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Application of inducible type regulatory T cell derived exosome

A technology of exosomes and cells, applied in the field of biomedicine, can solve problems such as life-threatening, side effects, secondary infection, secondary tumors, etc., and achieve the effect of relieving clinical symptoms and inhibiting inflammatory response

Active Publication Date: 2019-03-22
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the clinical treatment of RA is supportive and symptomatic therapy. The main treatment methods are: use of immunosuppressants, anti-inflammatory therapy and symptomatic treatment, etc., but none of them can cure RA, and may even bring serious side effects. Infection, secondary tumor, etc., threatening the patient's life

Method used

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  • Application of inducible type regulatory T cell derived exosome
  • Application of inducible type regulatory T cell derived exosome
  • Application of inducible type regulatory T cell derived exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of iTreg and isolation and identification of iTreg exo

[0033] 1. Preparation of inducible Treg

[0034] Mice were first obtained by magnetic bead sorting CD4 cells were then cultured for 3 days in the presence of TGF-β and IL-2, as figure 1 As shown in A, the proportion of CD4+CD25+Foxp3+ cells was detected by flow cytometry to confirm the induction of iTreg, and the establishment of CD4 control without adding TGF-β.

[0035] 2. Preparation and identification of iTreg exo

[0036] During the preparation of inducible Treg, the For CD4 control without adding TGF-β, the cells were cultured in exosome-free serum medium, and the cell supernatant was harvested after 3 days, and a certain proportion of Exo-quick was added to incubate overnight to extract exosomes. The identification of exosomes, using the method of transmission electron microscopy, such as figure 1 As shown in B, round vesicles with a diameter of about 100 nm having a bilayer ...

Embodiment 2

[0037] Example 2 Detection of iTreg exo inhibiting T cell proliferation and differentiation

[0038] Carboxyfluorescein diacetatesuccinimidyl ester (CFSE) is a fluorescent dye that can penetrate the cell membrane, and can directly enter the cell through the cell membrane, and once it enters the cell, its acetate group is absorbed by the intracellular esterase After removal, CFSE with fluorescence can be produced, and it is not easy to release out of the cell. At the same time, the succinimide ester of CFSE can be covalently combined with the primary amino group, so that it exists in the cell for a long time. As cells divide, CFSE is progressively partitioned into progeny cells, halving each time, so cell division over generations can be easily detected using flow cytometry. We used CFSE to label mouse T cells to evaluate the effect of iTreg exo on T cell proliferation.

[0039] figure 2 It was shown that iTreg exo significantly inhibited the proliferation of CD8+ and CD4+ ...

Embodiment 3

[0040] Example 3 Verification of the therapeutic effect of iTreg exo on rheumatoid arthritis

[0041] CIA is achieved by immunizing genetically susceptible mice with type II collagen mixed with complete Freund's adjuvant (CFA). The basic principle is that some amino acid fragments in the middle of type II collagen injected exogenously into animals can induce T cell activation as core antigen peptides that are bound by MHC type II molecules (such as mouse IAq, etc.) and recognized by T cell receptors (TCR) and hyperplasia, and C II is the main protein in cartilage, which is the target tissue of RA, so it will induce the occurrence of autoimmune diseases in experimental animals. The joints of mice were taken for HE staining, or the degree of bone destruction was measured by micro CT for the whole joint, and the difference in the onset time of arthritis, the severity of the disease, and the degree of bone and joint destruction were observed. The result is as Figure 4 As shown,...

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Abstract

The invention relates to application of inducible type regulatory T cell derived exosome (iTreg exo), and specifically, iTreg exo and application thereof in preparing medicines for treating rheumatoidarthritis and belongs to the technical field of biological medicine. The iTreg exo involved in the invention can carry miR-449a-5p to inhibit proliferation and differentiation of Th1 and Th17 cells,can reverse imbalance of Treg (inflammation inhibiting cells) / Th17 (inflammation cells) proportion in a mouse with rheumatoid arthritis, can inhibit inflammation reaction, can relieve clinical symptoms, can serve as a novel method for treating rheumatoid arthritis, and has an important clinical significance in further knowing exosome in occurrence and development of arthritis diseases and developing novel methods for treating arthritis.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to the application of exosomes (iTregexo) derived from inducible regulatory T cells in the preparation of drugs for treating rheumatoid arthritis. Background technique [0002] Treg cells (regulatory T cells) are a type of cell population that regulates the immune function of the body. They can maintain the tolerance of the immune system to its own components and maintain the immune homeostasis of the body. These cells are characterized by the expression of Foxp3, CD25, and CD4. In the past 20 years, studies have confirmed that Treg cells can suppress various pathways of pathophysiological immune responses in infection, tumor, organ transplantation, and allogeneic fetal immune-related diseases. Treg cells can be divided into naturally occurring natural regulatory T cells (nTregs) and induced adaptive regulatory T cells (aTregs or iTregs). The applicant first discovered that TGF-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/17A61P19/02A61P37/02
CPCA61P19/02A61P37/02A61K35/17
Inventor 郑颂国陈敬荣王菊华
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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