Culture system for simulating neutrophil extracellular trap net to intervene in oligodendrocytes in vitro

Abstract

The invention provides a culture system for simulating neutrophile granulocyte extracellular trap net intervention oligodendrocyte in vitro, and belongs to the technical field of cell and function detection. The invention establishes a stable and repeatable research system by screening and determining the optimal condition for in-vitro co-culture of the neutrophil extracellular trap net and the oligodendroglia. Based on the condition, the experiment can observe the obvious change of the oligodendroglia after the intervention, and the result shows that the condition can accurately simulate the endoplasmic reticulum stress regulation effect of the neutrophile granulocyte extracellular trap net on the oligodendroglia, thereby providing a necessary experimental basis for the subsequent mechanism research.

Description

Technical Field

[0001] The present invention relates to the technical field of cell and function detection, and particularly to a culture system for in vitro simulating neutrophil extracellular traps to intervene in oligodendrocytes. Background Art

[0002] Intracerebral Hemorrhage (ICH) is a subtype of hemorrhagic stroke with poor prognosis, and its secondary brain injury mechanism has not been fully clarified. Neuroimmune responses play a key role after intracerebral hemorrhage. In the past decade or more, it has been found that neutrophils have a special immune killing mechanism, that is, they can release neutrophil extracellular traps (NETs). NETs are mainly composed of decondensed chromatin DNA (containing double-stranded DNA - dsDNA), as well as antibacterial molecules such as histones and proteolytic enzymes. They wrap pathogens through a reticular structure, block the spread of pathogenic microorganisms and promote their clearance, and have a positive defensive significance in innate immune responses. For this acute injury of intracerebral hemorrhage, a large number of immune cells and blood components often accumulate in the local environment where the hematoma forms. Once a large amount of NETs are produced, it will cause secondary damage to the surrounding brain tissue. Oligodendrocytes are responsible for the myelin formation and repair of the central nervous system and play a key role in axonal signal conduction and neuron protection. After intracerebral hemorrhage, in addition to neurons facing mechanical injury, excitotoxicity, and attack by inflammatory factors, oligodendrocytes are also easily damaged functionally and even structurally due to the deterioration of the microenvironment. However, the specific role and mechanism of NETs in oligodendrocyte injury and myelin lesions after intracerebral hemorrhage are still unclear. Summary of the Invention

[0003] The purpose of the present invention is to provide a culture system for in vitro simulating neutrophil extracellular traps to intervene in oligodendrocytes, which can provide an effective means for studying the intervention mechanism of NETs.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a culture system for in vitro simulating neutrophil extracellular traps to intervene in oligodendrocytes, wherein the concentration of neutrophil extracellular traps in the culture system is 1 - 10 μg / mL.

[0006] Preferably, in the culture system, neutrophil extracellular traps and oligodendrocytes are co-cultured in vitro, and the time of the in vitro co-culture is 12 - 14 h.

[0007] Preferably, the concentration of neutrophil extracellular traps in the culture system is 4 - 6 μg / mL.

[0008] The present invention also provides the use of neutrophil extracellular traps in the preparation of endoplasmic reticulum stress inducers.

[0009] The present invention also provides the use of neutrophil extracellular traps in the preparation of Caspase3-dependent apoptosis pathway activators.

[0010] The present invention also provides the use of neutrophil extracellular traps in the preparation of reagents for regulating the unfolded protein response signaling pathway.

[0011] The present invention also provides the use of neutrophil extracellular traps in the preparation of CHOP-dependent endoplasmic reticulum stress apoptosis inducers.

[0012] The present invention also provides the use of neutrophil extracellular traps in the construction of oligodendrocyte injury models.

[0013] The present invention also provides the use of neutrophil extracellular traps in the preparation of reagents for detecting endoplasmic reticulum stress biomarkers.

