CN114752628A

CN114752628A - Construction method and application of C57BL/6 mouse multiple myeloma model

Info

Publication number: CN114752628A
Application number: CN202210600580.2A
Authority: CN
Inventors: 邱强
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2022-07-15
Anticipated expiration: 2042-05-30
Also published as: CN114752628B

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 a library-building sequencing and biological information analysis are carried out on plasma cells in spleens of a C57BL/6 mouse and a Balb/C mouse to screen out differential genes of the two, genes which can cooperate with C-MYC and KRAS12V to jointly convert IgM + B cells from C57BL/6 background into plasma cells are screened out according to the functions of the genes, then a retrovirus vector is constructed by utilizing the determined target genes, and the 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

Construction method and application of C57BL/6 mouse multiple myeloma model

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-lymphocyte lymphomas. Normally, plasma cells are cells that develop and mature from B cells through multi-organ, multi-stage, precise regulation, and antigen stimulation, and have antibody secretion function. In this process, various causes may cause abnormal expression of genes, eventually leading to MM. MM usually develops progressively from MGUS (monoclonal Gamma of advanced Significce), and in hematological malignancies, the incidence of MM is second, accounting for 10% -13% of hematological malignancies. Genome abnormality and epigenetic disorder are involved in the occurrence process of multiple myeloma, accumulation and expression abnormality of gene mutation can be accompanied in the occurrence and development process of multiple myeloma, the gene mutation comprises mutation of NRAS and KRAS, overexpression of MYC and inhibition of P53 gene, and with the development of sequencing technology, whole genome sequencing and whole exon sequencing show that more molecular genetic events participate in the occurrence and development of MM, but an ideal animal model is lacked to further research the specific mechanism of the gene participating in MM, so that the development of a fast and efficient MM animal model has important significance for mechanism research and clinical treatment of MM diseases.

Early MM animal models are established by injecting pristane into the abdominal cavity of a Babl/c mouse, the pathological phenotype of plasmacytoma appears in the mouse 200 or so days after injection, the mouse subcutaneous tumor transplantation MM model is a model which is widely applied at present, MM cell lines are planted under the skin of SCID (severe immunodeficiency) mice or MM patient samples are grown into tumors for modeling, and a plurality of MM mouse models are developed along with the maturation of transgenic mouse technology. For example, Ig light chain gene is used to drive cMYC expression, IgVH promoter and enhancer are used to drive XBP1, cMAF, cMYC expression. However, the mouse model has high cost and long modeling time, so the evaluation of MM mechanism and preclinical drugs is not an ideal model. Based on that retrovirus overexpression genes or suppressor genes induce blood tumor models in mice to be mature, at present, two protooncogenes of c-MYC and KRAS12V are introduced into B220+ IgM + mature B cells by retroviruses, and an MM mouse model can be quickly induced on Balb/c mice.

The background of the current transgenic mice mostly belongs to the C57BL/6 background, and is incompatible with the background of Balb/C mice, so that the application of the model is limited to a great extent, therefore, the scheme aims at inducing the MM model in the C57BL/6 background, and has important significance for mechanism research of MM diseases and screening and development of clinical prodrug.

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 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 biological information 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;

step S2, constructing a retrovirus vector by using the determined target gene, and packaging 293T cell viruses to obtain a target virus and a control virus;

in step S3, the cells of the C57BL/6 mouse are infected by the target virus retrovirus, and the infected cells are transplanted into the C57BL/6 mouse to induce the transformation of the primary cells of the C57BL/6 mouse 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 the difference genes of the C57BL/6 mouse and the Balb/C mouse by performing library construction, sequencing and biological information analysis on plasma cells in the spleen, screens out the IgM gene with the C57BL/6 background source together with the C-MYC and KRAS12V according to the functions of the genes⁺B cells are converted into genes of malignant plasma cells, and then retroviral vectors and virus-infected IgM are constructed using the determined target genes⁺B cells were inoculated into C57BL/6 background mice by bone marrow transplantation to successfully induce a multiple myeloma model.

