Use of bone marrow vascular endothelial cells in myelodysplastic syndrome

Abstract

The application discloses application of bone marrow vascular endothelial cells in myelodysplastic syndrome (MDS). The application provides application of bone marrow vascular endothelial cells as a marker in any one of the following: preparation of a product for detecting or assisting in detecting the disease progression of MDS, preparation of a product for diagnosing or assisting in diagnosing MDS, and preparation of a product for distinguishing or assisting in distinguishing MDS and non-MDS. Researches of the application find that the number of bone marrow vascular endothelial cells gradually increases from MDS-MLD, MDS-EB to AML patients, but the abnormal function gradually aggravates. In addition, with the disease progression, the supporting ability of bone marrow vascular endothelial cells of MDS patients to normal hematopoietic cells in vitro decreases, and the supporting ability to malignant hematopoietic cells increases. The application has important significance for detecting the occurrence and development of MDS, especially for monitoring the disease progression of MDS.

Description

[0001] This application is a divisional application of application No. 202111273168.6, with an application date of October 29, 2021, and an invention creation name of "Application of bone marrow vascular endothelial cells in myelodysplastic syndrome" TECHNICAL FIELD

[0002] The present application relates to the field of biomedicine, in particular to an application of bone marrow vascular endothelial cells in myelodysplastic syndrome. BACKGROUND

[0003] Myelodysplastic syndrome (MDS) is a group of heterogeneous myeloid clonal diseases characterized by abnormal development of myeloid cells and a tendency to transform into acute myeloid leukemia (AML). The pathogenesis of MDS is complex and diverse, and the current treatment strategy for MDS relies on allogeneic hematopoietic stem cell transplantation, hypomethylating drugs, immunosuppressive agents and chemotherapy, but the clinical efficacy of these treatments is limited. For example, only 40%-50% of patients can survive for 5 years with allogeneic hematopoietic stem cell transplantation. Therefore, further elucidation of the pathogenesis of MDS and establishment of new treatment strategies are important clinical scientific problems that need to be solved.

[0004] Ineffective hematopoiesis is the main pathophysiological process of MDS. MDS with multilineage dysplasia (MDS-MLD), MDS with excess blasts (MDS-EB) to acute myeloid leukemia (AML) is a typical process of MDS progression. At the same time, immune dysregulation as another important pathogenesis of MDS shows that different T cell subsets are associated with the progression of MDS.

[0005] Bone marrow vascular endothelial cells as an important component of the bone marrow microenvironment have unclear multiple effects on normal hematopoiesis, abnormal hematopoiesis and immune regulation in MDS patients at different clinical stages. In addition, the relationship between these effects of bone marrow vascular endothelial cells and the progression of MDS is still unknown. Therefore, it is of great significance for the clinical treatment of MDS to provide the correlation between bone marrow microenvironment cells and the progression of MDS. SUMMARY

[0006] The purpose of the present application is to provide an application of bone marrow vascular endothelial cells in myelodysplastic syndrome.

[0007] In a first aspect, the present application claims the application of bone marrow vascular endothelial cells as a marker in any of the following:

[0008] (A1) preparing a product for detecting or assisting in detecting the progression of myelodysplastic syndrome, or detecting or assisting in detecting the progression of myelodysplastic syndrome;

[0009] (A2) a product for diagnosing or aiding in the diagnosis of myelodysplastic syndrome, or diagnosing or aiding in the diagnosis of myelodysplastic syndrome;

[0010] (A3) a product for distinguishing or aiding in the distinction between a patient with myelodysplastic syndrome and a patient without myelodysplastic syndrome, or distinguishing or aiding in the distinction between a patient with myelodysplastic syndrome and a patient without myelodysplastic syndrome.

[0011] In a second aspect, the present application claims the use of substance A and / or substance B and / or substance C and / or substance D and / or substance E and / or substance F in any one of the following:

[0012] (A1) a product for detecting or aiding in the detection of the progression of myelodysplastic syndrome, or detecting or aiding in the detection of the progression of myelodysplastic syndrome;

[0013] (A2) a product for diagnosing or aiding in the diagnosis of myelodysplastic syndrome, or diagnosing or aiding in the diagnosis of myelodysplastic syndrome;

[0014] (A3) a product for distinguishing or aiding in the distinction between a patient with myelodysplastic syndrome and a patient without myelodysplastic syndrome, or distinguishing or aiding in the distinction between a patient with myelodysplastic syndrome and a patient without myelodysplastic syndrome.

[0015] The substance A is a substance for detecting the number of vascular endothelial cells in bone marrow.

[0016] The substance B is a substance for detecting the damage of vascular endothelial cells in bone marrow.

[0017] The substance C is a substance for detecting the functional state of vascular endothelial cells in bone marrow.

[0018] The substance D is a substance for detecting the support ability of vascular endothelial cells in bone marrow for hematopoietic stem cells.

[0019] The substance E is a substance for detecting whether vascular endothelial cells in bone marrow cause T cells to differentiate in the direction of immune tolerance.

[0020] The substance F is a substance for detecting the support ability of vascular endothelial cells in bone marrow for leukemia cells.

[0021] Furthermore, the damage to vascular endothelial cells in the bone marrow can be reflected in all or part of the following (the same applies below): the apoptosis rate of bone marrow vascular endothelial cells, the ROS level in bone marrow vascular endothelial cells, and the expression levels of apoptosis-related genes in bone marrow vascular endothelial cells. Higher apoptosis rates, higher intracellular ROS levels, and higher expression levels of apoptosis-related genes (such as CASP2, CASP3, and BAX) indicate more severe damage to bone marrow vascular endothelial cells.

[0022] Furthermore, the functional status of vascular endothelial cells in the bone marrow can be reflected in all or part of the following (the same applies below): the tube-forming ability and migration ability of bone marrow vascular endothelial cells. The stronger the tube-forming ability and migration ability of bone marrow vascular endothelial cells, the more severe the functional impairment of bone marrow vascular endothelial cells.