[0014] Advantages of the present invention:

[0015] By screening and determining the optimal conditions for co-culturing neutrophil extracellular traps with oligodendrocytes in vitro, the present invention establishes a stable and reproducible research system. Based on these conditions, significant changes in oligodendrocytes after intervention can be observed in the experiment, and the results show that these conditions can accurately simulate the regulatory effect of neutrophil extracellular traps on the endoplasmic reticulum stress of oligodendrocytes, providing a necessary experimental basis for subsequent mechanism research.

[0016] Through the above-mentioned condition system, the present invention systematically reveals the molecular mechanism by which neutrophil extracellular traps mediate the dysfunction of oligodendrocytes through the endoplasmic reticulum stress pathway. The research confirms that neutrophil extracellular traps can specifically activate two key signal axes of the unfolded protein response (IRE1 / XBP-1s and PERK / p-eIF2α), and significantly up-regulate the expression of the pro-apoptotic factor CHOP, ultimately driving the activation of the Caspase3-dependent apoptosis pathway, providing a new theoretical basis for analyzing the pathological mechanism of oligodendrocyte injury.

[0017] In addition, the condition system of the present invention provides a standardized model for various application scenarios. For example, based on the stable induction of endoplasmic reticulum stress markers (GRP78, XBP-1s, p-eIF2α), reagents for detecting the level of endoplasmic reticulum stress can be developed or a disease model of oligodendrocyte damage can be constructed; while the specific activation of the CHOP and Caspase3 pathways provides an experimental tool for exploring intervention strategies for apoptosis-related diseases. These applications rely on the clear action conditions of the present invention, and its quantifiable and controllable characteristics significantly improve the reliability of experimental results, providing important technical support for research in related fields. Description of the Drawings

[0018] Figure 1 It is a figure showing the results of neutrophil isolation and in vitro induction of NETs. Among them: A. The separation process by density gradient centrifugation; B. Giemsa staining shows highly pure neutrophils with typical multilobed nuclear structures; C. The reticular structure of NETs labeled with Sytox Green after PMA stimulation; D. Co-localization of PI and DAPI to verify the release of NETs; The scale bars of the Giemsa staining pictures are 100μm, 20μm and 5μm respectively; The scale bar of the Sytox Green staining picture is 50μm; The scale bar of the PI staining picture is 5μm;

[0019] Figure 2 It is a figure showing the results of quantitative analysis of MPO-DNA in NETs. Among them: ELISA detects MPO-DNA complexes in the brain tissues of mice with cerebral hemorrhage at different time points (12h, 24h, 48h, 72h) and purified NETs at different concentrations;

[0020] Figure 3 It is a figure showing the dose-dependent damage results of NETs on oligodendrocytes. Among them: A. Live / dead staining shows the cell death situation after 24h of NETs treatment; B. Statistical analysis of the proportion of PI-positive cells (n = 3); The scale bar of the immunofluorescence picture = 500μm; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001;

[0021] Figure 4 It is a figure showing the results of evaluating the effect of NETs intervention time on the survival of MO3.13 oligodendrocytes. Among them: A. The time-course change of AnnexinV / PI cell staining; B. Statistical analysis of the number of late apoptotic cells; The scale bar of the AnnexinV / PI cell fluorescence picture = 200μm; *P < 0.05, **P < 0.01, ***P < 0.001;

[0022] Figure 5Results of in vitro model study on the morphological and functional changes of NETs on oligodendrocyte apoptosis. Among them: A. Transmission electron microscopy shows apoptotic characteristics; B. DCFH-DA staining; C. Quantitative analysis of ROS fluorescence intensity (n = 3); D. Schematic diagram of JC-1 for detecting mitochondrial membrane potential mechanism; E. JC-1 mitochondrial staining; F. Statistical analysis of JC-1 red / green fluorescence ratio (n = 3); Scale bar of TEM image = 5μm, scale bar of DCFH-DA fluorescence image = 500μm, scale bar of JC-1 fluorescence image = 200μm; *P<0.05, **P<0.01, ***P<0.001, ****P<0.001;

[0023] Figure 6 Results of in vitro model study on the regulatory effect of NETs on oligodendrocyte gene expression. Among them: A. Cluster analysis of gene expression profiles; B. Volcano plot of differential genes; C. GO enrichment analysis; D. KEGG pathway enrichment bubble plot;

