Method for evaluating maturation and functional damage of CIAV virus to chicken bone marrow-derived dendritic cells

Abstract

The invention discloses a method for evaluating maturation and functional damage of CIAV virus to chicken bone marrow-derived dendritic cells, and belongs to the technical field of virus immunology. The method comprises the following steps: separating and culturing chicken bone marrow-derived dendritic cells in vitro, stimulating the chicken bone marrow-derived dendritic cells by using a CIAV virus, and detecting the phagocytic function of the cells, the expression of surface marker molecules CD40 and MHC II, the secretion level of cell factors IFN-beta, IL-10 and IL-12, and the induction capability of the dendritic cells on the proliferation of allogeneic T lymphocytes. And comprehensively evaluating the damage effect of the CIAV virus on the maturation, differentiation and function of the dendritic cells. The method is standard in operation and reliable in result, an effective tool is provided for deeply researching a CIAV-mediated natural immunosuppression mechanism, and meanwhile, a scientific basis is provided for development of a CIAV prevention and control strategy.

Description

Technical Field

[0001] This invention belongs to the field of viral immunology technology, specifically relating to a method for evaluating the damage of CIAV virus to the maturation and function of chicken bone marrow-derived dendritic cells. Background Technology

[0002] Chicken infectious anemia (CIAV) is a major pathogen causing aplastic anemia and immunosuppression in chicks. In recent years, its infection rate in chicken flocks in my country has reached 40%-70%, leading to decreased productivity and vaccine failure, resulting in severe economic losses. Current research mainly focuses on the epidemiology, pathogenicity, prevention, and diagnosis of CIAV, while its immunosuppressive mechanisms, particularly its impact on the function of key immune cells such as dendritic cells, remain unclear.

[0003] Dendritic cells, as the most important professional antigen-presenting cells in the body, play a central role in initiating the body's immune response. However, current research reports on how CIAV affects the maturation, differentiation, and function of dendritic cells are extremely limited, and systematic and standardized in vitro evaluation methods are lacking. Existing technologies cannot accurately quantify the damage that CIAV causes to dendritic cell phagocytosis, cytokine secretion, and T cell proliferation stimulation, thus hindering a deeper understanding of its immunosuppressive mechanisms.

[0004] In summary, the existing technical problem in this field is the lack of a comprehensive and quantitative method to evaluate the damage to the maturation and function of chicken bone marrow-derived dendritic cells caused by CIAV virus, which limits in-depth research on its immunosuppressive mechanism and the development of effective prevention and control strategies. Therefore, establishing an experimental method that can comprehensively evaluate the damage to the maturation and function of chicken dendritic cells caused by CIAV is of great significance for revealing its immunosuppressive mechanism and formulating effective prevention and control strategies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for evaluating the damage to maturation and function of CIAV virus on chicken bone marrow-derived dendritic cells, which solves the technical problem of lacking a systematic evaluation of the damage of CIAV to dendritic cell function in the prior art, and achieves the technical effect of quantitatively and multidimensionally assessing the immunosuppressive mechanism of CIAV.

[0006] To achieve the above objectives, the present invention provides a method for evaluating the damage of CIAV virus to the maturation and function of chicken bone marrow-derived dendritic cells, comprising the following steps:

[0007] (1) In vitro isolation and culture of chicken bone marrow-derived dendritic cells;

[0008] (2) Stimulate the dendritic cells cultured in step (1) with CIAV virus;

[0009] (3) Detect the maturation and functional indicators of dendritic cells stimulated by CIAV virus;

[0010] The maturity indicators include the expression levels of surface markers CD40 and MHC II;

[0011] The functional indicators include phagocytic function, cytokine secretion levels, and / or the ability to promote T cell proliferation.

