Application of dimethyl fumarate in promoting proliferation of amniotic fluid stem cells
By adding dimethyl fumarate (DMF) to the amniotic fluid stem cell culture medium as an NRF2 expression promoter, the problems of long culture cycle and high cost of amniotic fluid stem cells were solved, and rapid and economical cell proliferation was achieved.
Patent Information
- Application Number
- CN202511566533.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-30
- Publication Date
- 2026-01-20
AI Technical Summary
Current amniotic fluid stem cell culture methods are time-consuming and costly, making it difficult to meet the needs of rapid diagnosis, and growth factors are also expensive.
Dimethyl fumarate (DMF) was used as an NRF2 expression promoter in the culture medium of amniotic fluid stem cells at a concentration of 2.5–10 µM to promote cell proliferation and protect cells from oxidative damage.
It shortens cell culture time to 72 hours, reduces costs, and DMF is priced at 1/40th of growth factors, significantly improving cell proliferation efficiency.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medicine, in particular to application of dimethyl fumarate in promoting proliferation of amniotic fluid stem cells. BACKGROUND
[0002] Amniotic fluid prenatal diagnosis needs to obtain sufficient cells by in vitro culture, and the current scheme is: 1. using amniotic fluid special culture medium containing 10-20% fetal bovine serum (such as AmnioMAX-II); 2. adding growth factors such as bFGF and EGF (concentration 5-20 ng / mL); 3. culture period 7-14 days, and medium is replaced every 72 hours.
[0003] The prior art has the following disadvantages: 1. long period: cell adhesion needs 3-5 days, and expansion to the required number for diagnosis needs ≥7 days, which delays the diagnosis opportunity; 2. high cost: the price of growth factor additives is expensive (more than 60% of the cost of culture medium). SUMMARY
[0004] In order to solve the above technical problems, the embodiments of the present application provide application of dimethyl fumarate in promoting proliferation of amniotic fluid stem cells.
[0005] In order to achieve the above purpose, the embodiments of the present application adopt the following technical solutions: The present application provides application of dimethyl fumarate in promoting proliferation of amniotic fluid stem cells.
[0006] In some embodiments, the dimethyl fumarate is used as an NRF2 expression promoter.
[0007] In some embodiments, the concentration of the dimethyl fumarate is 2.5-10 µM.
[0008] In some embodiments, the structural formula of the dimethyl fumarate is: .
[0009] The present application has the following beneficial effects compared with the prior art: The dimethyl fumarate promotes NRF2 expression, protects amniotic fluid stem cells from oxidative damage, and accelerates proliferation of amniotic fluid stem cells, so that the cell culture time is shortened to 72 hours, and the unit price of DMF is 1 / 40 of that of growth factors. BRIEF DESCRIPTION OF DRAWINGS
[0010] Figure 1 Figure for DMF promoting NRF2 expression in amniotic fluid stem cells; Figure 2 Figure for viability of amniotic fluid stem cells after culture with different concentrations of DMF; Figure 3 Figure for proliferation of amniotic fluid stem cells after addition of DMF; Figure 4 For the identification of amniotic fluid stem cells after DMF action. DETAILED DESCRIPTION
[0011] The technical solutions in some embodiments of the present disclosure will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments provided in the present disclosure, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present disclosure.
[0012] Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.
[0013] Experimental materials Materials: DMEM medium, fetal bovine serum (American gibco company), DMF, NRF2 inhibitor ML385 (Shanghai selleck company), hydrogen peroxide (H2O2) solution (Guangdong Hengjian Pharmaceutical Co., Ltd.).
[0014] Main components of DMEM medium: (1) Inorganic salt: sodium chloride (NaCl): 6400 mg / L, potassium chloride (KCl): 400 mg / L, sodium dihydrogen phosphate (NaH2PO4·H2O): 125 mg / L, magnesium sulfate (MgSO4): 97.67 mg / L, Calcium chloride (CaCl2): 200 mg / L, sodium bicarbonate (NaHCO3): 3700 mg / L.
