Use of s1pr1 as a molecular marker for leukemia stem cells

Abstract

The present application relates to the field of leukemia diagnosis or prognosis evaluation, in particular to a kind of acute myeloid leukemia stem cell molecular marker S1PR1, it can be applied to the diagnosis and treatment of related diseases, and further relates to related diagnostic kit and treatment product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to S1PR1 as a regulatory AML leukemia stem cell surface marker. BACKGROUND

[0002] Leukemia is a malignant disease of abnormal proliferation of hematopoietic cells, which is ranked as one of the top ten high-incidence malignant tumors in China, and ranks first in children and adults under the age of 35. Among them, acute leukemia is a malignant clonal disease of hematopoietic stem cells, mainly due to the malignant proliferation caused by the change of gene expression profile of hematopoietic stem cells and progenitor cells, and the clinical manifestations are that the normal hematopoietic function of patients is inhibited, a large number of leukemia cells proliferate and accumulate in the bone marrow and infiltrate the extramedullary organs and tissues. Patients are prone to anemia, bleeding, infection, and organ infiltration.

[0003] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem / progenitor cells, which is one of the most common but also the most deadly types of leukemia. The diagnosis and treatment of refractory and relapsed AML face great challenges. According to the statistics of American Cancer Society (ACS), there were about 60,530 new cases of leukemia and 23,100 deaths from leukemia in the United States in 2020, of which about 19,940 were new cases of AML, and about 11,180 people died of AML. Leukemia is considered to be the result of the change of gene expression profile of normal hematopoietic stem / progenitor cells, and the existence of leukemia stem cells (LSCs) with self-renewal, differentiation and unlimited proliferation ability is considered to be the root of drug resistance and recurrence after leukemia treatment. Leukemia stem cells are a group of leukemia cells with self-renewal ability and unlimited replacement potential, and can produce a heterogeneous population of leukemia cells, which are found to be related to the resistance and recurrence of AML. Through research, some cell signaling pathways play an important role in the survival, proliferation and self-renewal of AML, and are abnormally activated or inhibited in LSCs, including NF-κB, Wnt / β-catenin, Hedgehog, Notch, EGFR, JAK / STAT, PI3K / AKT / mTOR, TGF / SMAD and PPAR pathways. Therefore, these signal transduction pathways have become a research hotspot for AML treatment, and LSC-related signal pathway inhibitors / activators are considered as a potential new type of anti-AML therapy, which can eliminate LSCs to guide the research and clinical treatment of acute myeloid leukemia.

[0004] After years of clinical practice, in general, the long-term survival rate of patients aged < 60 years is 35% ~ 45%, and the long-term survival rate of patients aged ≥ 60 years is only 10% ~ 15%. With the development of various treatment methods in recent years, the complete remission rate (CR) of leukemia chemotherapy has improved, but still 60% ~ 70% of patients are resistant or relapse after treatment, and leukemia relapse is the main bottleneck to improve the efficacy of AML.

[0005] Scientists first elucidated the existence of cancer stem cells (CSC) by isolating leukemia cells and injecting them into mice, and found that only a small number of cells could induce leukemia. Subsequently, scientists found that CD34+CD38- can be used as a surface marker of LSC. Since then, with the continuous in-depth study of scholars on LSC, more and more markers of LSC in AML have been found, such as HMGN1, JAM3, FOXM1, GPR56, CD200 and CD93, etc. Among them, many surface markers of LSC have been verified to have the function of regulating the biological characteristics of LSC self-renewal and leukemia reconstruction. Cytogenetic analysis provides prognostic information for different subtypes of AML, and more and more various molecular markers related to prognosis have been found recently. These findings have enabled the analysis of AML with different prognosis to enter the molecular level.

[0006] In clinical practice, molecular markers such as leukemia fusion genes RUNX1-RUNX1T1, CBFβ-MYH11 and PML-RARα, and NPM1 mutations can be used to predict AML relapse. AML patients who have achieved CR can detect pre-leukemia clone-related mutations such as DNMT3A, ASXL1 and TET2 in their bodies. Since these mutations can also be found in healthy populations and their incidence increases with age, they are also known as age-related clonal hematopoiesis or clonal hematopoiesis of unknown significance. Therefore, the prognostic significance of these mutations still needs to be confirmed. Other molecular markers, such as partial gene mutations of FLT3-ITD, FLT3-TKD, NRAS, KRAS, IDH1 and IDH2, have unstable phenomena when AML patients relapse (such as positive mutations at initial diagnosis, negative at relapse, or newly positive at relapse after initial negative), which will significantly increase the false positive and false negative rates. It can be seen that there is still a need to discover and study other types of leukemia-related markers with diagnostic and therapeutic effects.

