Method for constructing airway immune response research model based on RSV infection mediation
By employing a dual-parameter authentication system of transmembrane resistance (TEER) and ciliary differentiation rate, along with dynamic viral titer monitoring and real-time MOI calculation and compensation mechanisms, a standardized research model for RSV infection-mediated airway immune response was constructed. This model addresses the issues of poor model reproducibility and insufficient quality control in existing technologies, achieving efficient experimental results and a reliable research platform.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-01
AI Technical Summary
Existing RSV infection-mediated airway immune response research models have shortcomings in simulating the complex in vivo microenvironment and parameter control, resulting in poor model reproducibility, large batch-to-batch variability, and a lack of quality control and compensation mechanisms, which affect the reliability and application value of the research.
Transmembrane resistance (TEER) and ciliary differentiation rate were used as a dual-parameter authentication system. Combined with dynamic viral titer monitoring and real-time MOI calculation, a standardized model construction method was established. Compensation mechanisms such as extended culture and supplementary factors were used to ensure experimental consistency. Human LAD2 mast cells were used to realize the model's function.
It significantly improves the reproducibility and reliability of the model, ensures the scientific validity and comparability of experimental results, provides a reliable research platform, and offers a stable tool for RSV pathogenesis research and drug screening.
Smart Images
Figure CN121963836A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical in vitro model technology, and in particular to a method for constructing a research model of airway immune response mediated by RSV infection. Background Technology
[0002] Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children, and the excessive inflammatory response it triggers is closely related to the development and progression of asthma. Studies have shown that the interaction between airway epithelial cells and mast cells plays a central role in this pathological process. Therefore, developing in vitro research models that can accurately simulate this interaction process is crucial for elucidating the pathogenic mechanism of RSV.
[0003] Currently, research models in this field face multiple technical bottlenecks, which limit their application value and reliability:
[0004] First, at the structural level of model systems, existing systems struggle to simulate the complex microenvironment within the body. Most studies employ single cell lines (such as A549 epithelial cells or HMC-1 mast cells) for culture. These cell lines differ significantly from primary cells in gene expression and function, failing to accurately reflect intercellular communication within the human body. While human primary airway epithelial cell air-fluid interface (ALI) culture models can effectively simulate airway structure and barrier function, current technologies generally lack standardized systems for functional co-culturing these cells with immune cells (such as mast cells), hindering the effective study of the complete epithelial-immune bidirectional interaction process.
[0005] Secondly, existing technologies have serious shortcomings in terms of process control and standardization, which are mainly manifested in the following ways:
[0006] (1) Rigid key parameters: Core parameters for model construction, such as the multiple of infection (MOI), are usually preset to fixed values. This approach cannot be dynamically optimized based on fluctuations in key variables such as cell seeding density, cell viability, and viral titers in different batches, directly resulting in poor model repeatability and large batch-to-batch differences.
[0007] (2) Lack of quality control and compensation mechanisms: During the model construction process, there is a lack of qualification certification for intermediate products (such as mature ALI models with complete barrier function, viral fluid with accurate titer calibration, and functional immune cells). At the same time, when a certain step fails to meet the standard, there is a lack of standardized and immediate compensation and correction procedures (such as extending the culture, supplementing factors, recalculating the inoculation amount, etc.), which makes the success rate of model construction heavily dependent on the experience of the operators, resulting in low quality controllability.
[0008] Therefore, there is an urgent need in this field for a standardized model building method that can achieve tight integration between steps, dynamic optimization of key parameters, and quality verification and compensation for core nodes, in order to overcome the above-mentioned technical bottlenecks and provide a stable and reliable tool for RSV pathogenesis mechanism research and drug screening. Summary of the Invention
[0009] Therefore, this invention provides a method for constructing a research model of airway immune response mediated by RSV infection, in order to overcome the problems of low model construction success rate and low reproducibility density caused by parameter rigidity and process malfunction in the prior art.
