CN114209814B - Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion - Google Patents
Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion Download PDFInfo
- Publication number
- CN114209814B CN114209814B CN202111532973.6A CN202111532973A CN114209814B CN 114209814 B CN114209814 B CN 114209814B CN 202111532973 A CN202111532973 A CN 202111532973A CN 114209814 B CN114209814 B CN 114209814B
- Authority
- CN
- China
- Prior art keywords
- bone marrow
- macrophages
- tnfsf15
- protein
- stem cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000002540 macrophage Anatomy 0.000 title claims abstract description 70
- 101000830596 Homo sapiens Tumor necrosis factor ligand superfamily member 15 Proteins 0.000 title claims abstract description 59
- 102100024587 Tumor necrosis factor ligand superfamily member 15 Human genes 0.000 title claims abstract description 59
- 210000001185 bone marrow Anatomy 0.000 title claims abstract description 49
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 45
- 230000004069 differentiation Effects 0.000 title claims abstract description 27
- 230000001737 promoting effect Effects 0.000 title claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims description 48
- 210000002798 bone marrow cell Anatomy 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 239000001963 growth medium Substances 0.000 claims description 12
- 210000004979 bone marrow derived macrophage Anatomy 0.000 claims description 11
- 238000000338 in vitro Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- 230000035755 proliferation Effects 0.000 claims description 9
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 238000009472 formulation Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 6
- 206010028980 Neoplasm Diseases 0.000 description 23
- 239000006228 supernatant Substances 0.000 description 15
- 230000000694 effects Effects 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 8
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 7
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 6
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 4
- 102100022338 Integrin alpha-M Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000000834 fixative Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 3
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 3
- -1 F4/80 Proteins 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 238000010322 bone marrow transplantation Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000007873 sieving Methods 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940047120 colony stimulating factors Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000002025 microglial effect Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 210000004767 rumen Anatomy 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 1
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- 101100476779 Dictyostelium discoideum scaA gene Proteins 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000998132 Homo sapiens Interleukin-34 Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 102100033499 Interleukin-34 Human genes 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 210000001132 alveolar macrophage Anatomy 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000007894 caplet Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000004041 dendritic cell maturation Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003966 growth inhibitor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 210000001930 leg bone Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 101150053010 sca1 gene Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000006444 vascular growth Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides an application of TNFSF15 protein in promoting differentiation and expansion of bone marrow stem cells into macrophages, and provides a new idea for clinical macrophage-related disease treatment in the future after obtaining macrophages by using TNFSF15 protein and then treating related diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion.
Technical Field
Macrophages are important innate immune cells of the body, have a high degree of plasticity, are found in various tissues, such as osteoclasts (bones), alveolar macrophages (lungs), microglial cells (brain), histiocytes (connective tissue), cumic cells (liver) and langerhans cells (skin), etc., and exhibit a variety of functions. They play an important role in growth, maintenance of metabolic balance, tissue repair and immune response. The research finds that the injection of macrophages has an effect of promoting heart reconstruction after acute myocardial infarction; the implantation of microglial cells (macrophages), which can engulf useless substances or harmful proteins, into the brain can reduce amyloid beta and the like which cause Alzheimer's disease. Thus, a variety of diseases can be treated by transplanting macrophages. However, due to the immune function of the body itself, the injected macrophages are preferably derived from the individual itself, in response to rejection of the foreign species. Therefore, how to obtain macrophages of the body in a large amount without affecting the health of the body as much as possible is an important research direction. Bone marrow stem cells are an important source of macrophages, and by utilizing the proliferation characteristics of bone marrow stem cells and the existing technology for separating bone marrow cells, bone marrow cells can be taken out to induce the differentiation of bone marrow stem cells into macrophages in vitro so as to obtain a large number of macrophages. At present, cytokines which induce the differentiation of bone marrow stem cells into macrophages mainly comprise M-CSF and IL34, and because the cytokines promote the differentiation of the macrophages into the macrophages have specific phenotypes and have limitations in application to various disease models, the search for new proteins which promote the differentiation of the bone marrow stem cells into the macrophages is an important subject.
