Experimental method for researching influence of marrow supernatant of MPN patient on MPN disease progress

Abstract

The invention discloses an experimental method for researching the influence of marrow supernate of an MPN patient on MPN disease progression, and relates to the technical field of myeloproliferative tumors. A new thought is provided for MPN treatment; comprising the following steps: separating and culturing a primary monocyte (MNC) of an MPN patient or using a TF1 and HEL cell line as a substitution model; separating CD14 < + > mononuclear cells and CD34 < + > hematopoietic stem / progenitor cells through an immunomagnetic bead sorting technology; the bone marrow supernate of an MPN patient is used for treating TF1 and HEL cell lines, the influence of the TF1 and HEL cell lines on cell proliferation, apoptosis and phenotype is analyzed, and proteomics and transcriptomics analysis is carried out. According to the invention, the mononuclear cell subset change, especially the intermediate mononuclear cells (IM) and non-typical mononuclear cells (NCM), in the marrow of the MPN patient can be accurately separated and analyzed, and key targets influencing the MPN disease progress can be screened out through omics analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of myeloproliferative tumors, and in particular to an experimental method for studying the influence of bone marrow supernatant of MPN patients on the progression of MPN diseases. Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a type of chronic hematological malignancies caused by clonal proliferation of hematopoietic stem cells. Their complexity and diversity pose significant challenges in clinical treatment. Typical Philadelphia chromosome-negative MPNs mainly include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). These diseases not only affect the balance of blood components in patients, but also seriously threaten the life and health of patients due to their frequent bleeding and thrombotic events during the course of the disease, as well as the potential risk of acute leukemia transformation or bone marrow failure.

[0003] In recent years, due to changes in lifestyle and complex and changing environmental factors, the incidence of classic MPN has shown an increasing trend year by year, which makes the study of the pathogenesis of MPN and the development of effective treatment strategies particularly urgent. It is worth noting that MPN is not only an abnormal proliferative disease of the hematopoietic system, but its pathogenesis is also closely related to chronic inflammation. Inflammatory cells in the bone marrow microenvironment and the inflammatory factors they secrete play an important role in the occurrence and development of MPN. These inflammatory cytokines not only participate in regulating the hematopoietic function of the bone marrow, but also may promote the proliferation, invasion and metastasis of tumor cells, thereby accelerating the progression of the disease;

[0004] Existing studies have shown that both MPN-related tumor cells and normal cells can secrete a variety of pro-inflammatory cytokines, which are not only involved in the occurrence of systemic symptoms, but have also been confirmed to be key factors driving disease progression. However, although these findings provide a new perspective for our understanding of the pathogenesis of MPN, there is still a lack of in-depth research on how these cytokines specifically act on MPN cells and how they promote disease progression.

[0005] In view of this, this application aims to culture MPN cell lines (such as HEL and TF1) using bone marrow supernatants from MPN patients to simulate the effects of the in vivo microenvironment on tumor cells. By observing changes in the biological behavior of the cell lines, such as proliferation rate, apoptosis, migration and invasion ability, etc., we can more intuitively understand the effects of tumor-promoting factors that may exist in the bone marrow supernatant on MPN cells. At the same time, combined with transcriptome gene expression difference analysis, we can reveal how these tumor-promoting factors regulate the biological characteristics of MPN cells at the genetic level, thereby providing new clues for revealing the pathogenesis of MPN. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose an experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] An experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression comprises the following steps:

[0009] Isolate and culture primary mononuclear cells (MNCs) from MPN patients or use TF1 or HEL cell lines as alternative models;

[0010] CD14+ monocytes and CD34+ hematopoietic stem / progenitor cells were isolated by immunomagnetic bead sorting;

[0011] The TF1 and HEL cell lines were treated with bone marrow supernatant of MPN patients to analyze the effects on cell proliferation, apoptosis and phenotype;

[0012] The expression levels of TNF-α, TNFR1, and TNFR2 on the cell surface were detected by flow cytometry;

[0013] The treated cells were subjected to transcriptomic and proteomic analyses to screen key molecules and signaling pathways associated with MPN diseases.