The retrovirus-carried two proto-oncogenes, namely C-MYC and KRAS12V, can successfully induce a multiple myeloma model on a Balb/C mouse background, but the method cannot induce the multiple myeloma model on a C57BL/6 mouse due to the gene difference of a Balb/C mouse and a C57BL/6 mouse, the identification and the gene modification of the gene difference of the two mice are very complicated processes, the direct most related differential gene between the two is also very difficult to accurately find, different analysis methods can cause different analysis results of the differential gene, the multiple myeloma model cannot be induced on the C57BL/6 mouse at present, and the scheme has the advantages that the most directly related differential gene is expressed by identifying the plasma cells of the C57BL/6 and Balb/C background mice, a new way of a multiple myeloma model is found, the multiple myeloma model is successfully induced by using a C57BL/6 mouse, and the research field and the application rate of the multiple myeloma model are greatly improved.

Preferably, as an improvement, the differential genes include DENND2D, LYN, CD63, PTPRE, RAC1, E2F8, CYLD, CDK4, BTG2, and ATXN 3; the target gene is DENND 2D.

Has the beneficial effects that: by performing library-building sequencing and biological information analysis on plasma cells in spleens of a C57BL/6 mouse and a Balb/C mouse, the top 10 genes with high expression in the differential genes of the two are obtained, and DENND2D is determined to be a target gene according to differential expression sequencing. And the simultaneous high-expression DENND2D is determined to be a key factor for inducing a multiple myeloma model on a C57BL/6 mouse by C-MYC-KRAS12V, so that the induction success rate of the model is ensured.

Preferably, as an improvement, the retroviral vector is constructed such that DENND2D is simultaneously overexpressed on the basis of c-MYC and KRAS12V coexpression.

Has the advantages that: on the basis of co-expression of C-MYC and KRAS12V, DENND2D is over-expressed at the same time, 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 an improvement, the target virus is MSCV-cMY-2 a-GFP-IRES-DENND2D-IRES-KRAS 12V; the control virus was MSCV-cMY-2 a-GFP-IRES-KRAS 12V.

Has the advantages that: through the arrangement, three genes of C-MYC, KRAS12V and DENND2D are simultaneously overexpressed, multiple myeloma is induced in a C57BL/6 mouse, 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 can be clearly and directly judged.

Preferably, as a modification, step S3 further includes the following:

step S31, spleen cells of C57BL/6 mice were obtained, IgM in spleen was purified and enriched⁺Positive B cells, then IgM⁺Positive B cell proliferation;

step S32, two rounds of retrovirus infection are carried out successively;

step S33, IgM infection with virus⁺B cells were inoculated into C57BL/6 background mice by bone marrow transplantation;

step S34, carrying out tissue dissection and flow measurement on tumor cell types of the inoculated C57BL/6 background mice, and determining that the multiple myeloma model is successfully constructed.

Has the advantages that: through the steps, the C57BL/6 mouse and the target gene DENND2D can be fully utilized, and the multiple myeloma model can be quickly 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 an improvement, when the bone marrow transplantation mode is inoculated into C57BL/6 background mice, virus-infected IgM is inoculated through tail vein⁺B cells were injected into C57BL/6 recipient mice.

Has the advantages that: infection of the virus with IgM via the tail vein in the last stage⁺The B cell is transplanted into a C57BL/6 receptor mouse, an ideal in-vivo environment is provided for the differentiation and proliferation of the cell, and the actual occurrence and development conditions of multiple myeloma in real clinic are better simulated.

Preferably, as a refinement, the viral infection is determined by flow cytometry at a first interval after the first round of retroviral infection is performed.

Has the advantages that: the success rate of virus infection can be rapidly and accurately known by measuring the virus infection condition through a flow cytometer, thereby helping to determine the construction success rate of a multiple myeloma model.

Preferably, as a refinement, C57BL/6 background mice are irradiated multiple times with gamma radiation prior to inoculation.

Has the beneficial effects that: irradiating mice with gamma rays to thoroughly clear the marrow of the mice so that cell clones transplanted into the bone marrow have sufficient space for proliferation and transformation is a necessary condition for successfully inducing multiple myeloma models in C57BL/6 background mice.

