Establishment method of model for inflammation of mouse brain microvascular endothelial cells infected by haemophilus parasuis

Abstract

The invention discloses a method for establishing a model for inflammation of mouse brain microvascular endothelial cells infected by haemophilus parasuis, which is used for simulating the process of porcine meningitis caused by haemophilus parasuis infection by using the mouse brain microvascular endothelial cells infected by haemophilus parasuis and researching the pathogenesis of the porcine meningitis caused by haemophilus parasuis. The optimal MOI of mouse brain microvascular endothelial cells infected by haemophilus parasuis is 1, the optimal infection time is 6 h, under the condition, the inflammatory factor level of the cells is remarkably increased, and the success rate of the cell inflammation model construction method is higher.

Description

Technical Field

[0001] This invention belongs to the field of cell infection model technology, specifically relating to a method for establishing a mouse brain microvascular endothelial cell inflammation model infected with Haemophilus parasuis. Background Technology

[0002] Haemophilus parasuis (GPS) is a resident bacterium of the upper respiratory tract in pigs. It can enter the bloodstream, survive, and cause systemic infection, leading to meningitis, polyserositis, and arthritis, often presenting with pneumonia-like symptoms. It is the pathogen of Glasser's disease. With changes in pig farming practices, Haemophilus parasuis infection is a major cause of mortality in piglets worldwide, causing significant economic losses to the pig industry. During Haemophilus parasuis infection, the blood-brain barrier is disrupted, leading to further spread of the bacterium and exudative inflammation. Meningitis is a common symptom of Haemophilus parasuis infection, typically manifested as abnormal white blood cell counts in the cerebrospinal fluid and specific clinical symptoms, causing substantial economic losses to the pig industry. Its pathogenic mechanism requires further investigation.

[0003] Due to the high cost of pig farming and the long experimental cycle, and the lack of reports on in vitro cell models of porcine meningitis induced by Haemophilus parasuis infection, mouse brain microvascular endothelial cells are a good cell model for studying the in vitro blood-brain barrier, and meningitis is usually accompanied by changes in blood-brain barrier permeability. Therefore, this invention selects mouse brain microvascular endothelial cells as a model to construct an in vitro Haemophilus parasuis infection cell inflammation model for studying the pathogenesis and molecular mechanism of porcine meningitis caused by Haemophilus parasuis. Summary of the Invention

[0004] The purpose of this invention is to propose a method for constructing an inflammation model of mouse brain microvascular endothelial cells infected with Haemophilus parasuis, aiming to construct an in vitro infection model of mouse brain microvascular endothelial cells infected with Haemophilus parasuis SH0165, to simulate the process of swine meningitis caused by Haemophilus parasuis infection and to study the pathogenesis of swine meningitis caused by Haemophilus parasuis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for constructing a mouse brain microvascular endothelial cell model infected with Haemophilus parasuis includes the following steps: Haemophilus parasuis SH0165 standard strain is cultured to obtain a bacterial suspension; the Haemophilus parasuis suspension is added to mouse brain microvascular endothelial cells, and the infection is carried out for 3-12 h with a multiplicity of infection of 0.1-100.

[0007] Preferably, the infection time is 6 hours and the multiplicity of infection is 1.

[0008] Preferably, the relative expression levels of inflammatory factor genes in uninfected and infected mouse brain microvascular endothelial cells are detected. The inflammatory factors include IL-6, IL-8, IL-18, IL-1β, and TNF-α. If the expression levels of inflammatory factor genes in mouse brain microvascular endothelial cells infected with Haemophilus parasuis are significantly increased, then the cell inflammation model is successfully constructed.

[0009] The present invention has the following advantages and effects compared with the prior art:

[0010] (1) This invention provides a method for establishing a mouse brain microvascular endothelial cell model infected by Haemophilus parasuis, thereby establishing an inflammatory model of mouse brain microvascular endothelial cells infected by Haemophilus parasuis in vitro, and simulating the process of Haemophilus parasuis SH0165 in vitro infecting mouse brain microvascular endothelial cells.