[0024] Figure 7 Results of transcriptome evidence for NETs activating endoplasmic reticulum stress. Among them: A. Gene set enrichment profile of endoplasmic reticulum lumen; B. Enrichment profile of unfolded protein response;

[0025] Figure 8 Results of verification of endoplasmic reticulum stress in oligodendrocytes. Among them: A. TEM shows endoplasmic reticulum dilation; B. Western blot detects the expression of key proteins of endoplasmic reticulum stress; C-F. Semi-quantitative analysis of the gray value of protein expression of GRP78 (C), XBP-1s (D), p-eIF2α (E), and CHOP (F) (n = 3); Scale bar of large-field TEM image = 5μm, scale bar of locally magnified immunofluorescence image = 1μm; *P<0.05,

[0026] **P<0.01, ***P<0.001. Detailed implementation manners

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Embodiment

[0029] First, neutrophils were isolated from the bone marrow of mice, and neutrophils were separated and purified by density gradient centrifugation ( Figure 1 A), and the specific operation is as follows:

[0030] 1. Experimental preparation:

[0031] (1) Select male C57BL / 6 mice at 8-10 weeks old and weighing about 22-25g. Decapitate and soak in 75% ethanol for 25 min.

[0032] (2) Prepare neutrophil cell culture medium and bone marrow flushing solution:

[0033] RPMI1640 + 10% FBS + 1% PS.

[0034] 2. Bone marrow collection

[0035] (1) Place the mouse soaked in 75% ethanol on the cell operation table. Carefully cut open the abdominal skin with ophthalmic scissors and separate the lower limb skin on both sides. Use scissors to carefully remove the muscles of both legs, gradually peel and detach the acetabulum at the hip joint, taking care not to damage the femoral head.

[0036] (2) Collect the exposed femur and tibia into a sterile petri dish containing pre-cooled bone marrow flushing solution and place it on ice to maintain low temperature for subsequent operations.

[0037] (3) Before cutting both ends of each bone, rinse it in a sterile petri dish containing 75% ethanol and then rinse it three times with sterile 1×PBS to remove residual ethanol.

[0038] (4) Cut both ends of the femur and tibia. Assemble a 25G needle onto a 10 ml syringe, aspirate the bone marrow flushing solution, inject it into the bone marrow cavity, and collect the bone marrow cells into a 50 ml sterile centrifuge tube with a 100 μm cell sieve by gently flushing.

[0039] (5) Centrifuge the bone marrow cell suspension at 427×g for 5 minutes at 4°C, discard the supernatant, and retain the cell pellet.

[0040] (6) Resuspend the cell pellet in the red blood cell lysis solution to lyse red blood cells.

[0041] (7) Add 5 volumes of PBS, centrifuge at 427×g for 5 minutes at 4°C, and collect the bone marrow cell pellet. Finally, prepare a bone marrow single cell suspension of 2×10 8 -1×10 9 / ml for standby.

[0042] (8) Use the Solarbio mouse bone marrow neutrophil isolation kit to isolate neutrophils.

[0043] (9) Add 4 mL of reagent A to the centrifuge tube, and then stack 2 mL of reagent C on top of reagent A to form a gradient interface.

[0044] (10) Spread the cell suspension evenly above the surface of the separation liquid, maintaining a clear interface between the two liquid surfaces

[0045] (11) Centrifuge at a speed of 800 g for 25 minutes at room temperature using a centrifuge with a horizontal rotor.

[0046] (12) After centrifugation, two layers of annular milky white cell layers will form in the centrifuge tube. The first layer is the mononuclear cell layer, with 2 mL of reagent C in the middle, the second layer is the neutrophil layer, and then there is 4 mL of reagent C. Aspirate the neutrophil layer and transfer it to a clean 15 mL centrifuge tube.

[0047] (13) Add 10 mL of PBS to the centrifuge tube to wash the cells. Centrifuge at a speed of 250 g for 10 minutes.