The invention also provides application of the C57BL/6 mouse multiple myeloma model, which is used for researching pathogenesis and druggable target point of multiple myeloma by using the C57BL/6 mouse multiple myeloma model.

Has the advantages that: by using the C57BL/6 background transgenic mouse as a donor, after the C57BL/6 mouse successfully induces the multiple myeloma model, the induced model can be applied to the research on the pathogenesis of the multiple myeloma and druggable targets, so that the understanding of the occurrence and development of the multiple myeloma is improved, and a theoretical basis is provided for improving the clinical treatment of the multiple myeloma.

Preferably, as an improvement, the C57BL/6 mouse multiple myeloma model is used for the study of preclinical drug screening and effect evaluation.

Has the advantages that: by applying the model to the research of preclinical drug screening and effect evaluation, the most accurate judgment and analysis can be made on the reaction effect of the drug, so that a new drug with a better treatment effect is provided for the treatment of multiple myeloma.

Drawings

FIG. 1 is a schematic flow chart of a first embodiment of a method for constructing a C57BL/6 mouse multiple myeloma model according to the present invention.

FIG. 2 is a schematic diagram of an induction process of an embodiment of the method for constructing the C57BL/6 mouse multiple myeloma model according to the present invention.

FIG. 3 is a schematic diagram of the differential genes in the first embodiment of the method for constructing the C57BL/6 mouse multiple myeloma model according to the present invention.

FIG. 4 shows a method for constructing a model of multiple myeloma in mice C57BL/6 according to an embodiment of the present invention, IgM from a background source of C57BL/6 is infected with a virus⁺Percentage fluorescence 48 hours after B cells is shown.

FIG. 5 is a schematic diagram of a mouse survival curve according to a first embodiment of the method for constructing the C57BL/6 mouse multiple myeloma model of the present invention.

FIG. 6 is a diagram showing the results of flow cytometry analysis of mice according to the first embodiment of the method for constructing the C57BL/6 mouse multiple myeloma model of the present invention.

Detailed Description

The following is further detailed by way of specific embodiments:

the first embodiment is as follows:

this embodiment is substantially as shown in figure 1: the construction method of the C57BL/6 mouse multiple myeloma model comprises the following steps:

As shown in fig. 2, step S3 further includes the following steps:

step S31, obtaining IgM⁺Spleen cells of C57BL/6 mouse, purification and enrichment of IgM in spleen⁺Positive B cells followed by spleen IgM stimulation⁺Positive B cell proliferation;

step S32, performing a first round of retroviral infection, and then performing a second round of retroviral infection;

In performing the plasma cell differential gene screening in the spleens of C57BL/6 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 ranked first, thus determining DENND2D as the target gene.

When a retroviral vector is constructed, DENND2D is overexpressed on the basis of C-MYC and KRAS12V coexpression, so that a viral vector which meets the induction of a C57BL/6 mouse is constructed, then the 293T cell virus is packaged, the target virus is determined to be MSCV-cMYC-2 a-GFP-IRES-DEIRED 2D-KRAS 12V, and the control virus MSCV-YCcM-2 a-GFP-IRES-KRAS12V is determined for comparing the effects of the reference target virus.

The specific implementation process of this embodiment is as follows:

in the process of model construction, the differential gene screening of plasmacytes in C57BL/6 and Balb/C spleens is firstly carried out, and CD138 is obtained by separating and purifying through flow cytometry from spleens of C57BL/6 and Balb/C mice female at 8 weeks respectively⁺The cells of (4), CD138 to be obtained⁺The cells are cracked by Trizol, total RNA is extracted, library construction and sequencing are carried out, the sequencing result is subjected to bioinformatics analysis, a difference gene is found out, as shown in figure 3, and the target gene is the first-ranked DENND2D gene from the genes with the high expression top 10.