Tumor necrosis factor superfamily member 15 (Tumor necrosis factor superfamily-15, TNFSF15, also known as TL 1A) is a vascular growth inhibitor secreted primarily by mature vascular endothelial cells. Research shows that the negative blood vessel regulating factor can inhibit the angiogenesis and the tumor growth in tumor; can also be used as an immune activator for promoting T cell activation and dendritic cell maturation. At present, no report exists on the role of TNFSF15 protein in the differentiation of bone marrow stem cells into macrophages.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an application of TNFSF15 protein in promoting bone marrow stem cells to differentiate into macrophages and proliferate in vitro, so as to solve the defects of the prior art, and the protein can promote the bone marrow stem cells to differentiate into macrophages, increase the quantity of the macrophages and provide a treatment scheme for macrophage-related diseases.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in a first aspect of the invention there is provided the use of a TNFSF15 protein in the manufacture of a medicament or formulation for promoting differentiation and expansion of bone marrow stem cells into macrophages.
Preferably, in this use, the medicament or formulation increases the proportion of macrophages by promoting differentiation of bone marrow stem cells into macrophages.
Preferably, in this use, the medicament or formulation increases the number of macrophages by promoting proliferation of bone marrow stem cells/bone marrow derived macrophages.
In this use, TNFSF15 protein is capable of promoting differentiation of bone marrow stem cells into macrophages, either alone or in the presence of other related macrophage colony-stimulating factors (M-CSF/GM-CSF).
In a second aspect of the present invention, there is provided a method for inducing differentiation and expansion of bone marrow stem cells into macrophages in vitro using TNFSF15 protein, wherein the expansion medium used in the method is supplemented with TNFSF15 protein at a concentration of not more than 10. Mu.g/mL.
Preferably, the method comprises the steps of:
bone marrow of miceRemoving bone marrow cells, resuspension with culture medium containing RPMI 1640, 15% FBS and TNFSF15 protein, and adjusting cell concentration to 2×10 6 Adding into pore plate (six pore plates) with 2mL each, culturing in incubator at 37deg.C under 5% CO for 3-7d 2 。
Preferably, the composition of the amplification medium in the method is RPMI 1640, 15% FBS and 3. Mu.g/mL TNFSF15 protein.
TNFSF15 protein is detected in the in vitro culture process, and in the bone marrow stem cell differentiation process, the proportion of macrophages is improved, and the time and concentration dependence is presented.
Compared with the prior art, the invention has the following advantages:
(1) The TNFSF15 protein promotes differentiation and expansion of bone marrow stem cells to macrophages, and can be used for treating related diseases after obtaining the macrophages in vitro.
(2) TNFSF15 protein can play a role in promoting differentiation of bone marrow stem cells into macrophages when being used alone or in combination with other macrophage colony stimulating factors, has stable effect and is not easily influenced by other factors, and can be considered to be used in vivo.
(3) The TNFSF15 purifying method is simple and massive, has low economic cost, can be combined with other treatment modes for treatment, and has wide application prospect.
Drawings
Fig. 1 is: influence of TNFSF15 protein on differentiation of bone marrow stem cells into macrophages in vitro, wherein A is the change of the proportion of macrophages after the bone marrow stem cells are treated by TNFSF15 protein with different concentrations for 7d; b is the change of macrophage proportion after the bone marrow stem cells are treated by TNFSF15 protein for different days.
Fig. 2 is: influence of TNFSF15 protein on the proportion of macrophage differentiation in the presence of macrophage colony-stimulating factor M-CSF and granulocyte-macrophage colony-stimulating factor GM-CSF.
