| BM-MSCs can differentiate directly into a kind of adult stem cellsand adult cells or transversally differentiate into a variety of cells. Ananther study found, it may be a source of stem cells in the liver patients with end-stage and is expected to become a new type of seed cells. The in-depth study on the differentiation of mesenchymal stem cells to liver cells, is expected to bring new hope for patients with liver disease.Objective: To establish a method that separated and puffied BM-MSCs and Observe the morphology, growth characteristics and surface antigen of BM-MSCs. To explore the feasibility of combinati- on of hepatocyte growth factor and basic fibroblast growth factor induced BM-MSCs to differentiate into the hepatoeyte-like cells and provide foundation for further basic research and clinical applications.Methods:1 Separation and purification of BM-MSCs: BM-MSCs were extracted in aged 4 weeks, either male or female, under sterile conditions. BM-MSCs were separated and purified by whole bone marrow adherent culture method, inverted phase contrast microscopy of cell growth.2 Identification of BM-MSCs: the P3BM-MSCs were identiedby flow cytometry for cell surface antigen and induced into osteogeniccells.3 BM-MSCs differentiate into hepatocyte-like cells in vitro: (1) experimental groups:①M0 Group: as a negative control, with no any factor;②Ml Group: as the positive control group, with 20ng/ml HGF;③M2 Group: 20ng/ml HGF +5ng/ml bFGF;④M3 Group: 20ng/ml HGF +10ng/ml bFGF;⑤M4 Group: 20ng/ml HGF +20ng/ml bFGF; (2) induction of differentiation: P3 BM-MSCs, until 90% confluence, were digested and resuspended to the concentration of2x104/ml,then inoculated into 6-well culture plate according to the groups above. Induction liquid was changed once every 3 days;(3) Identification of hepatoeyte-like cells: Induction 7d, 14d, 21d sacrificing cells, RNA was extracted and detected of AFPmRNA and ALBmRNA by reverse transcription-polymerase chain reaction and AFP, ALB was detected expression by immunohistochemistry.Results:1 Differences in whole bone marrow adherent cells obtained after vaccination 6d see primary BM-MSCs showed a colony-like growth, at 812d colony touch with each other, and covered the bottom of the flask. radiating or swirling arrange to P2 trypan blue cell viability up to 98%.2 Flow cytometry BM-MSCs: CD29 positive cells ratio was 99.81%, CD34-positive cell ratio was 0.28%, CD45-positive cell ratio was 0.29%, CD90-positive cell ratio was 96.03%. Induced to osteogenic after 3 weeks, BM-MSCs can differentiate into osteoblasts and form a clear round or oval calcified nodules, Von-Kossa staining method to measure mineralization nodules, The group with no induction of cells is not mineralized nodules.3 Characteristic of hepatoeyte cell markers: M1M4 group were induced by the first 7d, 14d, 21d of the cell. RT-PCR: It was demonstrated that AFPmRNA positive expression emerged On day 7,decreased On day 14 and disappeared on day 21. ALBmRNA positive expression emerged on day 14 and arrived at peak on day 21. Immunocytochemistry results: AFP basically in the induction of the first 7d positive staining, in the induction of expression decrea-sed on day 14, and disappeared on day 21. ALB as a mature phenotype of liver cells mark, at the beginning of the first 14d of albumin expression appears, and then continue. M0 no positive staining of the control group. At the same time, M14 group rate of positive staining cells was significantly (P<0.05), where Ml and M2, The difference in M3 and M4 was not statistically significant (P>0.05), no significant difference between rates of positive staining cells, M3, M4 and M2 the difference was statistically significant (P<0.05), M3, M4 positive staining rate in group.Conclusions:1 Adherent whole blood culture method for isolating and culturing bone marrow mesenchymal stem cells with high viability and purity.2 HGF and bFGF ccould be induced and directed differentiation of BM-MSCs to express AFP,ALB of hepatocyte-like cells in vitro, and had ore effectively than using HGF alone.3 HGF and bFGF combined with BM-MSCs induced to different-iate into liver-like cells. In which the appropriate concentration of bFGF 10ng/ml, bFGF concentrations increased to 20ng/ml and would not improve BM-MSCs to differentiate into liver cells, low concentrations of bFGF (5ng/ml) can not play their joint induction of synergy.
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