[0007] The self-renewal and drug resistance of leukemia stem cells are maintained by the abnormal expression of genes. If specific genes can be found as targets for precise diagnosis, prognosis evaluation and treatment, it is of great significance for monitoring disease progression and achieving long-term survival for leukemia patients. Scientific evaluation of the diagnostic, therapeutic and prognostic related auxiliary indicators of acute leukemia has important clinical significance for selecting effective chemotherapy regimens or bone marrow transplantation. At present, the indicators for laboratory diagnosis of acute leukemia mainly include bone marrow smear cell morphology examination, flow cytometry immunophenotyping, peripheral blood complete blood count and genetic examination, etc. With the development of detection technology, there is a trend of using rapid detection indicators that are minimally invasive to patients, automatic detection, standardized and easy to promote. SUMMARY

[0008] The inventors of the present application found that S1PR1 has a regulatory effect on the self-renewal ability of leukemia stem cells in years of research. The present application clarifies the regulatory effect of S1PR1 on leukemia stem cells. The present application also provides a new type of leukemia stem cell marker, which can be used for the evaluation of the recurrence rate of related diseases, the risk assessment of diseases, the prognosis evaluation, the stratified diagnosis, the detection or auxiliary detection of disease progression, and the treatment or related drug screening of related diseases, etc. The marker can also be used to prepare leukemia model mice for scientific research.

[0009] The inventors found that sphingosine-1-phosphate receptor 1 (S1PR1) has a regulatory effect on the self-renewal and reconstruction ability of leukemia stem cells in years of research, and verified the regulatory effect of S1PR1 on leukemia stem cells. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite, which widely participates in the regulation of various biological functions by binding to specific G protein-coupled receptors. Currently, five members of the S1P receptor family have been found, which are S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5.

[0010] It has been shown that S1PR1 in human lung tissue, inhibition of S1PR1 gene expression can lead to increased lung capillary leakage, activated S1PR1 can regulate RacGTPase-dependent cortical actin rearrangement, enhance the barrier function of endothelial cells. S1PR1 can stabilize the localization of VE-Cadherin and inhibit VEGF-induced activation of VEGFR2, thereby inhibiting vascular sprouting and enhancing endothelial cell connectivity. In 2019, researchers (Fu Ying Chu, et al. Expression of LDB2 in lung cancer tissue and correlation analysis with S1PR1, Journal of Clinical Laboratory) studied the expression of S1PR1 gene in lung cancer tissue and analyzed its correlation with LIM domain binding protein 2 (LDB2) gene. The results showed that the expression of S1PR1 gene in lung cancer tissue was 0.710 (0.337-1.523), and the expression in the adjacent tissue was 1.582 (0.913-3.533), which was significantly lower and had a strong correlation with LDB2 gene, indicating that LDB2 and S1PR1 gene may have a certain synergistic effect in the biological process of lung cancer angiogenesis, but the specific regulation mechanism needs to be further verified. The study explains the expression of S1PR1 in lung cancer, but lung cancer is a solid tumor, which is completely different from the pathogenesis and treatment mechanism of leukemia. The molecular markers of these two diseases are also different.

[0011] In a first aspect, the present application provides the use of S1PR1, a molecular marker of leukemia stem cell, in the preparation of a reagent or a kit for the diagnosis or prognosis monitoring of leukemia. The use of S1PR1 as a molecular marker of leukemia stem cell is also provided. In particular, the use of S1PR1 as a molecular marker of leukemia stem cell, which can regulate the self-renewal ability of leukemia stem cells.

[0012] Further, the leukemia is acute leukemia, preferably acute myeloid leukemia.

[0013] Further, the diagnosis of disease progression or evaluation of prognosis includes: 1) S1PR1 is highly expressed in bone marrow blood mononuclear cells of acute myeloid leukemia patients than in normal donor-derived bone marrow blood mononuclear cells; 2) S1PR1 is highly expressed in acute myeloid leukemia stem cells than in non-stem leukemia cells; 3) S1PR1 is abnormally highly expressed in samples after refractory and relapse; or 4) S1PR1 is highly expressed in myeloid leukemia cell lines and drug-resistant cell lines with strong self-renewal and leukemia reconstruction ability.