[0023] Furthermore, the ability of the vascular endothelial cells in the bone marrow to support hematopoietic stem cells can be manifested in all or part of the following (the same applies below): the use of CD34 from bone marrow derived from healthy individuals... + After co-culturing cells with the tested bone marrow vascular endothelial cells, bone marrow CD34 + Apoptosis rate, bone marrow CD34 + ROS levels in cells, bone marrow CD34 + Cellular CFU-E, BFU-E, CFU-GM, and / or CFU-GEMM formation capacity. Bone marrow CD34. + The higher the apoptosis rate, the higher the intracellular ROS level, and the lower the ability to form CFU-E, BFU-E, CFU-GM, and CFU-GEMM, the lower the ability of vascular endothelial cells in the bone marrow to support hematopoietic stem cells.

[0024] Furthermore, whether the vascular endothelial cells in the bone marrow lead to the differentiation of T cells toward immune tolerance can be manifested in all or part of the following (the same applies below): CD3+ from bone marrow derived from healthy individuals... + After co-culturing T cells with the tested bone marrow vascular endothelial cells, bone marrow CD3 + T cell differentiation, including Th1 / CD4 + T cells, Th17 / CD4 + T cells, Th2 / CD4 + T cells, Treg / CD4 + T cells, Th1 / Th2 ratio. Bone marrow CD3. + T cells transform into Th1 / CD4 + T cells and Th17 / CD4 + The less T cell differentiation, the more likely the cells are to differentiate into Th2 / CD4. + T cells and Treg / CD4 +The more T cell differentiation and the lower Th1 / Th2 ratio, the more the bone marrow vascular endothelial cells cause T cells to differentiate in the direction of immune tolerance.

[0025] Further, the support ability of the bone marrow vascular endothelial cells for leukemia cells can be embodied by all or part of the following: the proliferation, apoptosis, intracellular ROS level, leukemia cell colony unit (CFU-leukemia, CFU-L) formation efficiency, intracellular apoptosis and / or expression of cell cycle-related genes of leukemia cells after co-culturing leukemia cells with the bone marrow vascular endothelial cells. The stronger the proliferation of leukemia cells, the lower the apoptosis rate, the lower the intracellular ROS level, the higher the leukemia cell colony unit (CFU-leukemia, CFU-L) formation efficiency, the higher the expression amount of cell cycle-related genes (such as CCNE1 and MCL1), the lower the expression amount of cell cycle-related genes (such as CASP2, CASP3, BAX, TP53 and CDKN1A), the stronger the support ability of the bone marrow vascular endothelial cells for leukemia cells.

[0026] In a specific embodiment of the present application, the leukemia cells are specifically HL-60 cells.

[0027] In the above aspects, the non-myelodysplastic syndrome patient needs to meet the following conditions: the number of bone marrow vascular endothelial cells is reduced, the damage of bone marrow vascular endothelial cells is reduced or does not occur, the dysfunction of bone marrow vascular endothelial cells is reduced or does not occur, the support ability of bone marrow vascular endothelial cells for hematopoietic stem cells is enhanced, bone marrow vascular endothelial cells do not cause T cells to differentiate in the direction of immune tolerance, and the support ability of bone marrow vascular endothelial cells for leukemia cells is reduced or the bone marrow vascular endothelial cells have no support ability for leukemia cells compared with myelodysplastic syndrome patients.

[0028] In a specific embodiment of the present application, the non-myelodysplastic syndrome patient is a healthy person.

[0029] In a specific embodiment of the present application, the progression of myelodysplastic syndrome can be embodied by the following three stages: MDS with multilineage dysplasia (MDS-MLD), MDS with excess blasts (MDS-EB) to acute myeloid leukemia (AML).

[0030] In (A3) of the above aspects, the myelodysplastic syndrome patient can be a MDS with multilineage dysplasia (MDS-MLD) patient, a MDS with excess blasts (MDS-EB) patient or an acute myeloid leukemia (AML) patient.

[0031] In the above two aspects, the product can be specifically a kit.

[0032] In the present application, each of the substances (substances A, B, C, D, E, F) described above can be a substance capable of specifically binding to a specific detection object or an instrument and a supporting reagent for detecting each index.

[0033] In a third aspect, the present application claims a system for detecting the progression of myelodysplastic syndrome, comprising:

[0034] (B1) reagents and / or instruments;

[0035] The reagents and / or instruments have all or part of the following functions: detecting the number of vascular endothelial cells in bone marrow, detecting the damage of vascular endothelial cells in bone marrow, detecting the functional state of vascular endothelial cells in bone marrow, detecting the support ability of vascular endothelial cells in bone marrow for hematopoietic stem cells, detecting whether vascular endothelial cells in bone marrow will cause T cells to differentiate in the direction of immune tolerance, and detecting the support ability of vascular endothelial cells in bone marrow for leukemia cells.

[0036] (B2) device;

[0037] The device comprises a data input module, a threshold value storage module, a data comparison module, and a judgment module.

[0038] The data input module is configured to input all or part of the following values detected by (B1): the number of vascular endothelial cells in the bone marrow to be tested from a myelodysplastic syndrome patient; the damage of vascular endothelial cells in the bone marrow to be tested; the dysfunction of vascular endothelial cells in the bone marrow to be tested; the support ability of vascular endothelial cells in the bone marrow to be tested for hematopoietic stem cells; the value of vascular endothelial cells in the bone marrow to be tested causing T cells to differentiate in the direction of immune tolerance; and the support ability of vascular endothelial cells in the bone marrow to be tested for leukemia cells.

[0039] The threshold value storage module is configured to store all or part of the following threshold values: threshold value A, threshold value B, threshold value C, threshold value D, threshold value E, and threshold value F; the threshold value A is the number of vascular endothelial cells in the bone marrow of a healthy person; the threshold value B is the damage of vascular endothelial cells in the bone marrow of a healthy person; the threshold value C is the dysfunction of vascular endothelial cells in the bone marrow of a healthy person; the threshold value D is the support ability of vascular endothelial cells in the bone marrow of a healthy person for hematopoietic stem cells; the threshold value E is the value of vascular endothelial cells in the bone marrow of a healthy person causing T cells to differentiate in the direction of immune tolerance; and the threshold value F is the support ability of vascular endothelial cells in the bone marrow of a healthy person for leukemia cells.