[0048] (14) Discard the supernatant, add 5 mL of PBS to resuspend the cells, and continue to centrifuge at a speed of 250 g for 10 minutes.

[0049] (15) After discarding the supernatant, resuspend the cells in an appropriate amount of neutrophil cell culture medium for standby.

[0050] Giemsa staining confirmed the typical multilobed nuclear structure of these cells, which was consistent with the characteristics of neutrophils ( Figure 1 B). Subsequently, after stimulation with PMA (phorbol ester) for 4 hours, neutrophils were successfully induced to release NETs, manifested as a significant increase in the reticular DNA structure labeled with Sytox Green ( Figure 1 C):

[0051] Induction of NETs production by neutrophils:

[0052] (1) Inoculate the cells at 5×10 6 / mL into a 10 cm cell culture dish, add PMA, and adjust the final concentration of PMA in the cell culture medium to 100 nM, and mix gently.

[0053] (2) Place the culture medium in an incubator at 37 °C and 5% CO2 for 4 hours to stimulate the generation of NETs by neutrophils.

[0054] (3) Discard the cell culture medium, aspirate 1 mL of PBS and repeatedly pipette the substances at the bottom of the culture dish. Centrifuge at 4 °C and 1000 g for 10 minutes, and the supernatant is the NETs suspension solution.

[0055] (4) Use Nanodrop to detect the concentration of dsDNA and quantify the NETs.

[0056] (5) Dilute the purified NETs of three batches to 0.1, 1, 5, and 10 ng / mL with PBS respectively, and measure the content of MPO-DNA.

[0057] 2. Sytox Green staining

[0058] (1) Prepare a 0.5 μM Sytox Green fluorescent dye solution.

[0059] (2) Add the dye solution to the neutrophils, ensuring that the dye fully covers the cell surface. Incubate in the dark at room temperature for 15 - 30 minutes to stain the NETs in the neutrophils.

[0060] (3) Wash the cells twice with PBS to remove the excess dye and ensure a clear fluorescence signal.

[0061] Meanwhile, the PI-positive cells increased significantly, and a large amount of cell content release was observed ( Figure 1 D).

[0062] To determine the content of NETs components in the intracerebral hemorrhage tissue and the purified NETs suspension, we collected the brain tissues of intracerebral hemorrhage mice at different time points (12 h, 24 h, 48 h, 72 h), and detected the concentration of MPO-DNA complex using ELISA (Jiangsu Enzyme Immunoassay Industry Co., Ltd.), with the sham group used for baseline correction:

[0063] 1. Add the MPO antibody to the 96-well plate and incubate overnight at 4°C to evenly bind the antibody to the bottom of the plate.

[0064] 2. Block with 1% BSA for 1 hour to reduce non-specific adsorption and improve the detection specificity.

[0065] 3. Add the sample supernatant and the standard product for making the standard curve to the 96-well plate, and add the anti-DNA antibody. Incubate on a shaker at room temperature for 2 hours to promote antigen-antibody binding.

[0066] 4. Wash 3 times with PBS to remove the unbound components.

[0067] 5. Add the peroxidase substrate and incubate at 37°C for 1 hour to catalyze the color reaction.

[0068] 6. Measure the absorbance of the sample at a wavelength of 405 nm for quantitative analysis of the level of MPO-DNA complex.

[0069] The results are as Figure 2 shown: The concentration of MPO-DNA complex in the intracerebral hemorrhage tissue reached the peak at 24 hours, showing no statistical difference from the purified NETs suspension group at 5 μg / mL. The above results provide a basis for the selection of NETs intervention concentration in the in vitro model.

[0070] Evaluate the effects of different concentrations of NETs on the survival of MO3.13 oligodendrocytes.

