CN114209814A - Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and amplification - Google Patents
Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and amplification Download PDFInfo
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Abstract
The invention provides application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and amplification, wherein after the TNFSF15 protein is used for obtaining the macrophages, related diseases are treated in an adoptive manner, and a new thought is provided for clinical treatment of the macrophage related diseases in the future.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and amplification.
Technical Field
Macrophages, which are important innate immune cells of the body, have high plasticity, are found in various tissues such as osteoclasts (bone), alveolar macrophages (lung), microglia (brain), tissue cells (connective tissue), kupffer cells (liver) and langerhans cells (skin), etc., and exhibit functional diversity. They play important roles in growth and development, maintenance of metabolic balance, tissue repair and immune response. Researches find that macrophage injection has a promoting effect on cardiac remodeling after acute myocardial infarction; transplantation of microglia (macrophages) which phagocytose unwanted substances or harmful proteins into the brain can reduce beta-amyloid which causes alzheimer's disease. Thus, various diseases can be treated by means of transplanted macrophages. However, due to the body's own immune function, it is preferable that the injected macrophages are derived from the individual itself in order to repel foreign species. Therefore, how to obtain a large amount of macrophages of the organism is an important research direction when the health of the organism is not influenced as much as possible. The marrow stem cells are an important source of macrophages, and by utilizing the proliferation characteristics of the marrow stem cells and the existing technology for separating the marrow cells, the marrow cells can be taken out and induced to differentiate towards the macrophages in vitro so as to obtain a large amount of macrophages. At present, the cytokines for inducing the differentiation of the bone marrow stem cells to the macrophages mainly comprise M-CSF and IL34, and because the macrophages for promoting the differentiation have specific phenotypes and have limitations in being applied to various disease models, the search for new proteins for promoting the differentiation of the bone marrow stem cells to the macrophages is a very important subject.
Tumor necrosis factor superfamily member 15(Tumor necrosis factor super family-15, TNFSF15, also known as TL1A) is a blood vessel growth inhibitory factor secreted mainly by mature vascular endothelial cells. Researches find that the compound is not only a blood vessel negative regulation factor, and can inhibit angiogenesis and tumor growth in tumors; can also be used as an immune activator to promote T cell activation and dendritic cell maturation. At present, the function of TNFSF15 protein in the differentiation of bone marrow stem cells into macrophages has not been reported.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention aims to provide a use of TNFSF15 protein for promoting differentiation of bone marrow stem cells into macrophages and proliferation thereof in vitro, so as to solve the above-mentioned deficiencies of the prior art, and the protein can promote differentiation of bone marrow stem cells into macrophages, increase the number of macrophages, and provide a treatment scheme for diseases related to macrophages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a use of TNFSF15 protein in the preparation 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 to 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, the TNFSF15 protein can promote differentiation of bone marrow stem cells to macrophages, either independently 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 amplification medium used in the method is supplemented with TNFSF15 protein at a concentration of not more than 10 μ g/mL.
Preferably, the method comprises the steps of:
removing bone marrow cells from mouse bone marrow, resuspending in culture medium containing RPMI 1640, 15% FBS and TNFSF15 protein, and adjusting cell concentration to 2 × 106Adding into a pore plate (six-pore plate) with 2mL per pore, and culturing in incubator at 37 deg.C and 5% CO for 3-7 days2。
Preferably, the composition of the amplification medium in the method is RPMI 1640, 15% FBS and 3. mu.g/mL TNFSF15 protein.
TNFSF15 protein was detected during in vitro culture to increase macrophage proportion during differentiation of bone marrow stem cells and was time and concentration dependent.
Compared with the prior art, the invention has the following advantages:
(1) the TNFSF15 protein can promote differentiation and expansion of bone marrow stem cells to macrophages, and can adoptively treat related diseases after macrophages are obtained in vitro.
(2) TNFSF15 protein can promote differentiation of bone marrow stem cell to macrophage, has stable effect, is not easily affected by other factors, and can be used in vivo.
(3) The TNFSF15 purification method is simple and mass, has low economic cost, can be used for combined treatment with other treatment modes, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of: the influence of the TNFSF15 protein on the differentiation of in vitro bone marrow stem cells to macrophages, wherein A is the change of macrophage proportion after the bone marrow stem cells are treated by TNFSF15 protein with different concentrations for 7 d; and B is the change of macrophage proportion after TNFSF15 protein treats the bone marrow stem cells for different days.