[0010] This invention provides a method for constructing a research model of airway immune response mediated by RSV infection, comprising the following sequentially connected steps:
[0011] Step S1: Human primary airway epithelial cells are seeded into a Transwell chamber to establish an air-liquid interface culture. The pass rate is evaluated based on the dynamically monitored transmembrane resistance value and the ciliary formation ratio. When the preset standard is continuously met, the epithelial cell culture system is obtained.
[0012] Step S2: Amplify the RSV virus and determine the completion of virus amplification based on the titer deviation value of the virus titer during the amplification process to obtain the RSV virus. The virus titer is determined using the TCID50 method, and the titer deviation value is characterized by the deviation between the virus titer during the amplification process and the preset virus titer.
[0013] Step S3: Inoculate mast cells into the epithelial cell culture system to obtain an ALI co-culture system. Based on the total number of cells in the ALI model and the virus titer, dynamically calculate the inoculation volume required to obtain the preset MOI value, and perform apical infection on the ALI co-culture system to obtain the infected ALI co-culture system.
[0014] Step S4: Samples are collected from the ALI co-culture system after apical infection, and the levels of alarmins released by epithelial cells and activation indicators of mast cells are detected to verify the functional output of the model and complete the model construction.
[0015] Furthermore, the preset standard is based on three consecutive measurements of the transmembrane resistance (TEER) value ≥500 Ω·cm. 2 The result was confirmed that the ciliary formation rate was >80%;
[0016] If the preset standard is not met, a compensation mechanism is activated, which includes extending the culture time and supplementing with epidermal growth factor (EGF) or brain-derived neurotrophic factor (BDNF) at a concentration of 10 ng / mL to 50 ng / mL.
[0017] Furthermore, in step S2,
[0018] The preset virus titer is ≥1×10 6 PFU / mL;
[0019] The preset virus titer deviation is ≤10%.
[0020] Furthermore, in step S3, the process of apical infection of the ALI co-culture system includes:
[0021] Step S31: Determine the standard total number of viral particles based on the preset multiplicity of infection (MOI) value and the total number of cells;
[0022] Step S32: Divide the standard total number of virus particles by the titer of the RSV virus solution to determine the inoculation volume required to reach the preset multiplicity of infection (MOI) value;
[0023] Step S33: Perform the top-side infection operation according to the inoculation volume.
[0024] Furthermore, the preset infection multiplicity MOI value is 1-5.
[0025] Furthermore, step S3, after inoculating mast cells, also includes a qualification assessment of the status of the ALI co-culture system following apical infection, including:
[0026] Based on the detection of cell density below 1×10 5 Based on the cell / mL result, it was determined that fresh cells from the same batch of culture should be added, and / or based on the result that the expression level of the activation marker CD117 based on the cell density is less than 70%, it was determined that human SCF at a concentration of 100 ng / mL should be added for maturation compensation before infection.
[0027] Furthermore, in S4, the alarm element includes ATP and IL-33.
[0028] Furthermore, in step S4, the activation indicators of the mast cells include at least one of histamine release rate, β-hexosidase release rate, and trypsin release rate.
[0029] Furthermore, the extended time corresponding to the extension of culture or activation time caused by the compensation mechanism activated in step S1 and / or step S3 is used to synchronously adjust the total experimental duration of subsequent steps.
[0030] Furthermore, the mast cells used in step S3 are human LAD2 mast cells.
[0031] Compared with existing technologies, the advantages of this invention are as follows: This invention establishes an ALI model maturity evaluation standard from two dimensions—barrier function and cell differentiation—by using transmembrane resistance (TEER) and ciliary differentiation rate as a dual-parameter certification system. When the TEER value is consistently ≥500 Ω·cm... 2 When the ciliary formation rate is >80%, it indicates that the epithelial model has established a complete polarization structure and barrier function. Based on this, the initiation time of the non-standard model was extended and the growth factor (EGF / BDNF) synergistic intervention program was launched. By regulating the EGFR and TrkB signaling pathways, the terminal differentiation of epithelial cells was promoted, thereby ensuring the biological relevance of the experimental system from the source, eliminating the experimental systematic error caused by the difference in the maturity of the epithelial model, and providing a standardized platform for subsequent research on viral infection and immune response.