[0011] (2) The application of Haemophilus parasuis infection model of mouse brain microvascular endothelial cells laid the foundation for studying the inflammatory ability of Haemophilus parasuis after in vitro infection of mouse brain microvascular endothelial cells and the interaction between Haemophilus parasuis and mouse brain microvascular endothelial cells. It also provided new research ideas and treatment methods for swine meningitis caused by Haemophilus parasuis infection.

[0012] (3) The optimal MOI (MOI=1:1) and infection time (6 h) for Haemophilus parasuis infection of mouse brain microvascular endothelial cells were obtained from the experiment, which made the construction method provided by the present invention have a higher success rate. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the implementation process of the method for constructing a mouse brain microvascular endothelial cell model infected with Haemophilus parasuis proposed in this invention.

[0014] Figure 2 This is a cell comparison image of mouse brain microvascular endothelial cells before and after infection with Haemophilus parasuis (6 h, MOI=1:1).

[0015] Figure 3 The mRNA expression levels of inflammatory factors IL-6, IL-8, IL-18, IL-1β, and TNF-α in the negative control group and the bacterial infection group when mouse brain microvascular endothelial cells were infected with Haemophilus parasuis. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can better understand and implement the present invention, but the embodiments of the present invention are not limited thereto.

[0017] Example 1: Activation, culture, and counting of Haemophilus parasuis

[0018] 1.1 Materials

[0019] The Haemophilus parasuis strain SH0165 was provided by the State Key Laboratory of Microbiology, Huazhong Agricultural University.

[0020] TSB and TSA culture media used for bacterial strain culture were purchased from BD Biosciences, Inc. (USA); fetal bovine serum used for bacterial strain culture was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; NAD used for bacterial strain culture was purchased from Beyotime Biotechnology Co., Ltd. Disposable bacterial culture dishes were purchased from Wuxi Nice Life Technology Co., Ltd.

[0021] 1.2 Methods

[0022] 100 μL of the standard strain of Haemophilus parasuis SH0165 stored at -80 ℃ was inoculated into TSB liquid medium and placed in a constant temperature shaker at 37 ℃ for 12 h at 180 rpm to obtain activated Haemophilus parasuis bacterial culture.

[0023] Activated Haemophilus parasuis bacterial suspension was streaked onto TSA plates and incubated upside down at 37°C for 24 hours to form colonies. Under aseptic conditions, a single colony was picked and inoculated into liquid TSB medium and incubated at 37°C with a shaker at 180 rpm. At 9, 10, and 12 hours of incubation, 100 μl of the bacterial suspension was added to 900 μL of TSB medium to prepare six different 10-fold serial dilutions. -1 10 -2 10 -3 10 -4 10 -5 10 -6 From 10 -4 10 -5 10 -6 100 μL of each of the three dilutions was spread onto TSA plates. After incubation at 37°C for 24 h, the number of colonies formed in each plate was recorded, and the total number of bacteria per milliliter of the original sample was calculated based on the dilution factor.

[0024] Under aseptic conditions, single colonies were picked and inoculated into TSB liquid medium, and incubated in a 37°C constant-temperature shaker at 180 rpm for 9 h to obtain the bacterial suspension required for modeling. The concentration of the bacterial suspension obtained after 9 h of incubation was 8 × 10⁻⁶. 7 CFU / mL.

[0025] Example 2: Resuscitation and Culture of Mouse Brain Microvascular Endothelial Cells

[0026] 2.1 Materials

[0027] Mouse brain microvascular endothelial cells (bEnd.3) were purchased from Wuhan Huanna Biotechnology Co., Ltd.

[0028] DMEM and sterile PBS used in cell culture were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; fetal bovine serum used in cell culture was purchased from EallBio; and 0.25% trypsin solution used in cell culture was purchased from Wuhan Saive Biotechnology Co., Ltd.

[0029] The cell culture dishes and six-well plates used for cell culture were purchased from Wuxi Nice Life Technology Co., Ltd.

