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Method for preparing megakaryocytic preparation by amplifying macronucleus ancestral cell and mature megacaryocyte and use

A technology of mature megakaryocytes and megakaryotic progenitor cells, applied in biochemical equipment and methods, extracellular fluid diseases, medical preparations containing active ingredients, etc., can solve the problems of poor clinical applicability and high price

Inactive Publication Date: 2007-02-28
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the expansion systems reported so far all use a combination of multiple cytokines, which is not only expensive, but also some of the factors have not yet obtained clinical approval, so the clinical applicability is not strong

Method used

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  • Method for preparing megakaryocytic preparation by amplifying macronucleus ancestral cell and mature megacaryocyte and use
  • Method for preparing megakaryocytic preparation by amplifying macronucleus ancestral cell and mature megacaryocyte and use
  • Method for preparing megakaryocytic preparation by amplifying macronucleus ancestral cell and mature megacaryocyte and use

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: In vitro induction and 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. See Table 1, Table 2 and Figure 1a, Figure 1b, Figure 2a, Figure 2b, Figure 3a, Figure 3b, and Figure 4 for the results of the tota...

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 was separated by magnetic bead affinity column (MACS) separation method + Cells were added to serum-free medium containing TPO+IL-11+Heparin for directional expansion for 7 days, and megakaryocytes were collected to make 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 was us...

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Abstract

The present invention relates to mainly one system of efficient culturing and directive inducing stem cell differentiation for culturing and proliferating megakaryocyte. The system has umbilical cord blood and marrow stem cell as seed cell, and the serum-free culture medium comprising TPO, IL-11 and heparin as the main component for megakaryocyte proliferation. The megakaryocyte preparation prepared based on the present invention contains great amount of macronucleus ancestral cells and mature megakaryocyte as well as CD34+ cells, and has the functions of treating thrombocytopenia and improving the blood-forming function after stem cell transplantation. The present invention provides novel efficient cell treating preparation for various thrombocytopenia and hemocyitopenia.

Description

technical field [0001] The invention relates to a method for preparing a megakaryocyte preparation, especially using stem cells derived from hematopoietic tissue (umbilical cord blood) as seeds, and combining thrombopoietin (TPO), interleukin 11 (IL-11) and heparin as megakaryocyte expansion stimulating factors The expansion system, the method and application of preparing megakaryocyte preparation by expanding megakaryotic progenitor cells and mature megakaryocytes. Background technique [0002] Platelets are blood cells necessary to maintain normal blood clotting and the integrity of blood vessel walls in the body. Abnormal numbers of platelets can lead to blood and thrombotic diseases. The incidence of thrombocytopenia is relatively high, and patients need regular treatment due to repeated bleeding, which seriously endangers their physical and mental health, and brings a heavy economic burden to patients, and even life-threatening bleeding in severe cases. Autologous or a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00C12N5/08A61K45/00A61P7/04A61P7/00
Inventor 韩忠朝冯义
Owner INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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