Human acute myelogenous leukemia cell strain with multiple poor prognosis gene mutations and application of human acute myelogenous leukemia cell strain

Abstract

The invention discloses an acute myelogenous leukemia cell strain with multiple adverse prognostic gene mutations in human and application thereof, the cell strain is named as human acute myelogenous leukemia cell strain YYXY-M14 and preserved in China Center for Type Culture Collection (Wuhan University, Wuhan, China) on October 12, 2024, and the preservation number is CCTCC NO: C2024350. The acute myelogenous leukemia cell strain simultaneously has three acute myelogenous leukemia poor prognostic gene mutations of TP53, RUNX1 and ASXL1, has the in-vitro natural proliferation capacity of acute myelogenous leukemia cells, and can be used as a cell material for researching the pathogenic mechanism of human acute myelogenous leukemia with positive mutation of human TP53, RUNX1 and ASXL1 in vitro. Meanwhile, the method can be used for in-vivo animal model construction and acute myelogenous leukemia drug screening and evaluation research.

Description

Technical Field

[0001] The present invention relates to the fields of biology and oncology, and in particular to a human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations and an application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a group of malignant clonal blood tumors originating from the bone marrow due to the disorder of differentiation and maturation of bone marrow hematopoietic function. The primitive cells in the bone marrow cannot further differentiate and mature, and a large number of primitive cells accumulate in the bone marrow or peripheral blood, and the normal blood cells decrease. Clinically, it often manifests as anemia, bleeding, infection and other symptoms. The clonal proliferation of leukemia cells can also cause clinical manifestations of tissue infiltration, such as hepatosplenomegaly. Epidemiological statistics show that AML mostly occurs in the elderly, with a median age of onset of about 68 years old, and no obvious gender difference. Due to the high heterogeneity of AML, its exact pathogenesis is currently unclear. Some studies have shown that environmental factors, such as benzene exposure, may lead to the occurrence of AML.

[0003] The treatment of AML includes induction remission therapy and post-remission therapy. Induction remission therapy with anthracyclines combined with cytarabine is still the first choice for AML treatment. In China, homoharringtonine is also commonly used for induction remission therapy of AML, with an overall remission rate of about 70%. After remission, based on the patient's risk stratification, their own conditions and supply factors, they can further choose consolidation chemotherapy or hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation is currently the only way to cure AML except for acute promyelocytic leukemia. With the continuous development of cytogenetics and molecular genetics, the risk stratification of AML has become more refined, and more cytogenetic markers and molecular genetic markers related to AML prognosis have been written into the diagnosis and treatment guidelines for AML. In addition to the more refined risk stratification of AML, some drugs targeting AML have also been used in the treatment of AML in the past decade, which not only opened a new stage of targeted treatment for AML, but also improved the treatment response of AML to a certain extent. The BCL-2-based targeted inhibitor venetoclax combined with azacitidine has improved the efficacy of elderly AML patients who are not suitable for intensive chemotherapy. The combination of venetoclax and cytarabine and daunorubicin, called "DAV", has increased the induced remission rate of AML to 90%. The future direction of efforts is to continuously reveal the pathogenic mechanism of AML, discover effective therapeutic drugs such as targeted drugs, optimize and promote individualized treatment plans, and improve the survival of AML.

[0004] In combination with the progress in the diagnosis and treatment of AML at home and abroad, hematology experts in my country have formulated the "Guidelines for the Diagnosis and Treatment of Adult Acute Myeloid Leukemia (Non-Acute Promyelocytic Leukemia) in China (2023 Edition)". Among them, AML is divided into three risk stratifications: good prognosis, moderate prognosis and poor prognosis based on the cytogenetic and subgenetic characteristics of AML. In terms of cytogenetics, chromosomal abnormalities such as t(8;21)(q22;q22) formed by chromosomes 8 and 21, and inv(16)(p13q22) or t(16;16)(p13,q22) formed by chromosome 16 abnormalities are classified as good prognosis risk stratification. Haploid karyotype, complex karyotype, -5, -7, t(8;16)(p11;p13) and other chromosomal abnormalities are classified as poor prognosis risk stratification. Normal karyotype, t(9;11)(p22;q23) and chromosomal abnormalities that are not classified as good prognosis and poor prognosis risk stratification belong to the moderate prognosis risk stratification. As the understanding of AML cytogenetics continues to deepen, the chromosomes for different stratifications are not static. Molecular genetics plays an increasingly important role in the risk stratification of AML, and more prognosis-related targets have been added to molecular genetics. In the risk stratification of AML with poor prognosis, TP53, RUNX1, ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 mutation-positive and FLT3-ITD high allele mutation ratio are written into the guidelines, among which AML patients with TP53 mutations have the worst prognosis, with an overall survival rate of only about 12%. Due to the high heterogeneity of AML, different AML patients have different chromosomal karyotypes and molecular characteristics, and most of the AML-specific chromosomal karyotype characteristics and molecular characteristics have not yet been discovered, and further technological innovation is needed to better understand the molecular characteristics of AML.