Then constructing a virus vector, over-expressing DENND2D on the basis of co-expression of c-MYC and KRAS12V to construct an MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V retrovirus vector, packaging 293T cell virus to obtain high-titer MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V virus, and preparing MSCV-cMYC-2a-GFP-IRES-KRAS12V virus as a control virus for constructing a multiple myeloma model, wherein the specific packaging process of the virus is as follows:

the recovered 293T cells are generally passaged for 3-4 generations until the cells grow well;

6cm dishes were plated with 4 x 10 the day before transfection⁶Cells, 5ml fresh DMEM +10% FBS medium added;

the next day (about 12-16 h), 5ml of fresh medium containing 25. mu.M chloroquine was replaced, and after about 20min, 1ml of infection solution was added dropwise to the medium.

The infection liquid configuration is shown in table 1 and table 2:

table 1: a infection liquid preparation table

Table 2: b infection liquid preparing table

Uniformly dripping 500ml of DNA calcium phosphate compound in the A into 500ml of 2 x HBS in the B, carrying out vortex while dripping to form 1ml of light atomized fine particles, and standing at room temperature for 5 min; replacing 4ml of fresh culture medium 10-12 hours after transfection, replacing 5ml of fresh culture medium once again 24 hours after transfection, collecting culture medium supernatant 48 hours after transfection, filtering out cell debris by using a 0.45 mu m filter head, subpackaging 2ml, and storing at-80 ℃ by freezing.

Then, the spleen cells of the C57BL/6 mice are obtained and cultured, and IgM is obtained first⁺The process of preparing the C57BL/6 mouse spleen cell is as follows:

1. with CO₂C57BL/6 donor mice were sacrificed, abdominal swab was performed with 70% ethanol under sterile conditions, abdominal cavity of the mice was opened with a dissecting tool, and the entire spleen of the mice was removed.

2. The spleens of mice were placed in a 6cm dish containing 5ml of 10% FBS in RPMI1640 pre-cooled medium, the spleens were rapidly ground to single cells using a 10ml sterile medical syringe handle tip, and the cells were filtered through a 75 μm filter into a 15ml centrifuge tube, centrifuged at 310g for 5 minutes, 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) with cell counting.

Spleen IgM was then purified and enriched⁺The specific process of the positive B cell is as follows:

1. according to the results of cell counting, according to 10⁷Resuspend cells in 100. mu.L Binding Buffer in 50ml centrifuge tube, add 2. mu.L anti-mouse lgM antibody (ebioscience, 12-5890-83) per 100. mu.L cell suspension, mix cells and antibody gently, incubate at 4 ℃ for 15 minutes in the dark, mix cells gently once every 5 minutes, let antibody and cells bind well.

2. According to each 10⁷Washing the cells with each 1ml Binding Buffer without Binding surplus antibody, centrifuging for 5 minutes at 310g, pouring out the supernatant, and adjusting the cells to 10 according to the counting result⁷Density of 80. mu.L.

3. According to each 10⁷20 μ L of anti-PE magnetic beads (Miltenyibitotec, # 130-.

4. Press 10⁷The cells were washed for excess unbound Beads per 2ml Bind Buffer, centrifuged again at 310g for 5 minutes and the supernatant decanted.

5. According to the cell number, the cell density was adjusted to 10 using Bind Buffer⁸500 μ L, LS Columns (Miltenyi: 130-.

6. Unbound cells that flowed through the Columns were collected and washed 3 times with 3ml Buffer, then 5ml Bind Buffer was added, the Columns were removed from the magnetic rack, the bound cells in the Columns were then quickly pushed into a 15ml centrifuge tube with a handle, the turbid cell suspension was confirmed by light transmission to enrich the cells, and finally the eluted cell suspension was centrifuged for 310g for 5 minutes and the supernatant was decanted.

