CN115281153A - Construction and application of primary central nervous system lymphoma mouse model - Google Patents
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- 208000016800 primary central nervous system lymphoma Diseases 0.000 title claims abstract description 63
- 206010007953 Central nervous system lymphoma Diseases 0.000 title claims abstract description 62
- 238000010172 mouse model Methods 0.000 title claims abstract description 52
- 238000010276 construction Methods 0.000 title claims abstract description 15
- 206010025323 Lymphomas Diseases 0.000 claims abstract description 35
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- 238000000034 method Methods 0.000 claims abstract description 30
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
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Abstract
The invention belongs to the field of transformation medicine and new medicine development, and particularly relates to construction and application of a primary central nervous system lymphoma mouse model. The invention discloses a method for constructing a primary central nervous system lymphoma mouse model; discloses a cell strain for constructing a primary central nervous system lymphoma mouse model; discloses tumorigenesis characteristics of a primary central nervous system lymphoma mouse model. The invention constructs and obtains a primary central nervous system lymphoma mouse model, lymphoma in the mouse model develops rapidly and mainly shows brain invasion and the like, has high similarity with the invasion characteristics of human primary central nervous system lymphoma, is an ideal in-vivo efficacy evaluation animal model for developing small molecule anticancer drugs, antibody therapy and cell therapy, and can also be used for molecular mechanism research of lymphoma invading the brain.
Description
Technical Field
The invention belongs to the field of transformation medicine and new medicine development, and particularly relates to construction and application of a primary central nervous system lymphoma mouse model.
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-hodgkin lymphoma, with an incidence rate of approximately 30%. Since diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphoma with very large heterogeneity in lymphomatogenic characteristics and drug response, approximately 40% of patients fail to benefit from standard chemotherapy regimen R-CHOP. In these relapsed refractory patients, lymphoma invasion of the central nervous system, such as the brain and spine, including primary and secondary central nervous system lymphomas, is considered to be the primary reason for the failure of these patients to benefit from chemotherapy. The prevention and treatment of PCNSL and SCNSL by intrathecal injection of high doses of MTX or Ara-C or other clinical trial drugs has not been clinically attempted for many years and has not achieved ideal therapeutic effects.
The lack of ideal experimental animal models of primary central nervous system lymphoma is a main limiting factor for developing molecular mechanism exploration and drug development of primary central nervous system lymphoma. The cell strain MA-2 derived from mouse B lymphoma cells is obtained by screening, and a lymphoma reconstruction model is constructed by adopting the cell strain through tail vein injection, so that a lymphoma mouse model invading brain characteristics can be quickly obtained, and the method is an ideal animal model for developing primary central nervous system lymphoma molecular mechanism research and drug development. The mouse model has short morbidity time and high consistency of brain invasion characteristics, is very similar to the brain invasion characteristics of human primary central nervous system lymphoma, is an ideal in-vivo efficacy evaluation animal model of small molecule anticancer drugs, antibody therapy and cell therapy, and can also be used for molecular mechanism research of lymphoma invading the brain.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide the construction and the application of a primary central nervous system lymphoma mouse model, and the primary central nervous system lymphoma mouse model can be used for in-vivo pharmacodynamic analysis of preclinical animals in the process of developing new drugs.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in a first aspect of the present invention, a method for constructing a mouse model of primary central nervous system lymphoma is provided, which comprises the following steps: (1) Analysis of MA cell lines by continuous transplantation experiments (genetic background E μ -Myc; cdkn2 a) Arf-/ -)Screening the tumorigenic characteristics of the original cell strain to obtain a cell strain MA-2 which tends to invade the brain; (2) Transplanting the cell strain MA-2 into a receptor mouse in a tail vein injection mode; (3) The receptor mice show a brain invasion phenotype about 20 days after receiving the transplantation, and a primary central nervous system lymphoma mouse model is obtained.
In one embodiment, the lymphoma of said mouse model of primary central nervous system lymphoma invades the central nervous system including the brain and spinal cord.
In a second aspect of the invention, there is provided MA-2, a cell line which is obtained by the aforementioned construction method and tends to invade the brain.
In a third aspect of the invention, a mouse model of primary central nervous system lymphoma obtained by the aforementioned construction method is provided.
In a fourth aspect of the present invention, there is provided the use of a mouse model of primary central nervous system lymphoma obtained by the aforementioned construction method for the in vivo efficacy evaluation of primary central nervous system lymphoma therapeutic agents and therapies.
In a fifth aspect of the invention, there is provided a method of performing an in vivo efficacy assessment of primary central nervous system lymphoma treatment drugs and therapies, comprising the steps of: the candidate drug or therapy is applied to the primary central nervous system lymphoma mouse model obtained by the construction method, if the lymphoma is regressed or the growth is slowed down, the drug or therapy is indicated to be effective, and the candidate drug or therapy can be determined as the candidate therapeutic drug or therapy of the primary central nervous system lymphoma.