[0014] In a second aspect, the present application provides the use of S1PR1 in the preparation of a drug for treating or preventing leukemia.

[0015] Further, the leukemia is acute leukemia, preferably acute myeloid leukemia.

[0016] Further, the drug is an inhibitor of S1PR1.

[0017] Preferably, the inhibitor of S1PR1 is an inhibitor that inhibits the activity or protein level of ZNF683 protein, or an inhibitor that inhibits the mRNA level of S1PR1, and the inhibitory activity is reversible or irreversible.

[0018] More preferably, the inhibitor that inhibits the activity or protein level of S1PR1 includes an antibody of S1PR1, a protein, a polypeptide, an enzyme, a natural compound, a synthetic compound, an organic substance, an inorganic substance that inhibits the activity or protein level of S1PR1; the inhibitor that inhibits the activity or protein level of S1PR1 refers to a substance that can bind to S1PR1 but does not produce a biological response when binding, or the inhibitor can block, inhibit or attenuate the response mediated by an agonist; the activity or protein level of S1PR1 refers to knocking out S1PR1.

[0019] Preferably, the inhibitor that inhibits the mRNA level of S1PR1 is an antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA thereof, or other proteins, polypeptides, enzymes, compounds that can inhibit the mRNA level of S1PR1.

[0020] The present application provides in a third aspect a method for preparing a leukemia model mouse, characterized in that the method comprises the following steps: 1) sorting out CD34+S1PR1+ cells in the bone marrow blood of an AML patient, and 2) obtaining the leukemia model mouse after feeding and monitoring the mouse for one week after tail vein injection of the CD34+S1PR1+ cell population.

[0021] Further, after transplantation of the CD34+S1PR1+ cells, the immunodeficient mouse with significantly increased bone marrow engraftment rate and significantly increased peripheral blood and liver and spleen leukemia cell infiltration is the leukemia model mouse.

[0022] The present application provides in a fourth aspect a method for screening a therapeutic agent for human acute myeloid leukemia, characterized in that the method comprises the following steps: preparing a leukemia model mouse according to the method described above, administering a test substance to the mouse, and evaluating the improvement of leukemia in the mouse, thereby screening a therapeutic agent for human acute myeloid leukemia.

[0023] The inventors of the present application performed gene expression differential analysis on the LSC-enriched cell population of CD34+CD38- and non-LSC cell population (including CD34+CD38+, CD34-CD38+ and CD34-CD38- cell population) by RNA-seq. The results showed that the expression amount of S1PR1 in LSC-enriched cells (CD34+CD38-) was higher than that in non-LSC. Under the condition of gene expression difference, the proportion of LSC-enriched cells of CD34+CD38- in S1PR1+ and S1PR1- populations in primary AML tumor cells was detected by flow cytometry, and it was further verified that the proportion of LSC-enriched cells of CD34+CD38- in S1PR1+ cell population was significantly higher than that in S1PR1- cell population. Therefore, S1PR1 can be used as a surface marker of LSC.

[0024] To further verify the regulatory effect of S1PR1 on the biological characteristics of LSC, the S1PR1 gene was knocked down in the AML cell line KG1, and a colony forming assay (CFU) was performed in vitro to evaluate the effect of S1PR1 on the self-renewal ability of AML cells in vitro. The results showed that after knocking down the S1PR1 gene, the colony forming ability of the KG1 cell line was significantly decreased. In addition, after sorting two groups of cells of CD34+S1PR1+ and CD34+S1PR1- in the bone marrow blood of AML patients, CFU experiment and flow cytometry experiment for detecting cell cycle were used to evaluate the effect of S1PR1 gene expression on the self-renewal ability and cell cycle of AML cells in an ex vivo manner. The results verified that compared with the cell population of CD34+S1PR1-, the number of CFU formed by CD34+S1PR1+ cells was significantly increased, the morphology was larger, and the number of cells in the division phase in the cell population was significantly increased. It can be seen that the expression of S1PR1 plays a key role in maintaining the self-renewal and leukemia reconstruction ability of LSC, which further proves that the gene can be used as a marker of leukemia stem cells, and there is a great relevance between the two.