Followed by stimulation of spleen IgM⁺Positive B cell proliferation, gently flicking the cells to loose, and suspending the cells with 10ml of stimulated medium with no more than 1.5X 10 in a 10cm dish⁷Petri dish, stimulation medium as shown in table 3:

table 3: stimulation medium configuration table

Name of reagent	Storage concentration	Final concentration	1 sample	4 samples
					RPMI1640 culture medium			8.22ml	32.88ml
Fetal bovine serum			1.5ml	6ml
					Penicillin/streptomycin	100×		100μL	400μL
L-Glutamine	100×		100μL	400μL
					Beta-mercaptoethanol	1000× (50mM)	50μM	10μL	40μL
Ciprofloxacin	1000× (2mg/ml)	2μg/ml	10μL	40μL
					Interleukin 4 (mIL4)	1000×(10μg/ml)	10ng/ml	10μL	40μL
Lipopolysaccharide (LPS)	200× (10mg/ml)	50μg/ml	50μL	200μL

The stimulation medium was then placed at 37 ℃ in 5% CO₂The cells were cultured in a conditioned cell incubator for 24 hours.

Then, the retrovirus infection is carried out, and the first round of retrovirus infection is as follows:

1. the cells were collected, all the cells in a 10cm dish were collected in a centrifuge tube with PBS, the cells were counted, and centrifugation was carried out for 310g and 5 minutes, and the supernatant was decanted.

2. Cells were resuspended in infection solution, which was prepared as shown in table 4, and transferred to 6-well plates:

table 4: infection liquid configuration table

Name of reagent	Storage concentration	Final concentration	1 sample of	4 samples
					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/streptomycin	100×		40μL	160μL
					L-Glutamine	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, the cells were subjected to centrifugal infection at 1000g for 90 minutes at 37 ℃ and then to 5% CO at 37 ℃₂Incubate for 3 hours under conditions.

4. Gently transfer the supernatant to a 15mL centrifuge tube with a 5mL pipette, centrifuge for 5 minutes at 310g, pour off the supernatant, and resuspend the cells to the original well plate with 4mL of a stimulating solution; the configuration of the stimulation medium is shown in table 5:

table 5: stimulation medium configuration table

Name of reagent	Storage concentration	Final concentration	1 sample	4 samples
					RPMI1640 medium			3.288ml	13.152ml
Fetal bovine serum			1.5ml	6ml
					Penicillin/streptomycin	100×		40μL	160μL
L-Glutamine	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 stimulation medium was then placed at 37 ℃ in 5% CO₂The cells were cultured overnight in a conditioned cell incubator.

And performing a second round of retrovirus infection, wherein the specific process is as follows:

1. gently aspirate with a 1mL pipette gun and discard 2mL of the stimulation medium supernatant and add 2mL of virus for a second infection as follows:

name of reagent	1 sample	4 samples
			Virus	2mL	N*2mL
Polyberen（2mg/ml）	8μL	32μL
			HEPES	40μL	160μL

2. The infection was performed by centrifugation at 1000g for 90 minutes at 37 ℃ followed by 5% CO at 37 ℃₂Incubate for 3 hours under conditions.

3. The cells were collected in a 50mL centrifuge tube, and the remaining cells were washed with 2mL PBS and collected together, and centrifuged at 300g for 10 minutes to remove the supernatant.

4. The cells were resuspended in the appropriate amount of PBS (5-10 mL) and cell counted (10. mu.l cell suspension + 40. mu.l pre-cooled red blood cell lysate, 10min on ice, counted with 2 × Trypan blue stain), centrifuged again at 300g for 10min and the supernatant decanted.

5. Primary cells were adjusted to 5.0 x 10 based on total cell number⁶The same batch of C57BL/6 recipient mouse bone marrow cells were taken simultaneously, and the cell density was adjusted to 5.0 x 10⁶And primary cells were mixed at a density of 1: 1, and uniformly mixing.

C57BL/6 recipient mice were irradiated with two lethal doses of gamma radiation 5.5Gy each, 3 hours apart, before induction, and after the irradiation was completed, the cells were injected into C57BL/6 recipient mice through the tail vein using a 1mL medical syringe (27G 1/2), 200. mu.L of cell suspension per mouse.

As shown in FIG. 4, the first round of MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V virus infected IgM from C57BL/6 background mouse⁺After 48 hours of B cells, the virus infection condition is determined by a flow cytometer, and the prepared retrovirus can successfully infect IgM⁺B cells.