[0032] Furthermore, viral titers were quantified using the TCID50 method, establishing a titer ≥1×10⁻⁶. 6 The quality standard of PFU / mL and the allowable range of ≤10% batch-to-batch variation ensure that the viral preparation has stable infectious efficacy, thereby providing reliable input parameters for the accurate calculation of MOI. This controls the accuracy of viral infection dosage from the source and ensures the consistency of infection intensity between different experimental batches.
[0033] Furthermore, by establishing a real-time MOI calculation model based on actual viral titers to determine the total required amount of standard viral particles, and then performing vector normalization calculations with the measured viral titers, the precise inoculation volume required to achieve the target infection intensity is finally derived. This enables adaptive control of the viral infection dose, overcomes the variation in infection efficiency caused by viral titer fluctuations in the traditional fixed inoculation volume method, and significantly improves the repeatability and accuracy of the experiment.
[0034] Furthermore, by limiting the MOI operation window to the range of 1-5, based on virological infection dynamics and cell tolerance studies, the optimal balance between infection efficiency and cell survival rate can be achieved within this range. This ensures sufficient pathogen stimulation while maintaining the metabolic activity of host cells, providing the best time window for observing complete epithelial-immune cell interactions.
[0035] Furthermore, by establishing mast cell density (≥1×10⁻⁶) 5 The system employs a dual quality control standard of cell count / mL and CD117 expression level (≥70%). Based on this standard, quantitative evaluation is performed by cell counting and flow cytometry. For systems that do not meet the standard, a correction strategy of cell supplementation or SCF maturation compensation is adopted to ensure that immune effector cells are in the best response state, avoid false negative results caused by insufficient effector cell number or low function, and ensure the strength and stability of the model response signal.
[0036] Furthermore, by establishing a multi-dimensional detection system targeting epithelial cell alarm release (ATP / IL-33) and mast cell activation (histamine / β-hexosidase / trypsin release rate), a complete chain of evidence is formed from the initial signal generation to the final effect output, thereby providing a quantitative and multi-parameter evaluation standard for model functional verification, and enabling the successful construction of the model to have a clear and objective basis for judgment.
[0037] Furthermore, a compensation time accumulation and transmission mechanism was established, incorporating the compensation operation time of each stage into the overall experimental time sequence adjustment. This ensured that different batches of models were at the same developmental stage for comparison, thereby eliminating the time variables introduced by quality control intervention, guaranteeing the time sequence comparability of data from different experimental batches, and enhancing the scientific rigor and reliability of the research.
[0038] Furthermore, by constructing a species-matched co-culture system using human LAD2 mast cell line and human primary airway epithelial cells, the species specificity of cytokine-receptor interactions was maintained, thereby maximally mimicking the immune microenvironment in the human body, significantly improving the clinical predictive value of experimental results, and providing a reliable platform for translational medicine research.
[0039] Furthermore, by establishing a standardized quality certification system, dynamic parameter control algorithm, and multi-level compensation mechanism, a highly stable research model with self-verification and self-correction functions was constructed, providing a reliable technical platform for RSV infection mechanism research and drug screening. Attached Figure Description
[0040] Figure 1 This is a flowchart illustrating the steps of constructing a research model for airway immune response mediated by RSV infection according to an embodiment of the present invention.
[0041] Figure 2 This is a flowchart illustrating the steps of apical infection in an ALI co-culture system according to an embodiment of the present invention.
[0042] Figure 3 These are morphological images of mast cells in each group of the embodiments of the present invention after toluidine blue staining, viewed under a microscope at ×1000x magnification.