Fig. 3 is: effect of TNFSF15 protein on proliferation and number of bone marrow stem cells/bone marrow-derived macrophages, wherein a is the change in proliferation of bone marrow stem cells/bone marrow-derived macrophages after TNFSF15 protein treatment; b is the change in cell number of bone marrow cells after 7d TNFSF15 protein treatment. .
Fig. 4 is: effects of TNFSF15 protein on macrophage progenitor cells, precursor cells and macrophages in tumor-bearing mouse bone marrow.
Fig. 5 is: effect of TNFSF15 protein on the number of macrophages differentiated from bone marrow stem cells in tumors.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concepts pertain. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The invention will be described in detail with reference to examples.
EXAMPLE 1 Effect of TNFSF15 protein alone on bone marrow Stem cell differentiation into macrophages in vitro
1) Acquisition of bone marrow cells
(1) Preparing: placing clean surgical instrument (at least two small scissors and small tweezers), gauze (8 layers, about 20 pieces), nylon membrane (70 μm aperture, 10 pieces) in lunch box, sterilizing under high pressure at 120deg.C for 20min;
(2) Bone marrow cells were removed: 5 weeks of male mice were sacrificed, the rear leg bones were removed, three sections of connected bones were removed from one leg, surrounding tissues were removed with gauze, joints at both ends of the bones were cut off, and bone marrow cells were flushed out with PBS using a 1mL syringe. The washed bone marrow cells are gathered together and are in red slices, and are blown by a 1mL syringe and a liquid-transferring gun to be dispersed into single-cell suspension;
(3) Filtering the collected bone marrow cells with 70 μm nylon mesh, centrifuging 400g at 4deg.C for 5min, and discarding the supernatant;
(4) Lysing erythrocytes: adding 2mL of erythrocyte lysate, re-suspending, placing on ice, adding 10mL of PBS solution containing 2% FBS after 3min, and uniformly mixing;
(5) 400g, centrifuging at 4 ℃ for 5min, discarding the supernatant, adding 10mL of PBS solution of 2% FBS, and uniformly mixing;
(6) Counting: mu.L of the above cell suspension was aspirated, diluted by a factor and counted under a microscope with a cell counting plate. 10mL total number of cells = total number of four large lattice cells/4 x10 4 X multiple x10 mL;
2) Effect of TNFSF15 protein on differentiation of bone marrow Stem cells into macrophages
(1) The cell suspension was centrifuged at 4℃and 400g for 5min, and the supernatant was discarded.
(2) Cell pellet was resuspended in (RPMI 1640+15% FBS) medium to adjust the cell concentration to 2X 10 6 2mL per well, added to an orifice plate (e.g., a 6-well plate);
(3) A, setting a control group and an experimental group in the experiment, wherein the experimental group is added with TNFSF15 protein of 0.3 mug/mL, 1 mug/mL, 3 mug/mL and 5 mug/mL in the culture medium, and the control group is added with buffer of corresponding dosage in the culture medium. Mixing with gentle shaking, and culturing in incubator at 37deg.C. Half-changing the liquid with corresponding culture medium for 3d and 5d, collecting cells until 7d, and labeling F4/80-PE-Cy7 antibody; b, control and 3. Mu.g/mL of the experimental group, cells were harvested at 3d,5d,7d, labeled F4/80-PE-Cy7 antibody, CD11b-FITC antibody, respectively. Each antibody was incubated at 4℃for 30min, protected from light, centrifuged, and unbound antibody was discarded. And resuspended with 200. Mu.L of cell fixative for flow detection.
3) Phenotypic analysis of bone marrow-derived macrophages induced by TNFSF15 protein differentiation
(1) The cell suspension was centrifuged at 4℃and 400g for 5min, and the supernatant was discarded.
