Screening kit for people susceptible to acute myelogenous leukemia or with poor prognosis

Abstract

The invention relates to the technical field of biological medicines, and provides a screening kit for people susceptible to acute myelogenous leukemia or with poor prognosis. The kit comprises an antibody for detecting the level of LILRB4 protein, and an antibody for detecting the number of CD3 + CD4 + T cells and the number of CD3 + CD4-T cells. By detecting the LILRB4 protein level, the number of CD3 + CD4 + T cells and the number of CD3 + CD4-T cells, whether acute myelogenous leukemia susceptible or poor prognosis people carrying the LILRB4 gene rs1048801 site G allele or not is judged, and the kit has the advantages that cost is saved, operation is easy, and the detection period is short.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and in particular relates to a screening kit for people susceptible to acute myeloid leukemia or with poor prognosis. Background Art

[0002] Acute myeloid leukemia (AML) is a highly aggressive hematological malignancy. Although existing immunotherapies have made some progress, their efficacy is limited. For example, the efficacy of immune checkpoint inhibitors (ICIs) targeting the PD-1 / PD-L1 and CTLA-4 pathways in myeloid malignancies is significantly inferior to that in solid tumors; new targets such as LAG-3, TIGIT, BTLA, VISTA, CD47 and TIM-3 limit the activity of immune cells through non-redundant mechanisms and have shown potential in preclinical and early clinical studies, but their efficacy is limited due to the heterogeneity of the tumor microenvironment and the complexity of immunosuppression mechanisms in acute myeloid leukemia.

[0003] It is worth noting that in recent years, the high expression of leukocyte immunoglobulin-like receptor B subfamily member 4 (LILRB4, also known as ILT3 or LIR-5) in acute myeloid leukemia and its immunosuppressive function have made it a potential immunotherapy target. LILRB4 plays a dual role in monocytic acute myeloid leukemia (M4 and M5 subtypes in FAB classification): on the one hand, it inhibits T cell activity through phosphorylation of ITIM motif (Y412 / Y422), and on the other hand, it promotes leukemia cell infiltration.

[0004] LILRB4 is an emerging immune checkpoint molecule, and the function and clinical significance of its genetic polymorphism in acute myeloid leukemia have been little studied. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a screening kit for people susceptible to acute myeloid leukemia or with poor prognosis, which aims to solve the problems mentioned in the background technology.

[0006] In one aspect, the present invention provides a screening kit for people susceptible to acute myeloid leukemia or with a poor prognosis, the kit comprising an antibody for detecting the level of LILRB4 protein, Cell number and Antibodies for cell number.

[0007] Furthermore, the antibody for detecting the LILRB4 protein level includes at least one of anti-human LILRB4-APC or anti-human LILRB4-PE.

[0008] Furthermore, the detection Cell number and Antibodies for cells include CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, and CD4-FITC.

[0009] Furthermore, the kit detects the sample of the subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be people with poor prognosis of acute myeloid leukemia who carried the G allele at the rs1048801 site of the LILRB4 gene.

[0010] Furthermore, the kit detects the sample of the subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be susceptible to acute myeloid leukemia carrying the GG genotype at the rs1048801 site of the LILRB4 gene.

[0011] Furthermore, the sample of the subject is a bone marrow sample or a peripheral blood sample.

[0012] In a second aspect, the present invention also provides the use of the rs1048801 site in the preparation of an auxiliary diagnosis or prognosis evaluation reagent for acute myeloid leukemia, wherein the reagent is used to detect the genotype of the rs1048801 site of the LILRB4 gene in a sample.

[0013] In a third aspect, the present invention further provides the use of the rs1048801 site in the preparation of a drug for treating acute myeloid leukemia, wherein the drug is a blocker that specifically blocks the G allele regulatory signal pathway at the rs1048801 site of the LILRB4 gene.

[0014] The present invention has the following beneficial effects: (1) By detecting the level of LILRB4 protein, Cell number and The number of cells can be used to determine whether the population is susceptible to acute myeloid leukemia or has a poor prognosis if it carries the GG genotype or G allele at the rs1048801 site of the LILRB4 gene, screen high-risk patients, guide early intervention, and gain more treatment time for patients; and more accurately determine the prognosis of patients, formulate personalized treatment plans for patients, and achieve precision medicine. Compared with the genotype detection of the rs1048801 site of the LILRB4 gene, it has the advantages of cost saving, simple operation, and short detection cycle; and it can detect the level of LILRB4 protein, Cell number and The number of cells can be reduced and conventional laboratory equipment can be used to carry out the test. No professional gene sequencing platform is required and the results can be obtained quickly.