[0040] The data comparison module is configured to receive all or part of the (B1) detected values (i.e. the number of vascular endothelial cells in the bone marrow to be tested; the damage condition of the vascular endothelial cells in the bone marrow to be tested; the function state of the vascular endothelial cells in the bone marrow to be tested; the support ability of the vascular endothelial cells in the bone marrow to be tested for hematopoietic stem cells; the differentiation of T cells to immune tolerance direction caused by the vascular endothelial cells in the bone marrow to be tested; and the support ability of the vascular endothelial cells in the bone marrow to be tested for leukemia cells) sent by the data input module, and call the thresholds in the threshold storage module, compare the number of vascular endothelial cells in the bone marrow to be tested with the threshold A, compare the damage condition of the vascular endothelial cells in the bone marrow to be tested with the threshold B, compare the dysfunction of the vascular endothelial cells in the bone marrow to be tested with the threshold C, compare the support ability of the vascular endothelial cells in the bone marrow to be tested for hematopoietic stem cells with the threshold D, compare the differentiation of T cells to immune tolerance direction caused by the vascular endothelial cells in the bone marrow to be tested with the threshold E, and / or compare the support ability of the vascular endothelial cells in the bone marrow to be tested for leukemia cells with the threshold F.

[0041] The judgment module is configured to receive the comparison results sent by the data comparison module, and then make the following result judgments: the number of vascular endothelial cells in the bone marrow to be tested is greater than the threshold A, the damage condition of the vascular endothelial cells in the bone marrow to be tested is greater than the threshold B (i.e. the damage condition of the vascular endothelial cells in the bone marrow to be tested is more serious), the dysfunction of the vascular endothelial cells in the bone marrow to be tested is greater than the threshold C (i.e. the dysfunction of the vascular endothelial cells in the bone marrow to be tested is more serious), the support ability of the vascular endothelial cells in the bone marrow to be tested for hematopoietic stem cells is less than the threshold D (i.e. the support ability of the vascular endothelial cells in the bone marrow to be tested for hematopoietic stem cells is weaker), the differentiation of T cells to immune tolerance direction caused by the vascular endothelial cells in the bone marrow to be tested is greater than the threshold E (i.e. the vascular endothelial cells in the bone marrow to be tested cause the differentiation of T cells to immune tolerance direction), and / or the support ability of the vascular endothelial cells in the bone marrow to be tested for leukemia cells is greater than the threshold F (i.e. the support ability of the vascular endothelial cells in the bone marrow to be tested for leukemia cells is stronger), and the greater the absolute value of the difference between each value and the corresponding threshold, the more serious the condition of the patient with myelodysplastic syndrome.

[0042] In a fourth aspect, the present application claims to protect a system for diagnosing or assisting in diagnosing myelodysplastic syndrome, comprising:

[0043] (C1) reagents and / or instruments;

[0044] The reagent and / or instrument has all or part of the following functions: detecting the number of vascular endothelial cells in bone marrow, detecting the damage of vascular endothelial cells in bone marrow, detecting the functional state of vascular endothelial cells in bone marrow, detecting the support ability of vascular endothelial cells in bone marrow to hematopoietic stem cells, detecting whether vascular endothelial cells in bone marrow will cause T cells to differentiate in the direction of immune tolerance, and detecting the support ability of vascular endothelial cells in bone marrow to leukemia cells.

[0045] (C2) the device;

[0046] The device comprises a data input module, a threshold value storage module, a data comparison module, and a judgment module.

[0047] The data input module is configured to input all or part of the following values detected in (B1): the number of vascular endothelial cells in the bone marrow of the subject to be tested; the damage of vascular endothelial cells in the bone marrow of the subject to be tested; the dysfunction of vascular endothelial cells in the bone marrow of the subject to be tested; the support ability of vascular endothelial cells in the bone marrow of the subject to be tested to hematopoietic stem cells; the differentiation of T cells in the direction of immune tolerance caused by vascular endothelial cells in the bone marrow of the subject to be tested; and the support ability of vascular endothelial cells in the bone marrow of the subject to be tested to leukemia cells.

[0048] The threshold value storage module is configured to store all or part of the following threshold values: threshold value A, threshold value B, threshold value C, threshold value D, threshold value E, and threshold value F; the threshold value A is the number of vascular endothelial cells in the bone marrow of a healthy person; the threshold value B is the damage of vascular endothelial cells in the bone marrow of a healthy person; the threshold value C is the dysfunction of vascular endothelial cells in the bone marrow of a healthy person; the threshold value D is the support ability of vascular endothelial cells in the bone marrow of a healthy person to hematopoietic stem cells; the threshold value E is the differentiation of T cells in the direction of immune tolerance caused by vascular endothelial cells in the bone marrow of a healthy person; and the threshold value F is the support ability of vascular endothelial cells in the bone marrow of a healthy person to leukemia cells.

[0049] The data comparison module is configured to receive all or part of the (C1) detected values (i.e. the number of vascular endothelial cells in the test bone marrow; the damage condition of the vascular endothelial cells in the test bone marrow; the function state of the vascular endothelial cells in the test bone marrow; the support ability of the vascular endothelial cells in the test bone marrow for hematopoietic stem cells; the differentiation of T cells to immune tolerance direction caused by the vascular endothelial cells in the test bone marrow; and the support ability of the vascular endothelial cells in the test bone marrow for leukemia cells) sent by the data input module, and call the threshold values in the threshold storage module, compare the number of vascular endothelial cells in the test bone marrow with the threshold value A, compare the damage condition of the vascular endothelial cells in the test bone marrow with the threshold value B, compare the dysfunction of the vascular endothelial cells in the test bone marrow with the threshold value C, compare the support ability of the vascular endothelial cells in the test bone marrow for hematopoietic stem cells with the threshold value D, compare the differentiation of T cells to immune tolerance direction caused by the vascular endothelial cells in the test bone marrow with the threshold value E, and / or compare the support ability of the vascular endothelial cells in the test bone marrow for leukemia cells with the threshold value F.

