Micromolecule allergen cell model for detecting potential allergenicity food-borne micromolecules and application of micromolecule allergen cell model in exploring small molecule allergen sensitization mechanism

Abstract

The technical scheme discloses a small molecule allergen cell model for detecting potential allergenicity food-borne small molecules and application of the small molecule allergen cell model in exploring a small molecule allergen sensitization mechanism. According to the micromolecule sensitizer cell model for detecting potential sensitization food-borne micromolecules, the cells are Raw264.7 macrophages, the micromolecule sensitizer regulates and controls the expression level of mRNA related to M1 / M2 of the Raw264.7 macrophages by changing gene expression of the Raw264.7 macrophages, polarization of the Raw264.7 macrophages can be regulated and controlled, and anaphylactic reaction is affected by increasing the expression level of the mRNA related to M2.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the construction of a small molecule allergen cell model for studying and detecting potential allergenic food-borne small molecules and its application in exploring the sensitization mechanism of small molecule allergens. Background Art

[0002] Food allergy is an immune disease. In recent years, food allergies have become more frequent and the number of people with allergies has increased, affecting nearly 8% of children and 5% of adults. Therefore, food allergy has become a major public health issue.

[0003] Erythritol, as a low-calorie functional sweetener, has been widely used in the food industry. According to statistics, erythritol has been approved for use in more than 60 countries around the world, and its demand has continued to expand at an average annual compound growth rate of 20.3%. In traditional cognition, erythritol is highly safe, has good intestinal tolerance, and is not likely to have a negative impact on health when consumed in moderation through food. However, in recent years, companies have received more and more complaints about erythritol allergies. Clinically, reports of cases related to erythritol allergies have also continued to increase, especially in Japan, which was the first country to approve the use of erythritol, revealing the possibility of long-term intake of erythritol inducing increased allergic reactions in the population. In addition, erythritol collinear contamination in the food production process has also been shown to cause severe allergic reactions such as respiratory distress and tachycardia, and the risk of hidden sensitization cannot be ignored. Although the allergenic risk of erythritol has attracted widespread attention, the sensitization mechanism behind it remains unclear.

[0004] Mannose is an organic compound with a molecular formula of C6H12O6 and a molecular weight of 180.156. It is a colorless or white crystalline powder. It is a sugar that plays an important role in the human body's metabolism, especially in the glycosylation of specific proteins.

[0005] Previous studies have found that allergic reactions are mainly caused by large molecular proteins, and compounds with a relative molecular weight of less than 1000Da are generally not easy to cause allergic reactions. Small molecule compounds that cause hypersensitivity reactions, such as penicillin and chlorogenic acid, often induce allergic reactions through a recognition pathway that is completely different from protein allergens. At present, there is still a lack of an effective cell model for studying small molecule allergens. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes to construct a small molecule allergen cell model for detecting potential allergenic foodborne small molecules for studying small molecule allergens. The cell model can accurately identify the allergenicity of small molecule allergens through the polarization state of macrophages, so that it can be used to evaluate the allergenicity of food allergens.

[0007] Another object of the present invention is to provide the application of the above cell model in exploring the sensitization mechanism of small molecule allergens.

[0008] A small molecule allergen cell model for detecting potentially allergenic food-borne small molecules, wherein the cells are Raw264.7 macrophages. The small molecule allergens can regulate the polarization of Raw264.7 macrophages by changing the gene expression of Raw264.7 macrophages to regulate the expression level of mRNA related to M1 / M2 of Raw264.7 macrophages, and affect allergic reactions by increasing the expression level of M2-related mRNA.

[0009] The small molecule allergen is erythritol or D-mannose.

[0010] A small molecule allergen cell model for detecting potentially allergenic foodborne small molecules is used in exploring the sensitization mechanism of small molecule allergens, which is used to evaluate whether small molecules are allergens using Raw264.7 macrophages.

[0011] The small molecule allergens are used to affect the phagocytosis of Raw264.7 macrophages.