FIG. 2 is a diagram of: the effect of TNFSF15 protein on the rate of macrophage differentiation in the presence of macrophage colony stimulating factor M-CSF and granulocyte-macrophage colony stimulating factor GM-CSF.
FIG. 3 is a diagram of: the effect of TNFSF15 protein on the proliferation and quantity of bone marrow stem cells/bone marrow-derived macrophages, wherein A is the proliferation change of bone marrow stem cells/bone marrow-derived macrophages after the bone marrow cells are treated by TNFSF15 protein; and B is the change of the cell number of the bone marrow cells after the TNFSF15 protein treatment for 7 d. .
FIG. 4 is a diagram of: effects of TNFSF15 protein on macrophage progenitors, precursors in tumor-bearing mouse bone marrow and macrophages in tumors.
FIG. 5 is a diagram of: effect of TNFSF15 protein on macrophage number of bone marrow stem cell differentiation in tumors.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. 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 the following examples.
Example 1 Effect of TNFSF15 protein alone on in vitro differentiation of bone marrow stem cells into macrophages
1) Obtaining bone marrow cells
(1) And (3) preparation: placing clean surgical instruments (at least two small scissors and small forceps), gauze (8 layers, 20 pieces), and nylon membrane (70 μm pore diameter, 10 pieces) in a lunch box, autoclaving at 120 deg.C for 20 min;
(2) and taking out bone marrow cells: the male mice were sacrificed for 5 weeks, the hind leg bones were removed, and three connected bones were removed from one leg, the surrounding tissues were removed with gauze, the joints at both ends of the bones were cut off, and the bone marrow cells were flushed out with PBS aspirated by a 1mL syringe. Collecting the flushed bone marrow cells together to form red sheets, and blowing and beating the red sheets by using a 1mL syringe and a pipette gun to disperse the red sheets into single cell suspension;
(3) filtering the collected bone marrow cells with a 70-micron nylon net, centrifuging the filtered bone marrow cells for 5min at 400g and 4 ℃, and removing supernatant;
(4) and lysing erythrocytes: adding 2mL of erythrocyte lysate, resuspending, placing on ice, adding 10mL of PBS solution containing 2% FBS after 3min, and uniformly mixing;
(5) centrifuging at 4 ℃ for 5min at 400g, removing the supernatant, adding 10mL of 2% FBS PBS solution, and mixing uniformly;
(6) counting: 10. mu.L of the cell suspension was aspirated, diluted by a certain factor, and counted under a microscope using a cell counting plate. 10mL of cells, total of four large cells/4X 104X multiple × 10 mL;
2) effect of TNFSF15 protein on differentiation of bone marrow Stem cells into macrophages
(1) The cell suspension was centrifuged at 400g for 5min at 4 ℃ and the supernatant was discarded.
(2) The cell pellet was resuspended in (RPMI 1640+ 15% FBS) medium and the cell concentration was adjusted to 2X 1062mL per well, into a well plate (e.g., a 6 well plate);
(3) and a, setting a control group and an experimental group for experiments, wherein the experimental group adds TNFSF15 protein of 0.3 mu g/mL, 1 mu g/mL, 3 mu g/mL and 5 mu g/mL into the culture medium, and the control group adds buffer with corresponding dose into the culture medium. Mixing the mixture by gentle shaking, and putting the mixture into an incubator at 37 ℃ for culture. Half-volume liquid change is carried out on the culture medium at 3d and 5d, and F4/80-PE-Cy7 antibodies are marked when cells are harvested at 7 d; b, control group and 3 ug/mL experimental group, cells were harvested at 3d, 5d, and 7d, respectively, and labeled F4/80-PE-Cy7 antibody and CD11b-FITC antibody. 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 assay.
3) Phenotypic analysis of bone marrow-derived macrophages induced to differentiate by TNFSF15 protein
(1) The cell suspension was centrifuged at 400g for 5min at 4 ℃ and the supernatant was discarded.