[0050] The judgment module is configured to receive the comparison results sent by the data comparison module, and then make a result judgment as follows: if the number of vascular endothelial cells in the test bone marrow is greater than the threshold value A, the damage condition of the vascular endothelial cells in the test bone marrow is greater than the threshold value B (i.e. the damage condition of the vascular endothelial cells in the test bone marrow is more serious), the dysfunction of the vascular endothelial cells in the test bone marrow is greater than the threshold value C (i.e. the dysfunction of the vascular endothelial cells in the test bone marrow is more serious), the support ability of the vascular endothelial cells in the test bone marrow for hematopoietic stem cells is less than the threshold value D (i.e. the support ability of the vascular endothelial cells in the test bone marrow for hematopoietic stem cells is weaker), the differentiation of T cells to immune tolerance direction caused by the vascular endothelial cells in the test bone marrow is greater than the threshold value E (i.e. the vascular endothelial cells in the test bone marrow cause the differentiation of T cells to immune tolerance direction), and / or the support ability of the vascular endothelial cells in the test bone marrow for leukemia cells is greater than the threshold value F (i.e. the support ability of the vascular endothelial cells in the test bone marrow for leukemia cells is stronger), then the testee is a patient with myelodysplastic syndrome.

[0051] The present application researches and finds that the number of bone marrow vascular endothelial cells (BM EC) gradually increases from MDS-MLD, MDS-EB to AML patients, but the dysfunction gradually aggravates. In addition, with the disease progression, the support ability of BM EC of MDS patients to normal hematopoietic cells in vitro decreases, and the support ability to malignant hematopoietic cells increases. After co-cultured with T cells, the proportion of Th2 and Treg increases, and the proportion of Th1 and Th17 decreases. Transcriptome sequencing shows that the expression profile of bone marrow vascular endothelial cells of MDS-MLD patients is more similar to that of healthy donors (HD), and MDS-EB is more similar to AML. Mechanically, the levels of hematopoietic regulation related genes CXCL12, SCF and NFKB of bone marrow vascular endothelial cells increase with the progression of MDS. The present application has important significance for detecting the occurrence and development of MDS, especially monitoring the disease progression of MDS. The present application provides a diagnostic service for MDS. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The figure is the proportion of bone marrow vascular endothelial cells in mononuclear cells of HD, MDS-MLD, MDS-EB and AML patients. Flow cytometry was used to detect the proportion of CD34, CD309 and CD133 positive endothelial cells in mononuclear cells of bone marrow of HD, MDS with multilineage dysplasia (MDS-MLD), MDS with excess blasts (MDS-EB) and acute myeloid leukemia (AML). P<0.05 was considered statistically different. * represents P<0.05; ** represents P<0.005; and *** represents P<0.001.

[0053] Figure 2 The figure is a double staining experiment of bone marrow vascular endothelial cells of HD, MDS-MLD, MDS-EB and AML patients. A is a typical graph; and B is a statistical analysis graph of the number of double staining of bone marrow vascular endothelial cells of HD, MDS-MLD, MDS-EB and AML patients.

[0054] Figure 3 The figure is the damage of bone marrow vascular endothelial cells of MDS patients. A is a statistical analysis graph of the apoptosis of bone marrow vascular endothelial cells of HD, MDS-MLD, MDS-EB and AML patients; B is a statistical analysis graph of the ROS level of bone marrow vascular endothelial cells of HD, MDS-MLD, MDS-EB and AML patients; and C is the relative mRNA level of CASP2, CASP3 and BAX in bone marrow vascular endothelial cells of HD, MDS-MLD, MDS-EB and AML patients.

[0055] Figure 4Figure 6. The function of bone marrow vascular endothelial cells in MDS patients is impaired. A is the tube formation ability of bone marrow vascular endothelial cells in HD, MDS-MLD, MDS-EB, and AML patients (original magnification, 10x); B is the migration ability of bone marrow vascular endothelial cells in HD, MDS-MLD, MDS-EB, and AML patients (original magnification, 10x); C is a statistical analysis chart of tube length; D is a statistical analysis chart of the number of cells that migrated across the membrane (tube pixels per field). Three fields were randomly counted, and the average value was taken for each sample. The scale bar represents 200 μιη.

[0056] Figure 5 Figure 7. The support ability of bone marrow vascular endothelial cells in MDS and AML patients for hematopoietic stem cells. A is the apoptosis rate of CD34+ cells after co-culture of bone marrow-derived vascular endothelial cells in HD, MDS-MLD, MDS-EB, and AML patients for 5 days; B is the ROS level in CD34+ cells after co-culture; C is the CFU formation ability of CD34+ cells after co-culture. + + +

[0057] Figure 6 Figure 8. Bone marrow vascular endothelial cells in MDS-MLD, MDS-EB, and AML patients cause abnormal differentiation of CD3+ cells. A is the proportion of Thl cells after co-culture; B is the proportion of Th2 cells after co-culture; C is the proportion of Thl 7 cells after co-culture; D is the proportion of Treg cells after co-culture; E is the Thl / Th2 ratio after co-culture.

[0058] Figure 7 Figure 9. Proliferation, apoptosis, and ROS level of HL-60 cells after co-culture with bone marrow vascular endothelial cells in MDS-MLD, MDS-EB, and AML patients. A is the proliferation of HL-60 cells; B is the apoptosis of HL-60 cells; C is a statistical analysis of the proportion of EdU-positive cells in HL-60 cells; D is a statistical analysis of the apoptosis proportion of HL-60 cells; E is a statistical analysis of the ROS level (mean fluorescence intensity) of HL-60 cells; F is the CFU-L formation efficiency of HL-60 cells after co-culture with bone marrow vascular endothelial cells in MDS-MLD, MDS-EB, and AML patients; G is the expression level of apoptosis and cell cycle-related genes in HL-60 cells after co-culture.

[0059] Figure 8 ​​​Transcriptome sequencing analysis results of bone marrow vascular endothelial cells of HD, MDS and AML patients. A is the principal component analysis chart of 12 transcriptome sequencing libraries; B is the gene expression heat map, and hierarchical clustering is performed according to Euclidean distance; C is the difference gene count between each group; D is the differential expression of apoptosis, hematopoiesis and immune related genes in the transcriptome sequencing results; E is the verification of hematopoietic related gene mRNA level by qRT-PCR; F is the verification of immune related gene mRNA level by qRT-PCR. DETAILED DESCRIPTION

[0060] The application will be further described in detail below in conjunction with the specific embodiments. The examples given are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0061] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0062] Example 1, Application of bone marrow vascular endothelial cells in MDS

[0063] I. Experimental materials

[0064] 1. EGM-2-MV-SingleQuots liquid medium: American Lonza Company.

