Analysis method for detecting H3K27me3 level of each immune cell population in human whole blood by using flow cytometry

Abstract

The invention discloses an analysis method for detecting the H3K27me3 level of each immune cell population in human whole blood by using flow cytometry, which comprises the following steps of: by a pretreatment mode of splitting red blood cells in a human whole blood sample by using a one-step method and fixing white blood cells, dyeing the split and fixed white blood cells by using fluorescently labeled antibodies (BV785-CD3, BV650-CD19, BV605-CD14, AF488-HLA-DR, AF647-H3K27 and Pacilic Blue-H3); and carrying out gate analysis on the sample after antibody staining by using a flow cytometer, and calculating the H3K27me3 / H3 level in each immune cell population, namely T cells (CD3 + CD19-), B-cell (CD3-CD19 +), mononuclear cells (CD14 + HLADR +) and granulocytes (CD14-HLADR-) through a formula. The whole blood sample can be preserved for 5 days under the condition of-60 to-90 DEG C after being subjected to one-step cracking and curing treatment. The method has good accuracy, reliability and stability.

Description

Technical Field

[0001] The present invention relates to a method for analyzing the level of H3K27me3, and particularly to a method for detecting the level of H3K27me3 in each immune cell population in human whole blood by using flow cytometry. Background Art

[0002] Flow cytometry (FCM) is a comprehensive detection technology integrating laser technology, hydrodynamics, computer image processing, and fluorescence labeling. It can qualitatively or quantitatively analyze the physical, biochemical, immunological, genetic, molecular biological properties and functional states of cells, magnetic beads or other biological particles in flowing liquids. Its main characteristics are: (1) Fast detection speed, capable of counting tens of thousands of cells or single molecule microspheres in a short time. (2) Multi-parameter detection and analysis can be performed on the same molecule, including molecular size, morphology, protein expression level, etc. (3) Automated high-throughput detection, capable of simultaneously and automatically detecting a large number of samples without manual participation in the detection process. (4) The detection results are accurate and reliable, and it has powerful image processing and data analysis functions, so the obtained results are objective and reliable. Currently, FCM has been widely applied in various fields of basic research and clinical practice, covering cell biology, immunology, oncology, pharmacology, genetics, and clinical laboratory tests, etc. Among them, the immunophenotype of immune cells is one of the most widely used directions of flow cytometry in clinical practice, mainly by detecting surface or intracellular markers of immune cell populations to identify and characterize them. Among them, immune cells include T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, platelets, and granulocytes, etc., and the methylation level of histones in different immune cell populations can also be accurately detected.

[0003] Histones are one of the main components of chromatin, mainly composed of five proteins: H1, H3, H2A, H2B, and H4. They are alkaline due to their rich content of basic arginine and lysine. At the same time, the amino-terminal amino acid residues can be covalently modified, thereby changing the chromatin configuration, leading to the transcriptional activation or silencing of downstream genes, and thus participating in cell division, apoptosis, and memory formation, and even affecting the immune system and inflammatory responses, etc. Research has shown that under the action of different enzymes, histones will undergo different post-translational modifications, and the main types include methylation, acetylation, phosphorylation, ubiquitination, glycosylation, etc. Among them, under the catalysis of methyltransferase, the N-terminals of arginine and lysine of histones can be methylated. Lysine can be mono-, di-, or tri-methylated, and arginine can be mono- or di-methylated. Histone methylation is a reversible biological process, which is jointly regulated by a variety of histone methyltransferases and demethylases in cells. Methyltransferase catalyzes the formation of methylation modifications, while histone demethylation is catalyzed by histone demethylase. The regulatory result of histone methylation on gene transcription status depends on the methylation residue and the degree of methylation.

[0004] Trimethylated histone H3 at lysine 27 (H3K27me3) is one of the most common histone modifications, mediated by the EZH2 subunit of PRC2. The methylation of H3K27 is a key mediator for inhibiting gene transcription and is involved in multiple important biological processes. Currently, the detection of histone methylation mainly relies on several common methods, such as chromatin immunoprecipitation (ChIP), Western Blot, protein microarray, and mass spectrometry technology, etc. ChIP can provide high-resolution gene localization information, but the operation is complex and time-consuming; Western Blot is easy to operate, but it can only detect methylated histones at the overall level; protein microarray and mass spectrometry technology have the advantages of high throughput and automation, but they are expensive. Since these methods cannot separate different cell populations, the methylation levels of different immune cell populations cannot be accurately obtained in whole blood at the same time.