[0043] Figure 4 This is a schematic diagram showing the comparison of histamine release rate levels in mast cells of different groups in this embodiment of the invention.
[0044] Figure 5 A schematic diagram showing the comparison of β-hexosidase release rates in mast cells of different groups;
[0045] Figure 6 This is a GO enrichment map of differentially expressed genes in mast cells from two groups of mast cells in the embodiments and comparative examples of the present invention;
[0046] Figure 7 This is a GO enrichment map of differentially expressed genes in airway epithelial cells of two groups in the embodiments and comparative examples of the present invention. Detailed Implementation
[0047] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.
[0048] Please see Figure 1 The diagram shows the steps of constructing a research model for airway immune response mediated by RSV infection according to an embodiment of the present invention.
[0049] The method for constructing a research model of airway immune response mediated by RSV infection according to embodiments of the present invention includes the following sequentially connected steps:
[0050] Step S1: Human primary airway epithelial cells are seeded into a Transwell chamber to establish an air-liquid interface culture. The pass rate is evaluated based on the dynamically monitored transmembrane resistance value and the ciliary formation ratio. When the preset standard is continuously met, the epithelial cell culture system is obtained.
[0051] Step S2: Amplify the RSV virus and determine the completion of virus amplification based on the titer deviation value of the virus titer during the amplification process to obtain the RSV virus. The virus titer is determined using the TCID50 method, and the titer deviation value is characterized by the deviation between the virus titer during the amplification process and the preset virus titer.
[0052] Step S3: Inoculate mast cells into the epithelial cell culture system to obtain an ALI co-culture system. Based on the total number of cells and viral titer of the ALI model, dynamically calculate the inoculation volume required to obtain the preset MOI value, and perform apical infection on the ALI co-culture system to obtain the infected ALI co-culture system.
[0053] Step S4: Samples were collected from the ALI co-culture system after apical infection, and the levels of alarmins released by epithelial cells and activation indicators of mast cells were detected to verify the functional output of the model and complete the model construction.
[0054] Specifically, in step S1, the preset standard is that the transmembrane resistance (TEER) value is ≥500 Ω·cm for three consecutive measurements. 2 And the proportion of cilia formation is >80%;
[0055] If the preset standard is not met, a compensation mechanism will be activated, which includes extending the culture time and supplementing with epidermal growth factor (EGF) or brain-derived neurotrophic factor (BDNF) at a concentration of 10 ng / mL to 50 ng / mL.
[0056] Specifically, in step S2,
[0057] Preset viral titer ≥ 1×10 6 PFU / mL;
[0058] The preset viral titer deviation is ≤10%.
[0059] Please see Figure 2 As shown, Figure 2 This is a flowchart illustrating the steps of apical infection in an ALI co-culture system according to an embodiment of the present invention.
[0060] Specifically, step S3, the process of apical infection of the ALI co-culture system includes:
[0061] Step S31: Determine the standard total number of viral particles based on the preset multiplicity of infection (MOI) value and the total number of cells;
[0062] Step S32: Divide the standard total number of virus particles by the titer of RSV virus solution to determine the inoculation volume required to achieve the preset multiplicity of infection (MOI) value.
[0063] Step S33: Perform the top-side infection procedure according to the inoculation volume.
[0064] Specifically, the MOI (Multiple Infections) value is preset to 1-5.
[0065] Specifically, step S3, after inoculating mast cells, also includes a qualification assessment of the ALI co-culture system following apical infection, including:
[0066] Based on the detection of cell density below 1×10 5 Results of cells / mL indicate the need to supplement with fresh cells from the same batch of culture, and / or results indicating that the expression level of the activation marker CD117 based on cell density is below 70%, indicate the need to add human SCF at a concentration of 100 ng / mL for maturation compensation before infection.
[0067] Specifically, in S4, alarm factors include ATP and IL-33.
[0068] Specifically, in step S4, the activation indicators of mast cells include at least one of histamine release rate, β-hexosidase release rate, and trypsin release rate.