(2) Cell pellet was resuspended in (RPMI 1640+15% FBS) medium to adjust the cell concentration to 2X 10 6 2mL per well, added to an orifice plate (e.g., a 6-well plate);
(3) The experimental group is provided with a control group and an experimental group, wherein the experimental group is added with 3 mug/mL TNFSF15 protein in the culture medium, and the control group is added with a buffer with a corresponding dosage in the culture medium. Mixing with gentle shaking, and culturing in incubator at 37deg.C. Half-volume exchanges were performed on the corresponding medium in 3d,5d cultures, and cells were harvested at 3d,5d,7d, labeled F4/80-PE-Cy7 antibody, MHC2-FITC, CD206-APC antibody. Incubate at room temperature for 30min, keep out of the sun, centrifuge, discard unbound antibody. And resuspended with 200. Mu.L of cell fixative for flow detection.
3) Analysis of results:
as can be seen from fig. 1, TNFSF15 protein increases the proportion of macrophages during in vitro differentiation of bone marrow stem cells compared to the corresponding buffer group, and exhibits a concentration (fig. 1A) and time (fig. 1B) dependence.
EXAMPLE 2 Effect of TNFSF15 protein on bone marrow-derived macrophage differentiation in the Presence of macrophage-related colony stimulating factor
1) Acquisition of bone marrow Stem cells
The same as in example 1
2) Effect of TNFSF15 protein on the ability of bone marrow Stem cells to differentiate into macrophages in the Presence of M-CSF/GM-CSF
(1) Centrifugation at 400g for 5min at 4℃and supernatant was discarded, resuspended in (RPMI 1640+15% FBS+50ng/mL M-CSF or GM-CSF factor) medium and the cell concentration was adjusted to 2X 10 6 2mL per well, added to an orifice plate (e.g., a 6-well plate);
(2) The experimental set is a control group and an experimental group, wherein 3 mug/mL TNFSF15 protein is respectively added into the culture medium, and the control group is added with buffer with corresponding dosage into the culture medium. Mixing with gentle shaking, and culturing in incubator at 37deg.C. Half-volume exchange of the corresponding medium was performed on the culture for 3d, and cells were harvested 5d, and F4/80-PE-Cy7 antibody was labeled. Incubate at room temperature for 30min, protected from light. Centrifugation, discarding unbound antibody. And resuspended with 200. Mu.L of cell fixative for flow detection.
As can be seen from FIG. 2, TNFSF15 protein enhances F4/80 in the in vitro differentiation of bone marrow stem cells in the presence of either M-CSF (FIG. 3A) or GM-CSF (FIG. 2B) as compared to the corresponding buffer group + Macrophage proportion. From these results, TNFSF15 protein still plays a role in promoting differentiation of bone marrow stem cells into macrophages in the presence of M-CSF and GM-CSF.
EXAMPLE 3 Effect of TNFSF15 protein on macrophage progenitor/bone marrow derived macrophage proliferation and quantity
1) Acquisition of bone marrow derived macrophages
The same as in example 1.
2) Effect of TNFSF15 protein on macrophage progenitor/bone marrow derived macrophage proliferation and quantity
(1) Centrifuging at 4deg.C for 5min at 400g, discarding supernatant, re-suspending with (RPMI 1640+10% FBS+3 μg/mL TNFSF15 protein/corresponding buffer) medium, and adjusting cell concentration to 2×10 6 2 mL/well, added to a well plate (e.g., 6 well plate), and placed in a incubator (37 ℃ C., 5% CO) 2 ) Culturing for 3d,5d and 7d;
(2) Half-volume exchange of the culture medium is performed in 3d and 5d, and the cells are harvested until 3d,5d and 7d, and the CD11b-APC and F4/80-PE-Cy7 antibodies are marked. Incubate at room temperature for 30min, protected from light. Stream analysis CD11b + F4/80 + Fluorescence intensity of macrophage CFSE; and the cell numbers of the experimental and control groups were observed and recorded under a microscope at 7 d.
