PDX model establishment method for TTMV-RARA mutant acute promyelocytic leukemia

Abstract

The invention discloses a method for establishing a TTMV-RARA mutant acute promyelocytic leukemia PDX model, which is characterized in that leukemia cells of a TTMV-RARA mutant acute promyelocytic leukemia patient are inoculated into a mouse body, and the TTMV-RARA mutant acute promyelocytic leukemia PDX model is established. The invention provides the leukemia PDX model construction method of the acute promyelocytic leukemia (APL) patient with the TTMV-RARA mutant genotype, the PDX model of the TTMV-RARA mutant APL is successfully constructed for the first time, and an important basis is provided for scientific research of acute promyelocytic leukemia with a rare genotype; in the model construction method, the mouse can be modeled without irradiation, the method is simple, the construction time is short, and the model is stable. By establishing the stable PDX model, a powerful experimental platform is provided for subsequent gene function research, drug screening and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of leukemia modeling, and more specifically, relates to a method for establishing a PDX model of TTMV-RARA mutant acute promyelocytic leukemia. Background Art

[0002] Acute promyelocytic leukemia (APL) is an aggressive hematological malignancy with a unique genetic abnormality, namely the t(15;17)(q22;q12) chromosomal translocation, which leads to the production of PML-RARA fusion protein. In recent years, with the rapid development of gene sequencing technology, more and more diseases have been found to be closely related to specific gene mutations. Among them, the TTMV-RARA mutant genotype plays an important role in the pathogenesis of acute promyelocytic leukemia (APL).

[0003] Torqueteno minivirus (TTMV) is a non-enveloped, circular, single-stranded DNA virus with a genome of about 2800 bases. Studies have found that TTMV genome fragments are inserted into the human genome and form a TTMV::RARA fusion gene. In 2011, it was first reported that TTMV can cause atypical APL in the form of specific genome fragment integration. So far, 6 cases of TTMV::RARA-APL have been reported, but the specific molecular mechanism of TTMV::RARA and APL has not yet been elucidated. Due to the complex and variable structure of the TTMV genome sequence, there are many types of TTMV-RARA fusion genes. At present, the treatment of APL with TTMV-RARA mutant genotype mainly relies on traditional chemotherapy, but the treatment effect is limited and the side effects are large. Therefore, in-depth research on APL with TTMV-RARA mutant genotype and the development of new treatment methods are of great significance to improve the survival rate and quality of life of APL patients, and related research requires an ideal animal model.

[0004] Mice are ideal animal models for studying tumors, and there are mouse PDX models for a variety of tumors. However, due to the heterogeneity and specificity of leukemia tumor cells, the conditions for constructing PDX models are variable, and it is difficult to construct mouse PDX models. Currently, there are not many PDX models for APL, and there are even fewer PDX models of leukemia cells with special genotypes. Therefore, in order to further study the mechanism of action of the TTMV-RARA mutant genotype in the pathogenesis of APL and explore new treatment methods, it is of great significance to construct a PDX model of APL with the TTMV-RARA mutant genotype. At present, there is no report on the establishment of a PDX model of APL with the TTMV-RARA mutant genotype at home and abroad. Summary of the invention

[0005] The present invention aims to solve the problem of the existing lack of PDX models of TTMV-RARA mutant genotype APL, and provides a method for establishing a TTMV-RARA mutant acute promyelocytic leukemia PDX model.

[0006] The purpose of the present invention is to provide a method for establishing a TTMV-RARA mutant acute promyelocytic leukemia PDX model and application of the model preparation method.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for establishing a TTMV-RARA mutant acute promyelocytic leukemia PDX model. Leukemia cells of a patient with the TTMV-RARA mutant acute promyelocytic leukemia are inoculated into mice to establish the TTMV-RARA mutant acute promyelocytic leukemia PDX model.

[0009] Specifically, the screening criteria for patients with TTMV-RARA mutant acute promyelocytic leukemia are:

[0010] a) Aged 0-60 years, with peripheral blood and bone marrow smear and cytochemistry examinations meeting the diagnostic criteria for acute myeloid leukemia of FBA and WHO; combined with the results of morphological testing, immunophenotyping, chromosome karyotype testing and genetic testing, the patient is diagnosed with TTMV-RARA mutation acute promyelocytic leukemia;

[0011] b) ECOG performance status score of 0-2;

[0012] c) Liver and kidney functions are basically normal.