In a sixth aspect of the present invention, a mouse model of primary cns lymphoma obtained by the above construction method is provided for developing molecular mechanism studies of primary cns lymphoma.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses a method for constructing a primary central nervous system lymphoma mouse model; discloses a cell strain for constructing a primary central nervous system lymphoma mouse model; discloses tumorigenesis characteristics of a primary central nervous system lymphoma mouse model.
The mouse model of primary central nervous system lymphoma is constructed and obtained, the lymphoma in the mouse model develops rapidly and mainly shows brain invasion and the like, has high similarity with the invasion characteristics of human primary central nervous system lymphoma, is an ideal in-vivo efficacy evaluation animal model for developing small molecule anticancer drugs, antibody therapy and cell therapy, and can also be used for molecular mechanism research of lymphoma invading the brain.
Drawings
FIG. 1: the identification of MA-2 cell strain and the construction of primary central nervous system lymphoma mouse model. The MA cell line is transplanted to a C57BL/6 mouse, namely a lymphoma transplantation model, by adopting a tail vein injection mode, when a recipient mouse is diseased, the recipient mouse is dissected to obtain the lymphoma, and the lymphoma of different recipient mouse sources is represented by MA-1L and MA-2L as shown in the figure. And (3) further culturing the single lymphoma in vitro to establish stable cell strains, wherein the stable cell strains established by the different lymphomas MA-1L and MA-2L are represented by MA-1 and MA-2 as shown in the figure, and then carrying out continuous transplantation experiments to analyze whether different cell strains have different invading organs in a receptor mouse or not, wherein the MA cell lines mainly invade organs outside lymph nodes such as kidney, lymph node and the like.
FIG. 2: invasion characteristics of a mouse model of primary central nervous system lymphoma. Survival curves of A, MA and MA-2 receptor mice. Wherein the median survival time of the MA receptor mouse is 27.5 days, and the median survival time of the MA-2 receptor mouse is 20 days. B, brain invasion characteristics of MA-2 receptor mice. The left picture is a photograph of the brain not invaded, the right picture is a photograph of the lymphoma invading brain, the statistical result shows that the brain invasion characteristic is a unique characteristic of the MA-2 receptor mouse, the p value is 0.015, and the difference is obvious. Other organ invasion characteristics of C, MA-2 receptor mice. The figure shows the picture of lymphoma invading thoracic spine, thoracic rib, stomach, kidney and lung, and the statistical result shows that the lung invading characteristic is the unique characteristic of MA-2 receptor mice, the p value is 0.0003, and the difference is very significant.
Detailed Description
The invention mainly discloses a primary central nervous system lymphoma mouse model which can be used for basic research of primary central nervous system lymphoma and in-vivo pharmacodynamic analysis of preclinical animals in the process of developing new drugs.
Compared with other lymphoma mouse models, the primary central nervous system lymphoma mouse model constructed based on the same transplantation model is considered to be closer to physiological microenvironment in the occurrence process of the lymphoma, and the occurrence process of the lymphoma is under a normal immune system and has stronger clinical relevance with human lymphoma. Moreover, in the intact immune system, the development of these lymphomas is in a normal biological and immune environment, and the functions of cytokines and effector cells can be more precisely studied.
The genetic background of the invention is E mu-Myc; cdkn2a Arf-/ On the basis of the B cell lymphoma cell line, a cell line MA-2 which is capable of invading the brain in a homograft model is further separated, and is an ideal mouse model of the primary central nervous system lymphoma of the human.
The primary central nervous system lymphoma mouse model is an ideal lymphoma mouse model for developing candidate drugs and therapy in-vivo efficacy evaluation, wherein a cell strain MA-2 is transplanted into a receptor mouse by adopting a tail vein injection mode, the model is simple and efficient in construction process and short in disease course, the disease course is generally 20 days of attack, and the disease occurrence characteristics similar to human primary central nervous system lymphoma can be shown.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, sambrook et al: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; (iii) METHODS IN ENZYMOLOGY, vol.304, chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.
Example 1
1. Material
The cell line MA-2 is B-cell lymphoma cell line (genetic background is E mu-Myc; cdkn2 a) Arf-/- ) Lymphoma was further isolated and primary cells were cultured to obtain stable cell lines in mice reconstituted with lymphoma obtained by tail vein transplantation. The B cell lymphoma cell strain is derived from Jiang Hai laboratory (Central advanced innovations in molecular cell science/institute of biochemistry and cell biology).
All strains of mice presented in the present invention were in the C57BL/6 background. All mice were housed in a specific microorganism-free (SPF) barrier according to standard experimental animal feeding methods.
2. Construction of Primary Central nervous System lymphoma mouse model
The core of the method for constructing the primary central nervous system lymphoma mouse model is to screen and obtain a cell strain capable of invading the brain in the allograft model and verify that the cell strain has stable invasion characteristics in the continuous transplantation model. The primary central nervous system lymphoma mouse model is obtained by injecting a cell strain MA-2 into tail vein by using an allograft model.
