CN114752628B - Construction method and application of C57BL/6 mouse multiple myeloma model - Google Patents
Construction method and application of C57BL/6 mouse multiple myeloma model Download PDFInfo
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- CN114752628B CN114752628B CN202210600580.2A CN202210600580A CN114752628B CN 114752628 B CN114752628 B CN 114752628B CN 202210600580 A CN202210600580 A CN 202210600580A CN 114752628 B CN114752628 B CN 114752628B
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Abstract
The invention relates to the technical field of multiple myeloma research, and discloses a construction method and application of a C57BL/6 mouse multiple myeloma model, wherein differential genes of the C57BL/6 mouse and the Balb/C mouse are screened out through library-building sequencing and biological information analysis on plasma cells in spleens of the C57BL/6 mouse and the Balb/C mouse, genes which can be used for converting C57BL/6 background source IgM+B cells into plasma cells together in cooperation with C-MYC and KRAS12V are screened out according to the functions of the genes, a retrovirus vector is constructed by utilizing a determined target gene, and virus-infected IgM+B cells are inoculated into the C57BL/6 background mouse in a bone marrow transplantation mode, so that the multiple myeloma model is successfully induced. The invention has the beneficial effects of improving the utilization rate of the existing transgenic mice, improving the application efficiency of the multiple myeloma model and improving the research field width.
Description
Technical Field
The invention relates to the technical field of multiple myeloma research, in particular to a construction method and application of a C57BL/6 mouse multiple myeloma model.
Background
Multiple Myeloma (MM) is a malignant plasmacytoma, belonging to the category of B-lymphocytic lymphomas. Normally, plasma cells are cells that develop and mature from B cells through multiple organ, multi-stage, precise regulation, and antigen stimulation, with antibody secretion. Several causes in this process may cause abnormal expression of genes, ultimately leading to MM. MM usually develops progressively from MGUS (Monoclonal Gammopathy of Undetermined Significance), with the incidence of MM being the second leading cause of hematological malignancy, accounting for 10% -13% of hematological neoplasms. The abnormal genome and epigenetic disturbance are involved in the process of multiple myeloma, and the accumulation and expression abnormality of gene mutation, including mutation of NRAS and KRAS, over-expression of MYC and inhibition of P53 gene are accompanied in the process of the disease development of multiple myeloma, and with the development of sequencing technology, whole genome sequencing and whole exon sequencing show that more molecular genetic events are involved in the development of MM, but an ideal animal model is lacking to further research the specific mechanism of the genes involved in MM, so that the development of an animal model of MM with high efficiency has important significance to the mechanism research and clinical treatment of MM diseases.
Early MM animal models were modeled by intraperitoneal injection of pristane into Babl/c mice, which developed a pathological phenotype of plasmacytoma in mice about 200 days after injection, and subcutaneous transplantation of tumor MM models into mice, which are currently widely used, by subcutaneous implantation of MM cell lines into SCID (severe immunodeficiency) mice or by tumor modeling of MM patient samples, and many MM mouse models were developed as transgenic mouse technologies matured. For example, ig light chain gene is used for driving cMYC expression, igVH promoter and enhancer are used for driving XBP1, cMAF and cMYC expression. However, this mouse model is costly and has a relatively long modeling time, so that the assessment of MM mechanism and preclinical drugs is not an ideal model. The induction of blood tumor models in mice based on retrovirus over-expressed genes or suppressor genes has been relatively mature, and currently, two protooncogenes, c-MYC and KRAS12V, are introduced into B220+IgM+mature B cells by retroviruses, and an MM mouse model can be rapidly induced in Balb/c mice.
The background of the current transgenic mice is mostly of C57BL/6 background and is not compatible with Balb/C mouse background, so that the application of the model is greatly limited, and the scheme aims at inducing an MM model in the C57BL/6 background and has important significance for mechanism research and preclinical drug screening development of MM diseases.
Disclosure of Invention
The invention aims to provide a construction method and application of a C57BL/6 mouse multiple myeloma model so as to improve the application efficiency of the multiple myeloma model.