3) Analysis of results:
as can be seen from fig. 3A, TNFSF15 protein promotes proliferation of macrophage progenitors or differentiated macrophages in bone marrow as compared to the control group; from fig. 3B, the number of bone marrow cells in TNFSF15 protein group was significantly higher than that in control group. From this, TNFSF15 protein can promote proliferation of bone marrow stem cells/bone marrow-derived macrophages and increase the number of bone marrow stem cells/bone marrow-derived macrophages; binding to TNFSF15 protein in fig. 1 significantly increased the proportion of macrophages during differentiation, indicating that TNFSF15 significantly increased macrophage numbers.
Example 4: effect of TNFSF15 protein on macrophage progenitor cells, precursor cells and macrophages in tumor-bearing mouse bone marrow
1) Obtaining a high expression TNFSF15 cell line
(1) Construction of plvx-puro-TNFSF15 plasmid.
(2) After transfection of plvx-puro-TNFSF15, pspAX2 and pMD.2G into 293T cells, supernatant virus was collected and LLC cells were transfected. Further screening the cell strain which over expresses TNFSF15 by a limiting dilution method;
2) Establishment of TNFSF15 over-expression tumor model
(1) Resuscitating and culturing the constructed LLC over-expressing hTNFSF15 with a control LLC cell line until the LLC is grown to a certain quantity, digesting,washed twice with PBS and then 5X 10 5 The density per mL was resuspended in serum-free medium.
(2) 5X 10 mice per mouse 5 Cells were plated subcutaneously into C57BL/6J mice in an amount of 100. Mu.L. The rumen size was measured every two days, and the mice body weight was recorded until tumors were harvested. Rumen volume (mm) 3 ) =length×width×width/2.
3) Analysis of macrophage progenitor cell and precursor cell ratio in bone marrow
(1) The method for obtaining bone marrow cells was the same as in example 1;
(2) CD45, F4/80, CD11b, CD86, CD115, CD117 (ckit) antibodies were diluted in PBS at a ratio of 1:100 according to antibody instructions; another group was prepared by diluting Lin (CD 5, CD11B, CD19, CD45R/B220, ly6G/C (Gr-1), TER119, 7-4), sca1, cKit, CD16/32, CD34 antibodies in PBS at a ratio of 1:50, adding 100. Mu.L of antibody dilution to each tube of samples, gently beating and mixing with a pipette, and incubating at 4deg.C for 30min in the absence of light;
(3) Centrifuging at 400g for 5min, discarding the supernatant, and washing with 1mL of PBS;
(4) And (3) centrifuging at 400g for 5min, discarding the supernatant, adding 200 mu L of cell fixing solution into the fluorescent direct-labeling sample group, re-suspending, sieving with a 70 mu m sieve, and transferring to a flow tube to perform on-machine detection analysis.
(5) APC-CY 7-strepitavidin is diluted in PBS according to the ratio of 1:100, 100 mu L of antibody diluent is added to each tube of fluorescent sample, and the mixture is incubated for 20min at room temperature;
(6) Centrifuging at 400g for 5min, discarding the supernatant, and washing with 1mL of PBS;
(7) Centrifuging at 400g for 5min, discarding supernatant, adding 200uL cell fixative, resuspending, sieving with 70 μm sieve, transferring into flow tube, and detecting on machine.
4) Analysis of macrophage proportion in tumor
(1) Tumor tissue was removed from mice, minced with small scissors, added with pancreatin, digested in a 37 ℃ water bath for 30min, and shaken every 10 min.
(2) Neutralizing with a culture medium containing 10% serum, and centrifuging;
(3) The cells were washed once with PBS, gently ground on a stainless steel 70 μm cell screen with an EP tube cap, and washed with PBS to disperse the cells into individual cells.