(2) The cell pellet was resuspended in (RPMI 1640+ 15% FBS) medium and the cell concentration was adjusted to 2X 1062mL per well, into a well plate (e.g., a 6 well plate);
(3) and an experimental group and a control group, wherein the experimental group adds TNFSF15 protein of 3 mu g/mL into the culture medium, and the control group adds buffer with corresponding dose into the culture medium. Mixing the mixture by gentle shaking, and putting the mixture into an incubator at 37 ℃ for culture. Half-volume change of the culture medium is carried out in 3d and 5d of culture, and cells are harvested in 3d, 5d and 7d of culture and labeled with F4/80-PE-Cy7 antibody, MHC2-FITC and CD206-APC antibody. Incubate at room temperature for 30min, protect from light, centrifuge, and discard unbound antibody. And resuspended with 200. mu.L of cell fixative for flow assay.
3) And (4) analyzing results:
as can be seen from FIG. 1, TNFSF15 protein increased the proportion of macrophages during the in vitro differentiation of bone marrow stem cells and was shown to be concentration (FIG. 1A) and time (FIG. 1B) dependent, compared to the corresponding buffer group.
Example 2 Effect of TNFSF15 protein on differentiation of myeloid-derived macrophages in the Presence of macrophage-associated colony stimulating factor
1) Obtaining bone marrow stem cells
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) Centrifuging at 400g for 5min at 4 deg.C, discarding the supernatant, resuspending in (RPMI 1640+ 15% FBS +50ng/mL M-CSF or GM-CSF factor) culture medium, adjusting cell concentration to 2 × 1062mL per well, into a well plate (e.g., a 6 well plate);
(2) and setting a control group and an experimental group, wherein the experimental group is added with TNFSF15 protein of 3 mu g/mL in the culture medium, and the control group is added with buffer of corresponding dosage in the culture medium. Mixing the mixture by gentle shaking, and putting the mixture into an incubator at 37 ℃ for culture. Half-volume change was performed with the corresponding medium at 3d of culture until 5d of harvest, labeled with F4/80-PE-Cy7 antibody. Incubate at room temperature for 30min, and protect from light. The unbound antibody was discarded by centrifugation. And resuspended with 200. mu.L of cell fixative for flow assay.
As shown in FIG. 2, TNFSF15 protein was able to increase F4/80 ratio during differentiation of bone marrow stem cells in vitro in the presence of M-CSF (FIG. 3A) or GM-CSF (FIG. 2B) compared to the corresponding buffer group+Proportion of macrophages. From this, it was found that TNFSF15 protein still exerts an effect of 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) Obtaining bone marrow-derived macrophages
Same as in example 1.
2) Effect of TNFSF15 protein on macrophage progenitor/bone marrow derived macrophage proliferation and quantity
(1) Centrifuging at 400g for 5min at 4 deg.C, discarding the supernatant, resuspending in (RPMI 1640+ 10% FBS + 3. mu.g/mL TNFSF15 protein/corresponding buffer) culture medium, adjusting the cell concentration to 2X 1062mL per well, into a well plate (e.g., 6 well plate), and in an incubator (37 ℃, 5% CO)2) Culturing for 3d, 5d and 7 d;
(2) and half-volume changing is carried out on the culture medium in 3d and 5d, and when the cells are harvested in 3d, 5d and 7d, the CD11b-APC and F4/80-PE-Cy7 antibodies are marked. Incubate at room temperature for 30min, and protect from light. Flow assay CD11b+F4/80+Fluorescence intensity of macrophage CFSE; and the number of cells in the experimental and control groups was observed and recorded under a microscope at 7 d.
3) And (4) analyzing results:
as can be seen from fig. 3A, TNFSF15 protein promoted proliferation of macrophage progenitors or differentiated macrophages in bone marrow compared to the control group; from fig. 3B, the number of bone marrow cells in the TNFSF15 protein group was significantly higher than that in the control group. Therefore, the TNFSF15 protein can promote the proliferation of bone marrow stem cells/bone marrow-derived macrophages and increase the number of the bone marrow stem cells/bone marrow-derived macrophages; combining TNFSF15 protein in fig. 1 significantly increased macrophage proportion during differentiation, indicating that TNFSF15 significantly increased macrophage numbers.
Example 4: effect of TNFSF15 protein on macrophage progenitors, precursor cells and macrophages in tumors in bone marrow of tumor-bearing mice
1) Obtaining a cell line with high expression of TNFSF15
(1) The plvx-puro-TNFSF15 plasmid was constructed.