[0069] Specifically, the extended time caused by the compensation mechanism initiated in step S1 and / or step S3 is used to synchronously adjust the total experimental time of subsequent steps.
[0070] Specifically, the mast cells used in step S3 are human LAD2 mast cells.
[0071] 1. Experimental Design
[0072] 1.1 Experimental Objective
[0073] To verify that in an RSV-infected airway epithelial-mast cell co-culture model, airway epithelial-derived ATP and IL-33 synergistically drive mast cell hyperactivation through dual pathways, and to evaluate the reliability of the model and the potential value of dual-target intervention.
[0074] 1.2 Model Construction
[0075] Example group (model of the present invention): RSV infection co-culture model with dynamic verification and compensation mechanism was constructed strictly following the method of the present invention.
[0076] Human primary airway epithelial cells (AECs) were cultured in Transwell chambers to establish an air-liquid interface (ALI), followed by TEER (≥500 Ω·cm) treatment. 2 The verification of the ratio of cilia formation (80%) was qualified.
[0077] Human LAD2 mast cells (MCs) were seeded in the lower chamber. Twenty-four hours post-seeding, the cell density in the lower chamber was measured to be 5.25 × 10⁻⁶. 4 The cell density was ≤1×10⁶ cells / mL, and the CD117 expression rate was 98% as detected by flow cytometry. 5 If the cell density in the lower chamber is 1.50 × 10⁶ cells / mL, but the CD117 expression rate is ≥70%, LAD2 cells from the same batch are added to the lower chamber, and cell counting and flow cytometry are performed again to detect the CD117 expression rate. 6 The CD117 expression rate was 99%, confirming the model's suitability.
[0078] Use a titer of 2.5 × 10⁻⁶ 6 RSV-A2 virus strain at PFU / mL. Based on the target MOI=3 and the total cell count of the ALI model, the total cell count in this example is 2.0 × 10⁻⁶. 5 Based on the number of samples and the measured viral titer mentioned above, the required inoculation volume was calculated to be 240 μL. The ALI co-culture system was then subjected to apical infection using this calculated volume.
[0079] Comparative Example 1 (Classical Pathway Activated Mast Cell Group): LAD2 cells were cultured alone, sensitized with anti-OVA IgE (1 μg / mL), and challenged with OVA (100 ng / mL).
[0080] Comparative Example 2 (Co-culture without infection): RSV infection was not performed, and the remaining steps were the same as in the Example.
[0081] Negative control group: LAD2 (MC) only, normal mast cell culture group.
[0082] 2. Experimental Results and Analysis
[0083] 2.1 Model Reliability Validation: Dual Alarm Factor Release
[0084] Table 1 shows a comparison of the dual alarmant release levels between Comparative Example 2 (simple co-culture without infection) and Example 1 (n=6). ±s).
[0085] Table 1: Comparison of Dual Alarmant Release Levels between Comparative Example 2 and Example Group (Table 1)
[0086]
[0087] Note: Compared with the example group, P<0.0001.
[0088] Results analysis:
[0089] The release of ATP and IL-33 in the Example Group was significantly higher than that in Comparative Example 2 (co-culture without infection), increasing by approximately 12.8 times and 2.6 times, respectively. This successfully reproduced the RSV-induced dual alarm storm, demonstrating that the method of the present invention has high stability and reproducibility.
[0090] 2.2 Functional Output: Mast Cell Hyperactivation
[0091] 2.2.1 The activation level of mast cells in each group was assessed morphologically by staining them with toluidine blue.
[0092] Please see Figure 3 As shown, these are morphological images of mast cells after toluidine blue staining under a 1000x microscope in each group of embodiments of the present invention. Among them, A is the image of the negative control group, B is the image of comparative example 1, C is the image of comparative example 2, and D is the image of the embodiment group.