[0005] Cell lines are the basic cell materials and tools for studying the causes of tumors, discovering molecular markers, exploring tumor therapeutic drugs, preclinical drug validation, and animal model construction. They play an irreplaceable role in promoting tumor research. In 1986, the National Cancer Center of the United States launched the human tumor cell line anti-tumor drug screening project NCI-60. This project used 60 human tumor cell lines to conduct cytotoxicity studies on more than 3,000 small molecule inhibitors. More than 100 drugs such as paclitaxel, cisplatin, and fludarabine were discovered as anti-tumor drugs in this study. Hematological tumor cell lines have also promoted basic and clinical research on hematological tumors. Among them, the HL-60 acute promyelocytic myeloid leukemia cell line with the PML-RARA fusion gene and the K562 chronic myeloid leukemia cell line with the BCR-ABL fusion gene provide unique cell materials for the pathogenesis and resistance mechanism, drug screening, and animal models of acute promyelocytic leukemia and chronic myeloid leukemia. There are also many AML cell lines used in research internationally. However, due to the high heterogeneity of AML's cytogenetics and molecular biology, the cell lines currently used in AML research cannot cover all AMLs with different genetic and molecular characteristics. There are also more new AML pathogenic mechanisms and molecular characteristics, which require the establishment of more AML cell lines with different molecular genetic characteristics for AML research.

[0006] In summary, the present invention provides a human acute myeloid leukemia cell line with positive mutations in three genes, TP53, RUNX1 and ASXL1, and has abnormal chromosome 16 and add(16)(q11.2) chromosome changes, which is used for the study of the pathogenesis of AML, drug screening and evaluation, and animal models, and provides a new AML cell line with abnormal chromosomes and abnormal gene mutations. Summary of the invention

[0007] The purpose of the present invention is to provide a human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations and application thereof.

[0008] The objective of the present invention is achieved through the following technical scheme: A human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations, the cell line is named human acute myeloid leukemia cell line YYXY-M14, the Latin name is Homo sapiens, and it was preserved in the China Center for Type Culture Collection (CCTCC) on October 12, 2024, address: Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C2024350.

[0009] The present invention also provides daughter cells of the above-mentioned human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations is human acute myeloid leukemia cell YYXY-M14.

[0010] The present invention also provides the above-mentioned human acute myeloid leukemia cells with multiple adverse prognosis gene mutations, which simultaneously have three TP53, RUNX1 and ASXL1 mutations that are clearly defined in the guidelines for AML adverse prognosis stratification, and an AML cell line with a chromosome karyotype of add(16)(q11.2). The human acute myeloid leukemia cell YYXY-M14 simultaneously has three acute myeloid leukemia adverse prognosis gene mutations of TP53, RUNX1 and ASXL1, and does not have BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and FLT3-ITD acute myeloid leukemia adverse prognosis gene mutations.

[0011] The chromosome karyotype of the human acute myeloid leukemia cell line YYXY-M14 is 46,XX,add(16)(p11.2) / 46,XX, which is a chromosome karyotype with a medium prognosis.

[0012] Cell lines are in a suspended state under cell culture conditions, growing individually or in clusters.

[0013] The present invention also provides the use of the acute myeloid leukemia cell line with multiple adverse prognosis gene mutations as described above, selected from any one or more of the following:

[0014] 1. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations is used in the study of the pathogenesis of TP53, RUNX1 and ASXL1 gene mutations in acute myeloid leukemia.

[0015] 2. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations is used in preparing a tumor cell model or a tumor animal model.

[0016] 3. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations is used in screening and / or evaluating tumor therapeutic drugs.

[0017] 4. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations is used in the preparation of tumor vaccines.