[0015] (2) Amino acid: L-arginine (L-Arg): 84 mg / L, L-cystine (L-Cys): 48 mg / L, L-glutamine (L-Gln): 584 mg / L, glycine (Gly): 30 mg / L, L-histidine (L-His): 42 mg / L, L-isoleucine (L-Ile): 105 mg / L, L-leucine (L-Leu): 105 mg / L, L-lysine (L-Lys): 146 mg / L, L-methionine (L-Met): 30 mg / L, L-phenylalanine (L-Phe): 66 mg / L, L-serine (L-Ser): 42 mg / L, L-threonine (L-Thr): 95 mg / L, L-tryptophan (L-Trp): 16 mg / L, L-tyrosine (L-Tyr): 72 mg / L, L-valine (L-Val): 94 mg / L.
[0016] (3) Vitamins: Choline: 4 mg / L, Folic acid: 4 mg / L, myo-Inositol: 4 mg / L, Nicotinamide: 4 mg / L, D-Ca Pantothenate: 4 mg / L, Pyridoxal-HCl: 4 mg / L, Riboflavin: 0.4 mg / L, Thiamine-HCl: 4 mg / L, i-Inositol: 7.2 mg / L.
[0017] (4) D-Glucose: 4500 mg / L.
[0018] Common medium: DMEM medium + 10% fetal bovine serum by volume. Add 2.5-10 µM dimethyl fumarate (DMF for short) in the common medium to form a "DMEM medium + DMF + 10% fetal bovine serum" system.
[0019] Culture and identification process: (1) Obtain amniotic fluid samples and centrifuge; (2) Resuspend the amniotic fluid stem cells in the common medium and inoculate in a culture flask, and place it in a 37°C, 5% CO2 incubator for culture; (3) After the cells adhere, replace the medium with the above-mentioned medium containing 2.5-10 µM DMF, and place it in the incubator for culture for 3 days, then harvest the cells.
[0020] (4) Flow cytometry identification of cells: amniotic fluid stem cells express CD29, CD44, CD147 and CD90 (percentage greater than 80%), and partially express CD105, but do not express CD106, CD133, CD117 and CD34 (percentage less than 5%).
[0021] Experimental process: After 1 mM H2O2 acts on amniotic fluid stem cells for 3 h, the culture medium is discarded, and the common medium without or with 5 µM or 10 µM DMF, 5 µM or 10 µM ML385 is added for culture for 24 h, and then WB is used to detect the expression of NRF2 in the cells. As shown in Figure 1 H2O2 inhibits NRF2 in amniotic fluid stem cells, and the addition of 10 µM DMF significantly promotes the expression thereof.
[0022] As shown in Figure 2 A of FIG. 1, after 1 mM H2O2 acts on amniotic fluid stem cells for 3 h, the culture medium is discarded, and the common medium containing 2.5-10 µM DMF is added for culture for 24 h, and then CCK8 is used to detect the cell viability.Figure 2 Figure B shows cells cultured in ordinary medium containing 0-10 µM DMF for 24 h, followed by CCK8 assay to detect cell viability. Figure 3 After amniotic fluid stem cells were treated with 1 mM H2O2 for 3 h, the culture medium was discarded, and the cells were cultured in ordinary culture medium containing 5 µM or 10 µM MDMF for 24 h. The cells were then photographed under a light microscope (independent samples or fields of view n≥3).
[0023] After the addition of DMF, the activity of amniotic fluid stem cells gradually increased with increasing DMF concentration. Figure 2 (Figure B) The same results were observed in cells treated with H2O2, showing increased cell viability. Figure 2 Figure A in the diagram shows increased cell density. Figure 3 ).
[0024] like Figure 4 As shown, after culturing in ordinary medium containing 10 µM DMF for 24 h, the expression of each molecule was detected by flow cytometry: CD105 9.59%, CD29 76.22%, CD106 2.74%, CD133 0.55%, CD44 86.96%, CD117 0.04%, CD147 76.72%, CD34 0.25%, and CD90 88.77%.
[0025] In summary, DMF promotes NRF2 expression, protects amniotic fluid stem cells from oxidative damage, accelerates amniotic fluid stem cell proliferation, shortens cell culture time to 72 hours, and has a DMF unit price of 1 / 40 that of growth factors.
[0026] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.
[0027] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. Use of dimethyl fumarate in promoting proliferation of amniotic stem cells.
2. Use of dimethyl fumarate as an NRF2 expression promoter.
3. Use according to claim 1 or 2, characterized in that, The dimethyl fumarate is administered at a concentration of 2.5-10 µM.
4. Use according to claim 3, characterized in that, The dimethyl fumarate has the following structural formula: 。