[0015] (2) Using inhibitors to specifically block the G allele regulatory signaling pathway at the rs1048801 locus of the LILRB4 gene, reduce the cloning ability of acute myeloid leukemia cells, and restore Cell function and prolong the survival of patients with acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings: Figure 1 This is a result diagram showing that the LILRB4 rs1048801 SNP (single nucleotide polymorphism) significantly affects AML susceptibility in Example 1 of the present invention; wherein: Figure 1 A in the figure is a schematic diagram showing the location of the LILRB4 gene in the chromosome 19q13.4 region; Figure 1 Figure B is the result of three SNP sites (rs3050015, rs3745871 and rs1048801) of LILRB4 identified by sequencing and sequence alignment.

[0017] Figure 2 This is a correlation diagram between the LILRB4 gene polymorphism and acute myeloid leukemia susceptibility in Example 1 of the present invention.

[0018] Figure 3 It is the association between the rs1048801 locus genetic model of Example 2 and the demographic and clinical pathological parameters of acute myeloid leukemia susceptibility; wherein the forest plot shows the association between the rs1048801 locus genetic model (based on additive, dominant and recessive models) and the age (≤60 years and >60 years) of acute myeloid leukemia (AML) patients, gender (male / female) and FAB typing (other subtypes, M4+M5 subtypes), Odd Ratios (95%CI) indicates that the true value has a 95% probability of falling within the current confidence interval, and the P value indicates the probability of observing sample data or more extreme data under the premise that the original hypothesis (usually the null hypothesis, H0) is true.

[0019] Figure 4 This is a result diagram of the correlation between the rs1048801 G allele and high expression of LILRB4 and poor prognosis in Example 3; wherein: Figure 4 A in the figure is the expression of LILRB4 mRNA in patients with acute myeloid leukemia with different LILRB4 SNP genotypes detected by RT-qPCR, ns indicates no significance, ** indicates P value < 0.01, *** indicates P < 0.001; Figure 4Figure B is a Kaplan-Meier analysis of the correlation between the genotype of the LILRB4 gene rs1048801 site and the overall survival of patients with acute myeloid leukemia; Figure 4 Figure C is a Kaplan-Meier analysis of the relationship between LILRB4 mRNA level and overall survival of patients based on the TCGA database (n=139).

[0020] Figure 5 This is a result diagram of Example 4 using MTS cell proliferation and soft agar clone formation experiments to demonstrate the effect of rs1048801 G allele on the growth of acute myeloid leukemia cells; wherein: Figure 5 A in the figure is the result of detecting the vitality of acute myeloid leukemia cells (AA, AG and GG genotypes at rs1048801 site) by MTS method, * indicates P < 0.05, ** indicates P < 0.01; Figure 5 B is a representative image of soft agar colony formation of acute myeloid leukemia cells (AA, AG, and GG genotypes at the rs1048801 site) under an inverted microscope, 100 times indicates a scale bar of 20 μm, and 200 times indicates a scale bar of 10 μm; Figure 5 C in the figure is the statistics of the colony number (>50 cells / field) of acute myeloid leukemia cells (AA, AG and GG genotypes at the rs1048801 site), ** indicates P < 0.01, and *** indicates P < 0.001.

