CN114287390A - Method for establishing mouse autoimmune myelofibrosis model - Google Patents
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Abstract
The invention provides a method for establishing a mouse autoimmune myelofibrosis model, and relates to the technical field of biology. The establishing method of the invention comprises the following steps: using C57BL/6 mouse as donor, collecting bone marrow of donor, washing bone marrow cavity with buffer solution, collecting and filtering washing solution to obtain single cell suspension, removing CD4+T cells and CD8+T cells to obtain a T cell-depleted bone marrow cell suspension; taking a spleen of a mouse, grinding the spleen, washing the ground spleen by using a buffer solution, collecting and filtering washing liquid to prepare a single cell suspension to obtain a spleen cell suspension; taking a BALB/c mouse as a mouse, carrying out whole-body ionizing radiation on the mouse, inputting the bone marrow cell suspension and the spleen cell suspension without the T cells into the mouse, and feeding for a period of time to obtain the mouse autoimmune myelofibrosis model. The invention canThe mouse autoimmune myelofibrosis model is successfully established, and the modeling success rate is high and the repeatability is good.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for establishing a mouse autoimmune myelofibrosis model.
Background
Bone marrow is the origin of the hematopoietic lineage and is also the microenvironment upon which hematopoietic stem cells live. Primary myelofibrosis is a hematopoietic disease, is often seen in the elderly, is characterized by myeloreticular fibroplasia, and is manifested by abnormal quantity and function of bone marrow hematopoietic stem cells, anemia, splenomegaly, extrahepatic and splenic hematopoiesis and other symptoms. Most patients with myelofibrosis incorporate mutations in genes such as JAK2, MPL, CALR, etc. It is considered that the primary myelofibrosis pathogenesis is mainly caused by differentiation dysfunction of hematopoietic stem cell gene mutation, megakaryocyte dysfunction activation and bone marrow stromal cell mass collagen production.
Autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, sjogren's syndrome, etc., also have myelofibrosis and hematopoietic dysfunction problems. Recent studies have shown that autoimmune myelofibrosis is a unique pathological feature different from primary myelofibrosis, and does not accompany gene mutation and megakaryocyte proliferation, but rather, a large amount of lymphocyte infiltration and autoantibody deposition occur in the bone marrow.
At present, the research on autoimmune myelofibrosis is less, and the pathogenesis of the autoimmune myelofibrosis is not clear, so that the establishment of a suitable animal model for researching the pathogenesis and a treatment scheme has important significance. Most of previous bone marrow fibrosis models reported in the research, such as JAK2V617 mice, Mybboo mice and the like, are primary bone marrow fibrosis models, and do not relate to autoimmune bone marrow fibrosis models.
Disclosure of Invention
Therefore, it is necessary to provide a method for establishing a mouse autoimmune myelofibrosis model aiming at the above problems, the established mouse model has obvious autoimmune myelofibrosis performance, and the method has high success rate of modeling and good repeatability.
A method for establishing a mouse autoimmune myelofibrosis model comprises the following steps:
preparing a bone marrow cell suspension: collecting bone marrow of mouse, washing marrow cavity with buffer solution, collecting and filtering washing solution to obtain single cell suspension, removing CD4+T cells and CD8+T cells to obtain a T cell-depleted bone marrow cell suspension;
preparation of spleen cell suspension: taking a spleen of a mouse, grinding the spleen, washing the ground spleen by using a buffer solution, collecting and filtering washing liquid to prepare a single cell suspension to obtain a spleen cell suspension;
treatment of the mice: and (3) carrying out whole-body ionizing radiation on the mouse, inputting the bone marrow cell suspension and the spleen cell suspension without the T cells into the mouse body, and feeding for 40-60 days to obtain the mouse autoimmune myelofibrosis model.
The establishment method successfully establishes the mouse autoimmune myelofibrosis model by infusing the small dose spleen cells and bone marrow cells of the mice supplied with incompatible transplanted MHC (major histocompatibility complex) antigens back to the mice subjected to lethal dose radiotherapy, and has the advantages of high success rate, good repeatability and simple and easy practical process.
In one embodiment, the donor mice are 7-9 week old mice.
In one embodiment, the subject is a 7-9 week old mouse.
In one embodiment, the cell concentration of the T-cell depleted bone marrow cell suspension is 0.8-1.2 × 107one/mL.
In one embodiment, the cell concentration of the spleen cell suspension is 0.8-1.2 × 106one/mL.
In one embodiment, the total radiation dose of the ionizing radiation is 750-850 cGy and the time duration is 8-10 h.