(4) Centrifuging, cleaning with PBS once, adding red blood cell lysate, and lysing for 5min on ice, wherein the red blood cell lysate is colorless, and the color of the solution can show the number of red blood cells;
(5) Neutralizing with PBS containing 2% serum, washing with PBS once, and passing through 70 μm nylon membrane to obtain tumor single cell suspension;
(6) The cells were then transferred to a 1.5mL EP tube, centrifuged at 400g for 5min and the supernatant discarded;
(7) Diluting CD45, F4/80 and CD11b antibodies in PBS according to the antibody specification in a ratio of 1:100, adding 100 mu L of antibody diluent into the centrifuged cells, gently blowing and mixing the cells by a pipette, and incubating the cells for 30min at 4 ℃ in a dark place;
(8) Centrifuging at 400g for 5min, discarding the supernatant, and washing with 1mL of PBS;
(9) Centrifuging at 400g for 5min, discarding supernatant, adding 200 μl PBS, resuspending, sieving with 70 μm sieve, and transferring into flow tube for on-machine detection analysis.
5) Construction of Red bone marrow transplantation model mice
(1) C57BL/6 female mice with a weight of 20-22 g for 8-10 weeks were selected as bone marrow transplantation model recipient mice. 9.0Gy Cs137 radiation (time about 10 min).
(2) Six hours after irradiation, 6-8 weeks of tdTomato fluorescence C57BL6 male mice were injected with red fluorescence (td-Tomato) + ) Bone marrow cells. tdTomato is required for recipient mice + Bone marrow cells are about 5X 10 6 Individual cells.
(3) Two weeks later were observed if the mice had a body weight above 18g and were in good condition, i.e. the bone marrow transplant model was successfully constructed.
6) Construction and administration of bone marrow transplantation tumor-bearing mouse model
(1) Tumor cells digested into single suspension cells were washed three times in 10-fold volume PBS, 200g,5min, the supernatant was discarded, and the cells were resuspended to 5X 106mL in serum-free and double antibody-free medium -1 . Each mouse (two weeks after receiving bone marrow cell transplantation) was inoculated subcutaneously with 100 μl of the resuspended cells described above.
(2) On day four of tumor inoculation time, mice were randomly assigned to two groups for paraneoplastic administration when tumors could be observed visually. Mice were dosed once every two days and monitored for body weight and tumor volume (vernier caliper to measure longest and shortest diameters). Tumor tissues were harvested on day 19 after tumor inoculation, respectively.
(3) Experimental group: 5mg/kg TNFSF15 was injected aside each murine tumor; control group: an equal volume of buffer was injected aside each murine tumor.
(4) Tumor volume (mm) 3 ) =l×w×w/2; wherein L represents the longest diameter of the tumor and W represents the shortest diameter of the tumor.
7) Immunofluorescence analysis of tumor tissue
(1) And slicing and recovering to room temperature. The sliced tissue was fixed at-20deg.C with ice-methanol (-20deg.C pre-cooling) for 20min. Tap water was flushed for 5min with slow water flow, taking care to avoid damaging tissue sections.
(2) PAP stroke waterproof circle, for subsequent incubation of antibody preparation. Note that the waterproof ring should not be too small to prevent the antibody from volatilizing. Approximately 30-50 μl of liquid is required in the tissue within each waterproof loop.
(3) Triton-X100 (0.25%) was diluted with PBST, at room temperature for 30min. PBST was washed 3 times for 5min each. The chip is thrown off lightly, and the filter paper sucks the redundant liquid. When the waterproof ring is obviously closed and surrounds the tissue, 5% BSA (PBST dilution) is dripped into the waterproof ring to seal the nonspecific antigen, and the waterproof ring is placed in a wet box at room temperature for 1h.
(4) Spin-drying and no washing. The diluted F4/80 antibody (1:100 dilution) was added dropwise into the waterproof ring to completely cover the tissue. Placing in a wet box at 37deg.C for 1 hr.
(5) PBST was washed three times for 5min each. And (3) dripping diluted FITC fluorescent secondary antibody into the waterproof ring, and incubating at room temperature for 1h. PBST was washed three times for 5min each.