[0021] Figure 6 The LILRB4 gene rs1048801 site G allele of Example 5 inhibits acute myeloid leukemia patients Cell proliferation results graph; wherein: Figure 6 The A in the figure is the genotype of different rs1048801 loci (GG, AG, AA) detected by flow cytometry in acute myeloid leukemia (M5 subtype). White blood cells and Results of LILRB4 expression intensity in monocytic leukemia cells; Figure 6 B is the flow cytometry detection of different rs1048801 locus genotypes (GG, AG, AA) in individuals with acute myeloid leukemia (M5 subtype) Cell ratio result diagram; Figure 6 C in the figure is the genotype of rs1048801 (GG, AG, AA) in patients with acute myeloid leukemia Cell ratio statistics, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; Figure 6 D in the figure is the co-culture of primary T cells (effector cells) and irradiated acute myeloid leukemia cells (target cells) (the ratio of effector cells to target cells was 1:1, 2:1 and 4:1, respectively, containing CD3 / CD28 activation magnetic beads and IL-2), and the cell morphology was observed under an inverted microscope; Figure 6 E in the figure is the flow cytometry proliferation analysis result after CFSE-labeled T cells were co-cultured with leukemia cells with different rs1048801 locus genotypes (GG, AG, AA); Figure 6 F in the figure is the flow cytometry quantification of the number and subsets of T cells in different genotype groups ( , ) Proportional statistical chart, ** indicates P < 0.01, *** indicates P < 0.001. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0024] In some embodiments, the present invention provides a screening kit for people susceptible to acute myeloid leukemia or with a poor prognosis, the kit comprising an antibody for detecting the level of LILRB4 protein, Cell number and Antibodies for cell number.

[0025] Specifically, the antibody for detecting the LILRB4 protein level includes at least one of anti-human LILRB4-APC or anti-human LILRB4-PE.

[0026] Specifically, detection Cell number and Antibodies for cells include CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, and CD4-FITC.

[0027] Specifically, the kit detects the sample of the subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be people with poor prognosis of acute myeloid leukemia who carried the G allele at the rs1048801 site of the LILRB4 gene.

[0028] Specifically, the kit detects the sample of the subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be susceptible to acute myeloid leukemia carrying the GG genotype at the rs1048801 site of the LILRB4 gene.

[0029] Specifically, the sample of the subject is a bone marrow sample or a peripheral blood sample.

[0030] In some embodiments, the present invention also provides the use of the rs1048801 site in the preparation of an auxiliary diagnosis or prognosis evaluation reagent for acute myeloid leukemia, wherein the reagent is used to detect the genotype of the rs1048801 site of the LILRB4 gene in a sample.

[0031] In some embodiments, the present invention further provides the use of the rs1048801 site in the preparation of a drug for treating acute myeloid leukemia, wherein the drug is a blocker that specifically blocks the G allele regulatory signal pathway at the rs1048801 site of the LILRB4 gene.

[0032] Experimental Materials: (1) Experimental reagents: Red blood cell lysis buffer and Ficoll-Paque density gradient centrifugation buffer were purchased from Sigma-Aldrich; DNA extraction kit and RNAprep Pure Blood kit were purchased from Tiangen Biotechnology; PrimeScript™ reverse transcription reagent and TB Green Premix Ex Taq II were purchased from Takara Bio; MTS cell proliferation was purchased from Promega; CD16 / 32 antibody, anti-human LILRB4-PE, and anti-CD3-APC were purchased from BioLegend; anti-human CD45-APC-Cy™7, CD33-FITC, CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, CD4-FITC, CD8-PC5, CD45-APC (BD), CD13-PerCP-Cy™5.5, anti-CD8-PE, and anti-CD4-PE were purchased from BD (Becton, Dickinson and Company); CD14-ECD was purchased from Beckman Coulter; anti-human LILRB4-APC was purchased from eBioscience; CFSE (carboxyfluorescein diacetate succinimidyl ester) cell dye and Dynabeads Human T-activatorCD3 / CD28 were purchased from Thermo Fisher Scientific; recombinant human IL-2 (50 U / ml) was purchased from Novoprotein; isotype control antibodies (mouse IgG-APC, 1:400; rabbit IgG-PE, 1:100; mouse IgG-PE, 1:100); healthy human peripheral blood (no.: PB009-1-0).

[0033] (2) Experimental primers: The primer sequences for DNA amplification are shown in Table 1; the primer sequences for real-time fluorescence quantitative PCR are shown in Table 2.