[0025] In addition, a patient-derived xenograft model of secondary transplantation was constructed. Two cell populations, CD34+S1PR1+ and CD34+S1PR1-, were injected into immunodeficient mice via tail vein injection. The effect of S1PR1 gene expression in AML cells on the maintenance of LSC stemness was then investigated in vivo. Results showed that, compared to mice carrying CD34+S1PR1- cells, immunodeficient mice injected with CD34+S1PR1+ cells exhibited significantly shorter survival times, significantly higher bone marrow engraftment rates, and significantly increased leukemia cell infiltration in peripheral blood and the liver and spleen in both primary and secondary transplantations. This experiment also validates that S1PR1 plays a crucial role in maintaining the self-renewal and leukemia remodeling capacity of LSCs.

[0026] This invention provides an excellent humanized AML model mouse, which is used to elucidate the relapse mechanism of AML and to develop therapeutic agents or treatments suitable for AML. Attached Figure Description

[0027] Figure 1 This is a heatmap of S1PR1 expression levels in LSCs and non-LSCs in primary AML cells.

[0028] Figure 2 The flow cytometry plot (A) and statistical graph (B) show the proportion of CD34+CD38- cells in S1PR1+ and S1PR1- primary AML cells.

[0029] Figure 3 These are colony morphology diagrams (A) and statistical diagrams (B) of AML cell lines after S1PR1 gene knockdown.

[0030] Figure 4 The images show the colony morphology (A) and statistical diagram (B) of AML cells with CD34+S1PR1+ and CD34+S1PR1-.

[0031] Figure 5 This is a statistical graph of the cell cycle in AML cells of CD34+S1PR1+ and CD34+S1PR1-.

[0032] Figure 6 This is a survival diagram of immunodeficient mice that underwent a second transplantation and were injected with AML cells containing CD34+S1PR1+ and CD34+S1PR1-. Detailed Implementation

[0033] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0034] Example 1: Gene expression differential analysis

[0035] In this embodiment, the inventors collected bone marrow blood from 3 AML patients, extracted bone marrow blood mononuclear cells, sorted LSC-enriched cell populations (CD34+CD38-) and non-LSC cell populations (including CD34+CD38+, CD34-CD38+and CD34-CD38- cell populations), and performed RNA sequencing and differential expression analysis on the two cell populations. The specific steps are as follows:

[0036] (1) Extraction of bone marrow mononuclear cells: After centrifugation of whole blood (parameters: 1500 rpm, 5 min), the upper plasma was removed; 4 mL of human leukocyte separation medium was placed in a 15 mL centrifuge tube, and the blood cells were diluted with PBS and placed on the upper layer of the human leukocyte separation medium; after centrifugation (parameters: 1800 rpm, 18 min, brake off), the middle mononuclear cell layer was taken and counted.

[0037] (2) Cryopreservation and recovery of mononuclear cells: Cryopreserve patient primary cells with serum-free cryopreservation solution, and transfer to liquid nitrogen for storage. After the patient is diagnosed with AML, recover in a 37-degree water bath, and transfer the cells to a 37-degree preheated recovery solution (consisting of 10% BIT and DNAase added to IMDM).

[0038] (3) Cell sorting: CD34-PE and CD38-APC were incubated with cells at room temperature for 15 minutes, washed twice with PBS, and then subjected to flow sorting. The sorting receiving tube was lubricated with serum in advance.

[0039] (4) After sorting, centrifugation, RNA extraction, the extraction kit brand is Qiagen, the model number is #74104, and the specific steps are described in the manufacturer's instructions. RNA sequencing was performed on the Illumina NovaSeq 6000 platform, and alignment was performed using Hisat2 with reference to the human genome GRCh38.87. Gene expression was calculated using Featurecount, and then R language function DEseq2 was used for differential gene analysis. The results showed that the expression of S1PR1 in LSC-enriched cells (CD34+CD38-) was higher than that in non-LSCs. Figure 1 ).

