oval cells was positive for c-kit,western blotting analysis detected the expression of c-kit protein.RT-PCR analysis provided evidence that the albumin and CK19 mRNA were expressed on the new isolated cells.The oval cells could be induced to differentiate into hepatocytes and bile epidermal cells in the present of DMSO and several growth factors.On the other hand, hepatic oval cells have markers of stem cells,hepatocytes and bile epidermal cells,may turn out hepatcytes and bile epidermal cells.2.Immunohistrochemical analysis indicatedβ-catenin expression both at membrane and in cytoplasm in WB cells line,L2 hepatocytes,SMCC and HepG2 cells were detected, however it is not easy to identify theβ-catenin accumulation in nucleus.Western blotting analysis detected theβ-catenin expression in cytoplasm of four cell lines and new isolated oval cells,over-expression ofβ-catenin were detected in the cytoplasm of SMCC and HepG2 cells,there was a littleβ-catenin expression in hepatic oval cells.There were significantlyβ-catenin accumulation in nucleus for SMCC and HepG2 cells,however,there was littleβ-catenin distribution in nucleus of WB cells line,L2 hepatocytes,there was notβ-catenin expression in nucleus of new isolated oval cells.These results indicated there were over-expression ofβ-catenin in the cytoplasm and theβ-catenin accumulation in nucleus of liver cancer cells and tissues.Immunohistrochemical staining suggested that there was theβ-catenin expression at cell membrane and in the cytoplasm in all cancer tissues of 20 cases.Contrasted with normal hepatic tissues and hepatocirrhosis tissues,there are three features for theβ-catenin expression in hepatic cancer tissues:①The expression and distribution ofβ-catenin at membrane of normal liver and hepatocirrhosis is continuous,clear and even.The distribution ofβ-catenin at the membrane of hepaticelluar carcinoma was not clear and continuous,or even lost,on another hand,the expression ofβ-catenin at the membrane was reduced.②β-catenin was over-expressed in the cytoplasm of the cancer cells.It is expressed significantly lower in low differentiated liver cancer than the high differentiated liver cancer.It is difficult for immunohistrocheminstry technique to observeβ-catenin localization in the nucleues.③There are significant different ofβ-catenin expression among different degree differentiated liver cancer.The distribution ofβ-catenin at membrane was clear and continuous in high differentiated liver cancer,and it is not clear and continuous for low differentiated liver cancer.Western blotting technique detected the distribution ofβ-catenin both in the cytoplasm and nucleues of hepatic carcinoma tissues, there wasβ-catenin expression in the cytoplasm of normal liver tissues,but not in the nucleues.β-catenin over-expressed both in the cytoplasm and nucleues of all 6 cases hepatic carcinoma(included one low differentiated liver cancer) were observed.Our results indicated thatβ-catenin expression at membrane decreased in liver cancer tissues,β-catenin was over-expressed in the cytoplasm,andβ-catenin was accumulation in nucleues of almost hepatic cancer cells.3.The proliferating rate of WB cells line,SMCC and HepG2 cells line increased one more time after inducing differentiation through observing their growth curve,cells proliferation was the fastest in firstly five days.The albumin amount which WB liver stem cells produced in 3 d,5d,and 7d was significantly more than HepG2 cells(P＜0.01,P＜0.05, P＜0.05),and more than SMCC cells only in 5d(P＜0.05),but it was no statistical difference among three kinds of cells in secreting AFP(P＞0.05).The cellar morphological changes of WB cells line,SMCC and HepG2 cells line after inducing differentiation were observed, the cells became polygon or long shuttle,after all,three cells could not produced mature hepatocytes just like oval cells.Immunohistrochemical staining shown there was little change in distribution ofβ-catenin at membrane and in the cytoplasm for these three cell lines.Mature hepatocytes presented on the 3th day when new isolated oval cells were induced,long shuttle cells-like bile epidermal was observed on the fifth day.As the time goes on,amount of hepatic stem cell decreased gradually,mature hepatocytes increased step by step.Hepatic oval cells proliferated quickly in three days after the freshly isolated oval cells was put in DMEM/F12 medium with lithium chloride,mature hepatocytes did not increased significantly.Western blotting analysis detected that theβ-catenin expression in the cytoplasm and nucleus of WB cells line,SMCC and HepG2 cells line did not changed significantly.There was a littleβ-catenin expression in the cytoplasm for oval cells, and not in its nucleus.Conclusions1.A new model combining N-2-fluorenylacetamide(AAF) treatment partial hepatectomy(PH) to activated oval cells was established,then developed a simple procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population.Diameter of the new isolated oval cells is about 1/4～1/6 of diameter of hepatocytes.Hepatic oval cells not only have the markers of stem cells,such as c-kit,but also have markers of mature hepatocytes and bile cells,on the hand they have bipotential capacity.2.There was obvious differentce of expression and distribution ofβ-catenin between liver stem cells and liver cancer cells.The distribution ofβ-catenin at membrane was clear and continuous.There was a little expression in the cytoplasm,and notβ-catenin accumulation in nucleus.The distribution ofβ-catenin at the membrane of hepaticelluar carcinoma was not clear and continuous,or lost,on another hand,the expression ofβ-catenin at the membrane was reduced,the lower differentiated,the smaller distribution of ]3-catenin at the membrane.β-catenin protein was over-expressed in the cytoplasm of the hepaticelluar carcinoma cells,β-catenin protein was accumulated in nucleus of hepaticelluar carcinoma.The difference ofβ-catenin distribution determined different biological action of hepatocelluar carcinoma,for example,the high metastasis rate of hepatic carcinoma through blood way may be related to decreasingβ-catenin distribution, the high growth rate and not well-differentiated of cancer cells may be related toβ-catenin accumulation in nucleus.3.The hepatic oval cells may be induced into mature hepatocytes and bile dect cells, and the size of stem cells changed from small to big in the course of inducing differentiation.There areβ-catenin expression in oval cells,and lithium chloride may make oval cells grow faster and differentiation suppressed.This result indicate that hepatic oval cells have wnt/β-catenin signal pathway,this signal pathway could controlled stem cell proliferation and differentiation.4.There is an eternal growth capacity for hepatic stem cell line from rat,and several growth factors,such as HGF and SCF in DMEM/F12 medium for inducing differentiation activatedβ-catenin signal in cytoplasm through their receptors and make stem cells grow faster.Our results indicated that there is the similarity ofβ-catenin expression and distribution at membrane and in cytoplasm between hepatic stem cells and hepatic stem cell line,and there is a similarity result between hepatic cancer tissues and hepatic cancer cell line.The proliferation and differentiation of hepatic stem cells and cancer cell are both regulated by wnt/β-catenin signal pathway.
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