[0065] 2. RPMI 1640 liquid medium: American Gibco Company.

[0066] 3. MethoCult H4434 Classic: Canadian STEMCELL Technologies Company.

[0067] 4. StemSpan SFEM: Canadian STEMCELL Technologies Company.

[0068] 5. Human peripheral blood lymphocyte separation medium (Ficoll): American GE Healthcare Company.

[0069] 6. Monoclonal antibodies: CD3-APC / H7 (Becton Dickinson (BD)), CD8-BV510 (Biolegend), CD25-PE / Cy7 (Invitrogen), IFN-γ-PerCP / Cy5.5 (Biolegend), IL-4-PE (BD), IL17A-FITC (BD), FOXP3-APC (Invitrogen), CD34-percp / cy5.5 (Biolegend), CD34-FITC (Biolegend), CD133-APC (Miltenyi), CD45-V500 (BD), vascular endothelial growth factor receptor2 (VEGFR2, CD309)-PE (BD Biosciences), Annexin-V and 7-amino-actinomycin D (7-AAD), mouse anti-human CD34 (BD), rabbit anti-human CD133 (Abeam).

[0070] 7. DAPI solution: Beijing Solabio Company.

[0071] 8. 2’,7’-dichlorofluorescein diacetate (DCFH-DA): China Biyun Tian Company.

[0072] 9. Reactive Oxygen Species Assay Kit: China Biyun Tian Company

[0073] 10. Fibronectin: Sigma Company, USA

[0074] 11. 5-Ethynyl-20-deoxyuridine (EdU): China RiboBio Company

[0075] 12. Recombinant human FLT-3: PEPROTECH Company, USA

[0076] 13. Recombinant human TPO: PEPROTECH Company, USA

[0077] 14. Recombinant human SCF: PEPROTECH Company, USA

[0078] 15. Human CD34 sorting magnetic beads: Miltenyi Company, Germany

[0079] 16. Human CD3 sorting magnetic beads: Miltenyi, Germany

[0080] 17. Hemolysin: BD FACSTM Lysing Solution, BD, USA

[0081] 18. Fetal bovine serum (FBS): Gibco, USA

[0082] 19. FITC-labelled Lectin from Ulex europaeus (FITC-UEA-I): Sigma-Aldrich, USA

[0083] 20. Diacetylated low-density lipoprotein: DiI-AcLDL, Life Technologies, USA

[0084] 21. Matrigel matrix: Corning, USA

[0085] 22. Transwell chamber: Corning, USA

[0086] 23. RNeasy Mini kit: QIAGEN, Germany

[0087] 24. RT reagent Kit with gDNA Eraser: TaKaRa, Japan

[0088] 25. SYBR-Green qRT-PCR kit: Thermo Fisher Scientific, USA

[0089] 26. HL-60 cell line: Human pro-myelocytic leukemia cell line, Cell Bank of Chinese Academy of Sciences / Stem Cell Bank of China

[0090] 27. This cohort study included 15 patients with newly diagnosed MDS-MLD, MDS-EB or AML and 15 bone marrow samples of HD as healthy controls. The diagnosis of MDS was made according to the WHO classification criteria in 2016 version; the newly diagnosed AML patients were primary AML, and were diagnosed as M2, M4 or M5 subtype. The basic characteristics of patients in each group were similar, such as gender and age.

[0091] II. Experimental methods

[0092] 1. Culture of bone marrow vascular endothelial cells

[0093] Bone marrow mononuclear cells isolation: Bone marrow was mixed with lymphocyte separation medium (GE Healthcare, USA) at a ratio of 1:1 and centrifuged at 1800 rpm for 18 min at room temperature. The white cloudy layer of mononuclear cells was carefully pipetted and washed twice with PBS. The bone marrow mononuclear cells were seeded in fibronectin-coated 6-well plates and cultured in endothelial cell induction medium (EGM-2-MV-SingleQuots) and 10% fetal bovine serum for 7 days at 37°C in a 5% CO2 incubator. The medium was changed every 4 days.

[0094] 2. Quantification of bone marrow endothelial cells

[0095] 1. Human bone marrow mononuclear cells were labeled with anti-human-CD45, anti-human-CD34, anti-human-CD133, and anti-human-CD309 for 15 min at room temperature in the dark. The cells were washed twice with PBS and centrifuged at 1500 rpm for 5 min. The cells were analyzed using BD LSR Fortessa software.

[0096] 2. After 7 days of culture in endothelial cell (EC) induction medium (EGM-2-MV-SingleQuots), the adherent cells were washed three times with PBS and incubated with 10 pg / ml Dil-Ac-LDL for 4 h at 37°C. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min. The fixed cells were washed twice with PBS for 5 min each. The cells were incubated with FITC-UEA-1 (10 pg / ml) for 1 h at room temperature in the dark. The stained cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan). Dil-Ac-LDL staining was red, FITC-UEA-1 staining was green, and cells with both stains were yellow, which were ECs. Three fields were randomly selected and the average value was calculated.

[0097] 3. Intracellular reactive oxygen species (ROS) level and apoptosis analysis

[0098] 1. ROS: 10 mM 2’,7’-dichlorofluorescence diacetate (DCFH-DA) was used to incubate the cells for 15 min at 37°C. After washing twice with PBS, the average fluorescence intensity of endogenous DCFH-DA (a ROS probe) was detected using BD LSR Fortessa software.

[0099] 2. Apoptosis: Annexin-V and 7-amino-actinomycin D (7-AAD) were used to incubate the cells for 15 min, and the apoptosis was directly detected using BD LSR Fortessa software. Annexin-V +7-AAD - Annexin-V is for early apoptosis. + 7-AAD + This indicates late-stage apoptosis.

[0100] All data were analyzed using BD LSRFortessa software.

