Method for preparing megakaryocytic preparation by amplifying macronucleus ancestral cell and mature megacaryocyte and use
A technology for mature megakaryocytes and megakaryotic progenitor cells, applied in biochemical equipment and methods, extracellular fluid diseases, medical preparations containing active ingredients, etc., can solve problems such as high price and poor clinical applicability
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Embodiment 1
[0041] Example 1: In vitro induced expansion of megakaryocytes
[0042] Freshly isolated cord blood CD34+ cells (1×10 5 / ml) inoculated in the serum-free medium containing thrombopoietin (TPO) of the present invention+recombinant human interleukin-11 (rhIL-11)+heparin (Heparin) in the final volume, wherein containing TPO: 50ng / ml ml, IL-11: 50ng / ml, Heparin: 25IU / ml, at 37°C, 5% CO 2 Cultured in an incubator in a humid environment, half of the medium was changed every 3.5 days, and various analyzes were performed on the 7th, 10th, and 14th day. At the same time, two serum-free culture expansion methods of TPO alone (50ng / ml) and TPO (50ng / ml)+IL-11 (50ng / ml) combination were used as controls to evaluate the efficacy of TPO+IL-11+heparin combination. The effect of serum culture expansion system on the preparation of megakaryocyte products. During the expansion process, the results of the total number of cells, progenitor cells at different stages, and the amplification facto...
Embodiment 2
[0055] Example 2. Application of megakaryocyte products expanded in vitro from umbilical cord blood CD34 positive cells
[0056] 1) In this example, umbilical cord blood from normal delivery was taken, and CD34+ cells were separated by magnetic bead affinity column (MACS) separation method, added to serum-free medium containing TPO+IL-11+Heparin for directional amplification for 7 days, and megakaryocytes were collected , made into 1×10 7 / 100ul spare.
[0057] 2) Non-obese diabetic / severe combined immunodeficiency mice (NOD / SCID) were randomly divided into two groups after Cs source irradiation (275cGy), one group was tail vein injection of PBS group, and the other group was tail vein injection of amplification The above megakaryocyte groups were all injected within 4 hours after irradiation, and transplanted 1×10 7 / 100ul of megakaryocytes, on the 7th day, 14th day, and 21st day after transplantation, the peripheral blood of the mice was measured, and the flow cytometry wa...
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