In order to achieve the above purpose, the invention adopts the following technical scheme: the construction method of the C57BL/6 mouse multiple myeloma model comprises the following steps:
step S1, performing library construction sequencing and bioinformatic analysis on plasma cells in spleens of a C57BL/6 mouse and a Balb/C mouse to screen out differential genes of the two, and determining a target gene from the screened differential genes;
s2, constructing a retrovirus vector by using the determined target gene, and obtaining a target virus and a control virus by packaging 293T cell viruses;
step S3, infecting cells of the C57BL/6 mice with the target virus retrovirus, and transplanting the infected cells into the C57BL/6 mice to induce the primary cells of the C57BL/6 mice to be converted into a multiple myeloma model.
The principle and the advantages of the scheme are as follows: in practical application, aiming at the condition that two protooncogenes of C-MYC and KRAS12V carried by retrovirus can successfully induce a multiple myeloma mouse model on a Balb/C mouse background, the scheme considers that the multiple myeloma mouse model is completed on a C57BL/6 mouse so as to improve the research mechanism of multiple myeloma and the application efficiency of the multiple myeloma model, specifically, the scheme screens out differential genes of the two genes by carrying out library-building sequencing and biological information analysis on plasma cells in spleens of the C57BL/6 mouse and the Balb/C mouse, screens out IgM from the C57BL/6 background according to the function of the genes, and can cooperate with the C-MYC and the KRAS12V + B cells are transformed into genes of malignant plasma cells, and retroviral vectors are constructed using the determined target genes, and virus-infected IgM + B cells were inoculated into C57BL/6 background mice by bone marrow transplantation, and a multiple myeloma model was successfully induced.
The method can successfully induce a multiple myeloma model on the background of Balb/C mice by utilizing two protooncogenes of C-MYC and KRAS12V carried by retrovirus, but the multiple myeloma model cannot be induced on the C57BL/6 mice due to the gene difference of the Balb/C mice and the C57BL/6 mice, the identification and the gene modification of the gene difference of the two mice are very complex processes, and the accurate finding of the direct most relevant differential genes between the two are very difficult, and different analysis methods also lead to the difference of the differential gene analysis results, so that the multiple myeloma model is not induced on the C57BL/6 mice at present.
Preferably, as an improvement, the differential genes include DENND2D, LYN, CD63, PTPRE, RAC1, E2F8, CYLD, CDK4, BTG2 and ATXN3; the target gene is DENND2D.
The beneficial effects are that: by carrying out library-building sequencing and biological information analysis on plasma cells in spleens of a C57BL/6 mouse and a Balb/C mouse, the first 10 of the two differential genes with high expression is obtained as a gene, and DENND2D is determined as a target gene according to the differential expression sequencing. And the simultaneous high-expression DENND2D is determined to be a key factor of inducing the multiple myeloma model on the C57BL/6 mouse by the C-MYC-KRAS12V, so that the induction success rate of the model is ensured.
Preferably, as a modification, a retroviral vector is constructed in which DENND2D is overexpressed simultaneously with c-MYC and KRAS12V co-expression.
The beneficial effects are that: on the basis of C-MYC and KRAS12V co-expression, DENND2D is simultaneously over-expressed, so that the gene can be applied to a construction system of a multiple myeloma model, and the research width of a C57BL/6 mouse and the application efficiency of the multiple myeloma model are improved.
Preferably, as a modification, the target virus is MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V; the control virus was MSCV-cMYC-2a-GFP-IRES-KRAS12V.
The beneficial effects are that: through the arrangement, three genes of C-MYC, KRAS12V and DENND2D are simultaneously over-expressed, multiple myeloma is induced in a C57BL/6 mouse body, and compared with a control virus, whether the induction of a model is successful or not can be detected after the induction is finished, so that the feasibility of the method is clearly and directly judged.
Preferably, as a modification, step S3 further includes the following:
step S31, obtaining spleen cells of the C57BL/6 mice, purifying and enriching IgM in the spleen + Positive B cells, followed by IgM + Positive B cell proliferation;
step S32, two rounds of retrovirus infection are carried out successively;
step S33, igM infected with virus + B cells are inoculated into a C57BL/6 background mouse through a bone marrow transplantation mode;
and step S34, performing tissue dissection and flow measurement on tumor cell types of the vaccinated C57BL/6 background mice, and determining that the multiple myeloma model is successfully constructed.