[0014] Preferably: the culture conditions of the TF1 and HEL cell lines are:

[0015] Use RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin;

[0016] TF1 cell line was supplemented with 2 ng / ml GM-CSF;

[0017] The cell culture density was 2×10 5 / ml, placed in a 37°C, 5% CO2 incubator, and passaged every 2 days.

[0018] Further: the immunomagnetic bead sorting step comprises:

[0019] Using Lymphoprep TM Isolation of mononuclear cells (MNC);

[0020] Monocytes were sorted by CD14 immunomagnetic beads, and hematopoietic stem / progenitor cells were sorted by CD34 immunomagnetic beads;

[0021] Before sorting, the samples were treated with dead cell removal reagent, and cell activity was tested after sorting to ensure sorting efficiency.

[0022] Further: The method for processing the bone marrow supernatant of the MPN patient comprises:

[0023] The bone marrow samples were centrifuged at 300 g for 10 min to separate the plasma, which was then aliquoted and stored at −80°C;

[0024] TF1 and HEL cell lines were treated with supernatants at different concentrations (0.5%, 1%, 2%, and 4%);

[0025] The supernatant was replenished on the third day of culture, and functional verification was performed on the fifth and seventh days.

[0026] As a preferred solution of the present invention: the flow cytometry analysis comprises:

[0027] Cell proliferation was detected using CFSE labeling;

[0028] ANNEXIN-V FITC / PI staining was used to detect cell apoptosis;

[0029] The expression levels of TNF-α, TNFR1 and TNFR2 on the cell surface were detected.

[0030] As a further embodiment of the present invention: the transcriptomics and proteomics analysis includes:

[0031] Transcriptome sequencing was performed on TF1 and HEL cells treated with 1% supernatant;

[0032] Perform proteomic sequencing on the supernatant of clinical samples;

[0033] Bioinformatics analysis was used to screen differentially expressed genes and proteins related to MPN diseases.

[0034] As a further solution of the present invention: the key genes screened out by transcriptomics analysis include:

[0035] CXCL8, CXCL10, CCR3, TNRSF10D and IL1R1.

[0036] The beneficial effects of the present invention are:

[0037] 1. An experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease, which can accurately separate and analyze monocyte subsets in the bone marrow of MPN patients, especially intermediate monocytes (IM) and non-classical monocytes (NCM), as well as the expression levels of TNF-α, TNFR1 and TNFR2 on their surfaces.

[0038] 2. An experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease. Bone marrow supernatant of MPN patients has a significant inhibitory effect on the proliferation of TF1 and HEL cell lines, and this inhibitory effect increases with the increase of supernatant concentration.

[0039] 3. An experimental method to study the effect of bone marrow supernatant of MPN patients on the progression of MPN disease. By performing transcriptomic analysis on HEL and TF1 cells treated with patient supernatant, genes closely related to MPN, such as chemokines such as CXCL8, were successfully identified.

[0040] 4. An experimental method to study the effect of bone marrow supernatant of MPN patients on the progression of MPN disease. The single-cell transcriptome sequencing part provides the possibility for in-depth study of the heterogeneity of MPN diseases and can more carefully depict the gene expression profiles of different cell subpopulations in MPN patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The present invention proposes an experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease, and shows an analysis diagram of the phenotype characteristics of monocytes in MPN patients (1);

[0042] Figure 2 This is a diagram (2) of the phenotype characteristics of monocytes in MPN patients, which is an experimental method proposed by the present invention for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN diseases;

[0043] Figure 3 This is a diagram (3) of the phenotype characteristics of monocytes in MPN patients, which is an experimental method proposed by the present invention for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN diseases;

[0044] Figure 4 The present invention proposes an experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease. The effect of bone marrow supernatant of MPN patients on the proliferation of TF1 and HEL cells is shown in Figure (1);

[0045] Figure 5 The present invention proposes an experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease, and the effect of bone marrow supernatant of PN patients on the proliferation of TF1 and HEL cells (2);

[0046] Figure 6 The present invention proposes an experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease. Transcriptomic analysis shows that chemokine CXCL8 and others are related to MPN disease (1).

[0047] Figure 7The present invention proposes an experimental method for studying the effect of bone marrow supernatant of MPN patients on the progression of MPN disease. Transcriptomic analysis shows that chemokine CXCL8 and others are related to MPN disease (Figure 2). DETAILED DESCRIPTION

[0048] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0049] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0050] Embodiment 1:

[0051] An experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression comprises the following steps:

[0052] 1: Isolation and culture of primary cells

[0053] Blood mononuclear cells (MNCs) were obtained from bone marrow blood samples of patients with polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF), or peripheral blood samples of normal volunteers;

[0054] The number of primary cells from MPN patients is very small and insufficient for research, so the following cells were purchased: TF1 human blood leukemia cells (cellcook, cat: cc1925), HEL human erythroleukemia cells (cellcook, cat: cc1916), cultured at a density of 2×105 / ml in RPMI 1640 medium (Gibco) (supplemented with 1% penicillin-streptomycin, 10% FBS), of which TF1 cell line was cultured with 2ng / ml GM-CSF (stemcell, Catalog: 78190), placed in a humidified incubator at 37°C and 5% CO2, and passaged every 2 days;

[0055] Before isolating mononuclear cells (MNCs), use ACK lysis buffer to lyse red blood cells from bone marrow or peripheral blood samples to reduce the interference of red blood cells on monocyte separation;

[0056] Use freezing solution containing 10% DMSO to freeze some cells for subsequent experiments;

[0057] The purchased TF1 and HEL cell lines need to undergo STR identification and mycoplasma testing to ensure the purity and contamination-free nature of the cell lines;

[0058] Add other cytokines (such as IL-3, SCF, etc.) to RPMI 1640 culture medium to simulate the microenvironment of MPN patients;

[0059] 2. Immunomagnetic separation of monocytes and hematopoietic stem / progenitor cells

[0060] Using Lymphoprep TM (stemcell, Catalog: 18061) Mononuclear cells (MNC) were isolated from peripheral blood and bone marrow. The obtained human monocytes were positively selected using CD14 immunomagnetic beads (Miltenyi Biotec), and human hematopoietic stem / progenitor cells were magnetically sorted using a CD34 immunomagnetic bead kit;

[0061] One-step selection method: After obtaining mononuclear cells, count the cells, centrifuge and wash, and then suspend them in a corresponding volume of buffer (80 μl / 107 cells) according to the number of cells, then add a corresponding amount of magnetic beads (20 μl / 107 cells), mix thoroughly, incubate at 4°C for 15 minutes, then add buffer and wash for 10 minutes, the concentration of the cell suspension should be 500 μl / 108 cells, and finally sort them through a separation column placed in a magnetic field;

[0062] Before magnetic bead sorting, use dead cell removal reagents such as Miltenyi's Dead Cell Removal Kit to remove dead cells to improve sorting efficiency; after sorting, use trypan blue staining or flow cytometry to detect cell activity to ensure that the sorting process does not damage the cells; after each sorting, thoroughly clean the sorting column to prevent residual cells from affecting the next sorting results; in addition to CD14 and CD34, other markers such as CD38, CD90, and CD45RA can be combined for more refined cell subset sorting;

[0063] 3. Treatment of MPN-related cell lines with patient supernatants

[0064] For the collected bone marrow samples from clinically confirmed MPN patients, before isolating mononuclear cells, the samples were subjected to slow-fall centrifugation at 300 g for 10 min to separate the plasma, which was then dispensed into 200 ul / vial and stored in a -80°C refrigerator;

[0065] The cell density of TF1 and HEL cell lines was adjusted to 1×106 / ml, and 2.5uM / ml eBioscienceTM CFSE (invitrogen, cat: 65-0850) was used to label TF1 and HEL cell lines. The obtained supernatants were treated with different concentrations (0.5%, 1%, 2%, 4%) of TF1 and HEL cell lines, and the supernatants were supplemented on the third day of culture. Functional verification was performed on the fifth and seventh days respectively.

[0066] Before the experiment, ELISA or proteomics were used to detect the concentration of key cytokines (such as TNF-α, IL-6, VEGF, etc.) in the supernatant to ensure the consistency of the supernatant quality; when CFSE labeling, the cell density should be moderate (1×10 6 / ml), and wash thoroughly after labeling to remove unbound dye; optimize the supernatant treatment time (such as 24 hours, 48 ​​hours, 72 hours) according to the preliminary experimental results to determine the best treatment conditions; perform proteomics or metabolomics analysis on the supernatant to identify the active ingredients that may affect cell function; increase the supernatant treatment concentration gradient such as 0.1%, 0.25%, 0.5%, 1%, 2%, 4% to more accurately evaluate the effect of the supernatant on cells;

[0067] 4. Analysis of cell phenotype, proliferation and apoptosis by flow cytometry

[0068] BD FACS Canto II was used to sample and analyze the human monocytes and bone marrow stem / progenitor cells, and the levels of TNF-α, TNFR1 and TNFR2 on the cell surface were analyzed. The proliferation of cells labeled with CFSE was directly analyzed by flow cytometry. ANNEXIN-V FITC / PI reagent was used to stain the cells and detect cell apoptosis.

[0069] Before flow cytometry, titration experiments were performed on antibodies to determine the optimal antibody dosage to avoid nonspecific binding; single-stained samples were used to adjust fluorescence compensation to ensure the accuracy of multicolor flow cytometry; when detecting TNF-α, TNFR1, and TNFR2, isotype controls and unstained controls were set to eliminate background interference; PI staining combined with flow cytometry was used to analyze cell cycle distribution and evaluate the effect of supernatant treatment on cell proliferation; combined with cell apoptosis, proliferation, and cell cycle results, the effect of supernatant treatment on cell function was studied;

[0070] 5. Transcriptomic and proteomic analysis

[0071] The obtained clinical sample supernatant was subjected to proteomic sequencing; TF1 and HEL cells were treated with 1% concentration of supernatant, and samples were collected on the third day of culture for transcriptome sequencing;

[0072] Before RNA and protein extraction, cell activity and purity are tested to ensure sample quality. In transcriptome and proteome data analysis, internal reference genes or proteins are used for normalization to reduce experimental errors. Transcriptome and proteome data are combined to perform pathway enrichment analysis and network construction, and key molecules and signaling pathways are mined. Single-cell transcriptome sequencing is performed on patient primary cells or supernatant-treated cell lines to study cell heterogeneity. CRISPR / Cas9 gene editing or RNA interference technology is used to verify the functions of key genes or proteins.

[0073] Embodiment 2:

[0074] An experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression, using the experimental steps of Example 1 to conduct an experiment, and the following experimental results were obtained:

[0075] 1. As Figures 1 to 3 As shown, clinical samples showed that the intermediate monocytes (IM) and non-classical monocytes (NCM) of MPN patients increased significantly, and the expression of mTNFα, TNFR1 and TNFR2 in MPN monocytes increased significantly.

[0076] CD14+ and CD34+ cells were sorted for the collected clinical samples, and subtype analysis of CD14+ cells revealed a significant increase in intermediate monocytes and nonclassical monocytes in MPN patients, and a significant increase in the expression of TNFα, TNFR1, and TNFR2 in monocytes in MPN patients. As for CD34 cells in MPN patients, Figure C shows that CD34 cells in MPN patients after treatment were significantly reduced, and the expression of mTNFα increased.

[0077] 2. If Figures 4 to 5 As shown, 1% concentration of patient supernatant had a significant inhibitory effect on the proliferation of TF1 cells, and 2% supernatant had a significant inhibitory effect on the proliferation of both HEL and TF1 cells.