[0093] Results analysis:
[0094] By comparing the example group with the negative control group, comparative example 1 group and comparative example 2 group, the mast cells in the example group showed the strongest hyperactivation phenotype, which was characterized by cell swelling, blurred edges and increased intracellular vacuoles; this proves that the model of the present invention can stably and reliably induce mast cell hyperactivation.
[0095] 2.2.2 The activation level of mast cells was assessed by detecting key indicators of mast cell degranulation, as shown in Table 2, which is a comparison table of mast cell activation index data for each group (n=3, ±s).
[0096] Table 2: Comparison of mast cell activation index data among groups
[0097]
[0098] Note: Compared with the example group, P<0.0001.
[0099] Please see Figures 4-5 As shown, Figure 4 This is a schematic diagram showing the comparison of histamine release rate levels in mast cells across different groups in this invention (n=3). ±s); Figure 5 This is a schematic diagram showing the comparison of β-hexosidase release rates in mast cells across different groups, where n=3. ±s; **** represents comparison with control group 1 (classical pathway activated mast cells group), P<0.0001.
[0100] Results analysis:
[0101] The mast cells in the example group exhibited the strongest hyperactivated phenotype, with histamine and β-hexosidase release rates significantly higher than those in the classical pathway activation group, demonstrating that the model of the present invention can stably and reliably induce mast cell hyperactivation.
[0102] Comparative Example 2: The mast cell activation level was significantly lower than that in the Example group. This again demonstrates that an imperfect model (difference in viral infection efficiency) cannot effectively transmit and amplify the damage signal of RSV, leading to a weakened subsequent immune response, thus verifying the accuracy and superiority of the model construction method of this invention from the opposite perspective.
[0103] 2.3 Transcriptomics Validation: Activation of IL-33 and P2R Signaling Pathways
[0104] By performing whole transcriptome sequencing and GO enrichment analysis on differentially expressed genes in mast cells in the Example Group and Comparative Example 2, compared with Comparative Example 2, RSV infection in the Example Group specifically activated mast cell antiviral response-related gene clusters. Notably, the expression of IL-33 and P2R signaling pathway genes was significantly upregulated. This transcriptome analysis confirms that in this example, RSV activates the P2R / IL-33 signaling axis through airway epithelial-mast cell interaction, driving an immune response cascade that leads to mast cell hyperactivation and the release of inflammatory mediators.
[0105] Please see Figure 6 As shown, Figure 6 This is a GO enrichment map of differentially expressed genes in mast cells from two groups of mast cells in the embodiments and comparative examples of the present invention, where n=3.
[0106] 2.4 Transcriptomic Validation: Activation of Core Signaling Pathways Regulating Epithelial Cell Inflammation
[0107] By performing whole transcriptome sequencing and GO enrichment analysis on differentially expressed genes in airway epithelial cells of the example group and the control group 2, the differentially expressed genes in epithelial cells of the example group were significantly enriched in the core pathway of inflammation regulation, where n=3.
[0108] Please see Figure 7 As shown, Figure 7 This is a GO enrichment map of differentially expressed genes in airway epithelial cells of two groups in the embodiments and comparative examples of the present invention.
[0109] Results analysis: The results confirm at the transcriptomic level that, in the examples, RSV infection successfully induced biological processes such as the release of inflammatory factors from airway epithelial cells, recruitment of immune cells, and extracellular matrix remodeling.
[0110] 3. Overall Conclusion
[0111] Model effectiveness: The airway immune response research model with dynamic verification and compensation mechanism provided by this invention can successfully simulate the in vivo microenvironment of RSV infection, stably induce the release of alarming factors from the airway epithelium, and drive mast cells to undergo hyperactivation.
[0112] Mechanism innovation: Experimental results confirm the ATP / IL-33 dual-pathway synergistic mechanism in the "RSV-epithelial-mast cell" tripartite dialogue. The two pathways complement each other functionally and synergistically amplify signals, jointly breaking through the activation threshold of mast cells.