The beneficial effects are that: through the steps, the C57BL/6 mouse and the target gene DENND2D can be fully utilized, and the multiple myeloma model can be rapidly and successfully induced in the C57BL/6 mouse through the steps, so that the application efficiency of the multiple myeloma model is effectively improved.
Preferably, as a modification, when bone marrow transplantation is inoculated into C57BL/6 background mice, virus-infected IgM is injected via tail vein + B cells were injected into C57BL/6 receptor mice.
The beneficial effects are that: at the final stage virus infection with IgM by tail vein + B cells of the strain are transplanted into a C57BL/6 receptor mouse, an ideal in-vivo environment is provided for cell differentiation and proliferation, and the occurrence and development real conditions of multiple myeloma in actual clinic are better simulated.
Preferably, as a modification, the virus infection is determined by flow cytometry at a first interval following a first round of retroviral infection.
The beneficial effects are that: the success rate of virus infection can be quickly and accurately known by measuring the virus infection condition through a flow cytometer, thereby helping to determine the construction success rate of the multiple myeloma model.
Preferably, as a modification, the C57BL/6 background mice are irradiated with gamma rays multiple times prior to inoculation.
The beneficial effects are that: the gamma rays are utilized to irradiate the mice, the mice are thoroughly myeloblasted, so that cell clones transplanted into bone marrow have enough space for proliferation and transformation, and the cell clones are a necessary condition for successfully inducing a multiple myeloma model in C57BL/6 background mice.
The invention also provides application of the C57BL/6 mouse multiple myeloma model, and the C57BL/6 mouse multiple myeloma model is used for researching pathogenesis of multiple myeloma and a patentable target.
The beneficial effects are that: after the C57BL/6 background transgenic mice are used as donors and the C57BL/6 mice successfully induce the multiple myeloma model, the induced model can be applied to research on pathogenesis of the multiple myeloma and a patent drug target, so that understanding of occurrence and development of the multiple myeloma is improved, and a theoretical basis is provided for improving clinical treatment of the multiple myeloma.
Preferably, as an improvement, the C57BL/6 mouse multiple myeloma model is used for preclinical drug screening and efficacy evaluation studies.
The beneficial effects are that: by applying the model to preclinical drug screening and effect evaluation research, the most accurate judgment and analysis can be made on the response effect of the drugs, so that a new drug with better treatment effect is provided for the treatment of multiple myeloma.
Drawings
FIG. 1 is a schematic flow chart of an embodiment of a method for constructing a C57BL/6 mouse multiple myeloma model according to the present invention.
FIG. 2 is a schematic diagram showing an induction process according to an embodiment of the method for constructing a C57BL/6 mouse multiple myeloma model according to the present invention.
FIG. 3 is a schematic diagram showing the differential genes of the first embodiment of the method for constructing a C57BL/6 mouse multiple myeloma model according to the present invention.
FIG. 4 shows a method for constructing a C57BL/6 mouse multiple myeloma model according to the present invention-virus infection of IgM derived from C57BL/6 background + Schematic of percent fluorescence 48 hours after B cells.
FIG. 5 is a schematic diagram showing survival curves of mice according to an embodiment of the method for constructing a C57BL/6 mouse multiple myeloma model of the present invention.
FIG. 6 is a schematic diagram showing the results of flow cytometry analysis performed on mice according to example one of the method for constructing a C57BL/6 mouse multiple myeloma model of the present invention.
Detailed Description
The following is a further detailed description of the embodiments:
embodiment one:
this embodiment is basically as shown in fig. 1: the construction method of the C57BL/6 mouse multiple myeloma model comprises the following steps:
step S1, performing library construction sequencing and bioinformatic analysis on plasma cells in spleens of a C57BL/6 mouse and a Balb/C mouse to screen out differential genes of the two, and determining a target gene from the screened differential genes;
s2, constructing a retrovirus vector by using the determined target gene, and obtaining a target virus and a control virus by packaging 293T cell viruses;
step S3, infecting cells of the C57BL/6 mice with the target virus retrovirus, and transplanting the infected cells into the C57BL/6 mice to induce the primary cells of the C57BL/6 mice to be converted into a multiple myeloma model.