[0012] Beneficial effects of the invention: The invention provides a low-cost method for judging food allergies based on the phenotypic changes of cells after the Raw264.7 macrophage model, which is animal-friendly; the invention is simple and fast to operate, and has a high accuracy rate for judging food allergies. It can be applied to evaluate whether small molecule allergens are allergens. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the effect of erythritol on the proliferation of Raw264.7 macrophages;

[0014] Figure 2 Neutral red was used to detect the effects of different treatments on the pinocytosis rate of Raw264.7 macrophages;

[0015] Figure 3 Schematic diagram of gating flow cytometry for detecting the effects of different treatments on the phagocytic rate of Raw264.7 macrophages;

[0016] Figure 4 Flow cytometry schematic diagram for detecting the effects of different treatments on the phagocytic rate of Raw264.7 macrophages;

[0017] Figure 5 Flow cytometry was used to detect the effects of different treatments on the phagocytic rate of Raw264.7 macrophages;

[0018] Figure 6 qPCR was used to detect the effect of erythritol Ery on the expression of related genes in mouse Raw264.7-M1 cells;

[0019] Figure 7 qPCR was used to detect the effect of erythritol Ery on the expression of related genes in mouse Raw264.7-M2 cells;

[0020] Figure 8 To detect the effect of D-mannose MAN2 on the expression of related genes (INOS) in mouse Raw264.7-M1 cells by qPCR;

[0021] Fig. 9 qPCR was used to detect the effect of D-mannose MAN2 on the expression of related genes (CD206) in mouse Raw264.7-M2 cells. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified. The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0023] The present invention constructs a cell model for studying small molecule allergens and its application. The following examples use erythritol (CAS: 149-32-6) and D-mannose (CAS: 3458-28-4) as small molecule allergens for illustration. It should be noted that other potential allergenic food-derived small molecules are also suitable for the Raw264.7 macrophage model.

[0024] Example 1: Construction of Raw264.7 macrophage model

[0025] After preparing the experimental equipment required for cell recovery, take the Raw264.7 macrophages out of the liquid nitrogen tank, shake them in a 37°C water bath for 1 minute to melt them, and then add them to the preheated DMEM high-glucose medium (containing 10% FBS, 100μg / mL streptomycin and 100U / mL penicillin) and mix them by blowing. After centrifugation, add new culture medium and mix them by blowing again. After inoculation in the culture bottle, place it in a 37°C, 5% CO2 incubator for 24 hours to observe the growth and replace the culture medium in time.

[0026] Raw264.7 macrophages in the logarithmic growth phase were inoculated in a 12-well plate at a density of 2×105 / mL and cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100U / mL penicillin, 100ug / mL streptomycin). The temperature was controlled at 37°C and the CO2 concentration was 5%. After 12 hours, the pretreatment was administered and grouped for culture. The specific groups are as follows:

[0027] 1. Blank control group: stage 1 (DMEM culture medium adherence culture for 12 hours) + stage 2 (PBS culture for 12 hours) + stage 3 (PBS culture for 24 hours);

[0028] 2. Erythritol-Ery induction group: stage 1 (DMEM culture medium adherence culture for 12 hours) + stage 2 (1mM Ery culture for 12 hours) + stage 3 (PBS culture for 24 hours)

[0029] 3. LPS-induced group: stage 1 (DMEM culture medium adherence for 12 h) + stage 2 (PBS culture for 12 h) + stage 3 (100 ng / mL LPS culture for 24 h);

[0030] 4. LPS & Ery combined induction group: stage 1 (DMEM culture medium adherence for 12 hours) + stage 2 (1mM Ery culture for 12 hours) + stage 3 (100ng / mL LPS culture for 24 hours);

[0031] 5. IL-4 induction group: stage 1 (DMEM culture medium adherence for 12 hours) + stage 2 (PBS culture for 12 hours) + stage 3 (20 ng / mL IL-4 culture for 24 hours);

[0032] 6. IL-4 & Ery combined induction group: stage 1 (DMEM culture medium adherence culture for 12 hours) + stage 2 (1mM Ery culture for 12 hours) + stage 3 (20ng / mL IL-4 culture for 24 hours);

[0033] The cell morphology was observed and photographed using an inverted microscope, and the cells and culture supernatant were collected for subsequent experiments.