[0018] The present invention also provides a method for constructing the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations, comprising the following steps: obtaining 5 ml of peripheral blood from an AML patient diagnosed by bone marrow Wright staining and multi-parameter flow cytometry into a vacuum blood collection tube anticoagulated with EDTA or heparin. The fresh blood sample is immediately centrifuged at 2000 rpm for 10 minutes. 2-3 ml of plasma is retained in a 1.5 ml sterile EP centrifuge tube in a biological safety cabinet and stored, and 2-3 ml of sterile 1xPBS is added to the remaining sample to resuspend the remaining sample. Further, a clean and sterile 15 ml centrifuge tube is taken and 5-6 ml of mononuclear cell separation solution is added. The resuspended sample is slowly added along the wall of the 15 ml centrifuge tube with a 1 ml pipette. Subsequently, centrifuge at 2000 rpm for 30 minutes. After the centrifugation, the white cell precipitate of the mononuclear cell layer is sucked into a new sterile 15 ml sterile centrifuge tube with a 1 ml pipette in the safety cabinet, and 5 ml of sterile 1xPBS is added to resuspend the cells, and centrifuge at 2000 rpm for 5 minutes. After discarding the supernatant, add 5 ml of IMDM complete medium (IMDM 90% + fetal bovine serum 10%) to resuspend the cells and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and add 5 ml of IMDM complete medium to resuspend the cells. Count the cells with an automatic cell counter and take 1-2 x 10 8 After adding 10 cells to a T25 cell culture flask, add 6 ml of IMDM complete medium to mix the cells and place them in a 37°C constant temperature and humidity carbon dioxide cell culture incubator to culture the cells. Observe the state of the cultured cells under an inverted optical microscope every day and count the cell concentration. According to the cell state and density, replace the new IMDM complete medium by centrifugation every 5 to 7 days until the cells begin to proliferate.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations of the present invention can be infinitely passaged in vitro without any external cytokine, and is cloned and proliferated. The cell shape in vitro is stable, which conforms to the biological characteristics of clinical AML tumor cells. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations originates from the peripheral blood of AML patients with a clear diagnosis. The pathogenic mechanism, targeted drug discovery and efficacy evaluation of the combined regimen of human acute myeloid leukemia with positive TP53, RUNX1 and ASXL1 mutations can be used to guide clinical drug use, and can also be used for the construction of AML animal models, providing good cell materials for the study of AML with multiple adverse prognosis mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with examples and drawings;

[0022] Figure 1The morphology of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations under culture conditions and the results after Wright-Giemsa staining;

[0023] Figure 2 The growth curve results of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations;

[0024] Figure 3 The chromosome karyotype and gene mutation results of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations;

[0025] Figure 4 The results of evaluating the sensitivity of human acute myeloid leukemia cell lines with multiple adverse prognosis gene mutations to chemotherapy drugs;

[0026] Figure 5 This is the animal model result of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations. DETAILED DESCRIPTION

[0027] The present invention is further described below with examples, but the present invention is not limited thereto. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions.

[0028] Example 1 Preparation of human acute myeloid leukemia cell line YYXY-M14 cell line

[0029] Primary cell culture: Leukemia mononuclear cells were immediately isolated from peripheral blood obtained from female patients with AML who were hospitalized in the People's Hospital Affiliated to Ningbo University. In the biosafety cabinet, 5 ml of separation solution was added to a 15 ml sterile centrifuge tube that had been pre-added with 5 ml of mononuclear cell separation solution, and centrifuged at 2000 rpm for 30 minutes. After centrifugation, the mononuclear cell layer was transferred to a new 15 ml sterile centrifuge tube, 5 ml of sterile 4°C pre-cooled 1xPBS was added to resuspend the cells, and centrifuged at 2000 rpm for 5 minutes. After discarding the supernatant, 5 ml of IMDM complete medium (IMDM 90% + fetal bovine serum 10%) was added to resuspend the cells, and centrifuged at 1500 rpm for 5 minutes. Discard the supernatant, add 6 ml of IMDM complete medium, and resuspend the cells. For the fully automated cell counting plate technology, 1 to 2x10 8 After adding cells to T25 cell culture flask, add IMDM complete medium to 6 ml and mix the cells. Then culture the cells in a 37°C constant temperature and humidity carbon dioxide cell culture incubator. According to the cell status and density, replace the IMDM complete medium every 5 to 7 days to remove cell debris and continue to culture. Replace the new medium every 5 to 7 days until the cells begin to proliferate.

[0030] Cell subculture: Cells begin to proliferate after 9 weeks of culture, growing in suspension or in clusters. At this time, the IMDM cell culture medium is replaced every 48 to 72 hours and subculture begins. So far, the cells have been subcultured for more than 40 generations, meeting the cell line establishment standards and becoming an immortalized cell line.

[0031] Cell freezing and thawing: After the cells have stabilized and proliferated, the cell density is read by the automatic cell counter. When the cell density is between 5 and 10 x 10 6 / ml, freeze the cells, take the cultured cells to a 15ml clean centrifuge tube in a biosafety cabinet, and centrifuge at 1500 rpm for 5 minutes. After centrifugation, remove the supernatant and adjust the concentration to 1-2x10 7 / ml cell concentration, add an appropriate amount of serum-free cell freezing solution to resuspend the cells, then transfer the cells to a cell cryopreservation tube, and place it in a -80℃ ultra-low temperature refrigerator for 6 to 8 hours. For long-term storage, place it in liquid nitrogen. When the cells are revived, take out the cryopreservation tube from liquid nitrogen, quickly transfer it to a 37℃ constant temperature water bath, thaw it quickly, and then transfer the thawed cells in the cryopreservation tube to a sterile 15ml centrifuge tube pre-added with 5ml IMDM in a biological safety cabinet, and mix them thoroughly with a pipette. Further centrifuge at 1500 rpm for 5 minutes, discard the supernatant, resuspend the cells with 1ml IMDM medium and transfer them to a T25 cell culture flask, and place it in a cell culture incubator to culture the cells.

[0032] In the present invention, the cell line cells grow in suspension individually or in clusters, the cells are round or oval, and the cell growth rate is stable. It is named YYXY-M14 and was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on October 12, 2024, with the deposit number CCTCC NO: C2024350.

[0033] Example 2 Biological properties and applications of human acute myeloid leukemia cell line YYXY-M14

[0034] The present invention adopts IMDM culture medium containing 10% fetal bovine serum to culture YYXY-M14 cells, so that the cells can be stably grown and passaged in vitro. Under a microscope, the cells are observed to grow in a suspended single or clumping manner, and are round or oval in shape. Wright-Giemsa staining shows that the cells are leukemia primitive cells, with large and darkly stained nuclei, less cytoplasm, and obvious nucleoli. In vitro proliferation experiments have shown that the cells can be stably proliferated in vitro. Chromosome karyotype analysis shows that the cells are 46,XX,add(16)(p11.2) / 46,XX, and three acute myeloid leukemia poor prognosis gene mutations, TP53, RUNX1 and ASXL1, are present at the same time, and BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and FLT3-ITD acute myeloid leukemia poor prognosis gene mutations are absent. This cell line can be used to prepare tumor cell models with mutations in three acute myeloid leukemia genes, TP53, RUNX1 and ASXL1, which are associated with poor prognosis; screen and / or evaluate targeted tumor therapeutic drugs; and develop tumor-related animal models and bioengineering products. The details are as follows:

[0035] Morphological observation

[0036] The cultured YYXY-M14 cell line was placed under an inverted microscope to observe whether the cells grew in suspension individually or in clusters. The cells were round or oval in shape. Figure 1 As shown in A. Take 1~5x10 6 Place the mixture in a 1.5 ml EP tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant and add 10 μ l medium to resuspend the cells, take 2.5 μ l Cell suspension onto a glass slide, and use another glass slide to slide at a 45-degree angle at a constant speed to make a cell smear. After the cell smear is naturally air-dried, add Wright-Giemsa stain to stain the cell smear for 5 minutes, rinse, and dry. Observe the cell morphology under an inverted microscope oil lens and take pictures. Figure 1 As shown in B, the cells are mainly primitive cells with relatively consistent morphology, large cell size, blue cytoplasm, pale areas around the nucleus, fine nuclear chromatin, and obvious nucleoli.

[0037] In vitro proliferation ability observation

[0038] The cultured YYXY-M14 cell line was cultured at 0.5, 1, 2, 4, 8x10 5 Five different cell concentrations of 100 μg / ml were plated in 96-well plates, with 100 μg / ml per well. μ l cell solution, add 20 μ l of MTS reagent, and the absorbance was detected by an enzyme-labeled instrument after 4 hours. The detection wavelength of the enzyme-labeled instrument was set to 492nm, and the proliferation curve of cells at different plating concentrations was drawn using graphpad software. Figure 2 As shown, it is proved that the YYXY-M14 cell line cells have good in vitro proliferation ability in vitro, and the proliferation rates of cells at different densities are different, and all have in vitro proliferation ability.

[0039] Chromosome karyotype examination

[0040] Count the cultured cells and take 1-2x10 6 The cell fluid of each cell was transferred to a 15 ml centrifuge tube, and colchicine was added to a final concentration of 0.05 μg / ml. After shaking, the mixture was incubated at 37°C for 2 to 3 hours, centrifuged at 1500 rpm for 10 min, and the supernatant was removed. The cells were fixed with a fixative solution, which was prepared with a ratio of 3:1 of methanol and glacial acetic acid. The fixation process was carried out through the steps of pre-fixation-fixation-fixation, and each time a fresh fixative solution was used for the steps of centrifugation-resuspending, and finally the cells were resuspended with the fixative solution. The chromosome R banding method was then used for banding, and manual analysis and photography were performed. The results are shown in the figure. Figure 3 As shown in AB, two chromosome karyotypes were detected in the cell line cells, namely: 46,XX,add(16)(p11.2) and 46,XX.

[0041] Whole exome sequencing

[0042] Count the cultured cells and take at least 5x10 6 The cell culture medium was centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, the cell pellets were retained, 1xPBS was added to resuspend the cells and centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cell pellets were retained. The genomic DNA in the sample was extracted, and the quality of the extracted DNA sample was detected by electrophoresis. Then the qualified DNA samples were randomly interrupted into 150bp-220bp fragments by Covaris, and the library was constructed and captured using the Agilent SureSelect Human All Exon V6 kit. The DNA fragments were repaired at the end, ployA tails were added, sequencing adapters were added, purified, magnetic beads were captured, PCR amplified, and other steps were performed to complete the library construction. After the library was qualified, double-end sequencing was performed using a sequencer. After the raw sequencing data (Raw data) was obtained from the sequencing machine, the bioinformatics analysis process was entered, which was divided into two stages: 1. Sequencing data quality assessment: mainly through statistics on sequencing error rate, data volume, alignment rate, coverage, etc., to assess whether the library construction sequencing has reached the standard, and if it meets the standard, subsequent analysis will be performed. 2. Variant information analysis: Align high-quality sequencing sequences to the reference genome, detect variant information in the sample, and analyze and interpret the detected variant information. Figure 3C. Based on the results of the whole exon gene analysis, all mutations in the introns were removed, and meaningful mutations were retained. The AML risk stratification defined in the 2023 version of the AML diagnosis and treatment expert consensus was analyzed for genes with poor prognosis. Figure 3 As shown in D, a total of 1 exon nonsynonymous mutation in the TP53 gene, 1 exon nonsynonymous mutation and 1 exon frameshift insertion mutation in the RUNX1 gene, and 2 exon nonsynonymous mutations in the ASXL1 gene were detected. No intentional mutation sites were detected in BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2, and FLT3-ITD.

[0043] Cell STR identification

[0044] The cultured cells were sent to Shanghai Yihe Biology for genotyping of cell STR loci and Amelogenin loci. The results showed that the cell line did not match the existing cell lines in the world and was unique. The genotyping degree of the STR loci and Amelogenin loci of the mother cells just separated from the patient was 100%, which meant that they were from the same source, that is, there was no cross contamination during the correct culture process of the cell source. The genotyping of the cell STR loci and Amelogenin loci is shown in Table 1.

[0045] Table 1: Genotyping results of STR loci and Amelogenin loci of YYXY-M14 cells

[0046]

[0047] Example 3 Evaluation of sensitivity of AML chemotherapy drugs in human acute myeloid leukemia cell line YYXY-M14 cells

