Detection method of megakaryocyte in bronchopulmonary dysplasia model

Abstract

The invention discloses a method for detecting megakaryocytes in a bronchopulmonary dysplasia model, and relates to the field of biomedicine. Comprising the following steps: constructing a BPD mouse model, carrying out HE staining, flow cytometry and immunofluorescence detection on lung tissues, and analyzing the number, proportion, ploidy and distribution of megakaryocytes, the construction of the BPD mouse model comprises the following steps: randomly dividing C57BL / 6j strain newborn mice into an experimental group and a control group one day after the newborn mice are born, feeding the experimental group in a 85-90% high oxygen concentration environment for 14 days, and feeding the control group in a C57BL / 6j strain newborn mice in a C57BL / 6j strain newborn mice in a C57BL / 6j strain newborn mice. The control group is fed in the air, and the female rats are interchanged among the groups every 24 hours. By integrating model construction, HE staining, flow cytometry and immunofluorescence, multi-dimensional detection of megakaryocytes in the BPD model is achieved, the method is standardized and good in repeatability, reduction and ploidy change of megakaryocytes in lung tissue can be accurately reflected, and a reliable tool is provided for research of a BPD mechanism.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a method for detecting megakaryocytes in a bronchopulmonary dysplasia model. Background Technology

[0002] Bronchopulmonary dysplasia (BPD) is a chronic lung disease in newborns, and reliable animal models are needed to study its pathogenesis. Changes in megakaryocytes, as precursor cells of platelets, in BPD models are crucial for understanding the disease's pathophysiology. Current techniques for detecting megakaryocytes include tissue staining, flow cytometry, and immunofluorescence, but a comprehensive detection method specifically for BPD models has not yet been systematized.

[0003] Therefore, this invention proposes a method for detecting megakaryocytes in a bronchopulmonary dysplasia model. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for detecting megakaryocytes in a bronchopulmonary dysplasia model.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for detecting megakaryocytes in a bronchopulmonary dysplasia model includes the following steps: S1: A BPD mouse model was constructed, and lung tissue was subjected to HE staining, flow cytometry, and immunofluorescence detection. S2: And analyze the number, proportion, ploidy and distribution of megakaryocytes.

[0006] Preferably, in step S1, the construction of the BPD mouse model includes: randomly dividing newborn C57BL / 6j mice into an experimental group and a control group one day after birth. The experimental group is fed in an environment with a high oxygen concentration of 85%-90% for 14 days, while the control group is fed in the air. The mother mice are switched between groups every 24 hours.

[0007] Preferably, in step S1, HE staining includes: fixing the tissue with 10% formaldehyde solution for one day, decalcifying with decalcifying solution for one month, preparing 5-micron sections by paraffin embedding, staining with hematoxylin and eosin after deparaffining, and counting the number of megakaryocytes, the number of secondary septa, and the mean linear intercept under a microscope.

[0008] Preferably, in step S1, flow cytometry includes: rinsing lung tissue with pre-cooled PBS, cutting it into 1 mm³ pieces, adding a digestive solution containing collagenase I and deoxyribonuclease I, and shaking and digesting at 37°C for 45 minutes; filtering and lysing red blood cells to prepare a single-cell suspension; incubating the suspension with antibodies against CD41, CD42d, CD3, CD11b, TER119, CD54R / B220, Ly6G / Ly-6C, and CD45 at 4°C in the dark for 30 minutes; staining the nucleus with Hoechst staining solution at room temperature in the dark for 60 minutes; resuspending in PBS and detecting by flow cytometry; and analyzing the megakaryocyte ratio and ploidy using software.

[0009] Preferably, in step S1, the immunofluorescence detection includes: fixing lung tissue with 4% PFA at 4°C for 4-6 hours, dehydrating it overnight with 30% sucrose solution at 4°C, embedding it with OCT, and then rapidly freezing it at -80°C to prepare 14μm sections; permeating the sections with 0.2% Triton X-100 at room temperature for 10 minutes, blocking them with 5% BSA and homologous serum for 1 hour, incubating them with CD41 primary antibody at 4°C overnight, incubating them with secondary antibody in the dark for 1 hour, staining them with DAPI for 5 minutes, mounting them, imaging them with a confocal microscope, and quantifying the distribution of megakaryocytes using software.

[0010] Preferably, the method also includes HE staining and flow cytometry analysis of bone marrow and spleen tissues to analyze the proportion, ploidy, and distribution of megakaryocytes.

[0011] Preferably, in step S1, the flow cytometry is performed using a FACS Celesta flow cytometer, the HE staining is performed using an Olympus BX51 microscope, and the immunofluorescence is performed using a confocal microscope.

[0012] Preferably, in step S1, the decalcification time is one month and the slice thickness is 5 micrometers.