In one embodiment, the number of bone marrow cells delivered to the subject is 4.8-5.2 × 106The number of spleen cells per mouse is 0.48-0.52 × 106One/only.
Compared with the prior art, the invention has the following beneficial effects:
the method for establishing the mouse autoimmune myelofibrosis model successfully constructs the mouse autoimmune myelofibrosis model by infusing the small-dose splenocytes supplied to the mouse with incompatible transplanted MHC antigens and bone marrow cells into a mouse body subjected to lethal dose radiotherapy, and has the advantages of high success rate, good repeatability and simple and easy time process. The mouse model constructed by the invention can provide an excellent experimental platform for clinical drug screening and treatment of autoimmune myelofibrosis.
Drawings
FIG. 1 shows the pathological results of bone marrow in the mouse in Experimental example 1.
FIG. 2 shows the results of the mouse lymphocyte infiltration assay in Experimental example 2.
FIG. 3 shows the results of detection of the mouse autoantibodies in Experimental example 3.
Detailed Description
To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
A method for establishing a mouse autoimmune myelofibrosis model comprises the following steps:
1. transplantation pretreatment of mouse
Transporting the model mouse to an animal irradiation chamber by using a special transportation cage for SPF animalsIn the clean area, BALB/c (H-2) with age of 8 weeks is selectedkd) Mice were used as recipients and placed side by side in sterile metal-free radiotherapy cassettes. Horizontally placing a radiotherapy box on an irradiation table of a Varian 600c linear accelerator, and setting irradiation parameters: the distance between the source skin and the target skin is 98cm, the irradiation depth is 2cm, the angle of the arm support is 0 degree, and the radiation field is opened to be large enough to target all mice. The dose rate was set at 100cGy per minute and a total dose of 850cGy was irradiated. After the sterilization, the surface of the radiotherapy box and the inner side wall of the animal transportation cage are disinfected by alcohol, and the animal transportation cage is properly sealed and then transported back to the SPF animal feeding room.
2. Treatment for rats
Healthy males C57BL/6 (H-2) with the age of 8 weeks are selectedkb) Mice, sacrificed by cervical dislocation, were completely immersed in a beaker of 75% alcohol for 7 minutes. Taking out the wood plate prepared in advance, and after disinfection, laying on a table top to be used as an operation table. The mice sterilized by alcohol are fixed on the pine board in the supine position by pins. A long vertical incision is made along the midline of the abdomen of a mouse by using sterile ophthalmic scissors and forceps, and then the skin is cut along the long axis of four limbs, so that the abdomen and the four limbs are fully exposed. The other set of ophthalmic scissors and forceps was replaced, the peritoneum was cut open, the abdominal cavity was opened, the intact spleen was removed after carefully peeling off the fat and connective tissue around the spleen, placed in a 50ml sterile centrifuge tube, added with pre-cooled transplantation buffer and stored in an ice box. The humerus, femur and tibia on both sides are cut off, muscle and fascia tissues attached to the long bones of the limbs are carefully stripped off along the bone edges by using an ophthalmic scissors and forceps, the obtained product is placed in a 50ml sterile centrifuge tube, precooled transplantation buffer solution is added, and the obtained product is placed in an ice box for standby.
3. Isolation of rat bone marrow cells
A. In a sterile superclean bench, open a 50ml centrifuge tube in which the mouse limb long bone is collected, carefully clamp the bone with sterile forceps, place in a sterile petri dish, add an appropriate amount of transplant buffer, and wash twice.
B. The metaphysis of the two ends of the long bone is cut off with ophthalmic scissors, and the marrow cavity is exposed. One hand held sterile forceps was used to grasp the long bone, and the other hand held 20ml syringe was used to aspirate the graft buffer. Inserting No. 5 fine needle into one end of marrow cavity, slowly pushing the syringe needle core, continuously flushing the marrow cavity with transplantation buffer solution under positive pressure, and stopping until the marrow cavity becomes white and transparent. All of the flushed liquid was collected and filled into a new 50ml centrifuge tube. After one long bone is treated, the next long bone is treated until all the long bones are washed. The tube containing the bone marrow-washing solution was placed in a centrifuge, centrifuged at 1500rpm for 5 minutes at 4 ℃ and the supernatant was removed.
C. The cell pellet left after centrifugation was resuspended by adding a small amount of the transplantation buffer, and the bone marrow tissue in the cell suspension was gently ground in a culture dish of 70 μm sterile cell mesh using a rubber tip of a stylet of a 5ml sterile syringe. The collected bone marrow cell suspension was filtered through a 70 μm cell screen, and then centrifuged at 1500rpm for 5 minutes at 4 ℃ to remove the supernatant.