[0030] 2.2 Methods

[0031] Mouse brain microvascular endothelial cells frozen in liquid nitrogen were rapidly thawed in a 37°C water bath. When small pieces of ice remained in the cryovial, the cells were removed and placed in a clean bench. They were then slowly added to a centrifuge tube containing 1 mL of complete culture medium (DMEM + 20% FBS + 1% antibiotics). The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and 1 mL of complete culture medium was added to prepare a cell suspension. The cell suspension was then added to a cell culture dish and incubated in a 37°C, 5% CO2 incubator.

[0032] When mouse brain microvascular endothelial cells have covered approximately 80% of the cell culture dish, discard the supernatant and gently rinse the cells three times with 2 mL of sterile PBS. Add 1 mL of trypsin and digest at 37°C for 5 min. Observe under a microscope; when cells are observed to detach in sheets, add 1 mL of complete culture medium to stop the digestion. Collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently resuspend the cells in 1 mL of complete culture medium.

[0033] The resuspended cells were passaged in a 1-to-2 manner, and the culture medium was added to about 8 mL. The cells were spread evenly by cross-shaking and then placed in an incubator for culture.

[0034] Example 3: Haemophilus parasuis infection of mouse brain microvascular endothelial cells

[0035] When mouse brain microvascular endothelial cells covered approximately 80% of the cell culture dish, discard the supernatant and gently rinse the cells three times with 2 mL of sterile PBS. Add 1 mL of trypsin and digest for 5 min. Observe under an inverted microscope; when cells are observed to detach in sheets, add 1 mL of complete culture medium to stop the digestion. Collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, and gently resuspend the cells in 1 mL of culture medium.

[0036] Mix 90 μL of cell suspension with 10 μL of 4% trypan blue solution and incubate at room temperature for 3 min. Using a pipette, slowly add 10 μL of the mixture along the edge of the counting chamber until it is full. Under a microscope, move the counting chamber to align the field of view with the central square. Count the number of cells in each of the four diagonal squares (counting only viable cells) and then take the average. The number of cells per milliliter of sample = average number of cells in each square × cell dilution factor × 10. 4 .

[0037] Mouse brain microvascular endothelial cells at 3×10 5 Seeds were inoculated into six-well plates, and 2 mL of DMEM culture medium containing 20% ​​fetal bovine serum (without 1% antibiotics) was added to each well. The plates were then incubated at 37°C and 5% CO2 for 24 h.

[0038] The bacterial infection group was set up as follows: Mouse brain microvascular endothelial cells were washed with sterile PBS buffer and then cultured in DMEM medium containing 20% ​​fetal bovine serum (without 1% penicillin-dip antibiotic solution). The bacterial suspension was added to the mouse brain microvascular endothelial cells at the MOI (Minimum Infection Point) for 3 h, 6 h, and 12 h, respectively, to obtain the cell infection group. The MOIs of *Haemophilus parasuis* in the bacterial suspension for infecting mouse brain microvascular endothelial cells were 1:10, 1:1, 10:1, and 100:1, respectively. MOI = number of bacteria per well / number of cells. The negative control group was also set up as follows: Mouse brain microvascular endothelial cells were washed with sterile PBS buffer and then cultured in DMEM medium containing 20% ​​fetal bovine serum (without 1% penicillin-dip antibiotic solution). The cells were then incubated in a CO2 incubator for 3 h, 6 h, and 12 h, respectively, to obtain the negative control group.

[0039] In addition, the negative control group and the bacterial infection group should be conducted simultaneously, using the same culture medium and incubation time, and both groups should use mouse brain microvascular endothelial cells from the same generation. Both the negative control group and the bacterial infection group have three replicate wells. Furthermore, because culture in DMEM medium containing 10% fetal bovine serum results in relatively slow growth of mouse brain microvascular endothelial cells, and because the antibiotic solution has a certain inhibitory effect on Haemophilus parasuis, the cell culture medium used in this invention is DMEM medium containing 20% ​​fetal bovine serum (without 1% antibiotic solution); the CO2 content of the carbon dioxide incubator is maintained at 5%, and the incubation temperature is 37°C.