[0034] Table 1 Primer sequences for DNA amplification

[0035] Table 2 Primer sequences for real-time fluorescence quantitative PCR

[0036] Example 1: Collecting clinical samples of acute myeloid leukemia to identify the effect of LILRB4 SNP (single nucleotide polymorphism) on susceptibility to acute myeloid leukemia To elucidate the potential functions of LILRB4 SNPs in immune regulation, we focused on the Ig domain and ITIM domain of LILRB4 (responsible for ligand binding and inhibitory signaling, respectively). A total of 151 newly diagnosed AML patients (AML group) and 203 healthy individuals (control group) were recruited from the First Affiliated Hospital of Nanchang University. All participants signed written informed consent in accordance with the Declaration of Helsinki, which was approved by the Institutional Ethics Review Committee of the hospital (ethics review number: CDYFYYLK-01-038). Bone marrow (BM) aspirates and peripheral blood (PB) samples were collected from AML patients during routine diagnosis. Bone marrow aspirates were treated with erythrocyte lysis buffer to remove erythrocytes and enrich leukocytes. Peripheral blood samples were separated into mononuclear cells (MNCs, including lymphocytes and monocytes) by Ficoll-Paque density gradient centrifugation. Genomic DNA was extracted from primary leukocytes using a DNA extraction kit and amplified by PCR. The PCR products were sequenced by ABI Prism 3100 Genetic Analyzer, and the sequences were aligned with the reference genome (GRCh38) using DNASTAR Lasergene v17.3 software.

[0037] The clinical characteristics of 151 newly diagnosed AML patients and 203 healthy individuals were collected and shown in Table 3 .

[0038] Table 3 Clinical characteristics of the population in the acute myeloid leukemia group and the control group

[0039] LILRB4 rs1048801 SNP significantly affects the susceptibility to acute myeloid leukemia. Figure 1 The results showed that the LILRB4 gene is located in the chromosome 19q13.4 region (such as Figure 1 ); and three SNP sites were identified by Sanger sequencing and sequence alignment of clinical samples: rs3050015, rs3745871 and rs1048801 (as shown in Figure 1 The rs3050015 site is a 7 bp repeat sequence, the rs3745871 site T>C is a synonymous mutation, and the rs1048801 site A>G leads to the substitution of arginine to glutamine at the second tyrosine residue in the ITIM domain, but does not affect its phosphorylation.

[0040] The distribution characteristics of LILRB4 gene polymorphism in acute myeloid leukemia and control populations (Chi-square test) are shown in Table 4. The results of the Chi-square test showed that there was a significant difference in the genotype of the rs1048801 locus between the acute myeloid leukemia group and the control group.

[0041] Table 4 Distribution characteristics of LILRB4 gene polymorphisms in the acute myeloid leukemia group and the control group

[0042] In Table 4, data are expressed as n (%), and the chi-square test is used to statistically analyze the genotype distribution of the acute myeloid leukemia group and the control group; OR (95%CI) is the probability that the true value falls within the current confidence interval with a 95% probability; P value is the probability of observing sample data or more extreme data under the premise that the null hypothesis (usually the null hypothesis, H0) is true.

[0043] Correlation between LILRB4 gene polymorphism and susceptibility to acute myeloid leukemia Figure 2 As shown, the data are expressed as n (%); the genotype distribution frequency of the control group was subjected to the HWE (Hardy-Weinberg equilibrium) test. If P>0.01, it means that the control group conforms to the Hardy-Weinberg equilibrium; P a The values ​​are the chi-square test of the distribution of LILRB4 single nucleotide polymorphisms between the acute myeloid leukemia group and the control group; the P value is the relationship between LILRB4 single nucleotide polymorphisms and acute myeloid leukemia susceptibility calculated using logistic regression analysis; P b The values ​​were adjusted for age and sex; AIC (Akaikeinformation criterion) and BIC (Bayesian Information Criterion) were used to select the optimal model; Crude OR (95%CI) was the odds ratio directly calculated by univariate analysis without adjusting other confounding variables (such as age, sex, etc.); Corrected OR (95%CI) b The calculated odds ratios are corrected for sex and age.

[0044] The results showed that after correction for confounding factors such as gender and age, multivariate logistic regression analysis showed that rs3050015 (adjusted OR=1.02, 95%CI=0.539-1.930, P=0.951) and rs3745871 (adjusted OR=1.125, 95%CI=0.578-2.154, P=0.745) were not significantly associated with the risk of acute myeloid leukemia; while rs1048 801 showed significant association in multiple genetic models. In the codominant model, the GG genotype of rs1048801 significantly increased the risk of acute myeloid leukemia compared with the AA genotype of rs1048801 (adjusted OR=4.996, 95%CI=2.645-9.403, P<0.001). In the dominant model, the risk of rs1048801 G allele carriers (AG+GG) was 2 times higher than that of rs1048801 AA homozygotes (adjusted OR=2.140, 95%CI=1.374-3.335, P<0.001). In the recessive model, the rs1048801 GG genotype had the highest risk compared with the rs1048801 AA+AG genotype (adjusted OR=4.054, 95%CI=2.273-7.230, P<0.001).