As shown in fig. 2, step S3 further includes the following:
step S31, obtaining IgM + Spleen cells of C57BL/6 mice of (C) and purifying and enriching IgM in spleen + Positive B cells, then stimulated spleen IgM + Positive B cell proliferation;
step S32, carrying out a first round of retrovirus infection and then carrying out a second round of retrovirus infection;
step S33, igM infected with virus + B cells are inoculated into a C57BL/6 background mouse through a bone marrow transplantation mode;
and step S34, performing tissue dissection and flow measurement on tumor cell types of the vaccinated C57BL/6 background mice, and determining that the multiple myeloma model is successfully constructed.
In performing plasma cell differential gene screening in the spleens of C57BL/6 mice and Balb/C mice, the top 10 genes were selected from a number of differential genes, including DENND2D, LYN, CD63, PTPRE, RAC1, E2F8, CYLD, CDK4, BTG2, and ATXN3, while DENND2D was ranked first, thus determining DENND2D as the target gene.
When a retrovirus vector is constructed, on the basis of C-MYC and KRAS12V co-expression, DENND2D is simultaneously over-expressed, so that a virus vector which accords with induction of a C57BL/6 mouse is constructed, then a 293T cell virus is packaged, the target virus is determined to be MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V, and a control virus MSCV-cMYC-2a-GFP-IRES-KRAS12V is also determined for comparison of the effect of a reference target virus.
The specific implementation process of this embodiment is as follows:
in the model construction process, firstly, screening plasma cell differential genes in C57BL/6 and Balb/C spleens, and separating and purifying the genes from spleens of 8-week female C57BL/6 and Balb/C mice by a flow cytometry to obtain CD138 + Is to obtain CD138 + Cells were lysed with Trizol, total RNA was extracted, library-built sequencing was performed, bioinformatic analysis was performed on the sequencing results to find differential genes, as shown in fig. 3, and the first-ranked DENND2D gene of the target genes was determined from the genes with high expression of top 10.
Then constructing a virus vector, and simultaneously over-expressing DENND2D on the basis of c-MYC and KRAS12V, constructing an MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V retrovirus vector, and then packaging 293T cell viruses to obtain high-titer MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V viruses, wherein the specific packaging process of the viruses is as follows:
the resuscitated 293T cells are generally passaged for 3 to 4 generations until the cell growth vigor is good;
spread 4×10 in 6cm dishes the day before transfection 6 Cells, 5ml fresh dmem+10% fbs medium was added;
the next day (about 12-16 h), 5ml of fresh medium containing 25. Mu.M chloroquine was changed, and 1ml of the infection solution was added dropwise to the medium after about 20 min.
The arrangement of the infection liquid is shown in table 1 and table 2:
table 1: a table for preparing infection liquid
Table 2: b table for preparing infectious liquid
Uniformly dripping 500ml of DNA (deoxyribonucleic acid) calcium phosphate compound in A into 500ml of 2 x HBS in B, and swirling while dripping to form light vaporific fine particles which are 1ml in total, and standing for 5min at room temperature; 4ml of fresh medium is replaced 10-12 hours after transfection, 5ml of fresh medium is replaced again 24 hours after transfection, culture medium supernatant is collected 48 hours after transfection, cell debris is filtered out by a 0.45 mu m filter head, 2ml of cell debris is split charging is carried out, and the cell debris is frozen at the temperature of minus 80 ℃ for preservation.
Then, the spleen cells of the C57BL/6 mice are obtained and cultivated, and IgM is obtained first + Spleen cells of C57BL/6 mice of (C) are prepared by the following steps:
1. by CO 2 C57BL/6 donor mice were sacrificed, the abdomen was wiped with 70% ethanol under sterile conditions, the abdomen was opened with an dissecting tool, and the whole spleen of the mice was removed.
2. Spleens of mice were placed in 6cm dishes with 5ml of RPMI1640 pre-chilled medium containing 10% fbs, the spleens were rapidly ground to single cells with 10ml of sterile medical syringe handle tip, and cells were filtered through 75 μm filter screens into 15ml centrifuge tubes, centrifuged for 310g, 5min, and the supernatant was decanted.
3. Erythrocytes were lysed with 5ml of sterile pre-chilled erythrocyte lysate, lysed on ice for 10min, and washed once with 5ml Binding Buffer (PBS with 0.5% FBS and 2mM EDTA) while cell counting was performed.
