Piglet weaning diarrhea in-vitro multi-cell co-culture model as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to a piglet weaning diarrhea in-vitro multicellular co-culture model as well as a preparation method and application thereof, and the piglet weaning diarrhea in-vitro multicellular co-culture model provided by the invention is obtained by co-culturing small intestine endothelial cells and IPEC-J2 cells and inducing through an inflammatory factor inducer. The piglet weaning diarrhea in-vitro multi-cell co-culture model provided by the invention solves the problems of incomprehensiveness of a single-cell model and heterogeneity and heterogeneity of an existing model, establishes a cell model closer to the physiological status of intestinal tracts, and is of great significance to research on piglet weaning diarrhea.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an in vitro multi-cell co-culture model of piglet weaning diarrhea, and a preparation method and application thereof. Background Art

[0002] Weaned piglet diarrhea is one of the most common and serious diseases in weaned piglets, and the mortality rate accounts for 50% to 70% of the total mortality rate in pig production. Due to the significant side effects of antibiotics, finding a drug or feed additive to prevent and control diarrhea has become a top priority in the management of weaned piglets. Establishing a suitable in vitro model is the key to screening and preventing diarrhea in weaned piglets and studying the disease.

[0003] The current multi-cell co-culture models mainly include: human colon adenocarcinoma cell line Caco-2, rat small intestinal crypt epithelial cell line IEC-6 / 17 / 18, human colon adenocarcinoma cell line HT-29, porcine small intestinal epithelial cell IPEC-J2, etc. However, these multi-cell co-culture models have problems such as differences in species sources and cannot better approximate the in vitro model of diarrhea in weaned piglets.

[0004] At present, the in vitro research model for piglet diarrhea is mainly porcine intestinal epithelial cells IPEC-J2. IPEC-J2 is a non-transformed cell line isolated from the ileum of newborn piglets. It can form a tight monolayer membrane and is therefore often used as a model of intestinal mucosal barrier. However, due to its limited secretory function, it cannot fully simulate the physiological function of intestinal cells. Summary of the invention

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: The invention provides an in vitro multi-cell co-culture model for piglet weaning diarrhea. The preparation method of the in vitro multi-cell co-culture model for piglet weaning diarrhea comprises the following steps: firstly inoculating and culturing IPEC-J2 cells in an upper chamber of a Transwell plate, then inoculating and culturing primary porcine small intestinal endothelial cells in a lower chamber of the Transwell plate, when the upper IPEC-J2 cells form a stable monolayer membrane structure and the fusion degree of the lower porcine small intestinal endothelial cells reaches 80% to 90%, adding 0.1 μg / ml to 1 μg / ml of lipopolysaccharide to the complete culture medium of the lower chamber for induction for 10 h to 12 h, so as to obtain the in vitro multi-cell co-culture model for piglet weaning diarrhea.

[0006] The present invention is the first to use primary porcine intestinal endothelial cells for in vitro culture and modeling, and the first to use porcine epithelial cells and primary endothelial cells for co-culture and establish an in vitro model of piglet weaning diarrhea. The model effect is evaluated by measuring the resistance of the upper epithelial cells with a monolayer membrane resistor, stimulating the lower endothelial cells with lipopolysaccharide solution, detecting the resistance of the epithelial cell monolayer and the concentration of inflammatory factors in the upper chamber culture medium.

[0007] Furthermore, the cell seeding density is 1×10 5 pcs / hole~5×10 5 Pieces / hole.

[0008] Furthermore, the IPEC-J2 cells are cultured for 15 to 19 days.

[0009] Furthermore, the time for culturing primary porcine small intestinal endothelial cells is 21 to 22 days.

[0010] Furthermore, the complete culture medium is a high-glucose DMEM solution containing fetal bovine serum, a penicillin-streptomycin mixture, and endothelial cell growth factor.