[0045] The SIFT (Sorting Intolerant From Tolerant) prediction results are shown in Table 5, which show that rs1048801 A>G is a deleterious mutation.

[0046] Table 5 SIFT prediction results

[0047] In summary, the GG genotype at rs1048801 of the LILRB4 gene significantly increases the susceptibility to acute myeloid leukemia.

[0048] Example 2: Analysis of the association between rs1048801 and acute myeloid leukemia susceptibility demographic and disease factors through genetic model: The results of the association between the rs1048801 genetic model and demographic and clinicopathological parameters of acute myeloid leukemia susceptibility are as follows Figure 3The results showed that the rs1048801 genetic model was associated with the gender, age and disease type of patients with acute myeloid leukemia. In the additive model, the rs1048801 GG genotype significantly increased the risk of acute myeloid leukemia in each subgroup: age > 60 years (OR = 6.706, 95% CI: 2.097-21.445), female (OR = 7.667, 95% CI: 2.841-17.575), M4 + M5 subtype (OR = 5.438, 95% CI: 2.653-11.147). In the dominant model, the risk of patients with AG+GG genotype was significantly higher than that of AA in patients aged ≤60 years (OR=2.355, 95% CI:1.354-4.009), females (OR=2.994, 95% CI:0.920-5.564), and M4+M5 subtypes (OR=2.285, 95% CI:1.337-3.906). In the recessive model, the risk of GG genotype was significantly higher than that of AA+AG genotype in patients aged >60 years (OR=6.405, 95% CI:2.615-18.953), females (OR=4.800, 95% CI:2.228-10.828), and M4+M5 subtypes (OR=4.417, 95% CI:2.317-8.433).

[0049] In summary, the GG genotype at the rs1048801 site of the LILRB4 gene significantly increases the susceptibility to acute myeloid leukemia, especially in women and the elderly, and they are more susceptible to M4 and M5 subtypes of leukemia.

[0050] Example 3: Verification of the correlation between the rs1048801 G allele and LILRB4 mRNA expression and poor prognosis by RT-qPCR (reverse transcription quantitative polymerase chain reaction) method The results of the correlation between the G allele of rs1048801 and high expression of LILRB4 and poor prognosis are as follows Figure 4 shown.

[0051] RT-qPCR detection of LILRB4 mRNA expression in patients with acute myeloid leukemia with different LILRB4 SNP genotypes (rs3050015, rs3745871, rs1048801) Figure 4 As shown in A in the figure; only single mutation was analyzed in each group: wild type / deletion type (n=10, 5 cases in each group), TT / TC / CC (n=15, 5 cases in each group), AA / AG / GG (n=15, 5 cases in each group). The results showed that LILRB4 mRNA expression was significantly increased in individuals carrying the G allele at rs1048801.

[0052] The survival analysis results are as follows Figure 4 As shown in B and C, Figure 4 B in the figure is the Kaplan-Meier analysis of the correlation between LILRB4rs1048801 genotype and overall survival in patients with acute myeloid leukemia (n=151), n=49 in the AA group, n=55 in the AG group, and n=47 in the GG group. The P value was calculated by the log-rank test. Figure 4 Figure C is the relationship between LILRB4 mRNA level and overall survival of patients based on Kaplan-Meier analysis of TCGA database (n=139), low expression group n=70, high expression group n=69. The results showed that patients with G allele carriers of rs1048801 of LILRB4 gene and patients with high expression of LILRB4 had worse prognosis.

[0053] Example 4: Using MTS cell proliferation and soft agar clone formation experiments to demonstrate the effect of rs1048801 G allele on the growth of acute myeloid leukemia cells (1) MTS cell proliferation assay: Primary acute myeloid leukemia leukocytes in the logarithmic growth phase (rs1048801 locus AA, AG, and GG genotypes) were cultured and cultured in the presence of The cells were seeded at a density of 10 μL / well in a 96-well plate. After 24, 48, 72, 96, and 120 hours of culture, 10 μL of MTS reagent was added to each well and incubated at 37 °C for 3 hours. Cell viability was calculated by absorbance at 490 nm.