[0013] Preferably, in step S1, the antibody incubation conditions are 4°C in the dark for 30 minutes, and the nuclear staining is performed using Hoechst staining solution at room temperature in the dark for 60 minutes.

[0014] The beneficial effects of this invention are as follows: This invention integrates model construction, HE staining, flow cytometry, and immunofluorescence to achieve multidimensional detection of megakaryocytes in a BPD model. The method is standardized, reproducible, and can accurately reflect the reduction and ploidy changes of megakaryocytes in lung tissue, providing a reliable tool for BPD mechanism research. Attached Figure Description

[0015] Figure 1 The results of BPD model validation show comparisons of HE staining of lung tissue, mean linear intercept, and number of secondary septa. Figure 2To compare the platelet count and mean volume in peripheral blood platelet count; Figure 3 The flow cytometry and immunofluorescence results of megakaryocytes in lung tissue show the proportion, ploidy, and distribution. Figure 4 The results of bone marrow megakaryocyte detection were compared in terms of proportion, ploidy, and distribution. Figure 5 The results of splenic megakaryocyte detection were compared in terms of proportion, ploidy, and distribution. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0017] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0018] Example 1: Experimental Materials and Methods: Main reagents: Phosphate-buffered saline (PBS), erythrocyte lysis buffer, 0.5 mol / L ethylenediaminetetraethylene (EDTA), 4% tissue cell fixative, bovine serum albumin (BSV), Triton X-100 (Shanghai Beyotime Biotechnology Co., Ltd.), Hoechst fluorescent staining solution (Beijing Solarbio Science & Technology Co., Ltd.), collagenase and deoxyribonuclease I (Sigma-Aldrich, USA), CD3, CD11b, TER119, CD54R / B220, Ly6G / Ly-6C, and CD41 (BD Biosciences, USA), CD42d and CD45 (BioLegends Biosciences, USA). Main instruments: FACS Celesta flow cytometer (BD Medical Systems, USA), Ts2R-FL inverted fluorescence microscope (Macody Industrial Group Co., Ltd.), XT-2000i fully automated animal blood analyzer (Sysmex Medical Electronics Shanghai Co., Ltd.), CM3050S cryostat (Leica GmbH, Germany), Olympus BX51 microscope (Olympus GmbH).

[0019] 1. Experimental animals: The donor newborn mice were of the C57BL / 6j strain. All experimental mice were SPF grade and provided by the Animal Center of Chongqing Medical University. They were bred from breeding mice. (1) Construction of BPD mouse model: 5-8 littermate C57BL / 6j newborn mice were randomly divided into experimental group and control group on day 1 (P1). The experimental group was fed in a high oxygen concentration environment, and the control group was fed in the air. The oxygen concentration in the high oxygen environment was 85%-90% for 14 days. To reduce stress response and avoid oxygen poisoning, the mother mice were exchanged between groups once every 24 hours. (2) HE staining: After 14 days of high oxygen exposure, 4-6 mice were randomly selected from each group to extract their femur, lung and spleen for testing. The samples were fixed with 10% formaldehyde solution for one day, and then decalcified with decalcification solution for one month. The organs were embedded in paraffin and prepared into 5-micrometer thick sections. After deparaffining, the sections were stained with hematoxylin and eosin (HE) and then analyzed using an Olympus BX51 microscope manufactured by Olympus Corporation. The field of view of each specimen was randomly photographed, and the number of megakaryocytes (MKs), the number of secondary septa, and the mean linear intercept were counted. (3) Flow cytometry analysis of the number and ploidy of MKs in mouse lungs: lung tissue sample processing and preparation of single cell suspension: mice were euthanized after anesthesia, and fresh lung tissue was taken and rinsed with pre-cooled PBS. The lung tissue was cut into small pieces of about 1 mm³. Digestion solution containing collagenase I and DNase I was added, and the mixture was shaken at 37°C for 45 minutes. After mixing, the mixture was filtered, red blood cells were lysed, and single cell suspension was prepared. Cell surface labeling and staining: the prepared cell suspension was dispensed into flow cytometry tubes. Add anti-CD41, CD42d, CD3, CD11b, TER119, CD54R / B220, Ly6G / Ly-6C, and CD45, and incubate at 4°C in the dark for 30 minutes. Label cell nuclei: Take another amount of cell suspension, treat it as described above, add Hoechst staining solution, and incubate at room temperature in the dark for 60 minutes. Resuspend the cells in PBS and immediately perform flow cytometry analysis. Flowjo 10.8.1 software is used to analyze the flow cytometry data. (4) Immunofluorescence detection of MKs distribution in the lungs of control group / BPD group mice: Sample preparation and frozen section: Tissue sampling and fixation: Quickly separate lung tissue and immerse tissue blocks in 4% PFA (4°C, 4-6 hours). Dehydration protection: Immerse in 30% sucrose solution (4°C, overnight). Embed the tissue with OCT gel to ensure complete coverage. Rapidly freeze at -80°C. Section: Section thickness: 14μm. Slide preparation: Use poly-L-lysine slides to prevent slide detachment. Immunofluorescence staining: Permeabilization and blocking: 0.2% Triton X-100, incubate at room temperature for 10 minutes. Blocking: 5% BSA + 5% secondary antibody homologous serum, block for 1 hour. Primary antibody incubation: CD41 (GPIIb, mouse) 1:100. Incubate overnight at 4℃. Secondary antibody counterstaining, incubate at room temperature in the dark for 1 hour. Nuclear staining: Stain the nucleus with DAPI for 5 minutes.Mounting film, anti-quenching mounting medium: ProLong Diamond, used for sealing nail polish edges. Imaging and analysis: confocal microscopy. Quantitative analysis: ImageJ software was used to statistically analyze the number and distribution of MKs.