(6) Sealing piece: the filter paper was blotted off excess liquid and an anti-fluorescence quenching caplet (about 20 μl per tablet) was added dropwise, lightly capping the slide, taking care to avoid air bubbles and complete tissue desiccation.
(7) And observing the expression and the positioning of the target antibody in the tissue under a confocal microscope.
As can be seen from fig. 4, TNFSF15 decreased the ratio of macrophage progenitor cells to precursor cells in the bone marrow of mice (fig. 4A, 4B), increased the ratio of macrophages in tumors (fig. 4C); from fig. 5, it can be seen that TNFSF15 increases the number of macrophages differentiated from bone marrow stem cells in tumors (fig. 5A), and thus TNFSF15 protein still has an effect of promoting differentiation of bone marrow stem cells into macrophages in vivo (fig. 5B).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
- Use of tnfsf15 protein in the manufacture of a medicament or formulation for promoting differentiation and expansion of bone marrow stem cells into macrophages.
- 2. Use according to claim 1, characterized in that: the medicament or preparation increases the proportion of macrophages by promoting differentiation of bone marrow stem cells into macrophages.
- 3. Use according to claim 1, characterized in that: the medicament or formulation increases the number of macrophages by promoting proliferation of bone marrow stem cells/bone marrow derived macrophages.
- 4. A method for inducing bone marrow stem cells to differentiate and expand into macrophages in vitro by using TNFSF15 protein, which is characterized in that: TNFSF15 protein with the concentration not higher than 10 mug/mL is added into the amplification culture medium used in the method.
- 5. The method of claim 4, wherein: the method comprises the following steps:bone marrow cells were removed from mouse bone marrow, resuspended in amplification medium containing RPMI 1640, 15% FBS and TNFSF15 protein,cell concentration was adjusted to 2X 10 6 mL, adding into the pore plate, culturing in incubator at 37deg.C under 5% CO for 3-7d in each pore 2mL 2 。
- 6. The method of claim 5, wherein: the composition of the amplification culture medium is RPMI 1640, 15% FBS and 3 mug/mL TNFSF15 protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111532973.6A CN114209814B (en) | 2021-12-15 | 2021-12-15 | Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111532973.6A CN114209814B (en) | 2021-12-15 | 2021-12-15 | Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114209814A CN114209814A (en) | 2022-03-22 |
| CN114209814B true CN114209814B (en) | 2024-02-23 |
Family
ID=80702312
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111532973.6A Active CN114209814B (en) | 2021-12-15 | 2021-12-15 | Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114209814B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116036242A (en) * | 2023-03-08 | 2023-05-02 | 南开大学 | Application of TNFSF15 protein in preparation of medicines for promoting tumor vascular normalization |
| CN116173186A (en) * | 2023-03-08 | 2023-05-30 | 南开大学 | Method for promoting differentiation of myeloid cells into pericytes by TNFSF15 protein and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040391A1 (en) * | 2003-10-27 | 2005-05-06 | Murdoch Childrens Research Institute | Compositions and methods for differentiating stem cells |
| CN104975041A (en) * | 2014-12-19 | 2015-10-14 | 中山大学 | Method for promoting differentiation of monocyte to macrophages |
| CN105101965A (en) * | 2013-03-15 | 2015-11-25 | 安皮奥制药股份有限公司 | Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same |
| CN110770335A (en) * | 2017-12-11 | 2020-02-07 | 韩国疾病预防控制中心 | Differentiation of immune cells using pluripotent stem cells |
| CN111918967A (en) * | 2018-02-12 | 2020-11-10 | 科迪亚克生物科学公司 | Methods and compositions for macrophage polarization |
| CN113521252A (en) * | 2021-07-29 | 2021-10-22 | 南开大学 | Application of TNFSF15 protein as macrophage immunopotentiator and activation method thereof |