[0054] The results of MTS assay for the viability of acute myeloid leukemia cells (rs1048801 site AA, AG and GG genotypes) are as follows Figure 5 As shown in A. The results showed that the absorbance (OD value) of acute myeloid leukemia cells with GG and AG genotypes at rs1048801 was significantly higher than that of acute myeloid leukemia cells with AA genotype at rs1048801 from the third day, and the absorbance of acute myeloid leukemia cells with GG genotype at rs1048801 was the highest.

[0055] (2) Soft agar colony formation assay: Single cells isolated from acute myeloid leukemia bone marrow / peripheral blood samples (rs1048801 locus AA, AG, and GG genotypes) ( The cells were suspended in 0.5% agarose and plated on a 0.8% agar substrate (12-well plate). After 18 days of culture, they were fixed with methanol and stained with 0.5% crystal violet. Representative images (5 per well) were acquired using a Nikon Eclipse TS100 microscope.

[0056] Representative images of soft agar colony formation of acute myeloid leukemia cells (AA, AG, and GG genotypes at rs1048801) under an inverted microscope Figure 5As shown in B; the number of colonies of acute myeloid leukemia cells (rs1048801 site AA, AG and GG genotypes) is shown in Figure 5 As shown in C. The results showed that the average diameters of cell clones of rs1048801 AG and GG genotypes were 40±3μm and 23±4μm, respectively, which were significantly larger than those of rs1048801 AA genotype (6±2μm); the average number of colonies of rs1048801 GG genotype reached 817±40 / well, which was significantly higher than those of rs1048801 AG genotype and rs1048801 AA genotype.

[0057] In summary, the G allele at rs1048801 of the LILRB4 gene mediates the poor prognosis of acute myeloid leukemia by upregulating LILRB4 expression.

[0058] Example 5: Using flow cytometry and mixed cell co-culture system to demonstrate the effect of rs1048801 G allele on T cells in acute myeloid leukemia (1) Flow cytometry analysis: To reduce nonspecific binding of antibodies, leukocytes were pre-blocked with anti-human CD16 / 32 antibodies for 15 minutes, followed by the addition of the following antibodies: anti-human LILRB4-APC, anti-human LILRB4-PE, CD45-APC-Cy™7, CD33-FITC, CD13-PerCP-Cy™5.5, CD14-ECD, CD3-PerCP-Cy™5.5, CD3-PE-Cy™7, CD4-FITC, CD8-PC5, and CD45-APC, as well as isotype control antibodies (mouse IgG-APC, 1:400; rabbit IgG-PE, 1:100; mouse IgG-PE, 1:100). Cells were analyzed by Calibur flow cytometer, and data were processed by Flowjo software.

[0059] Different rs1048801 genotypes (GG, AG, AA) in acute myeloid leukemia (M5 subtype) White blood cells and The expression intensity of LILRB4 in monocytic leukemia cells is shown in Figure 6 As shown in A; in individuals with acute myeloid leukemia (M5 subtype) with different rs1048801 locus genotypes (GG, AG, AA) The cell ratio results are as follows Figure 6 As shown in B; Patients with acute myeloid leukemia with different rs1048801 genotypes (GG, AG, AA) Cell ratio statistics Figure 6 The results showed that the rs1048801 site GG genotype White blood cells and LILRB4 expression was highest in monocytic leukemia cells and lowest in rs1048801 locus AA genotype, which was consistent with rs1048801 locus GG or AG genotype The proportion of cells decreased.

[0060] (2) Primary leukemia cell and T cell co-culture experiment: In the contact co-culture system, T cells from healthy donor peripheral blood ( / well) were labeled with CFSE, and recombinant human IL-2 (50 U / ml) and CD3 / CD28 activated magnetic beads ( Cells) were mixed with irradiated (28 Gy) leukemia cells at a specific effector-target ratio (E:T) in a U-shaped 96-well plate. After co-culture, cell morphology was observed under an inverted microscope, and proliferation was detected by flow cytometry (CFSE dilution method) after staining with anti-CD3-APC, anti-CD8-PE, and anti-CD4-PE. Unstimulated T cells were used as negative controls, and magnetic bead-activated cells were used as positive controls.