[0013] Specifically, the diagnostic criteria for acute myeloid leukemia of the FBA and WHO are as follows:

[0014] 1. Peripheral blood or bone marrow primitive cells ≥ 20%;

[0015] 2. Acute myeloid leukemia can be diagnosed when myeloid leukemia cells are ≥ 20% of bone marrow nucleated cells or non-erythroid cells are ≥ 20% (erythrocyte components > 50%), or in the case of recurrent cytogenetic abnormalities [t(8;21), t(15;17), inv(16) or t(16;16)], regardless of the percentage of primitive cells;

[0016] The basically normal liver and kidney functions are: blood bilirubin ≤34.2 μmol / L, AST / ALT below 2 times the upper limit of normal value, and blood creatinine ≤222 μmol / L.

[0017] As an alternative embodiment, the inoculation amount is 1×10 5 -1×10 6 cells.

[0018] As an alternative embodiment, the vaccination is carried out by injection through the rat tail vein.

[0019] Preferably, the mouse is an NCG mouse.

[0020] Specifically, the mice are 6-8 weeks old.

[0021] As an optional implementation scheme, the detection method of the TTMV-RARA mutant acute promyelocytic leukemia PDX model is: after inoculation, the blood of the mice is tested every week, and whether the model is successfully established is determined based on the test results. The test items include:

[0022] 1) Morphological detection of leukemia cells in blood smears and bone marrow smears;

[0023] 2) Detection of leukemia cells by flow cytometry immunophenotyping;

[0024] 3) Histopathological examination of the tissue infiltration characteristics of leukemia cells.

[0025] Specifically, the criteria for successful model building are:

[0026] 1) Human leukemia cells were found in peripheral blood smears and bone marrow smears;

[0027] 2) Detection of cell populations with human leukemia immune phenotype in peripheral blood, with the detection index being CD45+;

[0028] 3) The modeling was successful if histopathological observation revealed the presence of human leukemia cell infiltration in mouse tissues and organs.

[0029] As an optional embodiment, the blood of mice in the test is collected through the tail vein or submandibular vein.

[0030] As an optional embodiment, after the PDX model is established, the PDX model is also subcultured; the subculture includes: taking the established PDX model as the P1 generation, isolating leukemia cells from the Pn generation model mice, and then inoculating them into new NCG mice to obtain Pn+1 generation model mice, wherein n is an integer from 1 to 3.

[0031] The use of the leukemia PDX model prepared by the above method in the preparation of drugs for treating TTMV-RARA mutant acute promyelocytic leukemia should also be within the protection scope of the present invention.

[0032] The present invention has the following beneficial effects:

[0033] The present invention provides a method for constructing a leukemia PDX model of a patient with acute promyelocytic leukemia (APL) of a TTMV-RARA mutant genotype, and successfully constructs a PDX model of TTMV-RARA mutant APL for the first time, providing a suitable animal model for scientific research on TTMV-RARA mutant APL. In addition, the model construction method of the present invention is simple, and mice can be modeled without irradiation; the construction time is short, and the mouse model can be successfully constructed within 14 days; the mouse model constructed by the present invention is stable and can be stably passed from P1 to P3. The present invention provides a powerful experimental platform for subsequent gene function research, drug screening, etc. by establishing a stable PDX model of TTMV-RARA mutant APL genotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the method for establishing a leukemia PDX model for patients with TTMV-RARA mutant APL.

[0035] Figure 2 This is a bone marrow smear of the patient screened in the present invention.

[0036] Figure 3 This is the detection result of the TTMV-RARA mutant gene of the patients screened in the present invention.

[0037] Figure 4 This is a full transcriptome detection report of blood tumors of patients screened in the present invention.

[0038] Figure 5 The body weight of modeling mice was measured regularly.

[0039] Figure 6 Peripheral blood smear and bone marrow smear of modeling mice 14 days after P1 inoculation (Figure A is the peripheral blood smear of mice; Figure B is the bone marrow smear of mice).

[0040] Figure 7 The proliferation of CD45 human leukemia cells in the peripheral blood of modeling mice of generations P1, P2 and P3 was detected by flow cytometry 14 days after inoculation.

[0041] Figure 8 The results of histopathological detection of leukemia cell infiltration in the liver, spleen, and kidneys of modeling mice 14 days after P1 inoculation (Figure A is the spleen; Figure B is the liver; Figure C is the kidney). DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Figure 1 Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Mice were female NCG mice, 6-8 weeks old, purchased from Guangdong Yaokang Biotechnology Co., Ltd.

[0045] Example 1 Selection of clinical patients and pretreatment of leukemia samples

[0046] 1. Selection of patients with TTMV-RARA mutant acute promyelocytic leukemia (APL)

[0047] Specific screening criteria:

[0048] a) Aged 0-60 years, with peripheral blood and bone marrow smear and cytochemistry examinations meeting the diagnostic criteria for acute myeloid leukemia of FBA and WHO; combined with the results of morphological testing, immunophenotyping, chromosome karyotype testing and genetic testing, the patient is diagnosed with TTMV-RARA mutation acute promyelocytic leukemia;

[0049] b) ECOG performance status score of 0-2;

[0050] c) Liver and kidney functions are basically normal.