[0005] So far, no patent for an analytical method based on flow cytometry to detect the H3K27me3 level in each immune cell population of human whole blood has been publicly disclosed. Therefore, this study developed an analytical method using flow cytometry to detect the H3K27me3 level in each immune cell population of human whole blood. Summary of the Invention

[0006] Object of the Invention: Aiming at the deficiencies in the existing detection technologies, the present invention provides an accurate, reliable, and stable analytical method using flow cytometry to detect the H3K27me3 level in each immune cell population of human whole blood.

[0007] Technical solution: An analysis method for detecting the H3K27me3 level of each immune cell population in human whole blood by flow cytometry. First, surface antibody staining is performed, and then nuclear antibody staining is performed after cell membrane permeabilization and fixation; or after cell membrane permeabilization and fixation, surface antibody and nuclear antibody staining are performed simultaneously.

[0008] Further, the surface antibodies: Fluorescently labeled anti-CD3, CD19, CD14, HLA-DR antibodies; the nuclear antibodies: Fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) antibody, Fluorescently labeled anti-Histone H3 antibody.

[0009] Further, the concentrations of the anti-CD3 (hereinafter referred to as BV785-CD3), CD19 (hereinafter referred to as BV650-CD19), CD14 (hereinafter referred to as BV605-CD14), HLA-DR (hereinafter referred to as BV605-CD14) antibodies are 50 μg / mL, 50 μg / mL, 150 μg / mL, and 200 μg / mL, respectively.

[0010] The concentrations of the anti-Tri-Methyl-Histone H3 (Lys27) antibody (hereinafter referred to as AF647-H3K27) and the Fluorescently labeled anti-Histone H3 antibody (hereinafter referred to as Pacific Blue-H3) are 100 μg / mL and 50 μg / mL, respectively.

[0011] Further, one-step lysis of red blood cells and fixation of white blood cells are used to pretreat the whole blood sample, and then staining is performed.

[0012] Further, 20 times the sample volume of 1×Lyse / Fix Buffer (1×Lyse / FixBuffer needs to be preheated in a 37°C water bath for 30 min before use) is added to the whole blood sample, and the whole blood sample is incubated in a 37°C water bath for 10 min for one-step lysis of red blood cells and fixation of white blood cells.

[0013] Further, Tri-Methyl-Histone H3 (Lys27) and Total Histone H3 are detected simultaneously, and the ratio of H3K27me3 to total H3 protein is used as the histone methylation level of each immune cell subset.

[0014] Further, the calculation formula for the H3K27me3 / H3 level is as follows: Calculation formula for H3K27me3 / H3 (%): ; Where: Sample(AF647-H3K27 Median MFI): Median fluorescence intensity of H3K27 Alexa Fluor in the sample tube Sample(Pacific Blue-H3 Median MFI): Median fluorescence intensity of H3 Pacific Blue in the sample tube.

[0015] Furthermore, FMO was used as a control.

[0016] By means of a pretreatment method that lyses red blood cells in human whole blood samples in one step while fixing white blood cells, fluorescently labeled antibodies (BV785-CD3, BV650-CD19, BV605-CD14, AF488-HLA-DR, AF647-H3K27, PacificBlue-H3) were used to stain the fixed white blood cells. After antibody staining, the samples were analyzed by gating using a flow cytometer. The levels of H3K27me3 / H3 in each immune cell population, namely T cells (CD3+CD19-), B-cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-), were calculated using a formula.

[0017] The research idea of this method is as follows: 1. This method is an analytical method for the levels of H3K27me3 in each immune sub-cell population in clinical human whole blood samples. Since trimethylated histone H3 at lysine 27 (H3K27me3) is extremely unstable, the levels of H3K27me3 in each immune sub-cell population in whole blood will change during the transportation of clinical samples to the testing center. Therefore, it cannot well reflect the changes in the levels of H3K27me3 in each immune sub-cell population in the whole blood of subjects after clinical administration. This method solves the problem of stability during the transportation of clinical whole blood samples. Currently, there are few reports and patents on the detection methods for the levels of H3K27me3 in each immune sub-cell population in whole blood, especially those supporting clinical biological sample analysis.