[0034] Example 2: CCK-8 method to measure the survival rate of Raw264.7 macrophages

[0035] Take cells in logarithmic phase and adjust the concentration to 2×105 / mL to inoculate into 96-well plates. After the cells adhere to the wall, gently remove the culture medium. Only add the culture medium as the blank control well (Control). Add 100μL of erythritol samples with a concentration gradient of 1, 4, 16, and 64mM to each well for culture. After the 96-well plate is placed in an incubator for 24h, remove the culture medium and add 100μL of DMEM culture medium containing 10% CCK-8 reagent. After about 1h, place it in an ELISA reader to measure the absorbance value at 450nm. The cell survival rate is calculated according to the following formula:

[0036]

[0037] The results are as follows Figure 1 As shown, Figure 1Compared with the control group, there was no significant difference in the cell viability of Raw264.7 macrophages treated with 1mM Ery, that is, after treating Raw cells with 1mM Ery, the effect on cell viability was not significant. Later, 1mM erythritol was selected to treat Raw264.7 macrophages.

[0038] Example 3: Allergen detection by cell pinocytosis and phagocytosis rate

[0039] Determination of macrophage phagocytosis rate: FITC-labeled dextran was used to measure the phagocytosis rate of cells. The cells were inoculated into a 12-well cell culture plate (500 μL / well) at a density of 2×105 / mL and cultured in a cell culture incubator for 24 hours. Then, 50 μL of samples of different concentrations as well as blank controls (PBS buffer) and positive controls (LPS) were added to each well according to Example 3. The supernatant was discarded, washed with PBS, digested with trypsin, centrifuged at 300g for 5 minutes, resuspended with 1mL PBS, and 100 μL / well was spread on a 96-well plate. The cells were incubated in a 37°C constant temperature incubator for 3 hours, and 20 μL of neutral red was added to each well; the cells were continued to be cultured in a 37°C constant temperature incubator for 3 hours; the cells were taken out, washed with PBS 3 times, and 200 μL of cell lysis solution (glacial acetic acid: anhydrous ethanol = 1:1) was added, and the cells were gently shaken for 15 seconds; the cells were placed at room temperature for 2 hours until the cells were completely lysed, and the absorbance value was measured at a wavelength of 520 nm.

[0040] Determination of macrophage phagocytosis rate: FITC-labeled dextran was used to measure the phagocytosis rate of cells. The cells were inoculated into 12-well cell culture plates (500 μL / well) at a density of 2×105 / mL and cultured in a cell culture incubator for 24 hours. Then, 50 μL of samples of different concentrations as well as blank controls (PBS buffer) and positive controls (LPS) were added to each well according to Example 3. After incubation for 24 hours, the supernatant was aspirated, and 500 μL of 1 mg / mL FITC-labeled dextran solution was added to each well and incubated for 1 hour. Cold PBS (2% FBS) was added to terminate the reaction. Subsequently, the cells were washed 3 times with PBS, digested with trypsin, washed once with PBS by centrifugation, and then resuspended in PBS. MFI was detected by flow cytometry. The phagocytosis rate of the cells was calculated according to the following formula:

[0041]

[0042] Neutral red and FITC-labeled dextran were used to detect the effects of different treatments on the phagocytic rate of Raw264.7 macrophages. Figure 2 As shown, secondly, the results of FITC-labeled dextran detection were determined by flow cytometry and microplate reader to measure the fluorescence intensity. Figure 2 Flow cytometry was performed by gating to find FITC-positive cells. Figure 2The subsequent samples were tested by gating. The results are as follows Figure 4 As shown; the fluorescence intensity of FITC was detected by an enzyme marker, and the results were as follows Figure 5 As shown. The results of the above neutral red and FITC-labeled dextran detection methods showed that the phagocytic rate of cells in the LPS and Ery combined induction group was significantly decreased compared with the LPS group. This shows that erythritol can affect the macrophage endocytic activity of Raw264.7 macrophages, affect the intake of allergens, and thus affect allergies.