Then purifying and enriching spleen IgM + Positive B cells, the specific procedure is as follows:
1. according to the result of cell count, the ratio was 10 7 100. Mu.L Binding Buffer to resuspend cells in 50ml centrifuge tubes, 2. Mu.L of anti-mouse lgM antibody (ebioscience, 12-5890-83) was added per 100. Mu.L of cell suspension, the cells and antibody were gently mixed, incubated at 4℃for 15 minutes in the absence of light,cells were gently mixed once every 5 minutes to allow for adequate binding of antibodies to cells.
2. Every 10 7 Washing cells with 1. 1ml Binding Buffer of unbound surplus antibody, centrifuging for 310g and 5min, removing supernatant, and adjusting cell to 10 according to counting result 7 A density of 80. Mu.L.
3. Every 10 7 Cell density was added to 20 μl of anti-PE magnetic beads (miltenyibiocec, # 130-048-801), gently mixed, incubated at 4 ℃ for 15 min in the dark, gently mixed once every 5min to allow for adequate binding of antibody to the magnetic beads.
4. Press 10 7 The cells were washed with excess unbound Beads per 2ml Bind Buffer, centrifuged at 310g for 5min and the supernatant was decanted.
5. According to the cell number, the cell density was adjusted to 10 by using Bind Buffer 8 mu.L, LS Columns (Miltenyi: 130-042-401) were clamped onto a suitable MACs magnetic strip rack, LS Columns were rinsed with 3-5ml Buffer, and the above cell suspension was added to the rinsed LS Columns.
6. Unbound cells flowing through Columns were collected and washed 3 times with 3ml Buffer, then 5ml Bind Buffer was added, columns were removed from the magnetic rack, then the bound cells in Columns were quickly pushed by a handle into a 15ml centrifuge tube, cloudy cell suspension could be seen as evidence of enrichment of cells, finally the eluted cell suspension was centrifuged for 310g,5 minutes and the supernatant was decanted.
Followed by stimulation of spleen IgM + Positive B cells proliferate, flick the cells to a loose state, and resuspend the cells with 10ml of stimulation medium, 10cm dishes no higher than 1.5x10 7 Dish, stimulation medium is shown in table 3:
table 3: stimulation medium configuration table
| Reagent name | Storage concentration | |
1 sample | 4 samples |
| RPMI1640 medium | 8.22ml | 32.88ml | ||
| Fetal bovine serum | 1.5ml | 6ml | ||
| Penicillin/ |
100× | 100μL | 400μL | |
| L- |
100× | 100μL | 400μL | |
| Beta-mercaptoethanol | 1000× (50mM) | 50μM | 10μL | 40μL |
| Ciprofloxacin | 1000× (2mg/ml) | 2μg/ml | 10μL | 40μL |
| Interleukin 4 (mIL 4) | 1000×(10μg/ml) | 10ng/ml | 10μL | 40μL |
| Lipopolysaccharide (LPS) | 200× (10mg/ml) | 50μg/ml | 50μL | 200μL |
The stimulation medium was then placed at 37℃with 5% CO 2 The cells were cultured in conditioned cell culture boxes for 24 hours.
The first round of retroviral infection was performed as follows:
1. cells were collected, all cells were collected in a 10cm dish into a centrifuge tube with PBS, and the cells were counted, centrifuged at 310g for 5 minutes, and the supernatant was discarded.
2. Cells were resuspended with an infection solution and transferred to 6-well plates, the infection solution configuration is shown in table 4:
table 4: infection liquid allocation meter
| Reagent name | Storage concentration | |
1 sample | 4 samples |
| Virus (virus) | 2ml | |||
| 1M HEPES(PH7.4) | 40μL | 160μL | ||
| Polyberen | 2mg/ml | 8μL | 32μL | |
| RPMI1640 medium | 1.24ml | 4.96ml | ||
| Fetal bovine serum | 0.6ml | 2.4ml | ||
| Penicillin/ |
100× | 40μL | 160μL | |
| L- |
100× | 40μL | 160μL | |
| Beta-mercaptoethanol | 1000×(50mM) | 50μM | 4μL | 16μL |
| Ciprofloxacin | 1000×(2mg/ml) | 2μg/ml | 4μL | 16μL |
| mIL4 | 1000×(10μg/ml) | 10ng/ml | 4μL | 16μL |
| LPS | 200×(10mg/ml) | 50μg/ml | 20μL | 80μL |
3. Further centrifugal infection, 1000g, centrifugation at 37℃for 90 min followed by 5% CO at 37℃was performed 2 The culture was carried out in a incubator under the conditions for 3 hours.