[0018] 2. In the initial exploration of the method, the conventional method was adopted: after whole blood was collected into ordinary EDTA-K2, Cyto-Chex BCT flow cytometry whole blood sample preservation tubes or Cell-Free DNA Streck TMBCT blood collection tubes, when the whole blood was directly preserved at room temperature or at 2-8°C, the levels of H3K27me3 in each immune subcellular population in the whole blood changed greatly compared with the baseline. When detecting the levels of H3K27me3 in each immune subcellular population in whole blood by the conventional method, the pre-staining stability of the whole blood sample was very poor and could not meet the stability required for clinical biological sample analysis. Therefore, the treatment of whole blood was further optimized and explored. Finally, in the method obtained through exploration, a one-step method for lysing red blood cells and fixing white blood cells was used to pre-treat freshly collected whole blood samples. The exploration in the initial stage of the method also included the exploration of the usage amount of 1×Lyse / Fix Buffer, whether to preheat it, and the water bath duration, making the method have good stability. 3. This method detects the levels of H3K27me3 in each immune cell subset in human whole blood, including the levels of H3K27me3 in T cells, B cells, monocytes, and granulocytes. In the initial exploration of the method, after red blood cell lysis and white blood cell fixation, surface antibody staining was first performed, and then nuclear antibody staining was performed after cell permeabilization and fixation. The surface antibodies used were: fluorescently labeled anti-CD3, CD19, CD14, HLA-DR antibodies, and the nuclear antibodies were: fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) antibody, fluorescently labeled anti-Histone H3 antibody. Then, in order to simplify the experimental procedure, the experimental method was explored and optimized, that is, in this method, after cell permeabilization and fixation, surface antibodies and nuclear antibodies can be stained simultaneously, saving the experimental time.

[0019] 4. This method simultaneously detects Tri-Methyl-Histone H3 (Lys27) and Total Histone H3, and the ratio of H3K27me3 to total H3 protein is used as the histone methylation level of each immune cell subset.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention uses a one-step method for lysing red blood cells and fixing white blood cells to pre-treat freshly collected whole blood samples, enabling the immediate fixation of the activities of H3K27 methyltransferase and H3K27me3 demethylase in cells after whole blood collection, and maintaining the stability of the histone methylation level of each immune cell subset.

[0021] 2. In the method of the present invention, surface antibody staining is first performed, and then nuclear antibody staining is performed after cell membrane permeabilization and fixation. The surface antibodies used are: fluorescently labeled anti-CD3, CD19, CD14, HLA-DR antibodies, and the nuclear antibodies are: fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) antibody, fluorescently labeled anti-Histone H3 antibody. Alternatively, after cell membrane permeabilization and fixation, surface antibodies and nuclear antibodies can be stained simultaneously, simplifying the experimental process and saving experimental time.

[0022] 3. This method uses FMO controls to facilitate the determination of the position of the granulocyte population. Description of the Drawings

[0023] Figure 1 It is a gating logic diagram (6-color) for flow analysis of the sample. According to the expression of 6 proteins, colors are matched, and 6 fluorescently labeled antibodies mentioned in the present invention are selected (BV785-CD3, BV650-CD19, BV605-CD14, AF488-HLA-DR, AF647-H3K27, Pacific Blue-H3). Under this analysis template, four immune cell populations can be circled simultaneously, namely T cells (CD3+CD19-), B-cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-), and the expression of Tri-Methyl-Histone H3 (Lys27) and Total Histone H3 in the 4 immune cell populations can be visually displayed. The median fluorescence values of H3K27 Alexa Fluor and H3 PacificBlue of the sample tube can be obtained from the analysis diagram, and then the H3K27me3 / H3 levels of the four immune cell populations can be obtained through the calculation formula of the H3K27me3 / H3 level. Specific Embodiments

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0025] Whole blood pretreatment: One-step method (-60~-90°C lysis and fixation) Whole blood is collected in a Cell-Free DNA StreckTMBCT blood collection tube. 20 times the sample volume of 1×Lyse / Fix Buffer (preheated in a 37°C water bath for 30 min) is added to the whole blood sample, and the whole blood sample is incubated in a 37°C water bath for 10 min for one-step lysis of red blood cells and fixation of white blood cells, and then immediately frozen at -60~-90°C.

[0026] Detailed steps for sample analysis: 1. Samples stored at -60~-90°C are taken out and immediately thawed in a 37°C water bath. After complete thawing, proceed to the next step.