[0113] Treatment Implications: While blocking either the P2R or ST2 pathway alone can partially inhibit mast cell activation, its effect is not as significant as blocking both pathways simultaneously. This provides strong experimental evidence for developing bifunctional inhibitors that simultaneously target P2R and ST2, potentially becoming a novel strategy for treating RSV-related asthma.
[0114] Technical advantages: The failure of Comparative Example 2 highlights the key role of the dynamic verification and compensation mechanism in this invention in ensuring the physiological relevance of the model and the reliability of the data.
[0115] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.
[0116] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for constructing a research model of airway immune response mediated by RSV infection, characterized in that, include: Step S1: Human primary airway epithelial cells are seeded into a Transwell chamber to establish an air-liquid interface culture. The pass rate is evaluated based on the dynamically monitored transmembrane resistance value and the ciliary formation ratio. When the preset standard is continuously met, the epithelial cell culture system is obtained. Step S2: Amplify the RSV virus and determine the completion of virus amplification based on the titer deviation value of the virus titer during the amplification process to obtain the RSV virus. The virus titer is determined using the TCID50 method, and the titer deviation value is characterized by the deviation between the virus titer during the amplification process and the preset virus titer. Step S3: Inoculate mast cells into the epithelial cell culture system to obtain an ALI co-culture system. Based on the total number of cells in the ALI co-culture system and the viral titer of the RSV virus solution, dynamically calculate the inoculation volume of the RSV virus solution required to obtain the preset MOI value, and perform apical infection on the ALI co-culture system to obtain the ALI co-culture system after apical infection. Step S4: Samples are collected from the ALI co-culture system after apical infection, and the levels of alarmins released by epithelial cells and activation indicators of mast cells are detected to verify the functional output of the model and complete the model construction.
2. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 1, characterized in that, In step S1, the preset standard is based on three consecutive measurements of the transmembrane resistance (TEER) value ≥500 Ω·cm. 2 The result was confirmed that the ciliary formation rate was >80%; If the preset standard is not met, a compensation mechanism is activated, which includes extending the culture time and supplementing with epidermal growth factor (EGF) or brain-derived neurotrophic factor (BDNF) at a concentration of 10 ng / mL to 50 ng / mL.
3. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 2, characterized in that, In step S2 The preset virus titer is ≥1×10 6 PFU / mL; The preset virus titer deviation is ≤10%.
4. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 3, characterized in that, In step S3, the process of apical infection of the ALI co-culture system includes: Step S31: Determine the standard total number of viral particles based on the preset multiplicity of infection (MOI) value and the total number of cells; Step S32: Divide the standard total number of virus particles by the titer of the RSV virus solution to determine the inoculation volume required to reach the preset multiplicity of infection (MOI) value; Step S33: Perform the top-side infection operation according to the inoculation volume.
5. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 4, characterized in that, The preset infection multiplicity MOI value is 1-5.
6. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 5, characterized in that, Step S3, after inoculating mast cells, also includes a qualification assessment of the ALI co-culture system following apical infection, including: Based on the detection of cell density below 1×10 5 Based on the cell / mL result, it was determined that fresh cells from the same batch of culture should be added, and / or based on the result that the expression level of the activation marker CD117 based on the cell density is less than 70%, it was determined that human SCF at a concentration of 100 ng / mL should be added for maturation compensation before infection.
7. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 6, characterized in that, In S4, the alarm hormones include ATP and IL-33.
8. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 7, characterized in that, In step S4, the activation indicators of the mast cells include at least one of histamine release rate, β-hexosidase release rate, and trypsin release rate.
9. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 8, characterized in that, The extended time corresponding to the extension of culture or activation time caused by the compensation mechanism initiated in step S1 and / or step S3, and the extended time is used to synchronously adjust the total experimental time of subsequent steps.
10. The method for constructing a research model for airway immune response mediated by RSV infection according to claim 1, characterized in that, The mast cells used in step S3 are human LAD2 mast cells.