4. Gently transfer the supernatant to a 15mL centrifuge tube with a 5mL pipette, centrifuge at 310g for 5 minutes, pour the supernatant, and re-suspend the cells with 4mL of stimulation fluid for transfer to the original well plate; the stimulation medium was configured as shown in table 5:
table 5: stimulation medium configuration table
| Reagent name | Storage concentration | |
1 sample | 4 samples |
| RPMI1640 medium | 3.288ml | 13.152ml | ||
| Fetal bovine serum | 1.5ml | 6ml | ||
| Penicillin/ |
100× | 40μL | 160μL | |
| L- |
100× | 40μL | 160μL | |
| Beta-mercaptoethanol | 1000×(50mM) | 50μM | 4μL | 16μL |
| Ciprofloxacin | 1000×(2mg/ml) | 2μg/ml | 4μL | 16μL |
| IL-4 | 1000×(10μg/ml) | 10ng/ml | 4μL | 16μL |
| LPS | 200×(10mg/ml) | 50μg/ml | 20μL | 80μL |
The stimulated medium was then placed at 37℃with 5% CO 2 The cells were cultured overnight in a conditioned cell incubator.
The second round of retroviral infection was performed as follows:
1. 2mL of the stimulation medium supernatant was gently pipetted off using a 1mL pipette and 2mL of virus was added to perform a second infectious body as follows:
| |
1 sample | 4 samples |
| Virus (virus) | 2mL | N*2mL |
| Polyberen（2mg/ml） | 8μL | 32μL |
| HEPES | 40μL | 160μL |
2. Centrifugal infection was performed at 1000g for 90 minutes at 37℃followed by centrifugation at 5% CO at 37 ℃ 2 The culture was carried out in a incubator under the conditions for 3 hours.
3. Cells were collected in 50mL centrifuge tubes, and after washing the remaining cells with 2mL PBS, they were collected together, centrifuged at 300g for 10 minutes, and the supernatant was removed.
4. Cells were resuspended in an appropriate amount of PBS (5-10 mL) and cell counts were performed (10. Mu.l cell suspension+40. Mu.l pre-chilled red cell lysate, placed on ice for 10min, counted with 2 trypan blue staining), centrifuged for 300g, 10min, and the supernatant was decanted.
5. Primary cells were adjusted to 5.0 x 10 based on total number of cells 6 Simultaneously taking the same batch of C57BL/6 receptor mouse bone marrow cells and adjusting the cell density to 5.0 x 10 6 And primary cells according to a density of 1:1, uniformly mixing.
Before induction, C57BL/6 receptor mice were subjected to two lethal doses of gamma irradiation, 5.5Gy each, 3 hours apart, and after the end of irradiation, cells were injected into C57BL/6 receptor mice via the tail vein using a 1mL medical syringe (27G 1/2), 200 μl of cell suspension per mouse.
As shown in FIG. 4, the first round of MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V virus infected C57BL/6 background mice derived IgM + After 48 hours of B cells, the virus infection is measured by a flow cytometer, and the prepared retrovirus can successfully infect IgM + B cells.
After successful acquisition of sufficient titers of retrovirus, we infected the virus with IgM + B cells were inoculated into C57BL/6 background mice by bone marrow transplantation, the survival of the inoculated mice was monitored and a survival curve was drawn, as shown in FIG. 5, MSCV-cMYC-2a-GFP-IRES-KRAS12V mice did not die after 180 days of modeling, i.e., multiple myeloma models could not be successfully induced, while MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V mice were all succumbed to typical plasma cell symptoms within 150 days, and it was found that the spleen, lymph node and thymus of the mice showed obvious enlargement by tissue dissection and flow measurement of tumor cell types from the diseased mice.