[0051] The diagnostic criteria for acute myeloid leukemia of FBA and WHO are as follows:

[0052] 1. Peripheral blood or bone marrow primitive cells ≥ 20%;

[0053] 2. Acute myeloid leukemia can be diagnosed when myeloid leukemia cells are ≥ 20% of bone marrow nucleated cells or non-erythroid cells are ≥ 20% (erythrocyte components > 50%), or in the case of recurrent cytogenetic abnormalities [t(8;21), t(15;17), inv(16) or t(16;16)], regardless of the percentage of primitive cells;

[0054] Basically normal liver and kidney function means: blood bilirubin ≤34.2μmol / L, AST / ALT less than 2 times the upper limit of normal value, and blood creatinine ≤222μmol / L.

[0055] The bone marrow smear of the patient screened in the present invention is as follows Figure 2 As shown in the figure, the patient's bone marrow smear showed that the proportion of promyelocytes was 93%, and POX was strongly positive; bone marrow flow cytometry showed that the proportion of abnormal promyelocytes was about 91.6%, and the immunophenotype was inferred to be AML-M3. The test results of the TTMV-RARA mutant gene are as follows Figure 3As shown, the patient's blood tumor full transcriptome detection report is as follows Figure 4 shown.

[0056] The above results showed that the patient was a patient with TTMV-RARA mutant APL.

[0057] 2. Extract bone marrow fluid and separate blood tumor cells

[0058] (1) Select the appropriate part of the patient for bone marrow puncture, collect 1-2 mL of bone marrow fluid, add 3 times the volume of red blood cell lysis buffer to the bone marrow fluid, shake and mix; centrifuge at 1500 r / min for 5 min, discard the supernatant, and repeat the operation twice;

[0059] (2) adding phosphate buffered saline (PBS) to the precipitate and mixing; centrifuging at 1500 rpm for 5 min; and discarding the supernatant;

[0060] (3) Resuspend the cells in PBS and count the cells;

[0061] (4) Preparation of 1×10 5 -1×10 6 / 0.1-0.15mL concentration of mononuclear cell solution (the concentration of the mixture is calculated based on the total resuspension volume).

[0062] Example 2 Method for establishing and passage of NCG mouse PDX model

[0063] 1. Mouse selection and feeding conditions

[0064] Female NCG mice aged 6-8 weeks were housed in separate cages under SPF conditions. The room temperature was maintained at 18-25°C and the relative humidity was maintained at 40%-60%. The special cages, bedding, feed and drinking water were sterilized by autoclaving at 121°C for 30 minutes. The bedding was changed at least once a week.

[0065] 2. Modeling

[0066] The mononuclear cell solution prepared in Example 1 was inoculated via the tail vein of mice, with an inoculation volume of 1×10 5 -1×10 6 cells.

[0067] 3. Continuous inoculation and passage of leukemia cells

[0068] Blood or bone marrow was extracted from NCG mice (P1 generation) with successful primary cell modeling, and leukemia cells were isolated and cultured at a rate of 1×10 per mouse. 5 -1×10 6 New NCG mice (P2 generation) were inoculated with an inoculum of 10 cells per well via tail vein injection.

[0069] Blood or bone marrow was extracted from P2 NCG mice, and leukemia cells were isolated and expressed at 1 × 10 per mouse. 5 -1×10 6 New NCG mice (P3 generation) were inoculated with an inoculum of 10 cells per well via tail vein injection.

[0070] Example 3 Detection and identification after model establishment

[0071] 1. Modeling Success Evaluation Indicators

[0072] The detection and identification methods after the model is established include:

[0073] 1) Human leukemia cells were found in peripheral blood smears and bone marrow smears of NCG mice;

[0074] 2) Detection of cell populations with human leukemia immunophenotype in the peripheral blood of NCG mice, with the detection index being CD45+;

[0075] 3) Histopathological observation showed the presence of human leukemia cell infiltration in mouse tissues and organs.

[0076] (1) General observation indicators

[0077] After inoculation, the mice were observed for symptoms such as mental depression, poor appetite, emaciation, hunched back, diarrhea, hair loss, skin ulcers, hemiplegia, blindness, and growth of tumors. The mice were weighed weekly and the data were recorded.