[0040] (5) After primary cells were recovered, the labeled antibodies were as follows: CD34-PE, CD38-APC and S1PR1-BV421. The antibodies and cells were co-incubated for 15 minutes at room temperature in the dark, and then washed twice with PBS before flow cytometry analysis. The S1PR1+ and S1PR1- positive and negative groups were circled according to the S1PR1 expression, and the proportion of CD34+CD38- cells in the two groups of cells was analyzed, i.e. the proportion of LSC-enriched cells of CD34+CD38- in the S1PR1+ and S1PR1- groups in the primary AML tumor cells was detected by flow cytometry. The results showed that the proportion of LSC-enriched cells of CD34+CD38- in the S1PR1+ group was significantly higher than that in the S1PR1- group. Figure 2

[0041] Example Two Gene Knockout Verification

[0042] Based on the above gene expression differences, the S1PR1 gene was knocked down in the AML cell line KG1, and a colony forming assay (CFU) was performed in vitro to evaluate the effect of S1PR1 on the ability of AML self-renewal and leukemia reconstruction in vitro. The specific steps are as follows:

[0043] (1) Construction of AML cell line with S1PR1 gene knocked down: use interference RNA technology to obtain KG1 cell line with S1PR1 gene knocked down, and lentivirus is purchased from Shanghai Jikai Gene. The plating density of KG1 cell line is 1*10 5 / mL, 80 μL of transfection promoting solution is added, then virus solution is added, the MOI of transfection virus is 50, after centrifugation (parameter setting is 37 degrees, 1000g, 60 min), it is placed in a 37 degree incubator for culture, and after 15 hours, fresh culture medium is replaced. Cell sorting was performed after 3 days, and fluorescent positive cells were sorted out. The relative expression of S1PR1 gene was detected by real-time fluorescence quantitative qPCR method, and the knockdown efficiency was determined.

[0044] (2) Colony forming assay: colony forming medium is purchased from Stem Cell Technologies, item number #H04435. Resuspend the cells in IMDM containing 2% BIT and count, inoculate 1000 to 10000 cells. Take 1.1 mL of stem cell culture medium containing cells and place it in a 35 mm dish, plate 2 duplicate wells. Observe after 7-14 days of culture.

[0045] Figure 3 ​Figure 6 is a colony morphology chart (A) and a statistical chart (B) of AML cell lines after knocking down S1PR1 gene, the results show that after knocking down S1PR1 gene, the colony morphology of KG1 cell line becomes smaller, the number is reduced, and the colony formation ability is significantly reduced.

[0046] Example Three Self-renewal ability and cell cycle verification

[0047] In this example, two groups of CD34+S1PR1+and CD34+S1PR1- cells in the bone marrow blood of AML patients were sorted, and CFU experiments and flow cytometry were used to detect cell cycle experiments to evaluate the influence of S1PR1 gene expression on the self-renewal ability and cell cycle of AML cells in vitro ex vivo. The specific steps are as follows:

[0048] (1) Sort CD34+S1PR1+and CD34+S1PR1- cell groups: see the single nuclear cell extraction, freezing and recovery in Example One (1) and (2). Then resuspend the cells with 300 μL of PBS and filter into a single cell suspension, label the antibodies with 10 μL of CD34-PE and 5 μL of S1PR1-APC, and incubate at room temperature for 15 minutes in the dark, then wash twice with PBS. Then perform flow sorting.

[0049] (2) Colony formation experiment steps of CD34+S1PR1+and CD34+S1PR1- cells see Example Two (2).

[0050] (3) Measurement of cell cycle: 1*10 6 Resuspend the cells with 1 mL of Hoechst culture solution (composition: RPMI1640+2% BIT+10 μg / μL of Hoechst), and incubate for 60 minutes. Wash once with PBS, then resuspend the cells with 100 μL of PBS containing 10 μg / μL of Hoechst. Add 5 μL of CD34-PE and 5 μL of S1PR1-APC antibodies, incubate at room temperature for 15 minutes in the dark, wash twice with PBS, then resuspend the cells with 300 μL of PBS, and perform flow cytometry analysis.

[0051] Figure 4 Figure 8 is a colony morphology chart (A) and a statistical chart (B) of AML cells of CD34+S1PR1+and CD34+S1PR1-.

[0052] Figure 5 Figure 9 is a cell cycle statistical chart of AML cells of CD34+S1PR1+and CD34+S1PR1-.

[0053] The results show that compared with the CD34+S1PR1-cell group, the number of CFU formed by CD34+S1PR1+AML cells is significantly increased, and the morphology is largerFigure 4 ). Compared with the CD34+S1PR1- cell population, the number of cells in the division phase was significantly higher in the CD34+S1PR1+ cell population Figure 5 ).

[0054] Preparation of a transplantation model

[0055] This example constructed a patient-derived xenograft model of secondary transplantation, and injected two groups of CD34+S1PR1+ and CD34+S1PR1- cells into immunodeficient mice via tail vein injection to detect the effect of S1PR1 gene expression in AML cells on LSC self-renewal and the ability to reconstitute leukemia in vivo.