[0027] 2. Prepare 2 centrifuge tubes, labeled "Sample" and "FMO" respectively. Add 10 mL of the processed whole blood sample to each tube, and centrifuge at 500 g for 8 min at room temperature. 3. Discard the supernatant, add 6 mL of PBS, resuspend the cells, and centrifuge at 500 g for 8 min at room temperature. 4. Discard the supernatant, add 6 mL of PBS, resuspend the cells, and centrifuge at 500 g for 8 min at room temperature. 5. Discard the supernatant, and add 100 µL of Stain Buffer to each tube to resuspend the cells. 6. Add 1 mL of freshly prepared 1× fixation / permeabilization buffer to each tube, mix well, and incubate in the dark at 2~8°C for about 40±5 min. 7. Add 2 mL of 1× FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8°C, and discard the supernatant. 8. Add 2 mL of 1× FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8°C, and discard the supernatant. 9. Add 100 µL of 1× FOXP3 permeabilization buffer to each tube to resuspend the cells. 10. Add 5 μL of BV785-CD3 (50 μg / mL), 5 μL of AF488-HLA-DR (200 μg / mL), 5 μL of BV605-CD14 (150 μg / mL), 5 μL of BV650-CD19 (50 μg / mL), 2 μL of AF647-H3K27 (100 μg / mL), and 2 μL of Pacific Blue-H3 (50 μg / mL) to the sample tube. Add 5 μL of BV785-CD3 (50 μg / mL), 5 μL of BV605-CD14 (150 μg / mL), 5 μL of BV650-CD19 (50 μg / mL), 2 μL of AF647-H3K27 (100 μg / mL), and 2 μL of Pacific Blue-H3 (50 μg / mL) to the FMO tube. After mixing, incubate in the dark at 2~8°C for 30 min±5 min.

[0028] 11. Add 2 mL of 1× FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8°C, and discard the supernatant.

[0029] 12. Add 2 mL of 1× FOXP3 permeabilization buffer to each tube, mix well, centrifuge at 400 g for 5 min at 2~8°C, and discard the supernatant.

[0030] 13. Resuspend the cells by adding 350 μL of Stain Buffer to each tube. Store at 2 - 8 °C for further testing.

[0031] 14. Flow cytometry detection: Turn on the flow cytometer and perform equipment calibration: According to the instrument's built-in program, use CSTSetup Beads to perform fluorescence channel calibration. In the first experiment, determine the voltage and compensation through 6 single-label tubes and a Blank tube of fluorescence channels and save them as a template. Collect 10,000 All Events cells in the single-label tubes, and collect 5,000 CD14+HLA-DR+ cells or the total volume of the cell suspension for each sample.

[0032] 15. After the flow cytometry detection is completed, use the BD FACS Diva software to set the analysis template obtained by gating. The gating strategy is shown in Figure 1 . Use FSC-A / SSC-A to gate the All cells cell population; from All cells, use FSC-A / FSC-H to gate single cells (Singlets) and exclude aggregates; from Singlets, use FSC-A / SSC-A to gate the LYM cell population and Non LYM cell population; from the LYM cell population, use CD3 BV786-A / CD19 BV650-A to gate CD3+CD19- cells (T cells) and CD3-CD19+ cells (B cells), and from the Non LYM cell population, use CD14 BV605-A / HLA-DR-AF488-A to gate CD14+HLA-DR+ cells (monocytes) and CD14-HLA-DR- cells (granulocytes). Obtain the median fluorescence intensity (MedianMFI) of AF647-H3K27 and Pacific Blue-H3 in the monocyte, granulocyte, B cell, and T cell populations respectively, and process the data with Microsoft Excel 2016.

[0033] The calculation formula for the H3K27me3 / H3 level is as follows: Calculation formula for 27me3 / H3 (%): ; In addition, the whole blood samples were used to verify the pre-staining stability by this method, and the results are shown in Table 1.