[0043] Example 4: Allergen detection by qPCR

[0044] The cells were seeded into a 12-well cell culture plate (500 μL / well) at a density of 2×105 cells / mL and cultured in a cell culture incubator for 24 h. Each well was then cultured in groups according to Example 1. M1 macrophages (iNOS, MHCⅡ, TNF-α, IL-1β), M2 macrophages (CD206, TGF-β).

[0045] Extract RNA from cells according to the instructions of the Total RNA extraction kit, and detect RNA concentration using a nucleic acid protein concentration meter. The test results show that OD260 / OD280 is around 1.8-2.0. The extracted RNA should be immediately reverse transcribed to synthesize cDNA.

[0046] According to the instructions of the 1st Strand cDNA Synthesis reverse transcription kit, reverse transcription and synthesis of cDNA were performed as follows: ① Take 6μL of 5×gDNA digester Mix into a PCR tube, add an appropriate amount of RNA solution (final concentration is 50ng / μL) according to the measured concentration, and add RNase free ddH2O to make up to 30μL. Place the PCR tube in a PCR instrument and set the program: 42℃, 2min, to react to digest and remove the impurity DNA in the RNA solution. ② Add 10μL of 4× III SuperMix plus, placed in a PCR instrument, set the reverse transcription program: 25℃, 5min; 55℃, 15min; 85℃, 5min, and react. The reaction solution is cDNA, which is stored in a -80℃ refrigerator.

[0047] RT-qPCR was performed according to the instructions of the qPCR SYBR Green Master Mix kit, and the relative expression of the target gene was calculated according to the 2-ΔΔCt method. The primer sequences designed for the qPCR reaction are shown in Table 1.

[0048] Table 1: qPCR upstream and downstream primer sequences

[0049]

[0050] Quantitative real-time polymerase chain reaction (qPCR) technology was used to evaluate the effect of erythritol Ery on the expression of M1 / M2 polarization-related genes in mouse Raw264.7 macrophages. Figure 6 and Figure 7 As shown, Figure 6 and Figure 7 The results showed that erythritol Ery could regulate the phenotypic transformation of Raw264.7 macrophages and induce the phenotype of Raw264.7 macrophages to polarize toward M2 cells. In view of the potential role of M2 macrophages in food allergic reactions, the results suggested that erythritol Ery may be involved in or affect the mechanism of allergic reactions and have allergenicity.

[0051] Example 5: Allergen detection by qPCR

[0052] The cells were seeded into a 12-well cell culture plate (500 μL / well) at a density of 2×105 cells / mL and cultured in a cell culture incubator for 24 hours. Each well was then cultured in groups as follows to detect the gene expression of M1 macrophages (iNOS) and M2 macrophages (CD206).

[0053] 1. Blank control group: stage 1 (DMEM culture medium adherence culture for 12 hours) + stage 2 (PBS culture for 12 hours) + stage 3 (PBS culture for 24 hours);

[0054] 2. D-mannose MAN2 induction group: stage 1 (DMEM culture medium adherence for 12 h) + stage 2 (2 mM MAN2 culture for 12 h) + stage 3 (PBS culture for 24 h);

[0055] 3. LPS-induced group: stage 1 (DMEM culture medium adherence for 12 h) + stage 2 (PBS culture for 12 h) + stage 3 (100 ng / mL LPS culture for 24 h);

[0056] 4. LPS & MAN2 combined induction group: stage 1 (DMEM culture medium adherence for 12 hours) + stage 2 (2 mM MAN2 culture for 12 hours) + stage 3 (100 ng / mL LPS culture for 24 hours);

[0057] 5. IL-4 induction group: stage 1 (DMEM culture medium adherence for 12 hours) + stage 2 (PBS culture for 12 hours) + stage 3 (20 ng / mL IL-4 culture for 24 hours);

[0058] 6. IL-4 & MAN2 combined induction group: stage 1 (DMEM culture medium adherence for 12 hours) + stage 2 (2 mM MAN2 culture for 12 hours) + stage 3 (20 ng / mL IL-4 culture for 24 hours);

[0059] Extract RNA from cells according to the instructions of the Total RNA extraction kit, and use a nucleic acid protein concentration meter to detect RNA concentration, and the test results show that OD260 / OD280 is around 1.8 to 2.0. The extracted RNA should be immediately reverse transcribed to synthesize cDNA.

