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Medicine for promoting hematopoietic stem cell expansion, and application thereof

A technology for hematopoietic stem cells and drugs, which is applied in the field of drugs for expanding hematopoietic stem cells, can solve the problems of application limitation and scarcity of the number of hematopoietic stem cells, and achieve the effect of promoting the expansion of hematopoietic stem cells and the proliferation of hematopoietic stem cells.

Pending Publication Date: 2020-03-24
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a small molecular compound that can improve the expansion ability of hematopoietic stem cells, aiming to solve the problem of limited application of hematopoietic stem cells due to the scarcity of the number

Method used

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  • Medicine for promoting hematopoietic stem cell expansion, and application thereof
  • Medicine for promoting hematopoietic stem cell expansion, and application thereof
  • Medicine for promoting hematopoietic stem cell expansion, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: OF-1 promotes the ratio and quantity of LSK cells after expansion.

[0024] a) Kill the mouse (cervical dislocation method), soak it in a beaker with 200mL of 75% alcohol, put it in a 100mm dish, cut the skin along the inner thigh of the mouse, expose the muscle, remove part of the muscle, and put it in the middle of the calf Cut off the hip bone (do not cut the femur), put it in a petri dish with phosphate solution (PBS), carefully remove the lateral femoral muscle, cut off the joints at both ends of the femur, and put it in another petri dish with PBS. Cut a slant at each end of the femur, use a 1mL syringe to draw 1mL PBS to wash the bone marrow (note that the PBS with cells should be sucked lightly), until washed, and centrifuge at 300g for 6min at 4°C. Discard the supernatant, add 1mL erythrocyte lysate to each tube, invert to mix, lyse for 1min, centrifuge at 300g for 6min at 4°C. Discard the supernatant, add 1mL PBS to each tube to resuspend, and cent...

Embodiment 2

[0031] Example 2: OF-1 promotes the expansion of LSK cells by promoting the proliferation of LSK cells This example is based on Example 1, and the identification of the effect of the obtained LSK cells on OF-1 and the proliferation ability of LSK cells

[0032]The prepared EdU solution was added to the cultured LSK cells and incubated for 24 hours. Collect the cells into flow tubes, centrifuge at 1500rpm for 5min, and discard the supernatant. The cells were resuspended in PBS, centrifuged at 1500rpm for 5min, and the supernatant was discarded. After fixing with 4% paraformaldehyde at room temperature for 15-30 min, neutralize with 2 mg / mL glycine for 5 min, and wash once with PBS. Incubate with 0.5% TritonX-100 osmotic agent at room temperature for 10 minutes, and wash once with PBS. Add 200 μL 1x Apollo staining reaction solution (pre-prepared 469 μL deionized water + 25 μL reagent B + 5 μL reagent C + 1.5 μL reagent D + 5 mg reagent E) to resuspend the cells, incubate at r...

Embodiment 3

[0034] Example 3: The promotion of LSK cell expansion by OF-1 is the effect on the differentiation potential of LSK cells

[0035] Sorted the same number of LSK cells and planted them in SFEM medium, added 10 μM OF-1 or DMSO, removed OF-1 after 5 days and replaced with normal LSK expansion medium, collected and counted the cells after 2 days, and analyzed the cell lineage with FACS At the same time, the same number of cells were planted in a low-adhesion 24-well plate containing 700 μL of methylcellulose semi-solid medium (without erythropoietin), and additional EPO was added, mixed and placed at 37 ° C for 5 Cultured in a %CO2 constant temperature incubator for 7 days, observed the clone morphology and counted.

[0036] image 3 Myeloid cells shown in A (Gr1 + ), B cells (B220 + ) and red blood cells (Ter119 + ) was not significantly different from the control group. Such as image 3 BFU-E (Erythroid burst-forming units), CFU-GEMM (Colony-forming unit-granulocyte / erythr...

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Abstract

The invention discloses a medicine OF-1 for promoting hematopoietic stem cell expansion. The medicine OF-1 is a non-BET bromodomain inhibitor. It is proved for the first time that a non-BET bromodomain protein Brpf1 isomer Brpf1a is related to the expansion of hematopoietic stem cells. The OF-1 acts on the Brpf1a to remarkably promote in-vitro expansion of the LIN-Sca-1 + c-Kit + hematopoietic stem / progenitor cells LSKs, and does not affect the differentiation potential of the amplified cells, so that the OF-1 can become a novel medicine for promoting hematopoietic stem cell expansion. The targeted Braf1a is used for promoting hematopoietic stem cell expansion to provide a new idea for researching and developing hematopoietic stem cell expansion-promoting medicines.

Description

Technical field: [0001] The invention relates to a medicine for expanding hematopoietic stem cells, belonging to the technical field of biomedicine. Background technique: [0002] Expansion of hematopoietic stem cells (HSCs) for therapeutic purposes has always been a research hotspot in the field of stem cells. Expansion of hematopoietic stem cells in vitro can solve the shortage of materials for transplantation research and genetic modification research. It is important to maintain the long-term self-renewal and differentiation potential of stem cells while expanding. The most important and the main difficulty. [0003] At present, the methods for expanding HSCs in vitro mainly include cytokine co-culture method, simulated bone marrow microenvironment method, and gene regulation and expression technology. Although the use of these methods to expand HSCs in vitro has achieved certain results, a recent study showed that this combination of multiple growth factors may only re...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/65C12N2501/065
Inventor 赵蔚赵萌何秋萍
Owner SUN YAT SEN UNIV
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