[0061] Primary T cells were co-cultured with irradiated leukemia cells, and cell morphology was observed under an inverted microscope. Figure 6 As shown in D; the flow cytometry proliferation analysis results of CFSE-labeled T cells co-cultured with leukemia cells of different genotypes are shown in Figure 6 As shown in E; flow cytometry quantitative T cell number and subset ratio statistics of different genotype groups are shown in Figure 6 As shown in F. The results showed that at an effector-target ratio of 1:1 to 4:1, the formation of T cell colonies with the GG or AG genotype at rs1048801 was significantly inhibited. The CFSE dilution experiment confirmed that leukemia cells carrying the G allele at rs1048801 could inhibit T cell proliferation in a dose-dependent manner, and the inhibition rate of the GG genotype at rs1048801 was 2.6 times. It is worth noting that the GG genotype at rs1048801 mainly leads to The cells decrease, and There were no significant changes in cells.

[0062] In conclusion, the G allele at rs1048801 of the LILRB4 gene specifically inhibits the expression of LILRB4 in monocytic leukemia cells by upregulating the expression of LILRB4. Cell proliferation.

[0063] From the analysis of the results of Examples 1-5, the present invention draws the following conclusions: by detecting the level of LILRB4 protein, Cell number and The number of cells can be used to determine whether the population is susceptible to acute myeloid leukemia or has a poor prognosis if it carries the GG genotype or G allele at the rs1048801 site of the LILRB4 gene, screen high-risk patients, guide early intervention, and gain more treatment time for patients; and more accurately determine the prognosis of patients, formulate personalized treatment plans for patients, and achieve precision medicine. Compared with the genotype detection of the rs1048801 site of the LILRB4 gene, it has the advantages of cost saving, simple operation, and short detection cycle; and it can detect the level of LILRB4 protein, Cell number and The number of cells can be reduced, and conventional laboratory equipment can be used to conduct the test. No professional gene sequencing platform is required, and the results can be quickly obtained. In addition, the blocker is used to specifically block the G allele regulation signal pathway of the LILRB4 gene rs1048801 site, reduce the cloning ability of acute myeloid leukemia cells, and restore Cell function and prolong the survival of patients with acute myeloid leukemia.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A screening kit for people susceptible to acute myeloid leukemia or with poor prognosis, characterized in that: The kit includes an antibody for detecting the level of LILRB4 protein, Cell number and Antibodies for cell number.

2. The screening kit for people susceptible to acute myeloid leukemia or with poor prognosis according to claim 1, characterized in that: The antibody for detecting LILRB4 protein level comprises at least one of anti-human LILRB4-APC or anti-human LILRB4-PE.

3. The screening kit for people susceptible to acute myeloid leukemia or with poor prognosis according to claim 2, characterized in that: The detection Cell number and Antibodies to cells include , and CD4-FITC.

4. The screening kit for people susceptible to acute myeloid leukemia or with poor prognosis according to claim 3, characterized in that: The kit detects a sample of a subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be people with poor prognosis of acute myeloid leukemia who carried the G allele at the rs1048801 site of the LILRB4 gene.

5. The screening kit for people susceptible to acute myeloid leukemia or with poor prognosis according to claim 3, characterized in that: The kit detects a sample of a subject, and the LILRB4 protein level of the sample of the subject increases. Decreased cell number and There was no statistically significant difference in the number of cells, and the subjects were judged to be susceptible to acute myeloid leukemia carrying the GG genotype at the rs1048801 site of the LILRB4 gene.

6. The screening kit for people susceptible to acute myeloid leukemia or with poor prognosis according to claim 4 or 5, characterized in that: The sample of the subject is a bone marrow sample or a peripheral blood sample.

7. Use of the rs1048801 locus in the preparation of an auxiliary diagnosis or prognosis assessment reagent for acute myeloid leukemia, characterized in that: The reagent is used to detect the genotype of the rs1048801 site of the LILRB4 gene in a sample.

8. Use of the rs1048801 site in the preparation of a drug for treating acute myeloid leukemia, wherein the drug is a blocker that specifically blocks the G allele regulatory signal pathway at the rs1048801 site of the LILRB4 gene.

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