| CN113663056A (en) * | 2021-07-29 | 2021-11-19 | 南开大学 | Application of TNFSF15 protein as lymphocyte immunopotentiator and activation method thereof |
-
2021
- 2021-12-15 CN CN202111532973.6A patent/CN114209814B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040391A1 (en) * | 2003-10-27 | 2005-05-06 | Murdoch Childrens Research Institute | Compositions and methods for differentiating stem cells |
| CN105101965A (en) * | 2013-03-15 | 2015-11-25 | 安皮奥制药股份有限公司 | Compositions for the mobilization, homing, expansion and differentiation of stem cells and methods of using the same |
| CN104975041A (en) * | 2014-12-19 | 2015-10-14 | 中山大学 | Method for promoting differentiation of monocyte to macrophages |
| CN110770335A (en) * | 2017-12-11 | 2020-02-07 | 韩国疾病预防控制中心 | Differentiation of immune cells using pluripotent stem cells |
| CN111918967A (en) * | 2018-02-12 | 2020-11-10 | 科迪亚克生物科学公司 | Methods and compositions for macrophage polarization |
| CN113521252A (en) * | 2021-07-29 | 2021-10-22 | 南开大学 | Application of TNFSF15 protein as macrophage immunopotentiator and activation method thereof |
| CN113663056A (en) * | 2021-07-29 | 2021-11-19 | 南开大学 | Application of TNFSF15 protein as lymphocyte immunopotentiator and activation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114209814A (en) | 2022-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0812201B1 (en) | In vitro amplification of stem cells | |
| CN103937743B (en) | A kind of method that candidate stem cell is obtained using three-dimensional inducible system | |
| CN114209814B (en) | Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and expansion | |
| CN112280734A (en) | Preparation method of exosome and stem cell proliferation reagent containing exosome | |
| CN102643784B (en) | A kind of amplification in vitro system of hematopoietic stem/progenitor | |
| US20090305406A1 (en) | Method of cultivation of human mesenchymal stem cells, particularly for the treatment of non-healing fractures, and bioreactor for carrying out this cultivation method | |
| JP2010538681A (en) | Methods for extracting mesenchymal stem cells from human or animal embryos and their secretions | |
| CN109893541B (en) | Application of exosome derived from menstrual blood stem cells in preparation of medicine for treating intrauterine adhesion | |
| KR101430708B1 (en) | Stem cells from human salivary glands, a process for the preparation thereof, a culture solution thereof, and a use thereof for the treatment of salivary gland damage | |
| CN108685948A (en) | A kind of preparation method of new medical cell repair agent | |
| CN113667629A (en) | Tumor perivascular cell and separation method and application thereof | |
| CN115011561A (en) | Chimeric antigen receptor macrophage and preparation method and application thereof | |
| Bergwerf et al. | Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice | |
| CN106267413B (en) | AIDS plasma purification device | |
| CN106659560B (en) | The side group stem cell in gonad source | |
| CN106267425A (en) | AIDS immunoadsorption therapy instrument | |
| CN108495924A (en) | Method for generating mescenchymal stem cell | |
| CN107460170B (en) | Establishment and application of human pituitary adenoma cell line | |
| CN115399312A (en) | Preparation method of exosome normal-temperature storage protective agent in mesenchymal stem cell supernatant | |
| CN106267419A (en) | HIV immunologic purging device | |
| JP4809940B2 (en) | Method for amplifying mononuclear cells isolated from blood in vitro | |
| CN106178163A (en) | Acquired immune deficiency syndrome (AIDS) biological cell immunization therapy instrument | |
| CN106267409B (en) | AIDS biological therapy reactor | |
| CN106267417B (en) | AIDS therapeutic response device | |
| Karpenko et al. | Nestin-GFP transgene labels immunoprivileged bone marrow mesenchymal stem cells in the model of ectopic foci formation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |