A human acute myeloid leukemia cell line with normal karyotype and its use

Abstract

The present invention discloses a human acute myeloid leukemia cell line with normal karyotype and its use. The human acute myeloid leukemia cell line with normal karyotype is named as human acute myeloid leukemia cell line YYXY-M4, which was deposited in China Center for Type Culture Collection (Wuhan University, Wuhan, China) on July 24, 2023, with a deposit number of CCTCC NO: C2023217. The present invention obtains mononuclear cells by extracting and separating peripheral blood separated by a blood cell separator from a patient with normal AML karyotype, and obtains them by continuous natural passage in vitro culture. The leukemia cell line has a normal karyotype and is accompanied by mutations in FLT3, RUNX1, ASXL1, and IDH1. Good in vitro proliferation ability can be used as a cell material for studying the occurrence and development mechanism of human acute myeloid leukemia with normal human karyotype and in vitro research on individualized treatment, and can also be used for in vitro and in vivo research on drug screening and evaluation of human acute myeloid leukemia with normal human karyotype, and guide clinical medication.

Description

Technical Field

[0001] The invention relates to the fields of biology and oncology, and in particular to a human acute myeloid leukemia cell line with normal karyotype and a use thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant clonal proliferation tumor of the blood system. Due to the disorder of differentiation and maturation of primitive cells, primitive cells accumulate in the bone marrow or peripheral blood, inhibiting the production of normal blood cells. Clinically, it often manifests as anemia, bleeding, infection and other symptoms. AML is the most common hematological malignancy in adults, with a median age of onset of about 68 years old. Under current treatment conditions, the overall prognosis of AML is poor, with an overall survival of less than 30% in 5 years. The prognosis of elderly patients and relapsed and refractory patients is even worse, with an overall survival of less than 10% in 5 years. With the accelerated arrival of the aging of our society, the incidence of AML as an elderly disease is also increasing. Statistics show that the incidence, mortality and social burden of AML in my country are among the top three in major countries in the world. It is of great significance to improve the efficacy and long-term survival of AML.

[0003] With the advancement of detection technology, the diagnosis and prognosis stratification system of AML is becoming more accurate and personalized. The status of cytogenetics and molecular biology in the diagnosis, prognosis and stratification of AML is becoming increasingly consolidated. At present, the international prognosis stratification system of AML mainly relies on the cytogenetics and molecular biology of AML patients. Taking the most widely used AML2023 guidelines of the National Cancer Center (NCCN) of the United States as an example, AML is divided into three levels according to the cytogenetics and molecular biology of AML: good prognosis, moderate prognosis and poor prognosis. Among them, according to the cytogenetics characteristics, AML patients with chromosome karyotype t(8;21) translocation or inv(16) or t(16;16) translocation belong to the good prognosis group; those with t(6;9) translocation or t(8;16) translocation or t(9;22) translocation or inv(3) belong to the poor prognosis group; and AML with t(9;11) translocation or AML with normal karyotype (CN-AML) are classified as AML with moderate prognosis. Studies have shown that CN-AML patients account for more than 40% of AML patients. Before molecular biology was applied to the prognostic stratification of AML, it was difficult to further stratify CN-AML based solely on cytogenetics. Due to the high heterogeneity of AML, the prognosis of CN-AML varies greatly. With the widespread application of molecular biology technology in the prognostic stratification of AML, the prognostic stratification of AML has become more accurate. Based on the different molecular biological characteristics of AML patients, AML patients are also divided into three groups: good prognosis, moderate prognosis, and poor prognosis. The NCCN AML2023 guidelines indicate that AML patients with CEBPA containing NPM1 mutations without FLT3-ITD mutations or bZIP box mutations belong to the good prognosis group; NPM1 mutations with FLT3-ITD mutations or wild-type NPM1 mutations with FLT3-ITD mutations without adverse karyotypes belong to the moderate prognosis group; AML patients with ASXL1 or BCOR or EZH2 or RUNX1 or SF3B1 or STAG2 or U2AF1 or ZRSR2 or P53 mutations belong to the poor prognosis group. According to the latest AML risk stratification, it can be found that CN-AML can be further stratified. If AML with normal karyotype has different gene mutations, it can be further stratified, thereby achieving more accurate stratification of AML, which is of great significance for the personalized treatment of AML.

[0004] The significance of molecular biological targets in AML is becoming increasingly clear, and targeted drugs targeting specific targets of AML are increasingly entering clinical application. For example, drugs targeting FLT3 mutations (such as sorafenib, gilteritinib, etc.) have improved the therapeutic effect of AML with FLT3 mutations. Drugs targeting IDH1 / IDH2 and drugs targeting BCL2 are all used in the treatment of AML, making individualized treatment of AML possible. However, due to the high heterogeneity of AML, each AML patient may have unique molecular characteristics. Currently, most CN-AML patients have not yet found clear molecular changes based on current detection technology, and there are more molecular markers to be discovered. Cell lines are basic cell models for studying the causes of tumors, discovering molecular markers, exploring tumor therapeutic drugs, and preclinical validation of drugs. AML cell lines play an important role in promoting basic and clinical translational research on AML. At present, AML cells have also been established internationally, but due to the high heterogeneity of AML's cytogenetics and molecular biology, there are still many AMLs with different cytogenetics and specific molecular changes that have not yet been established. There are even fewer AML cell lines established in China. CN-AML accounts for more than 40% of AML patients, but currently only the GC-SH 1 strain has been found in foreign countries. Reinhold Munker et al. reported in 2009. Molecular biological analysis found that this CN-AML cell line has NRAS / KRAS, GATA2, EZH2, FLT3, RUNX1, and TET2 mutations. This CN-AML cell line is derived from primary cells after patients with myelodysplastic syndrome (MDS) transformed into acute myeloid leukemia. MDS transforms into AML only a small part of AML, and more are primary AML patients. Obviously, it is urgent to establish CN-AML cell lines that are primary to AML patients, and have different molecular characteristics, which is of great significance for the study of CM-AML.

[0005] In summary, the only reported CN-AML cell line is difficult to meet the research requirements of CN-AML, which accounts for more than 40% of AML. In addition, CN-AML cell lines have not yet been established in China, which limits the research of AML that lacks molecular characteristics of abnormal karyotype. Therefore, it is urgent to establish CN-AML cell lines with different molecular markers for the study of pathogenesis, drug screening, and chemotherapy regimen updates. Summary of the invention

[0006] The purpose of the present invention is to provide a human acute myeloid leukemia cell line with normal karyotype and its application.

[0007] The objective of the present invention is achieved through the following technical scheme: A human primary myelofibrosis cell line, the cell line is named human primary myelofibrosis cell line ZYXY-M2, which was deposited in the China Center for Type Culture Collection on July 24, 2023, with the deposit number CCTCC NO: C2023217.

[0008] The present invention also provides progeny cells of the human acute myeloid leukemia cell line with normal karyotype as described above.

[0009] The present invention also provides human acute myeloid leukemia cells with normal karyotype as described above, which simultaneously have FLT3, RUNX1, ASXL1, and IDH1 mutations. According to the NCCN2023 AML guidelines, AML with RUNX1 and ASXL1 mutations has a poor prognosis, and FLT3 mutations and IDH1 are also confirmed to be related to AML prognosis, but there is currently no AML cell line with CN-AML accompanied by FLT3, RUNX1, ASXL1, and IDH1 mutations.

[0010] Cells of the cell line grow individually or in clusters in suspension.

[0011] The present invention also provides the use of the human acute myeloid leukemia cell line with normal karyotype as described above, selected from any one or more of the following:

[0012] a. Used for the study of the pathogenesis of CN-AML with FLT3, RUNX1, ASXL1, and IDH1 mutations;

[0013] b. Prepare tumor cell models or prepare tumor animal models; wherein, the progeny cells established by this cell line or the progeny cells established by this cell line are transfected with fluorescently labeled genes. Prepare animal tumor models, and establish AML animal models by subcutaneous tumor loading or tail vein injection modeling.

[0014] c. Screening and / or evaluating / preparing tumor therapeutic drugs; wherein, the method for screening and preparing tumor therapeutic drugs can be: adding different tumor therapeutic drugs to the culture medium of the human acute myeloid leukemia cell line with normal karyotype, observing the changes in cell morphology, and obtaining preliminary effective candidate drugs. Then, the candidate drugs are administered to the cells, the half-maximal inhibitory concentration (I50) of the screened effective drugs is calculated, and the drug with the lowest IC50 is selected to further act on the animal model, and the survival period, tumor size, metastasis, etc. of the animals in the non-administered group are compared to screen for potential drugs for the treatment of CN-AML.

[0015] d. Develop tumor-targeted drugs, including drugs targeting any or combined mutations of FLT3, RUNX1, ASXL1, and IDH1;

[0016] e. Screening tumor biotherapeutic drugs; the tumor biotherapeutic drugs / reagents are tumor vaccines.

[0017] f. Develop tumor-related bioengineering products. The tumor-related bioengineering products can be CN-AML-specific molecular diagnostic PCR kits and fluorescence in situ hybridization kits.

[0018] The present invention also provides a method for constructing a human acute myeloid leukemia cell line with a normal karyotype, comprising the following steps: obtaining a fresh peripheral blood separation fluid separated by a blood cell separator from a patient with a normal karyotype and a clear diagnosis of AML. Take 6 ml of the peripheral blood separation fluid and add it dropwise to a 15 ml sterile centrifuge tube pre-added with 6 ml of mononuclear cell separation fluid, and centrifuge it at 2000 rpm for 30 minutes. After the centrifugation, take the white cell precipitate of the mononuclear cell layer to a new 15 ml sterile centrifuge tube, add 5 ml of sterile 1xPBS pre-cooled in a 4°C refrigerator to resuspend the cells, and centrifuge it at 2000 rpm for 5 minutes. After discarding the supernatant, add a sterile red blood cell lysis solution to lyse the cells at room temperature for 5 minutes and then centrifuge at 2000 rpm for 5 minutes. Discard the supernatant, add 5 ml of IMDM complete culture medium (IMDM 90% + fetal bovine serum 10%) to resuspend the cells, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, add 5 ml of IMDM complete culture medium, and resuspend the cells. Count the cells with a cell counting plate, and take 1*10 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 incubator. Depending on the cell proliferation status, replace with new IMDM complete medium after 3 to 5 days until the cells begin to proliferate.

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

[0020] The human acute myeloid leukemia cell line with normal karyotype of the present invention can be infinitely passaged, the cell shape in vitro is stable, and it conforms to the biological characteristics of clinical tumors. The human acute myeloid leukemia cell line with normal karyotype originates from CN-AML patients, and is positive for FLT3, RUNX1, ASXL1, and IDH1 mutations. The human acute myeloid leukemia cell line with normal human karyotype can be used for the study of the mechanism of the occurrence and development of CN-AML. The cells can also be used to analyze the efficacy of new drugs and combined regimens against CN-AML with FLT3, RUNX1, ASXL1, and IDH1 mutations, and to screen and evaluate CN-AML drugs, which can be used to guide clinical medication. It is of great significance to reveal the type of AML, CN-AML. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 1 The result of Wright-Giemsa staining of the human acute myeloid leukemia cell line with normal karyotype;

[0023] Figure 2 It is a growth curve diagram of the human acute myeloid leukemia cell line with normal karyotype;

[0024] Figure 3 The chromosome karyotype diagram of the human acute myeloid leukemia cell line with normal karyotype;

[0025] Figure 4 It is a Circos map of genome variation and positive molecular mutations of the human acute myeloid leukemia cell line with normal karyotype. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1 Preparation of YYXY-M4 cell line

[0028] Primary cell culture: Immediately separate leukemia mononuclear cells from the fresh peripheral blood (male, 49 years old, CN-AML) obtained from the People's Hospital Affiliated to Ningbo University separated by a blood cell separator. In the biological safety cabinet, take 6 ml of separation solution and add it dropwise to a 15 ml sterile centrifuge tube pre-added with 6 ml of mononuclear cell separation solution, and centrifuge at 2000 rpm for 20 minutes. After the centrifugation, take the mononuclear cell layer to a new 15 ml sterile centrifuge tube, add 5 ml of sterile 4°C pre-cooled 1xPBS 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, add 5 ml of IMDM complete medium, and resuspend the cells. For cell counting plate technology, take 1*10 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 incubator. According to the cell proliferation status, replace the new IMDM complete medium every 3 to 5 days to remove cell debris and continue to culture. Replace the medium every 3 to 5 days thereafter.

[0029] Cell subculture: Cells begin to proliferate at 4 weeks of culture, growing in suspension or in clusters. At this time, the cell culture medium is replaced every 72 hours and subculture begins. So far, the cells have been subcultured for more than 50 generations, and have become immortalized cell lines.

[0030] In the present invention, the cells grow individually or in clusters in a suspended state, the cells are round or oval, and the cell growth rate is stable. It is named YYXY-M4 and was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on July 24, 2023, with the deposit number CCTCC NO: C2023217.

[0031] Example 2 Biological characteristics and application of human acute myeloid leukemia cell line

[0032] The present invention uses IMDM culture medium containing 10% fetal bovine serum to culture cells YYXY-M4, so that it can grow stably in vitro and be stably passaged. Under a microscope, the cells are observed to grow in suspension alone or in clusters, and are round or oval in shape. Wright-Giemsa staining shows that the cells are acute leukemia primitive cells with large and darkly stained nuclei. In vitro proliferation experiments indicate that they can proliferate stably in vitro. Chromosome karyotype analysis shows that they are karyotype-normal AML (CN-AML) with FLT3, RUNX1, ASXL1, and IDH1 mutations. The cell line can be used to prepare tumor cell models or tumor animal models; screen and / or evaluate / prepare tumor therapeutic drugs; prepare tumor diagnostic products; screen tumor biological therapeutic drugs / reagents; and develop and detect tumor-related bioengineering products. The details are as follows:

[0033] Morphological observation

[0034] The cultured YYXY-M4 cell line was placed under an inverted microscope to observe the cells growing in suspension individually or in clusters. The cells were round or oval in shape. 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 Resuspend the cells in culture medium and slide the cells. After the cell smear is dry, add Wright-Giemsa stain for 5 minutes, rinse and dry. Observe the cell morphology under an inverted microscope and take pictures. Figure 1 As shown, the cells are mainly primitive cells with large cell size, blue cytoplasm, pale area around the nucleus, fine and granular nuclear chromatin, and obvious nucleoli.

[0035] In vitro proliferation ability observation

[0036] The cultured YYXY-M4 cell line was divided into 1, 2, 4, 8*10 5 Four concentrations of 100 μg / ml were plated in a 96-well plate. μ l cell solution, add 20 μ l of MTS reagent, set the absorbance of the microplate reader to 492nm after 4 hours, measure the absorbance of the 96-well plate, and use GraphPad software to draw the proliferation curve of cells at different plating concentrations. Figure 2 As shown, the cell line cells have good in vitro proliferation ability, and the proliferation rates of cells at different densities are different, showing malignant growth.

[0037] Chromosome karyotype examination

[0038] Count the cultured cells and take 1*10 6 Add a certain amount of cell solution to a 15ml centrifuge tube, add colchicine to a final concentration of 0.05μg / ml, shake well and incubate in a 37℃ incubator for 2-3 hours, centrifuge at 1500 rpm for 10min, remove the supernatant, add fixative to fix the cells, the fixative is a 3:1 ratio of methanol and glacial acetic acid, fixation is done through pre-fixation-fixation-fixation, use fresh fixative each time through centrifugation-resuspending, and finally resuspend the cells with the fixative. Then use the chromosome R banding method to develop the bands, analyze manually and take pictures, the results are as follows Figure 3 As shown, the cell line cells have normal karyotype chromosomes, namely 46 (XY).

[0039] Whole exome sequencing

[0040] Count the cultured cells and take 5*10 6 The cell culture medium was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed and the cell pellets were retained. The cells were resuspended in 1xPBS and centrifuged at 1500 rpm for 5 minutes, the supernatant was removed and the cell pellets were retained. The genomic DNA in the sample was extracted, and the qualified DNA samples were randomly sheared into 150bp-220bp fragments by Covaris for electrophoresis detection. The AgilentSureSelect Human All Exon V6 kit was used for library construction and capture. 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 inspection was qualified, the sequencer was used for double-end sequencing. After the raw sequencing data (Raw data) was obtained by sequencing, the bioinformatics analysis process was entered, which was divided into two stages: 1. Sequencing data quality assessment: mainly through the statistics of sequencing error rate, data volume, alignment rate, coverage, etc., to evaluate whether the library construction sequencing has reached the standard. If it meets the standard, subsequent analysis will be carried out.

[0041] 2. Analysis of variation information: Align high-quality sequencing sequences to the reference genome, detect variation information in the sample, and analyze and interpret the detected variation information. Figure 4 A. The results of the SNP and InDel mutation site detection and analysis of the YYXY-M4 cell genome are summarized in the NCCN2023 AML stratification guidelines and the mutation information of genes directly related to AML prognosis Figure 4B, it can be known that the cell line cells are FLT3, RUNX1, ASXL1, IDH1. Note: Figure 4 In B, M means that only mutant type was detected in the cell; WT / M means that both wild type and mutant type were detected in the cell.

[0042] Cell STR identification

[0043] The cultured cells were sent to Shanghai Yihe Biological 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 cell STR loci and Amelogenin loci was 100% with the cell STR loci and Amelogenin loci just isolated from the patient, indicating 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.

[0044] Table 1: Genotyping results of STR loci and Amelogenin loci of cells

[0045]

[0046]

[0047] 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 normal karyotype, characterized in that: The cell line was named human acute myeloid leukemia cell YYXY-M4 and was deposited in the China Center for Type Culture Collection on July 24, 2023, with the deposit number CCTCC NO: C2023217.

2. The progeny cells of the human acute myeloid leukemia cell line with normal karyotype as claimed in claim 1.

3. Use of the human acute myeloid leukemia cell line with normal karyotype as claimed in claim 1 or the progeny cells of the human acute myeloid leukemia cell line with normal karyotype as claimed in claim 2, characterized in that: Choose from any one or both of the following: a. For the study of the therapeutic mechanism of acute myeloid leukemia with FLT3, RUNX1, ASXL1, and IDH1 mutations; b. Preparation of tumor cell models; The tumor is human acute myeloid leukemia.

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