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The Role Of TPO Mediated Abnormal BM Niche In The Stemness Maintenance Of Leukemia Cells

Posted on:2017-04-22Degree:MasterType:Thesis
Country:ChinaCandidate:H ChenFull Text:PDF
GTID:2284330488967402Subject:Cell biology
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Objective:Abnormal BM niche participates in the initiation and development of leukemia, leukemia cells also can remodel the BM niche. In our previous research, we found that the TPO expression level in BM plasma of AML patients was higher than that in normal donors, which suggesting that the abnormal increase of TPO protein level may correlate with AML. In order to further demonstrate the role of abnormal BM niche in the sternness maintenance of leukemia cells and in the initiation and development of leukemia, we transfected mesenchymal stem cells with TPO and studied the role of abnormal BM niche in the sternness maintenance of leukemia cells in vitro. Furthermore, we applied the tissue engineer technology to construct ectopic human bone marrow niche in immunodeficiency mouse. This new model was then used to study the role of TPO mediated abnormal BM niche in the initiation and development of leukemia.Methods:The lentivirus vector carrying cDNA of TPO gene and the empty lentivirus vector were constructed and infected into HPL cultured mesenchymal stem cells respectively. Then GFP+ cells were sorted by flow cytometry. Western blot was applied to determine the expression level of TPO, confocal immunofluorescence was used to detect the location of TPO in MSC, TPO protein level in culture supernatant of mesenchymal stem cells was detected by ELISA. TF-1 cells were cocultured with mesenchymal stem cells infected with TPO lentivirus or empty lentivirus for 4 days. GO phase ratios in these two groups of TF-1 cells were then detected by flow cytometry. After these two groups of TF-1 cells were treated with VP16 for 24h or 48h, cell apoptosis was detected by flow cytometry. Cell proliferation capacity was measured by MTT, cell cycle in these two groups of TF-1 cells was analyzed by flow cytometry after TF-1 cells were stained with PI. In the construction of ectopic human bone marrow niche in immunodeficiency mouse, Human Platelet Lysate (HPL) was used as a substitute of fetal bovine serum(FBS) in the culture of MSC. Human Platelet Lysate (HPL) was obtained by repeatedly freezing and thawing of concentrated platelet. Bone marrow derived mesenchymal stem cells were cultured in a-minimal essential medium (a-MEM) containing 10% HPL or 10% FBS. We then compared the morphology, cell phenotype, multilineage differentiation potential in vitro and proliferation capacity between the mesenchymal stem cells cultured with HPL or FBS. To determine the human bone marrow formation capacity of HPL cultured MSC, MSC was seeded on β-TCP scaffolds for 12h, then MSC-coated scaffolds were implanted in a subcutaneous pocket on the dorsum of NOD/SCID mice. After 8-12W, the scaffolds were harvested from the mice, fixed, paraffin-embedded, and stained for HE. TPO transfected MSC were seeded on β-TCP scaffolds, then MSC-coated scaffolds were implanted in a subcutaneous pocket on the dorsum of NOD/SCID mice. After 8-12W, the scaffolds were harvested from the mice, human bone marrow formation capacity was determined by HE staining.Results:pCDH-EF1-TPO-T2A-copGFP lentivirus vector was constructed successfully and mesenchymal stem cells were infected. Mesenchymal stem cells infected with TPO lentivirus have higher TPO express level and secretion level than control cells, which was determined through Western blot and ELISA technology respectively. Most of TPO molecules were distributed in cytoplasm. TF-1 cocultured with mesenchymal stem cells infected with TPO lentivirus have higher GO phase ratio, higher drug resistance capacity, higer G0/G1 phase ratio but lower S phase ratio than mesenchymal stem cells infected with empty lentivirus. MTT analysis showed no obvious difference in cell proliferation capacity of TF-1 cells. In the construction of ectopic human bone marrow niche in immunodeficiency mouse, it showed that whether cultured in the presence of HPL or FBS, the MSC all displayed a spindle-shaped fibroblast-like morphology. Flow Cytometry analysis revealed no obvious differences in cell immunophenotype in these two groups, they all have the ability to differentiate towards osteoblasts, adipocytes, and chondrocytes in vitro. But mesenchymal stem cells cultured with HPL-contained medium showed stronger proliferation capacity and higher activity to differentiate towards osteoblasts. Mesenchymal stem cells cultured with HPL still have in vivo bone-forming capacity, so we successfully constructed ectopic human bone marrow niche in NOD/SCID mice.Conclusion:Overexpression of TPO in Mesenchymal stem cells increases the GO phase ratio of TF-1 leukemia cells and promotes leukemia cells in quiescence, thus endows TF-1 cells stronger drug resistance capacity. HPL cultured MSC have higher proliferation capacity and potential of differentiate towards osteoblasts, it can reconstruct ectopic human bone marrow niche in the NOD/SCID mice.
Keywords/Search Tags:bone marrow microenviroment, bone marrow mesenchymal stem cells, human platelet lysate(HPL), Thrombopoietin(TPO), leukemia cells, quiescence, drug resistance
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