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Application of histone methyltransferase inhibitor to preparation of product for promoting megakaryocyte proliferation or blood platelet formation

A methyltransferase and megakaryocyte technology, applied in the field of preparation of products that promote megakaryocyte proliferation or platelet production, histone methyltransferase inhibitors, can solve problems such as low platelet levels, and achieve low application cost and platelet production Efficient, maneuverable and reproducible results

Active Publication Date: 2019-12-20
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One aspect of the present invention is aimed at the lack of compounds and methods that simultaneously promote cell proliferation, megakaryotic differentiation and platelet production in the prior art, and the low level of the compounds in the prior art to promote platelet production provides a histone methyl Use of transferase inhibitors in the preparation of products that promote megakaryocyte proliferation or platelet production

Method used

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  • Application of histone methyltransferase inhibitor to preparation of product for promoting megakaryocyte proliferation or blood platelet formation
  • Application of histone methyltransferase inhibitor to preparation of product for promoting megakaryocyte proliferation or blood platelet formation
  • Application of histone methyltransferase inhibitor to preparation of product for promoting megakaryocyte proliferation or blood platelet formation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1) Cord blood CD34 + Cell Separation

[0062] a. Pour the cord blood into a plasma bottle or a 50ml centrifuge tube, follow V 脐带血 :V 分选buffer =1:1 Add sorting buffer (recipe as follows), add 1 / 4 of the above mixing volume of hydroxyethyl starch, mix well, and let stand at room temperature for 45 minutes;

[0063] Sorting buffer formula: phosphate buffered saline (PBS), 0.5% bovine serum albumin (BSA), 0.4% 500mM ethylenediaminetetraacetic acid (EDTA), 1% penicillin-streptomycin mixture (P / S)

[0064] b. Transfer the supernatant to a 50ml centrifuge tube, centrifuge at room temperature and 1500rpm for 10min; discard the supernatant, add sorting buffer to resuspend the cells; take a 15ml centrifuge tube, add 4.5ml Ficoll separation solution to each tube (V Ficoll :V 分选buffer =1:1), slowly add the above-mentioned resuspension to the Ficoll separation liquid, to avoid destroying the stratification of the liquid surface, 1800rpm, 15min (the centrifuge is set to 0 liters ...

experiment example 1

[0075] Experimental Example 1: Detection of Cell Proliferation

[0076] For the cell counts at each stage in Example 1, different control groups were set up simultaneously.

[0077] a. The control group (DMSO) and the experimental group (EHMTi) were cultured under the same conditions, and the cells were counted every three days to detect the effect of small molecule compounds on cell proliferation.

[0078] b. The result is as follows figure 1 As shown, compared with the DMSO control group, the addition of histone methyltransferase inhibitor (A366) on the 0-3 or 3-6 days of culture can significantly promote cell proliferation, and the cell expansion fold is increased by 15 times. above.

[0079] figure 1 On the 9th to 18th day of the mesomegakaryotic differentiation, the total number of cells in the control group (DMSO group) showed a downward trend; after treatment with histone methyltransferase inhibitors, the total number of cells still showed an upward trend on the 9th ...

experiment example 2

[0080] Experimental Example 2: Megakaryocyte Flow Cytometry Detection

[0081] The megakaryocytes in Example 1 were detected.

[0082] a. Collect about 1×10 cells on the 3rd, 6th, 9th, and 12th day of megakaryotic induction 5 The cell suspension was centrifuged at 300 g for 5 min, and the cell pellet was resuspended with 100 μL of 0.2% BSA.

[0083] b. Add 1 μL anti-CD41a-APC (BD) and 1 μL anti-CD42b-PE (BD) flow antibody to each group, incubate in the dark for 30 minutes, and detect CD41a by flow cytometry (FACS Canto II; BD Biosciences) + CD42b + The proportion of megakaryocytes.

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Abstract

The invention discloses an application of histone methyltransferase inhibitor to preparation of a product for promoting megakaryocyte proliferation or blood platelet formation. The histone methyltransferase inhibitor is a histone methyltransferase G9a inhibitor. The histone methyltransferase G9a inhibitor stated in the invention can be used for promoting single CD34+hematopoietic stem and progenitor cells to differentiate and form 400 CD41a+CD42b+platelet granules, and the platelet granules are increased by 10 times than the platelet granules of a control group; the thrombopoiesis efficiency is further higher than the general range in literature report obviously, and a foundation is laid for mass production of functional platelets for clinic treatment in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of histone methyltransferase inhibitors in the preparation of products for promoting megakaryocyte proliferation or platelet production. Background technique [0002] Platelets are one of the formed components of blood, and play an important role in the process of hemostasis and thrombus in the body through functions such as adhesion, aggregation, and particle release. Clinically, various etiologies such as malignant tumors of the hematopoietic system, aplastic anemia, malignant tumors, hematopoietic stem cell transplantation therapy, and radiotherapy and chemotherapy may lead to thrombocytopenia or dysfunction, resulting in varying degrees of bleeding in the body, and even endangering the body in severe cases. life. Donor-derived platelet concentrate transfusion is currently the only clinical treatment. According to statistics, the number of patients who need platelet tran...

Claims

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Application Information

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IPC IPC(8): C12N5/0786C12N5/078A61K35/19A61K35/28A61P7/06A61P7/04A61P1/16
CPCA61K35/19A61K35/28A61P1/16A61P7/04A61P7/06C12N5/0644C12N5/0645C12N2500/90C12N2501/125C12N2501/145C12N2501/2303C12N2501/72C12N2506/11
Inventor 周家喜刘懿莹刘翠翠苏培王洪涛
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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