[0060] According to the instructions of the 1st Strand cDNA Synthesis reverse transcription kit, reverse transcription and synthesis of cDNA were performed as follows: ① Take 6μL of 5×gDNA digester Mix into a PCR tube, add an appropriate amount of RNA solution (final concentration is 50ng / μL) according to the measured concentration, and add RNase free ddH2O to make up to 30μL. Place the PCR tube in a PCR instrument and set the program: 42℃, 2min, to react to digest and remove the impurity DNA in the RNA solution. ② Add 10μL of 4× III SuperMix plus, placed in a PCR instrument, set the reverse transcription program: 25℃, 5min; 55℃, 15min; 85℃, 5min, and react. The reaction solution is cDNA, which is stored in a -80℃ refrigerator.

[0061] RT-qPCR was performed according to the instructions of the qPCR SYBR Green Master Mix kit. The relative expression of the target gene was calculated according to the 2-ΔΔCt method. The primer sequences designed for the qPCR reaction are shown in Table 2 below.

[0062] Table 2: qPCR upstream and downstream primer sequences

[0063]

[0064] Quantitative real-time polymerase chain reaction (qPCR) was used to evaluate the effect of D-mannose on the expression of M1 / M2 polarization-related genes in mouse Raw264.7 macrophages. Figure 8 and Fig. 9 As shown, Figure 8 and Fig. 9The results showed that D-mannose could regulate the phenotypic conversion of Raw264.7 macrophages and induce the phenotype of Raw264.7 macrophages to polarize toward M2 cells. In view of the potential role of M2 macrophages in food allergic reactions, the results suggested that D-mannose may be involved in or affect the mechanism of allergic reactions and has allergenicity, indicating the application prospects of the above-established cell model for small molecule allergenicity detection.

[0065] It should be understood that the specific embodiments described above are only used to explain the present application and are not used to limit the present application. Based on the embodiments provided in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application. In addition, it can also be understood that although the efforts made in this development process may be complex and lengthy, for ordinary technicians in the field related to the contents disclosed in the present application, some changes such as design, manufacturing or production based on the technical contents disclosed in the present application are only conventional technical means, and should not be understood as the contents disclosed in the present application are insufficient.

[0066] Reference to "embodiments" in this application means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those of ordinary skill in the art that the embodiments described in this application may be combined with other embodiments without conflict.

Claims

1. A small molecule allergen cell model for detecting potential allergenic foodborne small molecules, characterized in that: The cells are Raw264.7 macrophages. The above-mentioned small molecule allergens can regulate the polarization of Raw264.7 macrophages by changing the gene expression of Raw264.7 macrophages to regulate the expression level of M1 / M2-related mRNA in Raw264.7 macrophages, and affect allergic reactions by increasing the expression level of M2-related mRNA.

2. A small molecule allergen cell model for detecting potential allergenic foodborne small molecules according to claim 1, characterized in that: The small molecule allergen is erythritol or D-mannose.

3. Application of the small molecule allergen cell model for detecting potential allergenic foodborne small molecules according to claim 1 in exploring the sensitization mechanism of small molecule allergens, characterized in that: It is used to evaluate whether small molecules are allergens using Raw264.7 macrophages.

4. The use of a small molecule allergen cell model for detecting potential allergenic foodborne small molecules according to claim 3 in exploring the sensitization mechanism of small molecule allergens, characterized in that: The small molecule allergens are used to affect the phagocytosis of Raw264.7 macrophages.

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