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Bone marrow mesenchymal stem cell exosome preparation and application thereof in promoting hematopoietic injury recovery

A technology for bone marrow mesenchymal and hematopoietic injury, applied in the field of cells, can solve the problems of affecting curative effect, low overall content of mesenchymal stem cells, short survival time, etc., achieve stable and long-lasting biological functions, promote the recovery of hematopoietic function, avoid The effect of related side effects

Active Publication Date: 2021-07-30
因诺伟(北京)生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high cost and long time of cell maintenance and expansion culture, the overall content of infused mesenchymal stem cells in the body is very low, and the survival time is very short, which restricts the curative effect of mesenchymal stem cells
It is also feasible to use soluble cytokines secreted by mesenchymal stem cells to achieve their biological effects, but after entering the human body, the cytokines will be diluted by the extracellular medium, affecting their curative effect, and it is difficult to separate and store cytokines , high cost, is also an important aspect restricting its use

Method used

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  • Bone marrow mesenchymal stem cell exosome preparation and application thereof in promoting hematopoietic injury recovery
  • Bone marrow mesenchymal stem cell exosome preparation and application thereof in promoting hematopoietic injury recovery
  • Bone marrow mesenchymal stem cell exosome preparation and application thereof in promoting hematopoietic injury recovery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Separation and purification of bone marrow mesenchymal stem cell exosomes

[0049] 1. Isolation, culture and identification of bone marrow mesenchymal stem cells

[0050] (1) Take 3 mL of fresh donor bone marrow, dilute it to 15 mL with PBS buffer in a 50 mL centrifuge tube, and make a cell suspension for use.

[0051] (2) Take a clean 50mL centrifuge tube again, and add 20mL of lymphocyte separation medium into the tube.

[0052] (3) Use a pipette gun to draw a certain amount of cell suspension, and gently superimpose it on the lymphocyte separation medium along the tube wall to keep the liquid surface separated. After trimming, centrifuge horizontally at room temperature for 400g×20min, increase slowly, and stop without braking.

[0053] (4) After the centrifugation, it can be seen that the layers in the centrifuge tube are clear, and the milky white buffy film-like interface is the mononuclear cell. From top to bottom in the tube are PBS, mononuclear cel...

Embodiment 2

[0067] Example 2 Identification, quantification and preparation of exosomes

[0068] 1. Identification of exosomes from peripheral blood hematopoietic stem cells by transmission electron microscopy

[0069] 1) Pipette 10 μL of spare exosomes onto the sterile copper mesh grid and let stand for 5 minutes.

[0070] 2) Gently blot the water dry with filter paper along the edge of the copper mesh.

[0071] 3) Add 20 μL of 2% sodium phosphotungstate to the copper mesh grid for negative staining, and place it at room temperature for 5 minutes.

[0072] 4) Gently blot the moisture with filter paper along the edge of the copper mesh.

[0073] 5) Bake the copper grid gently with an incandescent lamp.

[0074] 6) Put the processed copper mesh grid into the electron microscope, adjust the electron microscope, and observe.

[0075] 7) Transmission electron microscope results of exosomes collected from peripheral blood hematopoietic stem cells: the exosomes extracted from the culture su...

Embodiment 3

[0100] Example 3 Therapeutic experiment of exosome preparation

[0101] 1. Bone marrow mesenchymal stem cell exosome compound preparation promotes the proliferation of peripheral blood mononuclear cells.

[0102] By adding the exosome preparation prepared in Example 2 of the present invention to the co-culture of peripheral blood mononuclear cells after mobilization, it was explored whether the compound preparation of mesenchymal stem cell exosomes can promote the proliferation of peripheral blood mononuclear cells in vitro.

[0103] (1) In vitro isolation and culture of peripheral blood mononuclear cells:

[0104] After donor mobilization, 1 mL of peripheral blood hematopoietic stem cells was separated from mononuclear cells by human lymphocyte separation medium, washed and resuspended in 2 mL of PBS, and the cell concentration was adjusted to 1×10 6 / mL. Inoculate in culture flasks, add exosome compound preparations or exosomes in proportion (exocrine final concentration 4...

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Abstract

The invention belongs to the technical field of cells, and relates to an exosome preparation which comprises a bone marrow mesenchymal stem cell-derived exosome and a vitamin preparation. The mesenchymal stem cells derived from bone marrow separation are subcultured and purified to obtain the mesenchymal stem cells, proliferation of leukocytes, hemoglobin and platelets of patients after radiotherapy and chemotherapy can be promoted, proliferation of hematopoietic stem cells is promoted, and the proportion of CD34 + cells of mononuclear cells is promoted, so that the effect of promoting hematopoietic function recovery after radiotherapy and chemotherapy is achieved.

Description

technical field [0001] The invention belongs to the field of cell technology, and in particular relates to a bone marrow mesenchymal stem cell exosome preparation and its use for promoting the recovery of hematopoietic damage. Background technique [0002] Exosomes are special extramembrane vesicles with a diameter of 30-150 nm and a density of 1.13-1.19 g / mL. They have a stable bimolecular phospholipid structure and can protect their own DNA fragments, miRNA, mRNA and functional Bioactive substances such as proteins are not destroyed. Exosomes can be transported to target cells through interstitial fluid or blood, and after acting on target cells, the contents are released to activate intracellular signaling pathways, thereby exerting biological functions such as apoptosis, angiogenesis, regeneration and repair, inflammatory response, and immune response . [0003] Bone marrow mesenchymal stem cells are bone marrow stromal stem cells, a cell subpopulation found in the bon...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61P7/06A61K31/519A61K31/375A61K31/714
CPCA61K35/28A61K31/519A61K31/375A61K31/714A61P7/06A61K2300/00
Inventor 黄雅静
Owner 因诺伟(北京)生物医疗科技有限公司
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