[0012] To further explain, in step (1), the isolation and culture of the chicken bone marrow-derived dendritic cells includes: 1) rinsing bone marrow cells from the femur and tibia of healthy black-boned chickens aged 20-30 days;

[0013] 2) Isolate bone marrow mononuclear cells using density gradient centrifugation;

[0014] 3) Dendritic cells were induced and cultured to day 7 using a complete culture medium containing 50 ng / mL recombinant chicken GM-CSF and 50 ng / mL chicken rIL-4.

[0015] To further explain, in step (2), the inoculation amount of the CIAV virus is 20 μL / mL, and the stimulation time is 24 h.

[0016] To further explain, in step (3), the phagocytic function is detected by the following method: using FITC-labeled dextran as the phagocytic substrate, the proportion of positive fluorescent cells is detected by flow cytometry.

[0017] To further explain, in step (3), the final concentration of FITC-dextran in the phagocytic function detection is 1 mg / mL, and the incubation time is 30 minutes.

[0018] To further explain, in step (3), the levels of cytokine secretion are measured by ELISA at 6 hours, 12 hours and 24 hours after stimulation, respectively, for the contents of IFN-β, IL-10 and IL-12.

[0019] To further explain, in step (3), the ability to promote T cell proliferation is evaluated by a mixed lymphocyte reaction, which includes: co-culturing dendritic cells stimulated by CIAV virus with allogeneic T lymphocytes at a ratio of 1:1, and using the CCK-8 assay to detect T lymphocyte proliferation.

[0020] To further clarify, the dendritic cells were treated with mitomycin C before co-culturing.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. The research team of this application has confirmed through systematic experimental studies that CIAV stimulation can significantly inhibit the phagocytic function of dendritic cells, weaken their ability to stimulate T cell proliferation, reduce the expression of maturation molecules CD40 and MHCII on their surface, and regulate the cytokine secretion pattern (such as inhibiting IFN-β and IL-12 and promoting IL-10), thereby comprehensively revealing the damage mechanism of CIAV to the maturation and function of dendritic cells.

[0023] 2. The method of this invention is not only applicable to in-depth exploration of the immunosuppressive mechanism of CIAV in basic research, but can also be extended to fields such as vaccine potency evaluation, antiviral drug screening and immune adjuvant development, providing technical support and scientific basis for the prevention and control of avian immunosuppressive diseases. Attached Figure Description

[0024] Figure 1 The images show the morphological characteristics of chicken bone marrow-derived dendritic cells at different culture stages (ac corresponds to chicken DCs cultured on days 1, 4, and 7; 100X and 400X represent magnification of 100x and 400x, respectively).

[0025] Figure 2 Figure showing the results of flow cytometry detection of the expression of dendritic cell surface markers CD11c and MHCII.

[0026] Figure 3 A comparative diagram showing the ability of dendritic cells in different groups to phagocytose FITC-dextran.

[0027] Figure 4 The graph shows the changes in CD40 and MHCII expression after CIAV stimulation, as detected by flow cytometry.

[0028] Figure 5 A comparison of the Stimulation Indices (SI) for dendritic cells stimulating the proliferation of allogeneic T cells. Detailed Implementation

[0029] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0030] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0031] The experimental animals used in this invention were healthy female Silkie chickens aged 20-30 days, purchased from Guangxi Jinling Poultry Breeding Co., Ltd. Chicken infectious anemia virus (CIAV, AV1550) was purchased from the China Veterinary Microbiology Culture Collection Center. TCID was measured after 9-11 day old SPF chicken embryos were rejuvenated. 50 10-3.52 / 0.1mL.

[0032] The main reagents used in this invention are as follows: recombinant chicken rGM-CSF and chicken rIL-4 are both from Kingfisher Biotech; fetal bovine serum is a product of Hyclon; RPMI 1640 medium is a product of Gibco; lymphocyte separation medium Histopaque-1119 and LPS are both from Sigma; PE-labeled anti-human CD11c and PE-labeled anti-mouse CD40 are both from eBioscience; FITC-labeled anti-chicken MHC II is a product of Abcam; CCK-8 and FITC-labeled Dextran are both from Beyotime; and the detection kits for chicken interferon β (IFN-β), chicken interleukin 10 (IL-10), and chicken interleukin 12 (IL-12) are all from Shanghai Bosheng Biotechnology Co., Ltd.