[0071] MO3.13 oligodendrocytes were cultured in complete medium (DMEM + 20% FBS + 1% PS). When the cell density reached 70%, different concentrations of purified NETs (0.1, 1, 5, 10, 50 μg / mL) were added for intervention. After 24 hours of co-culture in vitro, cell cytotoxicity was detected by live / dead staining. The experimental results showed that NETs had a dose-dependent cytotoxic effect on MO3.13 cells ( Figure 3 A). When the concentration was 1 μg / mL, the proportion of PI-positive cells increased significantly (P < 0.05, Figure 3 B), and at 5 μg / mL, its toxic effect was more significant, and this concentration was close to the actual level of NETs at the intracerebral hemorrhage lesion site. It is worth noting that when the concentration of NETs increased to 10 μg / mL or 10 times the actual level of NETs at the intracerebral hemorrhage lesion site, the proportion of PI-positive cells increased sharply, suggesting that excessive NETs intervention could trigger extensive cell death, thereby affecting the cell physiological state and its interaction stability with NETs. Therefore, this study selected 5 μg / mL as the reference concentration for subsequent in vitro intervention to better simulate the cell-NETs interaction environment under pathological conditions, while avoiding excessive toxicity from interfering with the repeatability and controllability of the experimental system.

[0072] Evaluate the effect of NETs intervention time on the survival of MO3.13 oligodendrocytes.

[0073] MO3.13 oligodendrocytes were cultured in complete medium (DMEM + 20% FBS + 1% PS). When the cell density reached 70%, NETs intervention was added at a final concentration of 5 μg / mL. They were cultured for 4 h, 12 h, 24 h, and 48 h respectively, and the cell apoptosis status was detected. AnnexinV / PI double staining was used ( Figure 4 A):

[0074] 1. Directly perform AnnexinV / PI fluorescence detection in a 96-well plate.

[0075] 2. Aspirate the culture medium, add PBS buffer, gently wash once to remove the residual culture medium.

[0076] 3. Add 5 μL of AnnexinV-FITC to 195 μL of DMEM, add it to the 96-well plate and incubate at room temperature in the dark for 20 min. Add 10 μL of PI before observing under a fluorescence microscope and gently mix.

[0077] 4. Observe the cell apoptosis status under a fluorescence microscope.

[0078] The experimental data showed that after 4 hours and 12 hours of NETs intervention, Annexin V + PI +The proportion of late apoptotic cells was not significantly different from that of the untreated group (Blank group). Figure 4 B). However, when the intervention time was extended to 24 hours, the number of AnnexinV + PI + positive cells increased significantly, suggesting that NETs induced obvious late apoptosis of cells at this time point (P<0.05). When further extended to 48 hours, there was no statistical difference in the apoptosis level compared with the 24-hour group, indicating that the cell response to NETs tended to reach a plateau after 24 hours, which was the end time of cell damage in this system. To further explore the molecular mechanism of NETs-mediated cell damage, the intervention time of 12 hours was set as the time point for mechanism research in the experiment. At this time, the cells had not yet entered the late apoptosis stage on a large scale, but may have activated signal pathways such as stress and programmed cell death, which was conducive to identifying early key molecular events and revealing the core mechanism of NETs-induced cell damage.

[0079] Therefore, the concentration of NETs in the in vitro model was selected as 1-50 μg / mL, preferably 5 μg / mL, and the co-culture time was 12-24 hours.

[0080] The morphological changes of cells after NETs intervention were observed by TEM. The TEM results showed that oligodendrocytes treated with NETs presented typical apoptotic characteristics, including obvious nuclear condensation, chromatin margination and fragmentation, and the formation of apoptotic bodies in the cytoplasm. Figure 5 A). In addition, after NETs intervention, the fluorescence intensity of DCFH-DA in oligodendrocytes was significantly higher than that in the control group, showing a diffuse and enhanced fluorescence signal (P<0.0001, Figure 5 B and 5C). The results of the JC-1 experiment (5D) showed that the ratio of JC-1 red / green fluorescence in oligodendrocytes in the NETs intervention group decreased significantly (P<0.01, Figure 5 E and 5F)