D. The cell pellet was resuspended in 90. mu.L of MACS buffer, 30. mu.L each of CD4 and CD8 magnetic beads were added, mixed well and incubated at 4 ℃ for 15 minutes. Cells were washed by adding 2mL of MACS buffer and centrifuged at 4 ℃ at 1500rpm for 10 minutes, and the supernatant was removed. 500 μ L of MACS buffer resuspended cell pellet.
E.500. mu.L of MACS buffer washed the column, the cell suspension was added dropwise to the column placed in the magnetic field of the MACS sorter, the column was washed 3 times with 500. mu.L of MACS buffer, all the buffer passed through the column was collected, centrifuged at 4 ℃ at 1500rpm for 10 minutes, and the supernatant was removed.
F. The TCD-BM precipitation is resuspended in the transplantation buffer solution, repeatedly blown and fully mixed, 10 microliter is taken and placed in a new EP tube, 0.4 percent trypan blue working solution is added and fully mixed, and then the counting of the nucleated living cells is carried out in a cell counting plate under a microscope. Repeat 3 times and average. Adjusting the cell concentration of the bone marrow cell suspension to 1 × 10 according to the total number of the bone marrow cells7one/mL. The T-cell depleted bone marrow cell suspension (TCD-BM) was placed in an ice box for future use.
4. Isolation of splenocytes from mice
A. In a sterile clean bench, open a 50mL centrifuge tube containing the spleen. The spleen was gently removed with sterile ophthalmic forceps, placed in a sterile petri dish, washed twice with fresh transplantation buffer, and placed on a 70 μm sterile cell screen, taking care to keep the spleen tissue moist.
B. The spleen was lightly ground on a screen using a rubber tip of a 5mL sterile syringe. The filtered spleen cell suspension was collected into a new 50mL centrifuge tube and the screen was repeatedly washed with multiple additions of transplantation buffer until the spleen was completely dissociated leaving only white connective tissue on the screen. The tube was centrifuged at 1500rpm for 5 minutes in a 4 ℃ centrifuge to remove the supernatant.
C. Resuspending the splenocytes again with the transplantation buffer solution, mixing well, placing 10 μ L of splenocyte suspension in a new EP tube, adding 0.4% trypan blue working solution, mixing well again, counting the viable cells under microscope using cell counting plate, the cell concentration of splenocyte suspension is 1 × 106one/mL. Placing the spleen cell suspension in an ice box for later use.
5. Graft return
After receiving TBI for 8-10 hours, the mice are infused with bone marrow and spleen cell suspension from C57BL/6 donor mice back into BALB/C mice by tail vein injection. Each mouse was reinfused with 0.5ml of T cell-depleted bone marrow cell suspension and 0.5ml of spleen cell suspension, the number of bone marrow cells was 5X 106Spleen cells number 0.5X 106And (4) respectively. And feeding for 40 days to obtain model mice, continuing feeding normally, and observing pathological conditions of the mice.
The successful modeling is marked by: bone marrow massson staining and reticular fiber silver staining of mice showed massive collagen fiber and reticular fiber deposition in the bone marrow. By adopting the method for modeling, the success rate of different batches is over 60 percent.
Comparative example 1
A method for establishing a mouse autoimmune myelofibrosis model, which is substantially the same as in example 1, except that 0.5mL of T-cell depleted bone marrow cell suspension is returned, but spleen cell suspension is not returned.
Experimental example 1
And (5) carrying out pathological detection on the bone marrow of the mouse.
1. The bones of the limbs of the mice were removed and placed in a centrifuge tube containing 4% paraformaldehyde and fixed for 12 hours. After fixation, residual paraformaldehyde was washed with PBS. The bone after fixation is placed into 20% EDTA decalcification solution for decalcification for 3 days. During this period, a 1ml syringe needle can be used to penetrate the bone of the mouse, and decalcification is completed if the needle can puncture the cortical bone without resistance. The bones were washed with PBS to remove residual decalcifying solution. The bone after decalcification is dehydrated, transparent and the like, and then is embedded in paraffin for subsequent staining.