[0048] The cultured cells were divided into 2*10 5 / ml cell concentration was plated in 24-well plates to evaluate the sensitivity of conventional chemotherapy drugs for AML. The drugs were 4 first-line drugs for AML, namely, idarubicin, homoharringtonine, venetoclax, and azacitidine. For each drug, 1 blank control well and 6 to 7 different drug concentrations were plated. Specifically: homoharringtonine 0.5, 1, 2.5, 5, 10, 20, 40nM; idarubicin 10, 25, 50, 100, 200, 400, 600nM; azacitidine 1, 2.5, 5, 10, 25, 50, 100μM; venetoclax 2.5, 5, 10, 25, 50, 100μM. After counting the cells, the plates were plated, and 48 hours later, the plates were transferred to 96-well plates, MTS was added to measure the absorbance, and the half-inhibitory concentration of the drugs was calculated using Graphpad software. The results are as follows. Figure 4As shown in the figure, AML standard chemotherapy drugs, idarucizumab, homoharringtonine, cytarabine and venetoclax all have inhibitory effects on this cell line, but the half-inhibitory concentrations of different drugs are significantly different. The half-inhibitory concentrations of idarucizumab and homoharringtonine are at the nanomolar level, which are 20.08nM ( Figure 4 A) and 10.09nM( Figure 4 B). The half inhibitory concentrations of cytarabine and venetoclax are at the micromolar level, which are 20.55 μM ( Figure 4 C) and 4.57 μM ( Figure 4 D) The half inhibitory concentration of different concentrations of AML chemotherapy drugs differs by 1000 times. It is a good cell model for studying and evaluating AML drugs.

[0049] Example 4 Application of the human acute myeloid leukemia cell line YYXY-M14 cells in an animal model

[0050] The cultured YYXY-M14 cell line was cultured at 3*10 6 / cells were resuspended in 100 μL of sterile 1xPBS and injected into the immunodeficient 6-8 week old NCG mice produced by Jicui Yaokang through tail vein injection. The modeling effect was evaluated by detecting the proportion of humanized CD45 positive cells in the peripheral blood of mice and the survival time of mice. For details of the animal model process, see Figure 5 A. On the 36th and 44th days after the injection of cells, collect 100ul of mouse peripheral blood through the submandibular vein, add 2ml of red blood cell lysis solution to lyse the mouse peripheral blood at room temperature for 5-10min, centrifuge at 2000 rpm / 5min, remove the supernatant, resuspend the cells with 1ml 1xPBS, and centrifuge at 2000 rpm / 5min. After repeating once, the cells were divided into two equal parts, one with 300ul1xPBS and the other with 300ul1xPBS+anti-human PE-labeled CD45 monoclonal flow cytometry antibody. After incubation at room temperature in the dark for 20min, add 1ml1xPBS to mix, and centrifuge at 2000 rpm for 5min. Discard the supernatant, add 300ul of 1xPBS to each tube to resuspend the cells, and then measure CD45 expression on a flow cytometer. The results are as follows Figure 5 As shown in B, after the mice were injected with cells, the proportion of CD45 positive cells in the peripheral blood gradually increased over time. The mice were killed when they were dying, specifically showing hunched backs, ruffled hair, paralysis, and weight loss of more than 25%. The mice were killed and their survival time was recorded. The results are shown in Figure 5 As shown in C, all mice eventually became ill and died, with a median survival of 62.24 days, indicating that this cell line has good in vivo tumor capacity and is a good cell material for AML animal models.

[0051] The above embodiments are used to explain the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations, characterized in that: The cell line was named human acute myeloid leukemia cell YYXY-M14 and was deposited in the China Center for Type Culture Collection on October 12, 2024, with the preservation number CCTCC NO: 2024350.

2. Daughter cells of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations as claimed in claim 1.

3. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations according to claim 1, characterized in that: The human-associated multiple adverse prognosis gene mutations are the simultaneous presence of three acute myeloid leukemia adverse prognosis gene mutations: TP53, RUNX1 and ASXL1.

4. The human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations according to claim 1, characterized in that: The cell chromosome karyotype of the human acute myeloid leukemia cell line YYXY-M14 is 46,XX,add(16)(p11.2) / 46,XX.

5. Use of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations as claimed in any one of claims 1 to 4 in the study of the therapeutic mechanism of acute myeloid leukemia.

6. The use according to claim 5, characterized in that In the study of the pathogenesis of acute myeloid leukemia, multiple gene mutations associated with poor prognosis were found to be TP53, RUNX1 and ASXL1 gene mutations.

7. Use of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations as claimed in any one of claims 1 to 4 in preparing a tumor cell model or a tumor animal model.

8. Use of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations as claimed in any one of claims 1 to 4 in screening and / or evaluating tumor therapeutic drugs.

9. Use of the human acute myeloid leukemia cell line with multiple adverse prognosis gene mutations as claimed in any one of claims 1 to 4 in the preparation of tumor vaccines.

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