After successful acquisition of sufficient titer of retrovirus, we infected the virus with IgM⁺B cells were inoculated into C57BL/6 background mice by bone marrow transplantation and survival monitoring and survival curves were plotted, as shown in FIG. 5, mice in the MSCV-cMYC-2a-GFP-IRES-KRAS12V group did not die 180 days after modeling, i.e., failed to successfully induce multiple myeloma models, while mice in the MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS12V group died successively from typical plasma cell symptoms within 150 days, and significant swelling of spleen, lymph nodes and thymus was observed by tissue dissection and flow assay of tumor cell types in 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 revealed that all GFP-positive cells were CD 138-positive, while B220 and CD38 expression were negative, indicating that the diseased mouse tumor cells were plasma cell tumors, especially GFP in bone marrow⁺CD138⁺The proportion of the tumor cells is as high as 39.7 percent, and the tumor cells are hardly detected in peripheral blood, which is in accordance with the basic characteristics of the multiple myeloma disease. Therefore, we could confirm that the multiple myeloma model was successfully constructed 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 pathogenesis and druggable targets of multiple myeloma.

2. The C57BL/6 mouse multiple myeloma model was used in the study of preclinical drug screening and efficacy assessment.

3. The C57BL/6 mouse multiple myeloma model was used for immunotherapy evaluation and antibody therapy studies.

With the deterioration of ecological environment and living environment, and social problems such as food safety, the occurrence rate of tumors tends to increase. Multiple myeloma is a malignant plasma cell tumor with the second highest incidence among hematological tumors, and statistically has an average incidence of 0.003% and a male-female ratio of about 8:5, with most multiple myeloma patients older than 60 years. Multiple myeloma is a highly heterogeneous tumor, the disease cannot be cured by the existing medicines and treatment means, and relapse and drug resistance problems are frequently caused in clinical treatment. Therefore, the identification of new therapeutic targets and the evaluation of new therapeutic drugs and means through animal model research of diseases have important clinical significance for treating multiple myeloma.

The two protooncogenes, c-MYC and KRAS12V, are currently introduced into B220 using retroviruses⁺IgM⁺In immature B cells, a Balb/C mouse can quickly induce a multiple myeloma mouse model, but the same technical scheme cannot induce the multiple myeloma model in a C57BL/6 background, the background of the current transgenic mice mostly belongs to a C57BL/6 background, and the background is not compatible with the Balb/C mouse background, so that the application of the model is limited to a great extent, and particularly the application of the model to research on a disease mechanism of multiple myeloma and search of a therapeutic target point.

In the scheme, the background conditions of the current transgenic mice are fully considered, and how to induce the multiple myeloma model in the C57BL/6 background mice is considered. Based on the fact that multiple myeloma mouse models can be successfully induced on Balb/C mouse background by using two protooncogenes of C-MYC and KRAS12V carried by retrovirus in the early stage, the differences of the two protooncogenes of C57BL/6 and Balb/C background mice are further identified on the basis of the protooncogenes of C-MYC and KRAS12V, and the multiple myeloma mouse models can be successfully induced on C57BL/6 background mice by modifying specific difference genes. The plasma cells in the spleens of a C57BL/6 mouse and a Balb/C mouse are subjected to library-building sequencing and biological information analysis to screen out differential genes of the two, a DENND2D gene with high expression rank first is determined to be a target gene, and a DENND2D gene is simultaneously over-expressed on the basis of C-MYC and KRAS12V proto-oncogenes, so that a multiple myeloma model is successfully induced on a C57BL/6 background mouse.