[0020] Statistical analysis The experimental results were statistically analyzed using Prism 10.1.2 statistical software. All experimental data are expressed as mean ± standard deviation. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0021] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting megakaryocytes in a model of bronchopulmonary dysplasia, characterized in that, The method comprises the following steps: S1: constructing a BPD mouse model, performing HE staining, flow cytometry and immunofluorescence detection on lung tissue, S2: and analyzing the number, proportion, ploidy and distribution of megakaryocytes.

2. The method of claim 1, wherein the bronchopulmonary dysplasia model is characterized by, In the S1 step, constructing the BPD mouse model comprises: randomly dividing C57BL / 6j strain newborn mice into an experimental group and a control group at 1 day after birth, feeding the mice in the experimental group in an environment with a high-oxygen concentration of 85%-90% for 14 days, feeding the mice in the control group in air, and interchanging the mother mice between the groups every 24 hours.

3. The method of claim 1, wherein the bronchopulmonary dysplasia model is characterized by, In the S1 step, the HE staining comprises: fixing the tissue with 10% formaldehyde solution for one day, decalcifying with decalcifying solution for one month, embedding in paraffin to prepare 5-micron sections, performing hematoxylin and eosin staining after removing paraffin, and counting the number of megakaryocytes, the number of secondary septa and the average linear intercept under a microscope.

4. The method of claim 1, wherein the bronchopulmonary dysplasia model is characterized by, In the S1 step, the flow cytometry comprises: washing the lung tissue with pre-cooled PBS and cutting it into 1mm³ small pieces, adding a digestion solution containing collagenase I and deoxyribonuclease I to perform 37℃ oscillation digestion for 45 minutes, filtering and lysing red blood cells to prepare a single-cell suspension, incubating the suspension with anti-CD41, CD42d, CD3, CD11b, TER119, CD54R / B220, Ly6G / Ly-6C and CD45 antibodies at 4℃ in the dark for 30 minutes, incubating with hoechst staining solution for nuclear staining at room temperature in the dark for 60 minutes, resuspending with PBS and then detecting with a flow cytometer, and analyzing the proportion and ploidy of megakaryocytes by software.

5. The method of claim 1, wherein the bronchopulmonary dysplasia model is characterized by, In the S1 step, the immunofluorescence detection comprises: fixing the lung tissue with 4% PFA at 4℃ for 4-6 hours, dehydrating with a 30% sucrose solution at 4℃ overnight, embedding with OCT and then rapidly freezing at-80℃, and preparing 14-micron sections; permeabilizing the sections with 0.2% Triton X-100 at room temperature for 10 minutes, blocking with 5% BSA and homologous serum for 1 hour, incubating with CD41 primary antibody at 4℃ overnight, incubating with secondary antibody in the dark for 1 hour, performing DAPI nuclear staining for 5 minutes, and imaging with a confocal microscope after mounting, and quantifying the distribution of megakaryocytes by software.

6. The method of claim 1, wherein the bronchopulmonary dysplasia model is a mouse model. Further comprising performing HE staining and flow cytometry detection on bone marrow and spleen tissue, and analyzing the proportion, ploidy and distribution of megakaryocytes.

7. The method of claim 1, wherein the bronchopulmonary dysplasia model is characterized by, In the S1 step, a FACS Celesta flow cytometer is used in the flow cytometry, an Olympus BX51 microscope is used in the HE staining, and a confocal microscope is used in the immunofluorescence.

8. The method according to claim 3, wherein the megakaryocytes are detected in a model of bronchopulmonary dysplasia. In the S1 step, the decalcification time is one month, and the section thickness is 5 microns.

9. The method of claim 4, wherein the megakaryocytes are detected in a model of bronchopulmonary dysplasia. In the S1 step, the antibody incubation condition is 4℃ in the dark for 30 minutes, and the nuclear staining is hoechst staining solution at room temperature in the dark for 60 minutes.

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