(2) After plvx-puro-TNFSF15, pspAX2 and pMD.2G were transfected into 293T cells, the supernatant virus solution was collected and LLC cells were transfected. Further screening an over-expressed TNFSF15 cell strain by a limiting dilution method;
2) establishment of TNFSF15 overexpression tumor model
(1) Recovering and culturing LLC of the over-expressed hTNFSF15 and the control LLC cell line to certain amount, digesting, washing twice with PBS, and further washing with 5 × 105The density per mL was resuspended in serum-free medium.
(2) 5X 10 per mouse5At a rate of 100. mu.L, cells were seeded subcutaneously in C57BL/6J mice. Tumor size was measured every two days and mouse body weight was recorded until tumors were harvested. Tumor volume (mm)3) Length × width/2.
3) Analysis of macrophage progenitor 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 the antibody specification; the other group is prepared by diluting Lin (CD5, CD11B, CD19, CD45R/B220, Ly6G/C (Gr-1), TER119,7-4), Sca1, cKit, CD16/32 and CD34 antibodies in PBS according to the ratio of 1:50, adding 100 mu L of antibody diluent into each tube of sample, gently blowing and uniformly mixing by using a pipette gun, and incubating for 30min in a dark place at 4 ℃;
(3) centrifuging at 400g for 5min, discarding the supernatant, and adding 1mL PBS for washing;
(4) centrifuging at 400g for 5min, discarding the supernatant, adding 200 μ L of cell fixing solution into the fluorescent direct-labeled sample group, resuspending, sieving with a 70 μm sieve, transferring to a flow tube, and performing detection and analysis on the machine.
(5) APC-CY7-Streptavidin was diluted at a ratio of 1:100 in PBS, 100. mu.L of antibody dilution was added to each tube of fluorescent labeling sample, and incubated at room temperature for 20 min;
(6) centrifuging at 400g for 5min, discarding the supernatant, and adding 1mL PBS for washing;
(7) centrifuging at 400g for 5min, discarding the supernatant, adding 200uL of cell fixing solution for resuspension, sieving with a 70 μm sieve, transferring to a flow tube, and performing machine detection.
4) Analysis of macrophage proportion in tumors
(1) The tumor tissue was removed from the mice, minced with small scissors, added with pancreatin, digested in a 37 ℃ water bath for 30min, shaken every 10 min.
(2) After digestion, neutralizing the mixture by using a culture medium containing 10% of serum, and centrifuging;
(3) washed once with PBS, then gently ground on a stainless steel 70 μm cell screen with an EP tube lid, and washed with PBS to disperse into individual cells.
(4) Centrifuging, washing once with PBS, adding erythrocyte lysate, and performing lysis on ice for 5min, wherein the quantity of erythrocytes can be shown by the shade of the color of the erythrocyte lysate because the erythrocyte lysate is colorless;
(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) then transferring the cells into a 1.5mL EP tube, centrifuging at 400g for 5min, and discarding the supernatant;
(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 uniformly mixing the cells by using a pipette gun, and incubating the cells for 30min at 4 ℃ in a dark place;
(8) centrifuging at 400g for 5min, discarding the supernatant, and adding 1mL PBS for washing;
(9) centrifuging at 400g for 5min, discarding the supernatant, adding 200 μ L PBS for resuspension, sieving with 70 μm sieve, transferring to flow tube, and performing detection and analysis on the machine.
5) Construction of Red bone marrow transplantation model mouse
(1) And selecting a C57BL/6 female mouse with the body weight of 20-22 g for 8-10 weeks as a bone marrow transplantation model receptor mouse. 9.0Gy of Cs137 radiation (time approximately 10 min).
(2) Six hours after the radiation, red fluorescence (td-Tomato) of tdTomato fluorescence C57BL6 male mice was injected into tail vein for 6-8 weeks+) Bone marrow cells. The recipient mouse requires tdTomato+Bone marrow cells about 5X 106And (4) cells.
(3) Then, two weeks are observed, and if the body weight of the mouse is more than 18g and the state is good, the bone marrow transplantation model is successfully constructed.
