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Application of mesenchymal stem cell-derived small extracellular vesicles to preparation of drugs for treating autoimmune diseases

An autoimmune and mesenchymal stem cell technology, which is applied in small extracellular vesicles and the preparation of drugs for the treatment of autoimmune diseases, can solve the problems of mesenchymal stem cell dysfunction, unclear final differentiation fate, etc., to achieve The source is convenient to obtain, good rejection, low immunogenicity effect

Pending Publication Date: 2021-02-09
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] Mesenchymal stem cells (MSCs) provide a new idea for the treatment of autoimmune diseases. MSCs are derived from mesoderm adult stem cells. MSCs expanded in vitro have a strong immunosuppressive effect and can be used in the immune response at the same time. Multiple steps play a regulatory role, but there are some uncertainties in using these cells in clinical settings, for example, patients' mesenchymal stem cells are often dysfunctional, only cells of autologous origin can be used, and it is not clear whether these cells The final destination and differentiation fate after transplantation into the human body, other long-term side effects including infection and tumorigenesis are also considered as limitations and shortcomings of MSC cell therapy

Method used

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  • Application of mesenchymal stem cell-derived small extracellular vesicles to preparation of drugs for treating autoimmune diseases
  • Application of mesenchymal stem cell-derived small extracellular vesicles to preparation of drugs for treating autoimmune diseases
  • Application of mesenchymal stem cell-derived small extracellular vesicles to preparation of drugs for treating autoimmune diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, Isolation and cultivation of gingival mesenchymal stem cells (GMSC)

[0050] 1. Collection of samples: collection of human gum samples, which were obtained from tissues discarded by patients undergoing routine dental surgery in the Department of Stomatology, Sun Yat-sen University Third Hospital, approved by the Medical Ethics Committee of the Sun Yat-sen University Third Hospital Ethics Committee (IRB) (IRB 2018-02-195-01).

[0051] 2. Isolation and culture of gingival mesenchymal stem cells (GMSC): the collected human gingival samples were digested with Disase II (2mg / mL) at 4°C overnight or 2h at 37°C; then with a concentration of 4mg / mL The IV-type collagenase was digested at 37°C for 0.5h (the tissue was shaken every few minutes to avoid debris); the cell suspension was collected by filtration with a 70μm sieve, centrifuged at 300g for 5min to obtain the cell pellet, and the cell pellet was washed with α - MEM complete medium (containing 10% FBS, 100...

Embodiment 2

[0054] Example 2, Isolation and identification of GMSC-sEV

[0055] Take the supernatant of the resuspension prepared in Step 2 of Example 1 and centrifuge at 300g for 5min to remove cells; centrifuge at 3,000g for 15min to remove dead cells; centrifuge at 10,000g for 30min to remove cell debris; centrifuge at 110,000g for 70min to remove the supernatant, and The pellet is a mixture containing protein and EVs; then resuspend the pellet with a large volume of PBS and continue to centrifuge at 110,000g for 70min, the final pellet is small EVs (GMSC-sEV); the final sEV is resuspended in PBS and used immediately or Store at -20°C.

[0056] Obtained GMSCs were observed under a microscope, such as figure 2 -A, the diameter of GMSC is distributed in 10-30 μm, and the obtained GMSC-sEV is detected by transmission electron microscope (TEM), such as figure 2 -B, the diameter size distribution of GMSC-sEV is at 120nm, further adopts nanoparticle tracking analysis (NTA) technology to ...

Embodiment 3

[0057] Example 3, Detection of GMSC-sEV inhibiting T cell proliferation in vitro

[0058] Take the whole blood from healthy volunteers and dilute it with an equal volume of PBS and mix it evenly, then slowly add the diluted whole blood ( Be careful not to damage the interface of the separation liquid layer), room temperature (18-28°C), 400g, centrifuge for 20-30min (the centrifuge is set to acceleration 3, deceleration 0). After the centrifugation is over, carefully aspirate the cells in the buffy coat. All the collected cells were then centrifuged with 5 times the volume of PBS at room temperature at 300 g for 10 min, and washed 1-2 times with PBS to remove platelets as much as possible. The final obtained hPBMCs (human peripheral blood mononuclear cells) were used immediately or stored at -80°C with frozen storage solution for later use.

[0059] hPBMCs obtained freshly or revived one night in advance were placed in 1 μM CFSE (carboxyfluoresceindiacetate succinimidyl ester...

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Abstract

The invention relates to the technical field of cells, and particularly discloses an application of mesenchymal stem cell-derived small extracellular vesicles to preparation of drugs for treating autoimmune diseases. The mesenchymal stem cell-derived small extracellular vesicles provided by the invention have the advantages of extremely low immunogenicity, small side effects, small rejection immune response, stability in a living body, natural homing and targeting properties and long residence time, the mesenchymal stem cell-derived small extracellular vesicles are made into drugs which have avery good treatment effect on rheumatoid arthritis, and arthritis can be effectively resisted.

Description

technical field [0001] The invention relates to the field of cell technology, in particular to the application of a small extracellular vesicle derived from mesenchymal stem cells in the preparation of drugs for treating autoimmune diseases. Background technique [0002] Autoimmune diseases (ADs) are a class of chronic, heterogeneous diseases caused by the loss of immune tolerance to self-antigens, which can involve specific target organs or multiple systems. Common autoimmune diseases include autoimmune uveitis , multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, etc. [0003] Rheumatoid Arthritis (RA) is a self-systematic and heterogeneous disease with symmetrical multiple facet joint involvement as the main clinical manifestation, and chronic progressive destruction of synovium, cartilage, tendon, etc. in the affected joints. The prevalence of RA in different population areas at home and abroad is 0.18% to 1.07%, and the number of patients exceeds 5 ...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61K47/46A61P37/02A61P29/00A61P19/02A61P27/02A61P25/00
CPCA61K35/28A61K47/46A61P37/02A61P29/00A61P19/02A61P27/02A61P25/00
Inventor 郑颂国陈敬荣王菊华黄峰
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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