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Preparation method for plasma exosomes of mobilized hematopoietic stem cells and application of plasma exosomes

A technology of hematopoietic stem cells and exosomes, which is applied in the field of plasma exosome preparation of hematopoietic stem cells after mobilization, can solve the problems that the amount of exosomes extracted is not enough for the application, the cells cannot be used again, and the culture process is long, etc., so as to avoid The risk of complications, the promotion of hematopoietic injury recovery, and the effect of easy operation

Active Publication Date: 2020-10-16
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

At present, the extraction method of microbubbles is mainly used to extract mononuclear cells from the collected hematopoietic stem cells and culture them in vitro, and extract exosomes from the culture supernatant. The main disadvantages are as follows: long culture process, cell death and release of contents The problem; the culture conditions are not suitable for the growth of stem cells and immune cells at the same time, so there is a difference between the exosomes contained in the supernatant and the exosomes in the collection; the amount of cultured cells is small, and the amount of exosomes extracted is not enough for application Required; cells cannot be reused after culture, resulting in waste

Method used

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  • Preparation method for plasma exosomes of mobilized hematopoietic stem cells and application of plasma exosomes
  • Preparation method for plasma exosomes of mobilized hematopoietic stem cells and application of plasma exosomes
  • Preparation method for plasma exosomes of mobilized hematopoietic stem cells and application of plasma exosomes

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Extraction of Plasma Exosomes from Peripheral Blood Hematopoietic Stem Cell Collection

[0041] 1. Obtaining of donor peripheral blood hematopoietic stem cell collection

[0042] The collection of peripheral blood hematopoietic stem cells donated by healthy human donors was used as the research object. Give human healthy donors rhG-CSF 5ug / (kg.d)q 12h subcutaneous injection for 4.5 days (that is, rhG-CSF is injected subcutaneously once every 12 hours, each dose is rhG-CSF 5ug / kg body weight, continuous administration of 4.5 day) to mobilize peripheral blood hematopoietic stem cells, collect peripheral blood on the 5th day, and use a blood cell separator (purchased from Germany Fresenius, model Amicus) to collect peripheral blood hematopoietic stem cells (acquisition parameters are based on blood cell separation The machine recommends the collection of stem cell program settings), the general circulating whole blood volume is 10000ml, and the peripheral blood ...

Embodiment 2

[0071] Example 2 Exosomes collected from peripheral blood hematopoietic stem cells promote rapid recovery of hematopoietic damage after radiotherapy

[0072] The method of exosomes collected from peripheral blood hematopoietic stem cells to promote rapid recovery of hematopoietic injury after radiotherapy is as follows: image 3 As shown, the specific steps are as follows:

[0073] 1. Give healthy male C57 mice (6-8W age, SPF grade, purchased from Beijing Weitong Lihua Animal Technology Co., Ltd.) a subcutaneous injection of G-CSF (purchased from Beijing Shuanglu Pharmaceutical Co., Ltd., article number 87189190000087272782) 5ug / (kg.d)q 12h subcutaneous injection for 4.5 days (that is, subcutaneous injection of G-CSF once every 12 hours, each dose of G-CSF 5ug / kg body weight, continuous administration for 4.5 days) for mobilization, and making splenocyte suspension liquid (i.e. mouse peripheral blood hematopoietic stem cell collection), to simulate the method of embodiment 1...

Embodiment 3

[0076] Example 3 Plasma exosomes collected from peripheral blood hematopoietic stem cells promote the proliferation of peripheral blood mononuclear cells

[0077] In vitro experiments were conducted to verify whether plasma exosomes collected from peripheral blood hematopoietic stem cells can promote the proliferation of peripheral blood mononuclear cells. The specific steps are as follows:

[0078] Give human healthy donors rhG-CSF 5ug / (kg.d)q 12h subcutaneous injection for 4.5 days (that is, rhG-CSF is injected subcutaneously once every 12 hours, each dose is rhG-CSF 5ug / kg body weight, continuous administration of 4.5 day) to mobilize peripheral blood hematopoietic stem cells, and use a blood cell separator to separate the collected peripheral blood hematopoietic stem cells on the fifth day, and the volume of circulating whole blood is 10L. Take 1 mL of the isolated hematopoietic stem cell collection and separate the peripheral blood mononuclear cells with human lymphocyte ...

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Abstract

The invention discloses a preparation method for plasma exosomes of mobilized hematopoietic stem cells and application of the plasma exosomes. The invention firstly discloses an extracting method forthe exosomes, and the extracting method includes mobilizing peripheral blood hematopoietic stem cells by employing a mobilizing agent. The invention further provides an exosome preparation containingthe exosomes obtained by the extracting method and application of the exosome preparation. The extracting method for the plasma exosomes of the mobilized hematopoietic stem cells of the invention is simple, and is easy to operate, and the obtained exosomes have high concentration and good quality. By transfusing the plasma exosomes of the peripheral blood hematopoietic stem cells of the inventioninto a body with hematopoietic injury, recovery of the hematopoietic injury can be promoted, and the complication risk brought by implantation after transfusion of the peripheral blood hematopoietic stem cells is avoided; moreover, the exosomes can play a role of maternal cells, and avoid adverse reactions of the maternal cells; and in addition, in vitro experiments confirm that the plasma exosomes of the peripheral blood hematopoietic stem cell collected samples obtained by the invention have the function of promoting proliferation of peripheral blood mononuclear cells.

Description

technical field [0001] The invention relates to a preparation method and application of hematopoietic stem cell plasma exosomes after mobilization. Background technique [0002] At present, there are mainly two ways to promote the repair of hematopoietic damage: 1) Hematopoietic growth factors: granulocyte colony-stimulating factor, megakaryocyte growth factor, erythropoietin, thrombopoietin, etc. The role of these hematopoietic growth factors is mainly Promote the proliferation of hematopoietic cells in the bone marrow and restore normal hematopoiesis, but for the hematopoietic inhibition caused by severe radiation damage, it is difficult to play a role in promoting hematopoietic recovery; 2) Hematopoietic stem cell transplantation: hematopoietic stem cells can differentiate into various mature blood cells and immune cells. Cells, such as red blood cells, platelets, lymphocytes, etc., completely rebuild the entire hematopoietic system in the recipient, so hematopoietic stem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789A61K35/28A61P7/06
CPCC12N5/0647A61K35/28A61P7/06C12N2509/00
Inventor 黄雅静
Owner 因诺伟(北京)生物医疗科技有限公司
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