[0078] (2) Blood smear and bone marrow smear testing and flow cytometry testing

[0079] After inoculation, peripheral blood (blood was collected from the tail vein or submandibular vein) and bone marrow of mice were collected every week, and smears or prints were made, and the cell morphology was observed by Wright staining;

[0080] The proportion of leukemia immunophenotyping was monitored weekly by flow cytometric analysis of peripheral blood cells and bone marrow cell suspensions of mice.

[0081] (3) Histopathological examination

[0082] The mice with successful transplantation were selected and killed, and the tumor tissues, liver, spleen, and kidneys of the mice were taken. Conventional fixation, paraffin embedding, and sectioning were performed. The corresponding tissues of normal mice NCG were taken for histopathology and other controls. The tissues were stained with hematoxylin-eosin staining, and the morphology of tissues and organs was observed.

[0083] 2. Experimental Results

[0084] The results of regular body weight measurements of model mice are as follows Figure 5 And as shown in Table 1.

[0085] Table 1 Body weight data of modeling mice (unit: g)

[0086]

[0087]

[0088] (1) Peripheral blood smear and bone marrow smear of P1 generation model mice 14 days after inoculation Figure 6 As shown, the results showed that human leukemia cells were found in peripheral blood and bone marrow smears.

[0089] (2) Flow cytometry was used to detect the proliferation of CD45 human leukemia cells in the peripheral blood of modeling mice 14 days after inoculation of P1, P2, and P3 generations. Figure 7 As shown, the results showed that flow cytometry detected a large number of CD45+ cells in the peripheral blood and bone marrow of mice.

[0090] (3) Histopathological examination of the P1 generation mice 14 days after inoculation revealed infiltration of human leukemia cells in the liver, spleen, and kidneys. Figure 8 As shown, the results demonstrate the infiltration of human leukemia cells in the tissues and organs of the modeled mice.

[0091] Based on all the test results, the TTMV-RARA mutant APL / NCG mouse model was successfully established 14 days after P1 generation inoculation. The P2 generation mice inoculated with P1 generation peripheral blood and bone marrow tumor cells and the P3 generation mice inoculated with P2 generation mice peripheral blood and bone marrow tumor cells were also successfully modeled two weeks after inoculation and were able to be stably propagated.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing a TTMV-RARA mutant acute promyelocytic leukemia PDX model, characterized in that: The leukemia cells of patients with TTMV-RARA mutant acute promyelocytic leukemia were inoculated into mice to establish a TTMV-RARA mutant acute promyelocytic leukemia PDX model.

2. The method according to claim 1, characterized in that: The screening criteria for patients with TTMV-RARA mutant acute promyelocytic leukemia are: a) Aged 0-60 years, with peripheral blood and bone marrow smear and cytochemistry examinations meeting the diagnostic criteria for acute myeloid leukemia of FBA and WHO; combined with the results of morphological testing, immunophenotyping, chromosome karyotype testing and genetic testing, the patient is diagnosed with TTMV-RARA mutation acute promyelocytic leukemia; b) ECOG performance status score of 0-2; c) Liver and kidney functions are basically normal.

3. The method according to claim 1, characterized in that: The inoculation volume was 1×10 5 -1×10 6 cells.

4. The method according to claim 1, characterized in that: The inoculation method is injection through the rat tail vein.

5. The method according to claim 1, characterized in that: The mice are NCG mice.

6. The method according to claim 5, characterized in that: The mice were 6-8 weeks old.

7. The method according to claim 1, characterized in that: The detection method of the TTMV-RARA mutant acute promyelocytic leukemia PDX model is as follows: after inoculation, the blood of the mice is tested every week, and whether the model is successfully established is determined based on the test results. The test items include: 1) Morphological detection of leukemia cells in blood smears and bone marrow smears; 2) Detection of leukemia cells by flow cytometry immunophenotyping; 3) Histopathological examination of the tissue infiltration characteristics of leukemia cells.

8. The method according to claim 7, characterized in that: The criteria for successful model building are: 1) Human leukemia cells were found in peripheral blood smears and bone marrow smears; 2) Detection of cell populations with human leukemia immune phenotype in peripheral blood, with the detection index being CD45+; 3) The modeling was successful if histopathological observation revealed the presence of human leukemia cell infiltration in mouse tissues and organs.

9. The method according to claim 1, characterized in that: After the PDX model is established, the PDX model is also subcultured; the subculture includes: taking the established PDX model as the P1 generation, isolating leukemia cells from the Pn generation model mice, and then inoculating them into new NCG mice to obtain Pn+1 generation model mice, where n is an integer of 1-3.

10. Use of the leukemia PDX model prepared by the method according to any one of claims 1 to 9 in the preparation of a drug for treating TTMV-RARA mutant acute promyelocytic leukemia.

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