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A preparation method of exosomes and exosomes and its application in the preparation of drugs or preparations for treating sepsis

A technology of exosomes and sepsis, applied in the treatment of sepsis, the field of exosomes produced by mesenchymal stem cells, can solve the problems of limited survival, malignant transformation, MSCs inhomogeneity, etc. Bacterial clearance rate, enhancement of immunosuppressive function, strong effect of immunosuppressive function

Active Publication Date: 2019-07-23
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the clinical application of MSCs still faces many challenges, including heterogeneity of MSCs cultured in vitro, vascular occlusion caused by cell injection, limited survival in vivo after cell transplantation, genetic instability or loss of function caused by aging, etc.
What's more serious is that MSCs still have the possibility of malignant transformation and tumorigenesis when used in clinical practice.

Method used

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  • A preparation method of exosomes and exosomes and its application in the preparation of drugs or preparations for treating sepsis
  • A preparation method of exosomes and exosomes and its application in the preparation of drugs or preparations for treating sepsis
  • A preparation method of exosomes and exosomes and its application in the preparation of drugs or preparations for treating sepsis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Optimizing Umbilical Cord Mesenchymal Stem Cells with IL-1β

[0036] 1. Isolation and culture of human umbilical cord mesenchymal stem cells (MSCs) (the reagents and consumables used in the laboratory are all sterile)

[0037] With the informed consent of the puerpera, the umbilical cords of cesarean section fetuses with normal full-term pregnancy were collected. Before the umbilical cord is collected, the puerpera needs to undergo strict pathogen testing, including Treponema pallidum, HIV, cytomegalovirus, hepatitis B virus, hepatitis C virus, syphilis and mycoplasma, and use it after confirming safety.

[0038] Cut the umbilical cord near the placenta to 20-30 cm, store it in pre-cooled sterile phosphate buffered saline (PBS) at 4°C, and use it within 4 hours. In the ultra-clean workbench, the umbilical cord was cut into small sections of about 5 cm, and the residual blood on the surface of the umbilical cord was washed with PBS; DMEM / F12 medium (purchased f...

Embodiment 2

[0051] Example 2 Extraction and identification of IL-1β-optimized mesenchymal stem cell exosomes (βMSC-exo) and resting state mesenchymal stem cell exosomes (MSC-exo)

[0052] 1. Extraction of βMSC-exo and MSC-exo

[0053] Cell culture supernatants of βMSCs and resting MSCs were collected respectively in 500ml sterile centrifuge bottles or 50ml polypropylene centrifuge tubes (purchased from Beckman), and centrifuged at 4°C and 2000g for 10 minutes to remove dead cells and large debris. Carefully transfer the supernatant to a new sterile centrifuge tube and centrifuge at 10,000g for 30 minutes at 4°C to remove organelles and small particles. Carefully transfer the supernatant to a sterile ultracentrifuge tube, and centrifuge at 110,000g (Beckman ultracentrifuge) at 4°C for 70 minutes, discard the supernatant carefully, wash once with normal saline for injection, and store at 4°C, 110,000g ultracentrifugation for 70 minutes, the obtained pellet is exosomes. Depending on the vo...

Embodiment 3

[0059] Example 3 Inhibition of IL-1β-optimized mesenchymal stem cell exosomes (βMSC-exo) and resting state mesenchymal stem cell exosomes (MSC-exo) on T cells and macrophages cultured in vitro

[0060]1. Inhibitory effect on T cells

[0061] The methods for obtaining and processing mouse spleen T cells are the same as in Example 1. βMSC-exo and MSC-exo were added to T cells respectively, and the final concentration of exosomes was 10 μg / ml. At the same time, 5 μg / ml ConA was added to stimulate for 48 hours, and the intracellular protein transport inhibitor BFA was added 4 hours before the cells were collected, and T cells were collected, and the protein levels of IFN-γ in T cells and CD69 on the cell surface were detected by flow cytometry. The result is as Figure 7 As shown, βMSC-exo inhibited the protein levels of IFN-γ and CD69 more effectively than MSC-exo.

[0062] 2. Inhibitory effect on macrophages

[0063] The methods for obtaining and processing mouse bone marrow...

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Abstract

The invention discloses application of an exosome coming from umbilical cord mesenchymal stem cells optimized with interleukin-1beta (IL-1beta) in treating sepsis. Specifically, recombined IL-1beta of human is used for optimizing the umbilical cord mesenchymal stem cells to enhance the immunosupression function of the umbilical cord mesenchymal stem cells, and the exosome generated by the umbilical cord mesenchymal stem cells is extracted and used for treating sepsis. The exosome can effectively relieve the symptoms of sepsis, and increase the survival rate of mice suffering from sepsis; besides, the exosome has the advantages of being convenient to preserve and transport, and thus a new strategy is provided for treating diseases related to refractory inflammation such as sepsis.

Description

technical field [0001] The present invention relates to exosomes derived from mesenchymal stem cells, a preparation method of exosomes and uses thereof, in particular to the application of exosomes produced by mesenchymal stem cells after optimized treatment with IL-1β in the treatment of sepsis . Background technique [0002] Sepsis is a severe systemic inflammation caused by infection. Its pathogenesis includes a systemic inflammatory outbreak, accompanied by multiple organ dysfunction syndrome, acute respiratory distress syndrome, and circulatory system collapse, which eventually leads to death. There is still no effective treatment for this disease, and its mortality rate remains high among critically ill patients. Since the withdrawal of Xygris and Eritoran in 2011, there are currently no effective drugs for sepsis. Therefore, there is an urgent need to develop new and effective treatments. [0003] Mesenchymal stem cells (MSCs) are a group of pluripotent stem cells...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K35/28A61P31/04A61P29/00A61P37/06
CPCA61K35/28C12N5/0668C12N2501/2301
Inventor 侯亚义宋玉仙窦环泥艳红樊竑冶赵晓寅
Owner NANJING UNIV
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