Method for inducing megakaryoblast and megakaryocyte in vitro

A technology for megakaryocyte progenitor cells and megakaryocyte cells is applied in the field of inducing and differentiating mononuclear cells into megakaryocyte progenitor cells and megakaryocyte cells in vitro, and can solve the problems of cumbersome methods and low expression levels.

CN101864396BActive Publication Date: 2012-08-22FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2012-08-22

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Abstract

The invention discloses a method for inducing megakaryoblast and megakaryocyte in vitro and a special culture medium thereof. The special culture medium is a serum free medium containing 50ng / mL of SCF, 50ng / mL of TPO, 20ng / mL of IL-3 and 50ng / mL of IL-6 StemSpan. The method comprises the following steps of: 1) separating single karyocyte from umbilical cord blood by using the conventional Ficoll density gradient centrifugation method, wherein red blood cells in the umbilical cord blood are settled by 6 percent hydroxyethyl starch; and 2) culturing the single karyocyte in vitro for 4 to 14 days by using the special culture medium at 37 DEG C in the presence of 5 percent CO2 to obtain a mixture of the megakaryoblast and the megakaryocyte. The invention provides a source for supplementing the megakaryoblast and the megakaryocyte, and has the advantages of simple operation, low cost, high differentiation efficiency and the like. The method is about to play an important role in the field of medicaments, and has wide application prospect.
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Description

technical field

[0001] The invention relates to an in vitro induction method of mononuclear cells in the field of biotechnology, in particular to a method for inducing differentiation of mononuclear cells into megakaryotic progenitor cells and megakaryocytes in vitro. Background technique

[0002] Tumor is a new organism formed by the body under the action of various carcinogenic factors, a certain cell in the local tissue loses its normal regulation of its growth at the gene level, resulting in abnormal cell clonal proliferation. Tumors can be divided into benign and malignant. Malignant tumors are one of the major diseases that seriously endanger human health. The conventional treatment is still surgical treatment combined with radiotherapy and chemotherapy. After patients receive high-dose radiotherapy and chemotherapy, the hematopoietic function of the bone marrow is low, and thrombocytopenia often occurs, and in severe cases, the patient will die of hemorrhage. Platel...

Examples

Embodiment 1

[0022] Example 1: Determination of Factor Combinations in Medium

[0023] Experiment 1. Using domestic serum-free medium containing different cytokine combinations to induce cord blood mononuclear cells and observe the changes in the number of cells expressing CD41 / CD61

[0024] The domestic serum-free medium used in this experiment was BTN serum-free medium purchased from Beijing Botner Technology Co., Ltd.

[0025] Mononuclear cells (obtained from umbilical cord blood, see Example 2 for the process of isolating mononuclear cells) were seeded in 24-well plates, 1 ml per well, 10 6 cells / ml. The culture medium are:

[0026]Medium A: domestic serum-free medium, which contains 116t factor combination: 10ng / mL IL-11, 10ng / mL IL-6, 100ng / mL TPO.

[0027] Medium B: domestic serum-free medium, which contains st36 factor combination: 50ng / mL SCF, 50ng / mLTPO, 20ng / mL IL-3, 50ng / mLIL-6.

[0028] Medium C: domestic serum-free medium, which contains pt36 factor combination: 50ng / mL P...

Embodiment 2

[0039] Embodiment 2, in vitro induction of umbilical cord blood mononuclear cells to differentiate into megakaryotic progenitor cells and megakaryocytes and detection

[0040] Experiment 3. Comparison of the sedimentation effect of different concentrations of hydroxyethyl starch (HES) in sedimentation of cord blood red blood cells

[0041] Mix 6% hydroxyethyl starch and cord blood in a certain proportion, so that the final concentration of hydroxyethyl starch is 2.0%, 1.5%, 1.2%, 1.0% ("%" means "g / 100ml"), and settle at room temperature Red blood cells for 30 minutes to compare the sedimentation effect. Then, carefully aspirate the supernatant, centrifuge at 1800rpm for 5min, suspend the cells in saline, and slowly add the cell suspension to the surface of an equal volume of human lymphocyte separation medium (FICOLL, Institute of Bioengineering, Chinese Academy of Medical Sciences) at 22°C , Centrifuge at 2000rpm for 25min, collect the mononuclear cell layer, wash with norm...