[0056] (1) Construction of a patient-derived xenograft model (PDX): 6-8 week old immunodeficient NPG female mice were purchased from Beijing VitoDuo Biotechnology Co., Ltd. and were adaptively fed for at least 3 days. Antibiotic water was fed 1 day before transplantation. Irradiation was performed 4 hours before transplantation at a dose of 1 Gy. The cells to be injected were CD34+S1PR1+ and CD34+S1PR1- cell populations in primary AML cells, and the two cell populations were obtained by flow sorting, the specific steps of which are described in part (1) of Example III. Leukemia cells were injected into the tail vein of the mouse.

[0057] (2) Leukemia monitoring of mouse primary transplantation: 1 week after cell injection, the mouse peripheral blood flow was monitored by selecting the antibody hCD45. The survival of the mouse was recorded, and when the mouse was about to die, the bone marrow was immediately taken and the bone marrow cells were flushed out. Part of the bone marrow cells were used for flow detection, and the other part was frozen. The liver and spleen of the mouse were fixed for subsequent pathological experiments.

[0058] (3) Mouse secondary transplantation experiment: the bone marrow cells of the primary transplanted mouse were recovered, and the hCD45+ cell population was sorted by magnetic bead sorting. The immunodeficient mice were raised and irradiated as described in part (1) of this example, and leukemia cells were injected into the mouse body via the tail vein and raised and monitored. The mouse secondary transplantation monitoring scheme was the same as the primary transplantation monitoring method, as described in part (2) of this example.

[0059] Figure 6Figure 6 is a survival graph of immunodeficient mice injected with CD34+ S1PR1+ and CD34+ S1PR1- AML cells in secondary transplantation. Compared with mice carrying CD34+ S1PR1- cells, immunodeficient mice injected with CD34+ S1PR1+ cells showed significantly shorter survival time in both primary and secondary transplantation, higher bone marrow engraftment rate of leukemia cells, and more infiltration of leukemia cells in peripheral blood, liver and spleen.

[0060] In summary, the above examples show that S1PR1 can be used as a molecular marker of leukemia stem cells with the function of regulating LSCs, and can be used for the evaluation of the recurrence rate of related diseases, the risk assessment of diseases, the prognosis assessment, the stratified diagnosis, the detection or auxiliary detection of the progress of the disease course, the treatment of related diseases or the screening of related drugs, etc. In addition, the marker can be used to prepare a leukemia model mouse for scientific research.

[0061] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for some of the technical features. These modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. The application of a reagent for detecting the expression level of the molecular marker S1PR1 in leukemia stem cells in the preparation of a kit for leukemia diagnosis, characterized in that, The biomarker can regulate the self-renewal capacity of leukemia stem cells.

2. The application according to claim 1, characterized in that, The leukemia mentioned is acute leukemia.

3. The application according to claim 2, characterized in that, The acute leukemia mentioned is acute myeloid leukemia.

4. The application according to any one of claims 1-3, characterized in that, The kit diagnoses disease progression by detecting the expression level of S1PR1 in the bone marrow leukemia stem cells of subjects and comparing it with the average value of normal controls.

5. The application according to claim 4, characterized in that, The diagnosis of disease progression includes: 1) S1PR1 is highly expressed in bone marrow mononuclear cells of patients with acute myeloid leukemia compared to bone marrow mononuclear cells from normal donors; and 2) S1PR1 is highly expressed in acute myeloid leukemia stem cells compared to non-stem leukemia cells; or 3) S1PR1 is highly expressed in myeloid leukemia cell lines with strong self-renewal and leukemia regeneration capabilities and drug-resistant cell lines.

6. A method for preparing a mouse model of leukemia, characterized in that, The method includes the following steps: 1) Sorting CD34 from the bone marrow of AML patients. + S1PR1 + Cells, 2) Mouse tail vein injection of CD34 + S1PR1 + Leukemia model mice were obtained one week after cell population transplantation and feeding and monitoring.

7. The method according to claim 6, characterized in that, transplant CD34 + S1PR1 + After cell transplantation, immunodeficient mice with significantly increased bone marrow engraftment rate and significantly increased infiltration of leukemia cells in peripheral blood, liver, and spleen are designated as leukemia model mice.

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