Study on Human Peripheral Blood Stem Cells Differentiate into Liver Cells
|School||Dalian Medical University|
|Keywords||Peripheral blood monocytes Hepatocyte-like cells Transplantation|
Background : Viral hepatitis, alcoholic liver disease have an increasing incidence accompanied by a series of complications of liver cirrhosis , Not only a serious impact on health, And because of the chronic development of liver cirrhosis,Long-term treatment of patients also brought great economic pressure.Conventional medical treatment can improve symptoms,but the liver cell damage lead to liver dysfunction Can not be reversed .Currently,liver transplantation provides the only effective therapy for patients and solves the fundamental problems.However,this treatment brings a series of problems and constraints.China is a big country which have many people carry hepatitis B,especially lack of donor organs,high costs discourage a lot of people.Therefore, the search for an alternative treatment for imminent.Adult stem cells provide a promising treatment of end-stage liver disease .Recently,stem cell regeneration research has made great progress which provide new insights into therapies of liver diseases.Numerous studies in domestic and foreign show that Adult stem cells have the potential of self-renewal and terminal differentiation to replace peripheral mature cells continuously lost because of normal tissue turnover.Assume that the liver stem cells in the bone marrow was first proposed by Petersen,He proposed that lethally irradiated mice transplanted with bone marrow and found the liver in this mice can differentite intoliver stem cells and mature liver cells.Several studies have shown that BM-derived cells may differentiate into hepatocyte-like cells in vivo and in vitro It is unknown whether the cells staining positive for hepatocyte markers had functional characteristics of hepatocytes and whether cells that differentiate into hepatocytes can also differentiate into mesodermal cells, such as hematopoietic cells Lagasse et al. demonstrated that cKit+Thy1low Sca1+ Lin– cells present in murine BM differentiate into cells with not only hepatocyte phenotype but also hepatocyte function ,this is a historic beginning of the stem cell therapy for liver disease,and gives new hope for the treatment of end-stage liver disease and genetic metabolic liver diseases.Aims1.This study demonstrated that if G-CSF mobilized peripheral blood of patients with liver cirrhosis can increase the proportion of CD34 + .2.Separation and purification of PBMC if can proliferate in vitro and differentiate into liver-like cells by HGF,FGF-4 .After 0day 7 Days and 14days , cell morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including ALB AFP CK18 and phenobarbital-inducible cytochrome P450 activity by Immunofluorescence and PCR. 3.Calculation of in vitro differentiation of peripheral blood mononuclear cells efficiency, if the number of transplanted cells can provide a theoretical basis.Methods1. PBMCs were isolated from decompensated liver cirrhosis that mobilized by the administration of recombinant human granulocyte–colony-stimulating factor (G-CSF) by Blood cell separator and then cell surface phenotyping,CD34+ cells were detached by flow Cytometry ,these cells that experss CD34 CD14 CD45 were futher isolated by Ficoll-Hypaque density gradient centrifugation following the manufacturer’s protocol.2.We use M-CSF,IL-3? -Mercaptoethanol to stimulate Proliferation after six days use hepatocyte growth factor and FGF-4.to accelerate induction. After 0day 7 Days and 14days , cell morphology, which is characteristic of hepatocytes, was observed,We detected the human hepatocyte-markers genes by RT-PCR,such as Cytochrome P450 enzyme activity, We use common markers of hepatocyte. ALB、AFP and CK18 were expressed in the hepatocyte-like cells by Immunofluorescence, and compute differentiation efficiency . 3.A P value of <0.05 was considered as significant in all analyses .statistical analysis was done using the SPSS version 16.0 software.Results 1.After mobilization by G-CSF, CD34 positive cells increased form 0.107% to 0.401% by flow Cytometry,Granulocyte colony-stimulating factor (G-CSF) is routinely used to mobilize BMSC in the peripheral blood prior to allogeneic or autologous transplantation. 2.After the isolation procedure, PBMCs were resuspended in culture medium containing RPMI1640and 10%FBS, After that the floating cells were removed,and For colony formation,we use M-CSF,IL-3? -Mercaptoethanol,after six days, we use HGF and FGF-4 to induce PBMC, after 7days we observe that the Morphology chang form round to Polygon or Irregular shape and so on .14 days compared with 7days have an increase in morphological changes .ALB .AFP and CK18 were expressed in the hepatocyte-like cells by Immunofluorescence and PCR .We use Fluorescence microscope to calculate 5 high power field of cells for 7 days and 14 days induced, the cells expressed ALB, the analysis of P <0.001 was considered as significant in this analyses. Calculate the conversion efficiency obtained ALB, CK18 expression is proportional over time.The mRNA of PBMC was induced after 14 days and the control group liver cell line L02 analysis by PCR, calculate P <0.001 was considered as significant in this analyses.3.The control group were fibroblast-like cells and not experss CK18 but have low level of ALB.Conclusions1.G-CSF mobilized peripheral blood CD34 cell count can increase.2.stem cells in vitro cultured can be transformed into liver cells.3.Differentiation of stem cells into liver cells is proportional to the ratio of time.4.Mononuclear cell transplantation, nearly 18% of it may be transformed into liver cells. .5.Stem cells which are not induced in vitro can not be transformed into liver cells. In this study ,PBMC derived from the cirrhotic patients who mobilize by G-CSF could be not only differentiated into hepatocyte-like cells in vitro,but also could have liver cells functions .Although the differentiation efficiency is still low,lay a theoretical foundation for Clinical therapy.