[0101] 4. Tube formation experiment and migration experiment

[0102] ① Gel formation experiment: Add 200 μL of matrix gel solution to each well of a pre-cooled 24-well plate, then incubate at 37°C for 30 min to form a gel. Add 500 μL of endothelial cell suspension (induced for 7 days) to the gel surface, with a cell count of 5 × 10⁶ cells per well. 5 Each sample was added to EC induction medium (EGM-2-MV-SingleQuots) and observed and photographed under a microscope after 48 hours. A total of 4 × 10⁶ samples were collected. 4 One vascular endothelial cell was transferred to a matrix-coated plate and stored at 37°C in 5% CO2 for 48 hours. The relative tube length per field of view was measured using an inverted optical microscope.

[0103] ② Migration Assay: Cell migration assays were performed using Transwell chambers. Digested cells were introduced at a rate of 5 × 10⁶ cells per well. 4 Cells were seeded in the upper chamber, and 500 μl of EC induction medium (EGM-2-MV-SingleQuots) was added to the lower chamber. After culturing the cells at 37°C for 24 hours, the cells on the membrane were fixed with formaldehyde for 30 minutes, the upper layer of cells were wiped off with cotton swabs, and the cells at the bottom of the membrane were stained with crystal violet for 20 minutes. The stained cells were observed and photographed under a microscope (Olympus, Tokyo, Japan).

[0104] 5. CD34 + Co-culture of cells with bone marrow vascular endothelial cells

[0105] CD34 was isolated from bone marrow mononuclear cells of patients with hepatitis B using CD34 magnetic beads. + Cells, as non-adherent cells, were contact cultured with HD, MDS-MLD, MDS-EB, and AML bone marrow vascular endothelial cells cultured for 7 days in StemSpan™ SFEM and cultured for 5 days in an incubator at 37°C and 5% CO2 for subsequent detection.

[0106] 6. CD3 + Co-culture of cells with bone marrow vascular endothelial cells

[0107] CD3 was isolated from bone marrow mononuclear cells of (HD) using CD3 magnetic beads. +Cells, as nonadherent cells, were cocultured with HD, MDS-MLD, MDS-EB and AML bone marrow vascular endothelial cells on day 7 of culture in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated at 37°C in a 5% CO2incubator for 3 days for subsequent detection.

[0108] 7. Co-culture of HL-60 cells with bone marrow vascular endothelial cells

[0109] HL-60 cells, as nonadherent cells, were cocultured with HD, MDS-MLD, MDS-EB and AML bone marrow vascular endothelial cells on day 7 of culture in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated at 37°C in a 5% CO2incubator for 5 days for subsequent detection.

[0110] 8. Detection of colony-forming units (CFU) and leukemia colony-forming units (CFU-L)

[0111] Harvesting of suspended CD34 + Cells or HL-60 cells, 2 x 10 3 CD34 + cells / 10 3 HL-60 cells were mixed in 500 μL MethoCult TM H4434 Classic medium, and incubated at 37°C in a 5% CO2incubator for 14 days. After 14 days, the CD34 + erythroid colony-forming units (CFU-E), erythroid burst-forming units (BFU-E), granulocyte-macrophage colony-forming units (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM) of the CD34

[0112] 9. Analysis of T cell subsets

[0113] T cells were labeled with the following mouse anti-human monoclonal antibodies: anti-human-CD3, anti-human-CD4, anti-human-CD8, anti-human-CD25, anti-human-IFN-γ, anti-human-IL-4, anti-human-Foxp3, and anti-human-IL-17A, and lymphocyte subsets were quantified by flow cytometry. Th1, Th2, Th17 and Treg cells were CD3 + CD8 - IFN-γ + , CD3 + CD8 - IL-4 + , CD3 + CD8- IL-17A + and CD3 + CD8 - CD25 + Foxp3 + Data were analyzed using BD LSRFortessa software.

[0114] 10. EdU experiment

[0115] After co-culture, HL-60 cells were harvested and incubated with 50 mM EdU at 37 °C in a 5% CO2 incubator for 1 h, and the remaining steps were performed according to the product manual. Finally, the cell fluorescence intensity was detected and analyzed on BD LSRFortessa.

[0116] 11. RNA sequencing (RNA-seq) and real-time quantitative PCR (qRT-PCR)

[0117] RNA-seq analysis was performed on bone marrow-derived vascular endothelial cells of HD, MDS-MLD, MDS-EB and AML patients on day 7 of culture. Hierarchical clustering analysis and differential gene expression analysis were performed using the heatmap and DESeq2 packages in R (1.16.1). To verify the RNA-seq results, SYBR Green-based qRT-PCR (ViiA 7 Real-Time PCR System, Thermo Fisher Scientific, USA) was used to detect the mRNA levels of CASP2, CASP3, BAX, CCNE1, MCL1, p53, p21, CXCL12, KITLG and NFKB1, and the above genes were normalized to 18S mRNA levels.

[0118] 12. Statistical analysis

[0119] Statistical analysis was performed using GraphPad Prism 6.0 (USA). Mann- Whitney U test was used for comparison between two groups. The results were expressed as mean ± SEM, and P < 0.05 was considered statistically significant.

[0120] III. Results and analysis

[0121] 1. The number of bone marrow vascular endothelial cells in MDS patients gradually increased with disease progression

[0122] On the day of extraction (day 0), bone marrow mononuclear cells from HD, MDS-MLD, MDS-EB and AML patients were used to circumscribe vascular endothelial cells using flow cytometry antibodies CD34, CD309 and CD133. The proportion of bone marrow vascular endothelial cells in mononuclear cells is shown in Figure 1, MDS-EB group was significantly higher than MDS-MLD group, and AML group was significantly higher than MDS-EB group Figure 1

[0123] The cultured 7-day human bone marrow vascular endothelial cells were identified by Dil-ac-LDL / FITC-UEA-1 double-labeling experiment Figure 2 The double-labeling experiment is a classic experiment for identifying vascular endothelial cells, which simultaneously labels Dil-ac-LDL (red fluorescence) and FITC-UEA-1 (green fluorescence), and the cells with double positive staining are vascular endothelial cells (yellow fluorescence). The number of double positive cells under the field of fluorescence microscope was counted and statistically analyzed. The number of double positive bone marrow vascular endothelial cells in AML patients was significantly more than that in MDS-EB patients Figure 2

[0124] In summary, the number of bone marrow vascular endothelial cells in MDS and AML patients increased, and gradually increased with the progression of the disease from MDS-MLD, MDS-EB to AML.