As shown in FIG. 6, flow cytometry analysis of mouse bone marrow and peripheral organs including spleen, lymph node, thymus and peripheral blood cells showed that all GFP positive cells showed CD138 positive, while B220 and CD38 expression were negative, indicating that disease mouse tumor cells were plasma cell tumors, especially GFP in bone marrow + CD138 + The proportion of tumor cells is as high as 39.7%, and almost no tumor cells are detected in peripheral blood, which accords with the basic characteristics of the multiple myeloma diseases. Thus, we can confirm successful construction of multiple myeloma models in C57BL/6 background mice.
The application of the C57BL/6 mouse multiple myeloma model comprises the following aspects:
1. the C57BL/6 mouse multiple myeloma model is used for researching the pathogenesis of multiple myeloma and a patentable medicine target.
2. The C57BL/6 mouse multiple myeloma model is used for the study of preclinical drug screening and effect evaluation.
3. The C57BL/6 mouse multiple myeloma model was used for immunotherapy evaluation and antibody therapy study.
Along with the deterioration of ecological environment and living environment, and the social problems of food safety, the occurrence ratio of tumors is in an ascending trend. Multiple myeloma is the second highest incidence of malignant plasma cell tumors in blood tumors, and is statistically 0.003% average, with a ratio of about 8:5 in men and women, and most patients with multiple myeloma are older than 60 years. Multiple myeloma is a highly heterogeneous tumor, and the existing drugs and treatments cannot cure the disease, and the problems of relapse and drug resistance are frequently caused in clinic. Therefore, the identification of new therapeutic targets and the evaluation of new therapeutic drugs and means by animal model research of diseases are of great clinical significance for the treatment of multiple myeloma.
Currently, two protooncogenes, c-MYC and KRAS12V, are introduced into B220 using retroviruses + IgM + In immature B cells, a multiple myeloma mouse model can be rapidly induced on a Balb/C mouse, but the same technical scheme can not induce the multiple myeloma model in a C57BL/6 background, and the background of the current transgenic mouse belongs to the C57BL/6 background mostly and is not compatible with the Balb/C mouse background, so that the application of the model is greatly limited, and the application of the model in particular to the research of the disease mechanism of multiple myeloma and the exploration of treatment targets is greatly limited.
In the scheme, the background condition of the transgenic mice is fully considered, and how to induce the multiple myeloma model in the C57BL/6 background mice is considered. Based on the fact that the C-MYC and KRAS12V protooncogenes carried by the retrovirus can successfully induce the multiple myeloma mouse model on the Balb/C mouse background, the difference between the C57BL/6 and Balb/C background mouse genes is further identified on the basis of the C-MYC and KRAS12V protooncogenes, and the multiple myeloma mouse model can be successfully induced on the C57BL/6 background mouse by modifying specific differential genes. The differential genes of the C57BL/6 mice and the Balb/C mice are screened out by carrying out library-building sequencing and biological information analysis on plasma cells in the spleens of the mice, the DENND2D genes with the first high expression rank are determined to be target genes, and the DENND2D genes are simultaneously and excessively expressed on the basis of C-MYC and KRAS12V protooncogenes, so that a multiple myeloma model is successfully induced on the C57BL/6 background mice.
At the same time, the technical difficulty of the scheme is also that the C57BL/6 and Balb/C background mouse plasma cells (CD 138) + Cells) to express differential genes and screen IgM from C57BL/6 background sources in cooperation with C-MYC and KRAS12V according to the functions of the genes + The gene of the B cell converted into the tumor plasma cell is DENND2D gene, the process is not easy to realize, and meanwhile, the method is not easy to think, because the difference genes between different types of mice are very many, and the most direct related difference genes between the two are very difficult to find, not only by means of unique and advanced analysis equipment, but also by means of an accurate analysis method, so that the scheme can accurately find that the DENND2D gene is the difference gene of two mice is not easy to find, and has unobvious property; meanwhile, the scheme constructs the MM mouse model by utilizing a retrovirus system in a C57BL/6 background, so that on one hand, the existing various gene mice can be fully utilized to research the pathogenesis of MM and the target points of the MM, and on the other hand, the MM mouse model can be used as an ideal model for preclinical drug screening and effect evaluation, the application efficiency and the research field width of the multiple myeloma model are greatly improved, and the MM mouse model makes an important contribution to the subsequent research and treatment of multiple myeloma diseases.