[0081] In this invention, the regulatory effect of NETs on the gene expression of oligodendrocytes was deeply studied by transcriptomic analysis. The transcriptomic data showed that NETs treatment significantly changed the gene expression profile of oligodendrocytes, and cluster analysis showed a significant separation in the gene expression patterns between the two groups. Figure 6 A). The volcano plot of differential genes showed that 1399 genes were up-regulated in the NETs intervention group, and another 717 genes were down-regulated. Figure 6 B). Further GO analysis showed that NETs treatment mainly affected multiple biological processes including stress response, cell communication and intracellular signal transduction. Figure 6C). KEGG pathway enrichment analysis revealed the enrichment of multiple key signaling pathways in oligodendrocytes after NETs treatment, including the PI3K-Akt signaling pathway, extracellular matrix (ECM)-receptor interaction pathway, and TNF signaling pathway, etc.( Figure 6 D), and these pathways are closely related to cell proliferation, survival, migration, and immune response.

[0082] Endoplasmic reticulum stress (ER stress), as a key response mechanism of cells to external stress, plays a crucial role in maintaining protein homeostasis and cell survival. To systematically evaluate the impact of NETs on the endoplasmic reticulum stress pathway, enrichment analysis was first performed through transcriptome analysis. Through gene set enrichment analysis (GSEA), we found that the endoplasmic reticulum lumen-related pathway (GO:0005788) and the unfolded protein response pathway (GO:0030968) were enriched to a certain extent in the NETs intervention group (NES: 1.37; P < 0.01; Adjusted P < 0.5 and NES: 1.41; P < 0.05; Adjusted P < 0.5)( Figure 7 A and B). TEM ultrastructural observations verified the endoplasmic reticulum stress phenomenon induced by NETs. In oligodendrocytes of the NETs intervention group, the endoplasmic reticulum showed obvious dilation and swelling, the folded structure of the endoplasmic reticulum lumen became disordered or even disappeared, and some endoplasmic reticulum vesicles formed( Figure 8 A). To further clarify the endoplasmic reticulum stress signaling pathway induced by NETs, we detected key endoplasmic reticulum stress marker proteins( Figure 8 B). Western blot results showed that the expression level of the endoplasmic reticulum stress sensor GRP78 (UPR initiator factor) was significantly increased (P < 0.001, Figure 8 C). Analysis of the downstream UPR signaling axis showed that the IRE1 / XBP-1s pathway (abundance of XBP-1s spliceosome protein, P < 0.05, Figure 8 D) and the PERK / p-eIF2α pathway (phosphorylation level of eIF2α, P < 0.05, Figure 8 E) were both significantly upregulated. In addition, the expression level of the UPR effector factor CHOP related to apoptosis was significantly increased (P < 0.05, Figure 8 F).

[0083] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An in vitro culture system for simulating neutrophil extracellular traps to intervene in oligodendrocytes, characterized in that, The concentration of neutrophil extracellular traps in the culture system is 1-10 μg / mL.

2. The culture system for in vitro simulating neutrophil extracellular trap to interfere with oligodendrocytes according to claim 1, characterized in that, In the culture system, neutrophil extracellular traps and oligodendrocytes are co-cultured in vitro, and the time of the in vitro co-culture is 12-14 h.

3. The culture system for in vitro simulating neutrophil extracellular trap intervention of oligodendrocytes according to claim 2, characterized in that, The concentration of neutrophil extracellular traps in the culture system is 4-6 μg / mL.

4. Application of neutrophil extracellular traps in the preparation of endoplasmic reticulum stress inducers.

5. Application of neutrophil extracellular traps in the preparation of Caspase3-dependent apoptosis pathway activators.

6. Application of neutrophil extracellular traps in the preparation of reagents for regulating the unfolded protein response signaling pathway.

7. Application of neutrophil extracellular traps in the preparation of CHOP-dependent endoplasmic reticulum stress apoptosis inducers.

8. Application of neutrophil extracellular traps in the construction of oligodendrocyte injury models.

9. Application of neutrophil extracellular traps in the preparation of reagents for detecting endoplasmic reticulum stress biomarkers.

Images