Masson staining. And (3) soaking the slices in a potassium dichromate solution overnight, and then washing with double distilled water. And mixing the ferritin dyeing A and the ferritin dyeing liquid B in equal volume to obtain the ferritin dyeing liquid. And (3) soaking the specimen in the iron lignin dye solution for 3 minutes. And (5) cleaning with double distilled water after dyeing is finished. And (3) putting the cleaned slide into a dye vat filled with 70% alcohol (prepared by 1% hydrochloric acid) for differentiation, and cleaning the slide with double distilled water after the differentiation is finished. The iron lignin dye liquor A and the iron lignin dye liquor B are prepared immediately after use, and the dyeing capability is generally used after 24 hours of preparation. The differentiation time using 70% ethanol (formulated in 1% hydrochloric acid) should be determined according to specimen thickness and tissue type. And (3) placing the specimen into a dye vat with ponceau dyeing, soaking for 5-10 minutes, and then quickly rinsing by using double distilled water. And (3) spin-drying the residual liquid on the specimen as much as possible, and soaking the specimen in the phosphomolybdic acid aqueous solution for 1-2 minutes. When phosphomolybdic acid is differentiated, the myofibers and cellulose appear red and the collagen fibers appear light pink when observed under a microscope. And (3) directly putting the treated specimen into aniline blue dye liquor for dyeing for 3-6 minutes. Differentiating with 1% glacial acetic acid, and dewatering the differentiated specimen in 2 glass jars filled with anhydrous ethanol. The specimens are sequentially put into a dye vat containing absolute ethyl alcohol, normal butanol and dimethylbenzene, and the duration of each time lasts for 5 minutes. And after the transparence is finished, taking the specimen out of the xylene, slightly airing the specimen at room temperature or wiping redundant xylene completely by using a piece of lens wiping paper, and dropwise adding a little neutral resin sealing sheet. And (5) observing and image-collecting analysis under an upright microscope. Histopathological evaluation of bone marrow the area of collagen fiber deposition was assessed as a proportion of the total area of the marrow cavity as indicated by Masson staining.
3. And (4) silver dyeing of the reticular fibers. The specimen is placed on a staining rack, and the prepared Gordon-Sweets oxidant is dripped on the specimen to be fully oxidized. After 5 minutes the tissue oxidation was complete and it was placed in a dye vat and washed with double distilled water. And (4) bleaching the cleaned specimen in an oxalic acid solution dye vat for 1-2 minutes. After bleaching was completed, washing was carried out with flowing distilled water for 2 minutes. And (3) putting the specimen into an ammonium ferric sulfate solution, and soaking for 5 minutes. After the soaking, the paper was washed with distilled water and stained with Gomori silver ammonia solution for 3 minutes. And washing with distilled water after dyeing is finished. After the washing, the ammonia silver is reduced by using a Gomori reducing agent to develop color. And washing with distilled water for 10 minutes. And (6) transparent sealing sheets. The specimens are sequentially put into a dye vat containing absolute ethyl alcohol, normal butanol and dimethylbenzene, and the duration of each time lasts for 5 minutes. And after the transparence is finished, taking the specimen out of the xylene, slightly airing the specimen at room temperature or wiping redundant xylene completely by using a piece of lens wiping paper, and dropwise adding a little neutral resin sealing sheet. And (5) observing and image-collecting analysis under an upright microscope. The staining results showed that black was reticular fibers, yellow or tan was collagen fibers, and brown or dark brown was the nucleus. Histopathological evaluation of bone marrow was assessed as the proportion of area of reticular fiber deposition to the total area of the marrow cavity as indicated by silver reticulum staining.
4. The results showed that the color and luster of the marrow cavity of the mice of example 1 (model group, 0.5MSPL in the figure) were not uniform and the ratio of the deposition area of collagen fibers (FIG. 1B) and reticular fibers (FIG. 1C) in the marrow cavity was significantly increased in red and white phase distribution (FIG. 1A), and +40 and +60 days, compared with that of comparative example 1 (control group, TCD-BM in the figure).
Experimental example 2
And (5) detecting the infiltration of the mouse bone marrow lymphocytes.
1. After the mice were decapped and sacrificed, the tibia of the mice was taken, both ends of the tibia were carefully subtracted with an ophthalmic scissors, the tibia was inserted from the fractured end of the tibia using a 1ml syringe needle, and the cells in the marrow cavity were carefully blown into the centrifuge tube until the marrow cavity became white. Then the cells are put on a filter screen for grinding and filtering. The filtered cells were centrifuged, the supernatant was discarded, 3ml of 1 × erythrocyte lysate was added, and the cells were lysed at room temperature for 15 minutes. After the lysis time was over, 20ml of flow buffer was added to the centrifuge tube and counted. Bone marrow cells were made to 1 × 10 by counting, PBS resuspension6Single cell suspension per ml, 1ml of single cell suspension was put into a flow tube and centrifuged.
