Biological Properties Of Hepatic Progenitors In Rat Developing Fetal Liver

Liver diseases, for example, hepatitis and cirrhosis, which finally lead to liver function failure(LFF), have a high incidence rate in China. Now we still face with great challenge to how to treat them. Although liver transplantation is generally accepted as the first selection for the treatment of LFF, it is not extensively applied because of deficiency of donor and immunological rejection after transplantation. Since 1970s later, hepatocyte transplantation has gradually flourished. It has many virtues: 1) One donator can provide hepatocyte for many recipients. 2) Transplantation technique is comparatively simple. 3) There would be little influence on recipients, even if the operation failed. So much more attention was paid on hepatocyte transplantation. Amid a large number of transplanted cell - denominations, liver stem cell, which has multi- differential and highly proliferation ability and plenty source, follows with much more interest. Now, hepatic oval cell, marrow hemopoietic stem cell, pancreatic progenitor and hepatic progenitor of fetal liver have been granted as putative hepatic stem cell. Objective: Rat hepatic progenitor of fetal liver on ED 13.5 day are served asexperimental material to investigate: (1)regularities about isolation and culture of HPFL. (2)multi- differentiation of HPFL in vitro. (3)proliferation of HPFL after transplantation into spleen.(4)effect of cryopreservation on HPFL. All study aim is to lay a foundation for clinic appliance.Methods: 1) HPFL was isolated by enzymatic digestion derived from rat fetal liver on ED13.5d. Furthermore, erythrocyte and other cell were removed from HPFL by erythrocyte-cracking solution and different attachment method. Finally, that effect of different culture mediums and matrix on the proliferation and differentiation of HPFL has investigated by MTT assay. 2) Adopted micromanipulation cell cloning and immunohistochemistry staining, ALB CK-19 clones of HPFL were choosed and seeded in inducing medium to show whether the ALB + CK-19+ cells appear. If ALB+ CK-19- cells would be induced into ALB + CK-19+ cells, we can prove that HPFL contain bipotential stem cells.3) Primary HPFL seeded on PREF feeder layer were collected and engrafted into the spleen of SCED mice to explore the propagation of HPFL in vivo. 4) After primary HPFL derived from rat fetal liver on ED 13.5d have been cryopreserved for 3 weeks and 6 weeks, they were recovered to observe the functional change of HPFL.Result:1) DMEM:F12 would enhance the proliferation of HPFL, especially present with a concentration of 20ng/ml EOF; PREF feeder layer can defer the differentiation of HPFL in vitro, but collagen-VI and gelatin can not do. 2) HPFL was able to induce into hepatic-like and biliary-like cells in vitro. 3) After HPFL are transplanted into the spleen of SCED mouse, it was found that hepatic cord-like constitution appeared and showed G-6-P positive. 4) Cryopreservation had a little influence on HPFL'function, and the influence aggravated ascryopreservation time went on. When HPFL were recovered, their activity and attachment efficiency declined, and the excretion peak value of ALB descended. Conclusion: HPFL shows the characterization of stem cell: excellent capability of proliferation and bipotential differentiation. Although cryopreservation hurts it a little, it is possible that HPFL could be deposited over a long period of time in "Liver Stem Cell Bank". As a potential candidate, getting an advantage over adult hepatocyte, HPFL provides a novel choice in theory for treatment of liver disease.