Specifically, as shown in fig. 1, the process of constructing the mouse model of primary central nervous system lymphoma includes the steps of: (1) Analysis of MA cell lines by continuous transplantation experiments (genetic background E μ -Myc; cdkn2 a) Arf -/-) the tumorigenic characteristics of the primary cell strain, and screening to obtain a cell strain MA-2 which tends to invade the brain; (2) Transplanting the cell strain MA-2 into a receptor mouse in a tail vein injection mode; (3) The receptor mice show a brain invasion phenotype about 20 days after receiving the transplantation, and a primary central nervous system lymphoma mouse model is obtained. Compared with the MA cell line, the cell line MA-2 has unique brain invasion characteristics.
3. Invasion characteristics of primary central nervous system lymphoma mouse model
In the invention, the lymphoma invasion characteristics and the total survival time of a lymphoma mouse model obtained by constructing the MA cell line and the MA-2 cell line are analyzed. The MA cell line mainly invades tissues and organs such as kidney in recipient mice transplanted with lymphoma, and the MA-2 cell line mainly invades brain. Statistical analysis showed significant differences in lymphoma invasion characteristics in the two models. Survival curve analysis showed a significant difference in the overall survival time for both models (p = 0.0003), a median survival time for MA of 27.5 days, and a median survival time for MA-2 of 20 days.
Specifically, as shown in fig. 2: FIG. 2A shows the survival curves of lymphoma transplantation models constructed using MA cell line and MA-2 cell line. FIG. 2B, representative picture and statistical difference of brain invasion for MA cell line and MA-2 cell line model. P <0.05 was a significant difference. Figure 2c, representative pictures of other invasion features of ma-2 cell line, including thoracic spine, thoracic rib, stomach, kidney, lung, where lung invasion is statistically different. P <0.05 was significantly different, P <0.01 was very significantly different, and P <0.001 was very significantly different. There were 8 MA receptor mice and 6 MA-2 receptor mice.
Claims (9)
1. A method for constructing a primary central nervous system lymphoma mouse model comprises the following steps: mouse B cell lymphoma cell line MA (genetic background E mu-Myc; cdkn2 a) Arf-/- ) Constructing an allografting model, culturing primary lymphomas with different invasion parts in vitro and establishing stable cell strains after the lymphomas occur, then carrying out continuous transplantation experiments by adopting the primary cell strains, and screening to obtain a cell strain MA-2 with brain invasion tendency. The cell strain MA-2 is transplanted into a receptor mouse in a tail vein injection mode, and the receptor mouse has a phenotype of brain invasion about 20 days after receiving the transplantation, namely a primary central nervous system lymphoma mouse model is successfully constructed.
2. The method for constructing a mouse model of primary central nervous system lymphoma according to claim 1, wherein the cell line MA-2 is derived from mouse B-cell lymphoma cell line (genetic background is E μ -Myc; cdkn2 a) Arf-/- )。
3. The method of claim 1, wherein cell line MA-2 is selected from the group consisting of a primary central nervous system lymphoma, and a primary central nervous system lymphoma.
4. The method for constructing a mouse model of primary central nervous system lymphoma according to claim 1, wherein the cell line MA-2 is transplanted into a recipient mouse by tail vein injection, and the recipient mouse has a phenotype of brain invasion about 20 days after receiving the transplantation.
5. The method for constructing a primary central nervous system lymphoma mouse model according to claim 1, wherein the primary central nervous system lymphoma mouse model is constructed by using cell line MA-2, and the main phenotype of the receptor mouse is brain invasion.
6. The method of constructing a mouse model of primary central nervous system lymphoma according to claim 1, comprising the steps of: (1) Analyzing the tumorigenic characteristics of the primary cell strain from the MA cell line by adopting a continuous transplantation experiment, and screening to obtain a cell strain MA-2 tending to invade the brain; (2) Transplanting the cell strain MA-2 into a receptor mouse in a tail vein injection mode; (3) The receptor mice show a brain invasion phenotype about 20 days after receiving the transplantation, and a primary central nervous system lymphoma mouse model is obtained.
7. A mouse model of primary central nervous system lymphoma prepared by the method of any one of claims 1 to 6.
8. Use of the method of construction according to any one of claims 1 to 6 or the mouse model of primary central nervous system lymphoma according to claim 7 for screening a mouse model of primary central nervous system lymphoma for the treatment of a drug.
9. A method for screening and evaluating the in vivo efficacy of a primary cns lymphoma treatment agent comprising the steps of: a candidate drug or therapy is applied to the mouse model of the primary central nervous system lymphoma obtained by the construction method as described in any one of claims 1 to 6, if the occurrence of lymphoma is reduced and the survival time of a receptor mouse is prolonged, the drug or therapy is indicated to be effective, and the candidate drug or therapy can be used as a candidate therapeutic drug or therapy of the primary central nervous system lymphoma.
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