6) Construction and administration of bone marrow transplanted tumor-bearing mouse model
(1) Washing the tumor cells which are digested into single suspension cells in PBS with 10 times of volume for three times, 200g and 5min, abandoning supernatant, adding serum-free and double-antibody-free culture medium to resuspend the cells to 5 multiplied by 106mL-1. Each mouse (two weeks after receiving bone marrow cell transplantation) was inoculated subcutaneously with 100. mu.L of the above resuspended cells.
(2) And on the fourth day of tumor inoculation time, when tumors can be observed by naked eyes, the mice are randomly distributed into two groups for paratumoral administration. Mice were dosed every two days and monitored for body weight and tumor volume (longest and shortest diameters measured with a vernier caliper). Tumor tissues were harvested on day 19 post tumor inoculation, respectively.
(3) And experiment group: 5mg/kg TNFSF15 was injected para-tumorally to each mouse; control group: each mouse was injected peritumorally with an equal volume of buffer.
(4) Tumor volume (mm)3) L × 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) The sections were returned to room temperature. The sliced tissue was fixed with ice methanol at-20 deg.C (-pre-cooled at-20 deg.C) for 20 min. The water is slowly flushed for 5min, and the tissue slices are prevented from being damaged.
(2) PAP strokes are taken to form waterproof rings to prepare for subsequent antibody incubation. Note that the waterproof ring should not be too small to prevent antibody volatilization. Approximately 30-50 μ L of liquid is required in the tissue within each waterproof ring.
(3) PBST diluted Triton-X100 (0.25%), room temperature, 30 min. PBST washing 3 times, each time for 5 min. Lightly throw the slices and filter paper sucks away the excess liquid. When the waterproof ring obviously forms a closed surrounding tissue, 5% BSA (PBST dilution) is dripped into the waterproof ring to block the nonspecific antigen, and the waterproof ring is placed in a wet box at room temperature for 1 hour.
(4) Spin-drying and no washing. The diluted F4/80 antibody (1:100 dilution) was added dropwise to the water-tight ring to completely cover the tissue. Placing in a wet box at 37 deg.C for 1 h.
(5) PBST was washed three times for 5min each. And (3) dropwise adding the diluted FITC fluorescent secondary antibody into the waterproof ring, and incubating at room temperature for 1 h. PBST was washed three times for 5min each.
(6) And sealing sheet: the filter paper sucks away the excess liquid, adds the anti-fluorescence quenching sealing tablet (about 20. mu.L of each tablet) dropwise, and covers the glass slide lightly, taking care to avoid 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 in fig. 4, TNFSF15 decreased the proportion of macrophage progenitors and precursors in mouse bone marrow (fig. 4A, 4B), and increased the proportion of macrophages in tumors (fig. 4C); as shown in fig. 5, TNFSF15 increased the number of macrophages differentiated from bone marrow stem cells in tumors (fig. 5A), and thus TNFSF15 protein still promoted differentiation of bone marrow stem cells into macrophages in vivo (fig. 5B).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
- Use of TNFSF15 protein in the preparation 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 medicine or preparation increases the proportion of macrophages by promoting the differentiation of bone marrow stem cells to macrophages.
- 3. Use according to claim 1, characterized in that: the medicine or preparation increases the number of macrophages by promoting the proliferation of bone marrow stem cells/bone marrow-derived macrophages.
- 4. A method for inducing differentiation and expansion of bone marrow stem cells to macrophages in vitro by using TNFSF15 protein is characterized in that: the amplification medium used in the method is supplemented with TNFSF15 protein at a concentration of no more than 10. mu.g/mL.
- 5. The method of claim 1, wherein: the method comprises the following steps:removing bone marrow cells from mouse bone marrow, resuspending with amplification medium containing RPMI 1640, 15% FBS and TNFSF15 protein, and adjusting cell concentration to 2 × 106Adding into a pore plate (six-pore plate) with 2mL per pore, and culturing in incubator at 37 deg.C and 5% CO for 3-7 days2。
- 6. The method of claim 1, wherein: the composition of the amplification medium is RPMI 1640, 15% FBS and 3 mug/mL TNFSF15 protein.
- The application of the TNFSF15 protein in preparing an antitumor drug, wherein the antitumor drug contains TNFSF15 protein, and the TNFSF15 protein achieves an antitumor effect by promoting bone marrow stem cells to be differentiated into macrophages and amplified.
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Cited By (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
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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 |
Cited By (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 |
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