[0078] Since there are many abnormal cytokines in the bodies of MPN patients, in order to explore the role of these factors in MPN diseases, we used different concentrations of MPN patient serum to culture two MPN cell lines, HEL and TF1. We found that low concentrations of patient serum below 1% had no significant effect on the proliferation of HEL cell lines, but could significantly inhibit the proliferation of TF1 cells. However, high concentrations of patient serum could significantly cause the death of both cells.

[0079] 3. Such as Figure 6 to Figure 7 As shown in Figure 2, transcriptomic analysis showed that chemokines such as CXCL8 are closely related to MPN diseases.

[0080] Since the patient supernatant has different effects on the HEL cell line and the TF1 cell line, transcriptome analysis was performed on HEL&TF1 cells treated with 1% patient supernatant. Preliminary analysis revealed some MPN-related genes, such as TF and CXCL10 downregulated by TF1. In addition, there are 100 overlapping differentially expressed genes between TF1 and HEL cells, such as CCR3, CXCL8, TNRSF10D recombinant protein, and IL1R1, which may be closely related to this experiment.

[0081] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. An experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression, characterized in that: The following steps are involved: Isolate and culture primary mononuclear cells (MNCs) from MPN patients or use TF1 or HEL cell lines as alternative models; CD14+ monocytes and CD34+ hematopoietic stem / progenitor cells were isolated by immunomagnetic bead sorting; The TF1 and HEL cell lines were treated with bone marrow supernatant of MPN patients to analyze the effects on cell proliferation, apoptosis and phenotype; The expression levels of TNF-α, TNFR1, and TNFR2 on the cell surface were detected by flow cytometry; The treated cells were subjected to transcriptomic and proteomic analyses to screen key molecules and signaling pathways associated with MPN diseases.

2. The experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression according to claim 1, characterized in that: The culture conditions of the TF1 and HEL cell lines are: Use RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin; TF1 cell line was supplemented with 2 ng / ml GM-CSF; The cell culture density was 2×10 5 / ml, placed in a 37°C, 5% CO2 incubator, and passaged every 2 days.

3. The experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression according to claim 2, characterized in that: The immunomagnetic bead sorting step comprises: Using Lymphoprep TM Isolation of mononuclear cells (MNC); Monocytes were sorted by CD14 immunomagnetic beads, and hematopoietic stem / progenitor cells were sorted by CD34 immunomagnetic beads; Before sorting, the samples were treated with dead cell removal reagent, and cell activity was tested after sorting to ensure sorting efficiency.

4. The experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression according to claim 3, characterized in that: The method for processing bone marrow supernatant of MPN patients comprises: The bone marrow samples were centrifuged at 300 g for 10 min to separate the plasma, which was then aliquoted and stored at −80°C; TF1 and HEL cell lines were treated with supernatants at different concentrations (0.5%, 1%, 2%, and 4%); The supernatant was replenished on the third day of culture, and functional verification was performed on the fifth and seventh days.

5. The experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression according to claim 4, characterized in that: The flow cytometry analysis includes: Cell proliferation was detected using CFSE labeling; ANNEXIN-V FITC / PI staining was used to detect cell apoptosis; The expression levels of TNF-α, TNFR1 and TNFR2 on the cell surface were detected.

6. The experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression according to claim 5, characterized in that: The transcriptomic and proteomic analyses include: Transcriptome sequencing was performed on TF1 and HEL cells treated with 1% supernatant; Perform proteomic sequencing on the supernatant of clinical samples; Bioinformatics analysis was used to screen differentially expressed genes and proteins related to MPN diseases.

7. The experimental method for studying the effect of bone marrow supernatant of MPN patients on MPN disease progression according to claim 6, characterized in that: The key genes screened out by the transcriptomic analysis include: CXCL8, CXCL10, CCR3, TNRSF10D and IL1R1.

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