| Backgrounds and aimsBasic treatment and prescribe medicine according to the disease are often used to treat liver diseases of terminal stage. Though orthotopic liver transplantation is an effective method of treating acute and chronic liver failure, due to shortage of organs, high cost and immunologic rejection, restrict wider application of orthotopic liver transplantation. Bioartificial liver has been used in clinic and play a role in improving syndromes of terminal liver disorders and prolonging survival time of patients with liver failure, as the same with hepatocytes transplantation, the most important obstacle is the shortage of hepatocytes which are the key part of these two methods. It is urgent to find a high effective method to treat liver diseases or solve the problem of hepatocytes shortage. In recent years, the research of stem cells give the possibility of using stem cells transplantation and /or stem cells derived hepatocytes to treat liver diseases.The research of bone marrow stem cells is now given more regarding duing all the studies of stem cells. There are two types of stem cells in bone marrow, one is hematopoietic stem cells (HSCs) , the other is mesenchymal stem cells (MSCs) . MSCs derive from mesoderm, has strong ability of self-reproduction, can produce filial generation of kinds of phenotype. They can differentiate into some cell types including osteoblasts, chondroblasts, adipocytes, myoblasts and neurocytes under certain conditions. MSCs have two advantages, first, it is convenient to get MSCs and the process do little harm to body, second, self stemcells derived organs are not restricted by MHC, therefore, researchs of MSCs are increasingly becoming a hot topic. The aim of this study is to culture human MSCs (hMSCs) and one of their subpopulation-HLA-DR-C-Kit - cells in vitro, observe the effects of HGF and/or FGF4 on the differentiation of hMSCs and HLA-DR-C-Kit- cells, look for the best conditions that influence and adjust the hepatocyte-like differentiation progress of these two kinds of cells, discuss the possibility of obtaining hepatocyte-like cells which have specific phenotype and function in vitro by inducing differentiation. If this differentiation process success, we will have another source of modern cell enginerring and give hopes to the treatment of terminal liver diseases in clinic.Materials and methodsBone marrow cells were collected from healthy volunteers aged from 2 to 35 years old. This study was divided into two groups: group A, hMSCs; group B, HLA-DR-C-Kit-cells. The cells in group A were separated by gradient centrifugation (1.077g/ml) , the purity of hMSCs is tested by flow cytometry at passage 3. Cells in group B were separated by magnetic cell sorting method from group A which reached logarithmic stage. Cells in the two groups were precultured in DMEM-LG including 10% FBS,100 U/ml penicillin and 100 U/ml streptomycin. When these cells reached logarithmic stage and began to induce, inoculated them into 24-well plates with glass coverslips, the glass coverslips were treated with 1ug/ml fibronectin beforehand, the medium is also changed by DF medium supplemented with 5% FBS, 1 xinsulin/transferring/selenium, 4.7ug/ml linoleic acid, 1mg/ml BSA, 10-8M/L DEX, 10-4M/L ascorbic acid, 100 U/ml penicillin, 100 U/ml streptomycin and 10ng/ml EGF. After plating on 24-well plates, cells in the two groups were divided into four groups respectively according to the growth factor added in the medium: HGF, FGF4, HGF+FGF4, no growth factor. Each group include 12 wells.Cells in each group were plated at a density of 2 X 105/cm2. Cells in each group were collected on day 0, 7, 14, 21, 28 after inducing and freshly separated after gradient centrifugation. Use immunocytochemistry to examine cell phenotype after inducing, markers including CK18, a-fetoprotein and albumin, Periodic acid Schiff (PAS) method for glycogen stain were used for functional test after inducing. AFP C-met and FGFR2 mRNAwere examined by RT-PCR in freshly separated cells after gradient centrif...
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