[0033] The experimental data in the following examples were analyzed using SPSS 23.0 with one-way ANOVA and Duncan's method for multiple comparisons. The results are expressed as mean ± standard error, and P < 0.05 indicates a significant difference.

[0034] Example 1: This example demonstrates the isolation and culture of dendritic cells.

[0035] Healthy female Silkie chickens aged 20-30 days were euthanized and then placed in a sterile operating table for 5 minutes of alcohol immersion followed by 5 minutes of PBS (containing 2% antibiotics). The skin of the chicken legs was cut open with sterile scissors, and all femurs and tibias were completely removed. Before rinsing the medullary cavity, the flesh outside the femurs and tibias was removed. Then, an appropriate amount of sterile PBS (containing 2% antibiotics) was drawn up with a syringe and the bone marrow was repeatedly rinsed from the ends of the femurs and tibias until the medullary cavity turned white. All rinsing fluid was collected, centrifuged, and the supernatant was discarded. The cell pellet was resuspended in an appropriate amount of PBS for later use. 3-4 ml of Histopaque-1119 was added to a 15 ml glass centrifuge tube, followed by the cell suspension added slowly in an equal volume ratio. After centrifugation for 30 minutes, the cells in the middle white haze-like boundary layer were collected. An appropriate amount of PBS was added, and the cells were centrifuged again, discarding the supernatant. The cells were resuspended in RPMI 1640 medium (containing 10% FBS and 1% antibiotics). The isolated bone marrow cells were divided into 2×10 6 The cells were seeded at a concentration of 1 cell / mL into six-well cell culture plates, with each well containing 3 mL of complete culture medium (containing 50 ng / mL rGM-CSF and 50 ng / mL rIL-4). The plates were then incubated at 37 °C in a 5% CO2 incubator, and half of the medium was replaced every other day.

[0036] After 7 days of induction and culture of chicken bone marrow-derived dendritic cells (DCs), the cell slurry was collected by pipetting the bottom of the cell plate and centrifuged. The cell pellet was resuspended in PBS. Flow cytometry was used to detect the markers CD11c and MHCII on the surface of the chicken bone marrow-derived DCs to identify cell purity. In addition, cell morphology was observed and photographed on days 1, 4, and 7 of cell culture using an optical microscope.

[0037] The results are as follows: Figure 1 As shown, after 1 day of in vitro culture, chicken bone marrow-derived DCs stimulated by GM-CSF and IL-4 existed as single or several cells, with varying morphology and size, and were irregular in shape. Figure 1 -a); After 4 days of culture, many colonies appeared at the bottom of the cell plate, at which point most of the cells were accumulated in the colonies ( Figure 1 -b). After 7 days of culture (24 hours after LPS stimulation), it was found that the colonies adhered very loosely, and many cells detached from the colonies. The detached cells were larger, with more spikes becoming larger, exhibiting a typical dendritic cell morphology. Figure 1 -c). Furthermore, by Figure 2 It was found that after 7 days of cell culture, the expression levels of the molecular markers CD11c and MHCII on the surface of chicken DCs without LPS stimulation were 75.9% and 68.10%, respectively.

[0038] Example 2: This example demonstrates CIAV stimulation and phagocytic function detection.

[0039] After 6 days of induction culture of chicken bone marrow-derived DCs, the cell concentration was adjusted to 2×10⁻⁶. 6 CIAV cells were seeded at a concentration of 20 μL / mL in 6-well cell culture plates. 200 ng / mL LPS was added as a positive control, and an equal volume of cell culture medium was added as an unstimulated group. After culturing for 24 h, FITC-dextran was added to a final concentration of 1 mg / mL, and the plates were incubated at 37 °C with 5% CO2 for 30 min. After washing twice with pre-cooled PBS, positive fluorescent cells were detected by flow cytometry.