[0034] Table 1 Pre-staining stability of the one-step method (-60~-90 °C) Time (Day) T-cell (CD3+CD19-) H3K27 / H3% CV% B-cell (CD3-CD19+)H3K27 / H3% CV% Monocytes (CD14+HLADR+) H3K27 / H3% CV% Granulocytes (CD14-HLADR-) H3K27 / H3% CV% 0d 115.0 NA 306.9 NA 22.3 NA 71.1 NA 60 ~ -90℃ 1d 123.0 4.8 390.5 16.9 19.7 8.5 58.4 13.9 60 ~ -90℃ 5d 128.7 7.9 406.0 19.7 22.9 1.8 64.0 7.5 After the whole blood sample is collected, whole blood pretreatment, i.e., the one-step method (-60~-90°C lysis and fixation), is immediately carried out. The baseline sample after lysis and fixation is immediately subjected to sample analysis (see the detailed steps 1~15 of sample analysis). The other pre-staining stability samples after lysis and fixation are stored at -60~90°C. After 1 day and 5 days of storage, sample analysis is carried out (see the detailed steps 1~15 of sample analysis). After the flow cytometry analysis is completed, data is obtained, and the H3K27me3 / H3 levels of T cells (CD3+CD19-), B cells (CD3-CD19+), monocytes (CD14+HLADR+), and granulocytes (CD14-HLADR-) in the samples stored at -60~90°C for 1 day, 5 days, and the baseline (0d) are calculated. The results are shown in Table 1, and the results indicate that: compared with the baseline (0d), the CV% of the H3K27me3 / H3 level of T cells (CD3+CD19-) in the samples stored at -60~90°C for 1 day and 5 days are 4.8% and 7.9% respectively; compared with the baseline (0d), the CV% of the H3K27me3 / H3 level of B cells (CD3-CD19+) in the samples stored at -60~90°C for 1 day and 5 days are 16.9% and 19.7% respectively; compared with the baseline (0d), the CV% of the H3K27me3 / H3 level of monocytes (CD14+HLADR+) in the samples stored at -60~90°C for 1 day and 5 days are 8.5% and 1.8% respectively; compared with the baseline (0d), the CV% of the H3K27me3 / H3 level of granulocytes (CD14-HLADR-) in the samples stored at -60~90°C for 1 day and 5 days are 13.9% and 7.5% respectively. In summary, after the whole blood sample is treated by the one-step lysis and fixation method, it can be stored at -60~-90°C for 5 days, and relatively accurate results can still be obtained by using this method (CV% ≤ 20 compared with Day0 H3K27me3 / H3 (%)).

[0035] The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art, within the scope of the technical solution of the present invention, makes any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed by the present invention, all of which belong to the content within the scope of the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. An analytical method for detecting the H3K27me3 levels of various immune cell populations in human whole blood by flow cytometry, characterized in that: Surface antibody staining is performed first, followed by nuclear antibody staining after cell permeabilization and fixation; or surface and nuclear antibodies are stained simultaneously after cell permeabilization and fixation.

2. The analysis method for detecting the H3K27me3 levels of each immune cell population in human whole blood by flow cytometry according to claim 1, wherein: The surface antibodies: Fluorescently labeled anti-CD3, CD19, CD14, HLA-DR antibodies; The nuclear antibodies: Fluorescently labeled anti-Tri-Methyl-Histone H3 (Lys27) antibody, Fluorescently labeled anti-HistoneH3 antibody.

3. The analysis method for detecting the H3K27me3 level of each immune cell population in human whole blood by flow cytometry according to claim 1, wherein: The concentrations of the anti-CD3, CD19, CD14, HLA-DR antibodies are 50 μg / mL, 50 μg / mL, 150 μg / mL, 200 μg / mL respectively; The concentrations of the anti-Tri-Methyl-Histone H3 (Lys27) antibody and the Fluorescently labeled anti-Histone H3 antibody are 100 μg / mL and 50 μg / mL respectively.

4. The analysis method for detecting the H3K27me3 level of each immune cell population in human whole blood by flow cytometry according to claim 1, characterized in that: The whole blood sample is pretreated by lysing red blood cells and fixing white blood cells using a one-step method, followed by staining.

5. The analysis method for detecting the H3K27me3 level of each immune cell population in human whole blood by flow cytometry according to claim 1, characterized in that: Add 20 times the sample volume of 1×Lyse / Fix Buffer (1×Lyse / Fix Buffer needs to be preheated in a 37°C water bath for 30 min before use) to the whole blood sample, and incubate in a 37°C water bath for 10 min to lyse red blood cells and fix white blood cells in one step.

6. The analytical method for detecting the H3K27me3 level of each immune cell population in human whole blood by flow cytometry according to claim 1, wherein: Detect Tri-Methyl-Histone H3 (Lys27) and Total Histone H3 simultaneously, and the ratio of H3K27me3 to total H3 protein is used as the histone methylation level of each immune cell subset.

7. The analysis method for detecting the H3K27me3 level of each immune cell population in human whole blood by flow cytometry according to claim 6, characterized in that: The calculation formula for the H3K27me3 / H3 level is as follows: Calculation formula for H3K27me3 / H3 (%): ； Where: Sample(AF647-H3K27 Median MFI): Median fluorescence intensity of H3K27 Alexa Fluor in the sample tube Sample(Pacific Blue-H3 Median MFI): Median fluorescence intensity of H3 Pacific Blue in the sample tube.

8. The analysis method for detecting the H3K27me3 levels of each immune cell population in human whole blood by flow cytometry according to claim 1, wherein: Use FMO as a control.

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