Meanwhile, the technical difficulty of the scheme is that the identification of C57BL/6 and Balb/C background mouse plasma cells (CD 138)⁺Cells) express differential genes, and C57BL/6 background IgM can be selected by cooperating with C-MYC and KRAS12V according to the functions of the genes⁺The gene for converting B cells into tumor plasma cells is DENND2D gene, the process is not easy to realize, and the method is not easy to think, because the difference genes among different types of mice are very many, and the difference gene which is most directly related to the two types of mice is very difficult to find, and the method not only depends on unique advanced analysis equipment, but also depends on an accurate analysis method, so that the method can accurately find the difference gene of DENND2D gene which is not easy to find but also has non-obvious property; meanwhile, the MM mouse model is constructed by utilizing a retrovirus system in the C57BL/6 background, so that on one hand, the existing various gene mice can be fully utilized to research the pathogenesis and druggable target points of MM, and on the other hand, the model can be used as an ideal model for screening and effect evaluation of clinical prodrugs, the application efficiency and the research field width of the multiple myeloma model are greatly improved, and the model makes important contribution to the subsequent research and treatment of multiple myeloma diseases.

The foregoing are embodiments of the present invention and are not intended to limit the scope of the invention to the particular forms set forth in the specification, which are set forth in the claims below, but rather are to be construed as the full breadth and scope of the claims, as defined by the appended claims, as defined in the appended claims, in order to provide a thorough understanding of the present invention. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be defined by the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

A method for constructing a C57BL/6 mouse multiple myeloma model, characterized in that: the method comprises the following steps:

step S1, performing library construction sequencing and biological information 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;

step S2, constructing a retrovirus vector by using the determined target gene, and packaging 293T cell viruses to obtain a target virus and a control virus;

in step S3, the cells of the C57BL/6 mouse are infected by the target virus retrovirus, and the infected cells are transplanted into the C57BL/6 mouse to induce the transformation of the primary cells of the C57BL/6 mouse into a multiple myeloma model.
2. The method of claim 1 for constructing the C57BL/6 mouse multiple myeloma model, wherein the method comprises: the differential genes include DENND2D, LYN, CD63, PTPRE, RAC1, E2F8, CYLD, CDK4, BTG2, and ATXN 3; the target gene is DENND 2D.
3. The method of claim 1 for constructing a C57BL/6 mouse multiple myeloma model, wherein the method comprises: the retroviral vector is constructed by simultaneously over-expressing DENND2D on the basis of c-MYC and KRAS12V co-expression.
4. The method of claim 1 for constructing the C57BL/6 mouse multiple myeloma model, wherein the method comprises: the target virus is MSCV-cMYC-2a-GFP-IRES-DENND2D-IRES-KRAS 12V; the control virus is MSCV-cMY-2 a-GFP-IRES-KRAS 12V.
5. The method of claim 1 for constructing a C57BL/6 mouse multiple myeloma model, wherein the method comprises: the step S3 further includes the following steps:

step S31, obtaining spleen cells of a C57BL/6 mouse, purifying and enriching IgM + positive B cells in the spleen, and then proliferating the IgM + positive B cells;

step S32, two rounds of retrovirus infection are carried out successively;

step S33, inoculating virus-infected IgM + B cells into C57BL/6 background mice by bone marrow transplantation;

in step S34, the inoculated C57BL/6 background mice are subjected to tissue dissection and flow measurement of tumor cell types, and the successful construction of the multiple myeloma model is determined.
6. The method of claim 5 for constructing a C57BL/6 mouse multiple myeloma model, wherein the method comprises: when the bone marrow transplant was inoculated into C57BL/6 background mice, virus-infected IgM + B cells were injected into C57BL/6 recipient mice via the tail vein.
7. The method of claim 5 for constructing a C57BL/6 mouse multiple myeloma model, wherein the method comprises: at a first interval after the first round of retroviral infection, viral infection was measured by flow cytometry.
8. The method of claim 5 for constructing a C57BL/6 mouse multiple myeloma model, wherein the method comprises: the C57BL/6 background mice were irradiated multiple times with gamma radiation prior to inoculation.
Use of a C57BL/6 mouse multiple myeloma model, characterized by: the C57BL/6 mouse multiple myeloma model is used for researching pathogenesis and druggable targets of multiple myeloma.
10. The use of the C57BL/6 mouse multiple myeloma model of claim 9, wherein: the C57BL/6 mouse multiple myeloma model was used in the study of preclinical drug screening and effect evaluation.