[0040] The results are as follows: Figure 3 As shown, unstimulated chicken bone marrow-derived dendritic cells (DCs) exhibited strong phagocytic capacity, with 64.3% being FITC-dextran positive. After stimulation with LPS and CIAV, the phagocytic rates of chicken bone marrow-derived DCs decreased to 40.8% and 32.3%, respectively, both significantly lower than those of unstimulated cells (P < 0.05). The phagocytic rate of DCs in the CIAV group was lower than that in the LPS group, but the difference was not statistically significant (P > 0.05).

[0041] Example 3: This example demonstrates the detection of surface markers and cytokines.

[0042] (1) After 6 days of induction culture of chicken bone marrow-derived DCs, they were inoculated with CIAV and cultured for another 24 hours before the cells were collected. Flow cytometry was used to detect the co-stimulatory molecules MHCII and CD40 on the surface of chicken bone marrow-derived DCs after various treatments.

[0043] The results are as follows: Figure 4 As shown, flow cytometry analysis revealed that the expression of maturation molecules CD40 and MHCII on the surface of chicken dendritic cells (DCs) stimulated by CIAV was higher than that of the control group, but lower than that of chicken DCs stimulated by LPS, indicating that CIAV has a certain inhibitory effect on dendritic cell maturation.

[0044] (2) After 6 days of induction culture of chicken bone marrow-derived DCs, CIAVs were inoculated according to method 1.12. At 6h, 12h and 24h after inoculation, 300ml of cell supernatant was collected. The collected supernatants were centrifuged at 3000r / min for 15min. The supernatants were collected and stored in a -80℃ refrigerator for later use. The levels of IFN-β, IL-12 and IL-10 were detected according to the instructions of the ELISA kit.

[0045] The results are as follows: After CIAV stimulation of chicken DCs, IFN-β levels showed a trend of first increasing and then decreasing. At 12 h, the IFN-β protein expression levels in both stimulation groups were significantly higher than those in the unstimulated group (P < 0.05), while at 24 h, the IFN-β protein expression levels in the CIAV stimulation group were significantly lower than those in the unstimulated group (P < 0.05). After CIAV stimulation of chicken DCs, IL-10 levels showed a trend of first decreasing and then increasing. From 6 h to 24 h, the IL-10 protein expression levels in both stimulation groups were significantly higher than those in the unstimulated group (P < 0.05). From 6 h to 12 h, the IL-12 protein expression levels in the CIAV stimulation group were significantly higher than those in the unstimulated group (P < 0.05), while at 24 h, the IL-12 protein expression levels in the CIAV stimulation group were lower than those in the unstimulated group, but the difference was not significant (P > 0.05).

[0046] Table 1. Changes in cytokine protein levels after CIAV stimulation of chicken bone marrow-derived dendritic cells.

[0047]

[0048]

[0049] Example 4: This example is a mixed lymphocyte reaction.

[0050] Preparation of T lymphocytes: After euthanizing 30-day-old chicks, spleen cells were extracted from the chicks after soaking them in 75% alcohol for 5 minutes under sterile conditions. The spleen cells were then ground in sterile petri dishes, passed through a sieve, and the cell suspension was collected for later use. Lymphocytes were separated from the cell suspension using a lymphocyte separation solution. Allogeneic T lymphocytes were then separated by nylon capillary column filtration, and the T cell concentration was adjusted to 2 × 10⁻⁶. 6 Cells / mL, for later use. Preparation of chicken bone marrow DCs: DCs after different treatments were incubated with 50ug / ml mitomycin C for 45 min, washed twice with PBS, and set aside. The procedure for stimulating allogeneic T lymphocyte proliferation with chicken bone marrow DCs was carried out according to the method of Fu Jia et al., in which the ratio of stimulating cells (DCs stimulated by LPS, CIAV, or unstimulated) to reacting cells (allogeneic T lymphocytes) was 1:1, 1:10, and 1:100, respectively. After co-culturing for 72 h, they were incubated with CCK-8 for 2 h, and the OD value of each well was measured at a wavelength of 450 nm. Finally, the stimulation index (SI) value of each group was calculated.