2. Add 50. mu.l/tube live/dead dye and anti-CD 16/CD32 to label bone marrow and block cell surface FcR to reduce subsequent antigen-antibody non-specific binding. After 10 minutes, flow fluorescent antibody 50 ul/tube was added, anti-mouse CD8 APC (1: 100 dilution); anti-mouse CD4 pacific blue (1: 100 dilution). Shaking and mixing the mixture by using a turbine shaking instrument, and incubating the mixture for 30 minutes at 4 ℃. After the incubation was complete, 2 ml/tube flow buffer was added to wash away unbound flow antibody. Centrifuge at 1500rpm for 5 minutes. After discarding the supernatant, 400. mu.l of flow buffer was added, and the expression of CD4/CD8 antigen in bone marrow cells was detected by flow cytometry.
3. The results showed that CD4 in the marrow cavity of mice in the model groups at +40 and +60 days after transplantation, compared with the control group+T lymphocytes (left side of FIG. 2) and CD8+The number and proportion of T lymphocytes (right side of FIG. 2) were significantly increased.
Experimental example 3
And detecting the mouse bone marrow autoantibody.
1. Sections were hydrated using 200 μ l PBS solution and incubated at room temperature for 5min for full hydration. The PBS solution was removed using a pipette gun, leaving a small portion of the PBS solution to prevent the sections from drying. Immediately after removal of the PBS solution, 200ul of permeabilizing reagent was added to the perimeter of the specimen and the sections were incubated for 20min at room temperature. The permeabilizer was removed around the specimen, 200. mu.l of freshly prepared blocking solution was added around the specimen, and incubated for 30 minutes at room temperature. And (4) dropwise adding an anti-mouse IgG antibody diluted solution to the periphery of the specimen, and placing the specimen in a wet box for incubation at 4 ℃. After the antibody incubation was completed, the cells were washed with PBS at room temperature for 5 minutes each for 3 times. 200ul of freshly prepared DAPI solution was added dropwise to the perimeter of the specimen and incubated for 15 minutes at room temperature. After completion of DAPI counterstaining, the staining solution was removed and washed 3 times with PBS at room temperature. Mounting, and adopting a fluorescence microscope for image collection and analysis. Bone marrow autoantibody deposition evaluation was assessed as the proportion of anti-mouse IgG positive area to total area of field.
2. The results showed that the proportion of anti-mouse IgG deposition area in the bone marrow cavity of mice in the model group was significantly increased at +40 and +60 days after transplantation, compared to the control group (FIG. 3).
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. A method for establishing a mouse autoimmune myelofibrosis model is characterized by comprising the following steps:
preparing a bone marrow cell suspension: using C57BL/6 mouse as donor, collecting bone marrow of donor, washing bone marrow cavity with buffer solution, collecting and filtering washing solution to obtain single cell suspension, removing CD4+T cells and CD8+T cells to obtain a T cell-depleted bone marrow cell suspension;
preparation of spleen cell suspension: taking a spleen of a mouse, grinding the spleen, washing the ground spleen by using a buffer solution, collecting and filtering washing liquid to prepare a single cell suspension to obtain a spleen cell suspension;
treatment of the mice: and (3) taking a BALB/c mouse as a mouse, carrying out whole-body ionizing radiation on the mouse, inputting the bone marrow cell suspension and the spleen cell suspension without the T cells into the mouse, and feeding for 40-60 days to obtain the mouse autoimmune myelofibrosis model.
2. The method for constructing a mouse according to claim 1, wherein the donor mouse is a 7-9 week old mouse.
3. The method for constructing a mouse according to claim 1, wherein the mouse is a 7-9 week old mouse.
4. The method according to claim 1, wherein the cell concentration of the T-cell depleted bone marrow cell suspension is 0.8-1.2X 107one/mL.
5. The method of claim 1, wherein the spleen cell suspension has a cell concentration of 0.8-1.2X 106one/mL.
6. The method of claim 1, wherein the ionizing radiation is irradiated at a total dose of 750-850 cGy for a period of 8-10 hours.
7. The method according to any one of claims 1 to 6, wherein the number of bone marrow cells introduced into the body of the subject is 4.8 to 5.2X 106The number of spleen cells per mouse is 0.48-0.52 × 106One/only.
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| US20080305085A1 (en) * | 2004-11-19 | 2008-12-11 | Scadden David T | Compositions And Methods For Stem Cell Expansion |
| CN103114074A (en) * | 2013-02-21 | 2013-05-22 | 广东省人民医院 | Method for building cGVHD (chronic graft-versus-host disease) model of mouse allogene after bone marrow transplantation |
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