| Background:End-stage liver disease is the final stage of all kinds of the liver disease, whichmainly include acute liver failure, Chronic liver failure and Congenital metabolic liverdisease. Although liver transplantation is the most effective treatment, the surgical traumaand the lack of organ supplier caused the requirement of replacement treatments.Treatment based on cell transplant is a very hopeful substitution short. Compare with livertransplant, there are several advantages in cell transplant therapy, for instances, the lowerinvasive, lower immunogenicity and repeatability. For treatment of end-stage liver diseaseby cell plant, the key point is the cell type chosen. Although earlier researches provedfeasibility of the method, several defects prevented the clinic application of it. Thesedefects include: lack of the characteristic cell origin, persistent perfusion and cellamplification. That’s why stem cell was chosen as sort of replacement cell. Fetal Liver Stem/progenitor Cells (FLSPCs) has a broad prospect in application in end-stage liverdisease. The research on FLSPCs isolation, identification and differentiation potencyanalysis is the foundation of understanding of the liver development and clinic applicationof FLSPCs.Aims：Isolate FLSPCs from mice C57BL/6J-EGFP and rats F344, optimize the cultureprotocol to get high-purify FLSPCs, investigate the potential of self proliferation and theinduced differential potential to the liver cell and bile duct cell.Methods:1.Harvest embryonic liver from13days embryo from mice C57BL/6J-EGFP or ratsF344. To get the cells, processing the liver by combination digestion of mechanicalenforcement and Collagenase. Collecting the cell and proliferation.2. Identify the expression of stem cell biomarkers by Flow cytometry3. Measure the expression level ofAFP by RT-PCR and Western4. Induced FLSPCs by HGF or TGF-β, expression level of ALB, CK-7,8,9beforeand after induction was measured by RT-PCR and Western blot to identify the differentialpotency of FLSPCs to liver cell or bile duct cell.5. Through tail vein, inject FLSPCs to liver resection wild type C57BL/6J mice,observe the capacity of liver construction.Results:1. FLSPCs were separated by flow cytometry with the expression of stem cellbiomarkers, such as EpCAM、CD133、CD49f.2.AFP expression level was higher in FLSPCs than in mature liver cell，remarkabledifference was observed between them.（t=8.77，5.35，P＜0.05）3. FLSPCs and mature hepatocytes were both cultured in Ultra-low attachmentdishes，the clone formation rate was higher in FLSPCs than in mature liver cell, significantdifference was observed between them.(t=12.71，P<0.01). After induced differentiationby HGF in vitro, the mRNA expression level of ALB and CK-8in FLSPCs peaked at the8th day and the10th day respectively, compared with the2nd day, there was a significant difference between them（t=42.55，25.17，P＜0.05）；the protein expression level ofALBand CK-8in FLSPCs peaked at the the10th day, compared with the2nd day, there was asignificant difference between them（t=175.08，41.69，P＜0.05）；After the induction ofdifferentiation by TGF-β in vitro, the mRNA expression level of CK-7and CK-19inFLSPCs peaked at the12th day, compared with the2nd day, there was a significantdifference between them（t=12.32，9.09，P＜0.05）；the protein expression level of CK-7and CK-19in FLSPCs peaked at the the14th day, compared with the2nd day, there was asignificant difference between them（t=53.89，39.71，P＜0.05）4. After FLSPCs transplant, mature liver cells and bile duct cells with the EGFPexpression can be observed in liver resection wild type C57BL/6J mice in vivo.Conclusion:In the experiments, FLSPCs were harvested successfully from embryonic liver bycombination digestion. The stable expression of stem cell biomarker prove that the cellshave the remarkable features of liver stem cell and possess the dual differential potency.Also, the cells can be easily monitored in vivo. |
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