[0051] SI calculation formula = ,

[0052] The negative control wells contained only T cells, while the blank control wells contained only an equal volume of cell culture medium.

[0053] The results are as follows: Figure 5 As shown, when the cell concentrations were 1:1, the stimulation index (SI) value of the CIAV group was significantly higher than that of the unstimulated group (P < 0.05); however, when the cell concentrations were 1:10 or 1:100, there was no significant difference in the SI values ​​between the CIAV group and the control group (P > 0.05). For the LPS group, the SI value was significantly higher than that of the control group at all cell ratios (P < 0.05).

[0054] In summary, the applicant has systematically verified the feasibility and effectiveness of the method of this invention. The results show that CIAV virus can significantly inhibit the phagocytic function, mature surface molecule expression, and normal cytokine secretion of chicken bone marrow-derived dendritic cells, and weaken their ability to stimulate proliferation, thereby comprehensively damaging dendritic cell function. The method of this invention is standardized in operation and comprehensive in its indicators, providing a reliable technical platform for the study of the CIAV immunosuppressive mechanism, and has significant theoretical value and promising prospects for widespread application.

[0055] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for evaluating the damage to maturation and function of chicken bone marrow-derived dendritic cells caused by CIAV virus, characterized in that, Includes the following steps: (1) In vitro isolation and culture of chicken bone marrow-derived dendritic cells; (2) Stimulate the dendritic cells cultured in step (1) with CIAV virus; (3) Detect the maturation and functional indicators of dendritic cells stimulated by CIAV virus; The maturity indicators include the expression levels of surface markers CD40 and MHC II; The functional indicators include phagocytic function, cytokine secretion levels, and / or the ability to promote T cell proliferation.

2. The method as described in claim 1, characterized in that, In step (1), the isolation and culture of the chicken bone marrow-derived dendritic cells includes: 1) Rinse bone marrow cells from the femur and tibia of healthy black-boned chickens aged 20-30 days; 2) Isolate bone marrow mononuclear cells using density gradient centrifugation; 3) Dendritic cells were induced and cultured to day 7 using a complete culture medium containing 50 ng / mL recombinant chicken GM-CSF and 50 ng / mL chicken rIL-4.

3. The method as described in claim 1, characterized in that, In step (2), the inoculation amount of CIAV virus is 20 μL / mL, and the stimulation time is 24 h.

4. The method as described in claim 1, characterized in that, In step (3), the phagocytic function is detected by using FITC-labeled dextran as the phagocytic substrate and detecting the proportion of positive fluorescent cells by flow cytometry.

5. The method as described in claim 1, characterized in that, In step (3), the final concentration of FITC-dextran in the phagocytic function detection is 1 mg / mL, and the incubation time is 30 minutes.

6. The method as described in claim 1, characterized in that, In step (3), the levels of cytokine secretion are measured by ELISA at 6, 12 and 24 hours after stimulation, respectively, for the contents of IFN-β, IL-10 and IL-12.

7. The method as described in claim 1, characterized in that, In step (3), the ability to promote T cell proliferation is evaluated by mixed lymphocyte reaction, including: co-culturing dendritic cells stimulated by CIAV virus with allogeneic T lymphocytes at a ratio of 1:1, and detecting T lymphocyte proliferation using the CCK-8 method.

8. The method as described in claim 7, characterized in that, The dendritic cells were treated with mitomycin C prior to co-culture.

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