[0040] Example 4: The gene expression levels of inflammatory factors in the negative control group and the bacterial infection group were measured and compared.

[0041] 4.1 Materials

[0042] Trizol Universal Total RNA Extraction Reagent was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Primescript ® RT Reagent Kit with gDNA Eraser Reverse Transcription Kit, TB Green ® Premix Ex Taq TM All reagent kits were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0043] 4.2 Methods

[0044] Remove the culture medium from the negative control group and the bacterial infection group, and wash three times with PBS buffer. Add 800 μL of Trizol Universal Total RNA Extraction Reagent to the washed cells, digest and lyse at room temperature for 5 min, pipette the cells, and collect the lysate into a sterile 1.5 mL centrifuge tube. Store at -80°C. Extract total RNA from the cells according to the Trizol Universal Total RNA Extraction Reagent instructions (TIANGEN, Tiangen Biotech (Beijing) Co., Ltd.). The specific steps are as follows: Remove the cell lysate from the -80°C freezer and thaw it on ice. After thawing, let it stand at room temperature for 5 min. Add 160 μL of chloroform to the lysate from the above step, tighten the centrifuge tube cap, and shake vigorously by hand for 15 s. After the solution is fully emulsified, let it stand at room temperature for 5 min, centrifuge at 12000 rpm at 4°C for 15 min, and carefully transfer the supernatant to another new 1.5 mL centrifuge tube (do not aspirate the white intermediate layer). Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube to mix thoroughly, let stand at room temperature for 10 min, then centrifuge at 12000 rpm at 4℃ for 10 min. Carefully discard the supernatant, keeping only the precipitate. Slowly add 800 μL of 75% ethanol along the wall of the centrifuge tube, centrifuge at 10000 rpm at 4℃ for 5 min, then discard the ethanol. Dry the precipitate at room temperature for 3 min, add 10 μL of enzyme-free water to dissolve the precipitate completely, and store the RNA at -80℃.

[0045] Using a cDNA synthesis kit and following Primescript ® The RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian Takara Bio Inc.) is used to reverse transcribe cellular RNA into cDNA. TB Green is employed. ® Premix Ex Taq TMReal-time quantitative PCR was performed using the kit (TaKaRa, Dalian Takara Bio Inc.). Primers were designed using Primer Premier 5.0 software (Table 1). Each reaction was performed in quadruplicate, with GAPDH as the internal control gene. －△△Ct The relative expression levels of inflammatory factor (IL-6, IL-8, IL-18, IL-1β, TNF-α) genes were calculated using a method.

[0046] The gene expression levels of inflammatory factors in the negative control group and the bacterial infection group were compared. The results showed that the gene expression levels of five inflammatory factors were significantly increased in the bacterial infection group (p < 0.05), indicating successful modeling. By comparing the increase in inflammatory factors at different MOIs and infection times, the infection condition with the highest proportion of increased inflammatory factors (MOI = 1:1, 6 h of infection) was selected as the subsequent modeling condition. Figure 3 ).

[0047] Table 1 Primer information for real-time quantitative PCR

[0048]

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for establishing an in vitro cellular inflammation model infected with Haemophilus parasuis, characterized in that, Includes the following steps: A standard strain of Haemophilus parasuis SH0165 was cultured to prepare a bacterial suspension. The Haemophilus parasuis suspension was added to mouse brain microvascular endothelial cells and infected for 3-12 hours, with a multiplicity of infection of 0.1-100.

2. The method according to claim 1, characterized in that, The study also includes detecting the relative expression levels of inflammatory factor genes in uninfected and infected mouse brain microvascular endothelial cells, including IL-6, IL-8, IL-18, IL-1β, and TNF-α. If the expression levels of inflammatory factor genes in mouse brain microvascular endothelial cells infected with Haemophilus parasuis are significantly increased, then the cell inflammation model is successfully constructed.

3. The method according to claim 1 or 2, characterized in that, The infection time was 6 hours, and the multiplicity of infection was 1.

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