[0125] 2、MDS patients' bone marrow vascular endothelial cell damage gradually increased with the progression of the disease

[0126] In order to study the damage of bone marrow vascular endothelial cells, we detected the apoptosis, ROS and expression level of apoptosis-related genes of bone marrow vascular endothelial cells in HD, MDS-MLD, MDS-EB and AML patients. The apoptosis and ROS were determined by flow cytometry on the 0th day of bone marrow vascular endothelial cells. The expression level of apoptosis-related genes was determined by culturing 7-day human bone marrow vascular endothelial cells.

[0127] The apoptosis rate of bone marrow vascular endothelial cells in MDS-EB group was significantly higher than that in MDS-MLD group Figure 3 The ROS level of bone marrow vascular endothelial cells in AML patients was significantly higher than that in MDS-EB patients Figure 3 Further, qRT-PCR was used to analyze the expression level of CASP2, CASP3 and BAX apoptosis-related genes of bone marrow vascular endothelial cells. Compared with MDS-EB patients, the mRNA level of CASP2 of bone marrow vascular endothelial cells in AML patients was significantly increased Figure 3 CASP3 and BAX in MDS-EB patients were significantly higher than MDS-MLD Figure 3 In summary, these data suggest that MDS and AML bone marrow vascular endothelial cells are damaged, and the damage of bone marrow vascular endothelial cells gradually increases with the progression of MDS.

[0128] 3、MDS patients' bone marrow vascular endothelial cell dysfunction gradually increased with the progression of the disease​​

[0129] The function of bone marrow-derived vascular endothelial cells from patients with HD, MDS-MLD, MDS-EB, and AML will be assessed from their angiogenic capacity. Tube-forming capacity of bone marrow-derived vascular endothelial cells will be analyzed on day 7 of culture. Figure 4 (A) and transferability ( Figure 4 (Middle B). Three visual fields were randomly selected for statistical analysis, and the average value of each sample was taken. Statistical analysis of tube formation length showed that bone marrow vascular endothelial cells in MDS-EB patients had significantly higher tube formation capacity than those in MDS-MLD patients. Figure 4 (C). Human bone marrow vascular endothelial cells exhibit transmembrane migration ability in chamber migration assays; bone marrow vascular endothelial cells from AML patients demonstrate stronger migration ability than those from MDS-MLD patients. Figure 4 These results indicate that bone marrow vascular endothelial cells in patients with MDS and AML exhibit dysfunction, and that this dysfunction worsens with the progression of MDS.

[0130] 4. The ability of bone marrow vascular endothelial cells to support hematopoietic stem cells weakens as MDS progresses.

[0131] To investigate the effects of bone marrow vascular endothelial cells on hematopoietic stem cells, we sorted donor-derived bone marrow mononuclear cells, removing CD34 cells from HD cells. + Cells were co-cultured with bone marrow-derived vascular endothelial cells from patients with HD, MDS-MLD, MDS-EB, and AML on day 7, and CD34 levels were measured after 5 days. + Apoptosis, intracellular ROS levels, and hematopoietic stem cell colony-forming unit (CFU) formation capacity were assessed. Apoptosis and intracellular ROS levels were detected by flow cytometry. CFU formation capacity was evaluated using a 2×10⁻⁶ cell line. 3 CD34 cells were co-cultured for 5 days. + Cells were uniformly seeded in a special semi-solid culture medium for 14 days, and colonies of different types were counted. Compared with the MDS-MLD group, when co-cultured with MDS-EB bone marrow vascular endothelial cells, bone marrow CD34 levels were significantly increased. + The level of apoptosis was significantly increased. Figure 5 (A) AML Group CD34 + Cellular ROS levels were higher than in the MDS-MLD group. Figure 5 (Middle B). Compared with the MDS-MLD group, the formation abilities of CFU-E, BFU-E, CFU-GM, and CFU-GEMM were all reduced in the MDS-EB and AML groups. Figure 5 (C). These data indicate that bone marrow vascular endothelial cells in MDS and AML patients lead to hematopoietic stem cell dysfunction, and that as MDS progresses, the ability of vascular endothelial cells to support normal hematopoietic stem cells becomes increasingly weak.

[0132] 5. With the progression of MDS, bone marrow vascular endothelial cells cause T cells to gradually differentiate in the direction of immune tolerance

[0133] Considering the immunologically related pathogenesis of MDS, we further studied the in vitro immunomodulatory effect of bone marrow vascular endothelial cells, and we co-cultured HD-derived bone marrow CD3 + T cells with bone marrow vascular endothelial cells from MDS-MLD, MDS-EB and AML patients, and analyzed the differences in T cell subsets after 3 days. The proportion of Th1 in CD4 + T cells in the AML group was significantly lower than that in the MDS-EB group Figure 6 A). The proportion of Th2 in CD4 + T cells in the AML group was higher than that in the MDS-MLD group Figure 6 B). The proportion of Th17 in CD4 + T cells in the MDS-EB group was significantly lower than that in the MDS-MLD group Figure 6 C). The proportion of Treg in CD4 + T cells in the MDS-EB group was higher than that in the MDS-MLD group Figure 6 D). The Th1 / Th2 ratio in the AML group was lower than that in the MDS-EB group, and the Th1 / Th2 ratio in the MDS-EB group was significantly lower than that in the MDS-MLD group Figure 6 E). These data suggest that bone marrow vascular endothelial cells from MDS, AML patients are the cause of immunoregulatory abnormalities in MDS, and with the progression of the disease, bone marrow vascular endothelial cells may be more inclined to induce T cells to differentiate into immune tolerant cells.