The foregoing is merely an embodiment of the present invention, and a specific structure and characteristics of common knowledge in the art, which are well known in the scheme, are not described herein, so that a person of ordinary skill in the art knows all the prior art in the application day or before the priority date of the present invention, and can know all the prior art in the field, and have the capability of applying the conventional experimental means before the date, so that a person of ordinary skill in the art can complete and implement the present embodiment in combination with his own capability in the light of the present application, and some typical known structures or known methods should not be an obstacle for a person of ordinary skill in the art to implement the present application. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (8)
- The method for constructing the C57BL/6 mouse multiple myeloma model is characterized by comprising the following steps of: the method comprises the following steps:step S1, performing library construction sequencing and bioinformatic analysis on plasma cells in spleens of a C57BL/6 mouse and a Balb/C mouse to screen out differential genes of the two, and determining a target gene from the screened differential genes; the target gene is DENND2D;s2, constructing a retroviral vector by utilizing the determined target gene, wherein the constructed retroviral vector is obtained by simultaneously overexpressing DENND2D on the basis of c-MYC and KRAS12V co-expression; and obtaining target virus and control virus by packaging 293T cell virus;step S3, infecting cells of the C57BL/6 mice with the target virus retrovirus, and transplanting the infected cells into the C57BL/6 mice to induce the primary cells of the C57BL/6 mice to be converted into a multiple myeloma model.
- 2. The method for constructing a C57BL/6 mouse multiple myeloma model according to claim 1, wherein: the differential genes include DENND2D, LYN, CD63, PTPRE, RAC1, E2F8, CYLD, CDK4, BTG2, and ATXN3.
- 3. The method for constructing a C57BL/6 mouse multiple myeloma model according to claim 1, wherein: the target virus isMSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V; the control virus is MSCV-cMYC-2a-GFP-IRES-KRAS12V.
- 4. The method for constructing a C57BL/6 mouse multiple myeloma model according to claim 1, wherein: the step S3 further includes the following:step S31, spleen cells of a C57BL/6 mouse are obtained, igM+ positive B cells in the spleen are purified and enriched, and then the IgM+ positive B cells are proliferated;step S32, two rounds of retrovirus infection are carried out successively;step S33, inoculating IgM+B cells infected by viruses into a C57BL/6 background mouse through a bone marrow transplantation mode; when the bone marrow transplantation mode is inoculated into a C57BL/6 background mouse, virus-infected IgM+B cells are injected into a C57BL/6 receptor mouse through tail veins;and step S34, performing tissue dissection and flow measurement on tumor cell types of the vaccinated C57BL/6 background mice, and determining that the multiple myeloma model is successfully constructed.
- 5. The method for constructing a C57BL/6 mouse multiple myeloma model according to claim 4, wherein the method comprises the following steps: at a first interval following the first round of retroviral infection, the viral infection is determined by flow cytometry.
- 6. The method for constructing a C57BL/6 mouse multiple myeloma model according to claim 4, wherein the method comprises the following steps: the C57BL/6 background mice were irradiated with gamma rays multiple times prior to inoculation.
- An application of a c57bl/6 mouse multiple myeloma model, characterized in that: the method for constructing the C57BL/6 mouse multiple myeloma model according to any one of claims 1 to 6 is used for researching pathogenesis of multiple myeloma and a patentable target.
- 8. The use of the C57BL/6 mouse multiple myeloma model according to claim 7, wherein: the C57BL/6 mouse multiple myeloma model is used for research of preclinical drug screening and effect evaluation.
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| CN111518835A (en) * | 2020-04-28 | 2020-08-11 | 王季石 | Primary multiple myeloma mouse model construction method and application thereof |
| CN114848653B (en) * | 2021-02-03 | 2023-05-30 | 四川大学 | Application of RORa protein and agonist thereof in preparation of acute DNA damage repair agent |
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| WO2014109696A1 (en) * | 2013-01-10 | 2014-07-17 | Alf Grandien | Method for immortalization of b cells and uses thereof |
| WO2019084493A1 (en) * | 2017-10-27 | 2019-05-02 | The Trustees Of The University Of Pennsylvania | Methods and compositions for treating diseases associated with exhausted t cells |
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