[0134] 6. The supporting ability of bone marrow vascular endothelial cells from MDS patients for leukemia cells is enhanced with the progression of the disease

[0135] To investigate the effect of bone marrow vascular endothelial cells on AML cells in vitro, we detected the proliferation, apoptosis, intracellular ROS level of HL-60 cells after co-culturing with bone marrow vascular endothelial cells for 5 days, the efficiency of leukemia colony-forming units (CFU-L) formation, and the relative expression of apoptosis and cell cycle-related gene mRNA in HL-60 cells after co-culturing. The proliferation of HL-60 cells was detected by EdU (5-Ethynyl-20-deoxyuridine) experiment. The apoptosis of HL-60 cells and the intracellular ROS level were detected by flow cytometry. The CFU-L formation ability was detected by plating 10 3HL-60 cells co-cultured for 5 days were uniformly seeded on a special semi-solid medium for 14 days, and the number of leukemia cell colonies was counted. The EdU positivity rate of HL-60 cells in the MDS-EB group was significantly higher than that in the MDS-MLD group. Figure 7 (A and C). Apoptosis rate ( Figure 7 The ROS levels (relative to HD) in the AML group (B and D) were significantly lower than those in the MDS-MLD group, and the ROS levels (fold relative to HD) in the MDS-EB group were significantly lower than those in the MDS-MLD group. Figure 7 In addition, the CFU-L formation efficiency in the AML group was significantly higher than that in the MDS-EB group (E). Figure 7 Compared with MDS-MLD, the MDS-EB group showed significantly decreased TP53 and CDKN1A, and significantly increased MCL1, while the AML group showed significantly decreased CASP2, CASP3, BAX, TP53, and CDKN1A. Compared with MDS-EB, the AML group showed significantly decreased CASP2, TP53, and CDKN1A, and significantly increased CCNE1 and MCL1. Figure 7 (G). These data suggest that the bone marrow vascular endothelial cells in MDS and AML patients have enhanced support for leukemia cells. More importantly, this support gradually increases as the disease progresses.

[0136] 7. Heterogeneity of bone marrow vascular endothelial cells and potential damage mechanisms in the progression of MDS

[0137] To confirm the heterogeneity of bone marrow vascular endothelial cells in MDS and AML patients at the transcriptome level, and to further explore the mechanisms of bone marrow vascular endothelial cell damage in MDS and AML patients, we performed transcriptome sequencing and principal component analysis on bone marrow-derived endothelial cells cultured for 7 days. Figure 8 (A), hierarchical cluster analysis ( Figure 8 (B) and differential gene analysis ( Figure 8 The results showed that the 12 samples were clearly divided into two distinct subgroups: HD and the disease group (C). Figure 8 (A, B). More importantly, the heatmap ( Figure 8 (B) shows the progression of bone marrow vascular endothelial cell RNA expression profiles from HD to AML. The differential gene counts between HD and patients ( Figure 8The C values ​​(e.g., HD vs. MDS-MLD (3448)) were significantly higher than those between patients (e.g., MDS-MLD vs. MDS-EB (722)). These results revealed the heterogeneity of bone marrow-derived endothelial cells at the transcriptomic level in HD and MDS-MLD, MDS-EB, and AML. Further analysis of transcriptomic sequencing results revealed that apoptosis (CASP2, CASP3, BAX), hematopoietic (CXCL12, KITLG, NFKB1), and immune (HAVCR2, LGALS9, CIITA)-related genes were elevated in bone marrow-derived endothelial cells of MDS and AML patients, such as ( Figure 8 (D). The elevated levels of hematopoietic-related genes CXCL12, KITLG, and NFKB1 were verified using qRT-PCR. Figure 8 E) and immune-related genes (HAVCR2, LGALS9, CIITA) increase with disease progression. Figure 8 These data further reveal the role of bone marrow vascular endothelial cells in regulating hematopoiesis and immunity in MDS and AML, and suggest that related genes may be the cause of bone marrow vascular endothelial cell damage in MDS and AML.

[0138] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of substance F in the preparation of a product for detecting or assisting in detecting the progression of myelodysplastic syndrome; The substance F is a substance for detecting the supporting ability of vascular endothelial cells in bone marrow for leukemia cells; The detection of the support ability of vascular endothelial cells in bone marrow for leukemia cells is carried out by detecting the proliferation, apoptosis, intracellular ROS level, leukemia cell colony unit formation efficiency, intracellular apoptosis and / or expression of cell cycle-related genes of leukemia cells after co-culturing leukemia cells with vascular endothelial cells in the test bone marrow; and the substance F is composed of leukemia cells and at least one of the following substances : a substance for detecting the proliferation of leukemia cells, a substance for detecting the intracellular ROS level of leukemia cells, a substance for detecting the leukemia cell colony unit formation efficiency, and a substance for detecting the expression of apoptosis and / or cell cycle-related genes in leukemia cells.

2. A system for detecting the progression of myelodysplastic syndrome, comprising: (B1) reagents and / or instruments; The reagents and / or instruments have the following functions: detecting the support ability of vascular endothelial cells in bone marrow for leukemia cells; the detection of the support ability of vascular endothelial cells in bone marrow for leukemia cells is carried out by detecting the proliferation, apoptosis, intracellular ROS level, leukemia cell colony unit formation efficiency, intracellular apoptosis and / or expression of cell cycle-related genes of leukemia cells after co-culturing leukemia cells with vascular endothelial cells in the test bone marrow; (B2) a device; The device comprises a data input module, a threshold value storage module, a data comparison module, and a judgment module; The data input module is configured to input the following values detected by (B1): the support ability value of vascular endothelial cells in the test bone marrow from a myelodysplastic syndrome patient for leukemia cells; The threshold value storage module is configured to store a threshold value F; the threshold value F is the support ability value of vascular endothelial cells in the bone marrow of a healthy person for leukemia cells; The data comparison module is configured to receive the values detected by (B1) sent from the data input module, and to call the threshold value F in the threshold value storage module, and to compare the support ability value of vascular endothelial cells in the test bone marrow for leukemia cells with the threshold value F; The judgment module is configured to receive the comparison result sent by the data comparison module, and then make a result judgment according to the following: the greater the absolute value of the difference between the support ability value of vascular endothelial cells in the test bone marrow for leukemia cells and the threshold value F, the more serious the condition of the myelodysplastic syndrome patient.

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