[0011] Furthermore, in the complete culture medium, serum accounts for 10% to 15% of the volume of the high-glucose DMEM solution, the penicillin-streptomycin mixture accounts for 1% to 2% of the volume of the high-glucose DMEM solution, and the endothelial cell growth factor accounts for 1% to 5% of the mass of the high-glucose DMEM solution.

[0012] The present invention also provides application of an in vitro multi-cell co-culture model for weaning diarrhea in piglets in screening drugs for treating weaning diarrhea in piglets.

[0013] The present invention has the following beneficial effects: The multi-cell co-culture model of piglet weaning diarrhea provided by the present invention solves the incompleteness of single-cell models and the heterogeneity problem of existing models, and establishes a cell model that is closer to the physiological state of the intestine, which is of great significance for the study of piglet weaning diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 TEER diagram of cell transmembrane resistance values ​​for single cell culture model and in vitro multi-cell co-culture model of piglet weaning diarrhea.

[0015] Figure 2 This is a graph showing the expression of TNF-α in a single cell culture model and an in vitro multi-cell co-culture model of piglet weaning diarrhea under lipopolysaccharide induction. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0017] Example 1: Establishment of an in vitro multi-cell co-culture model for piglet weaning diarrhea.

[0018] 1. Culture of porcine small intestinal microvascular endothelial cells.

[0019] 1. Preparation of common liquids.

[0020] Washing solution I is a PBS buffer containing a penicillin-streptomycin mixture, wherein the penicillin-streptomycin mixture accounts for 5% of the volume of PBS. The penicillin-streptomycin mixture was purchased from Beijing Solebow Technology Co., Ltd., with a product number of P1400.

[0021] Washing solution II is a PBS buffer containing a penicillin-streptomycin mixture, wherein the penicillin-streptomycin mixture accounts for 2% of the volume of PBS.

[0022] Wash solution III is a PBS buffer containing a penicillin-streptomycin mixture, wherein the penicillin-streptomycin mixture accounts for 1% of the volume of PBS.

[0023] The complete culture medium for primary porcine small intestinal endothelial cells PIMVEC is a high-glucose DMEM solution containing fetal bovine serum, a penicillin-streptomycin mixture, and endothelial cell growth factor, wherein the fetal bovine serum accounts for 15% of the volume of the high-glucose DMEM solution, the penicillin-streptomycin mixture accounts for 1% of the volume of the high-glucose DMEM solution, and the endothelial cell growth factor accounts for 5% of the mass of the high-glucose DMEM solution. The solution is filtered through a 0.22 μm filter and stored at 4°C.

[0024] 0.05% pancreatin solution is a PBS buffer containing pancreatin, wherein the pancreatin accounts for 0.05% of the volume of PBS.

[0025] Collagenase solution: Dissolve 1g of type II collagenase in 50ml of PBS, mix thoroughly to make a 2% collagenase solution, aliquot and store at -20℃. Dissolve 1g of type IV collagenase in 50ml of PBS, mix thoroughly to make a 2% collagenase solution, aliquot and store at -20℃. Dilute 10 times with PBS before use.

[0026] 2. Obtaining primary cells.

[0027] (1) Take 1 cm of ileum from a 2-month-old piglet and transfer it to the clean bench.

[0028] (2) Place the tissue in pre-cooled washing solution I, rinse the contents thoroughly, then cut the intestine longitudinally to expose the intestinal mucosa. Wash thoroughly three times until the intestinal contents are clean.

[0029] (3) Place the intestinal tube in 0.05% pancreatic enzyme solution and gently shake it for 3 minutes.

[0030] (4) After 0.05% trypsin digestion, wash with wash solution II three times and use ophthalmic scissors to cut the tissue into pieces that can pass smoothly through the 1 mL pipette tip.

[0031] (5) Collect the liquid containing tissue fragments into a 10 mL centrifuge tube, pipette 50 times, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 5 mL of washing solution II, shake upside down and continue centrifugation until the supernatant is clear and discard the supernatant.

[0032] (6) Add a 0.2% mixed solution of type II collagenase and type IV collagenase into the tissue, mix well by blowing moderately, and place in a 37°C incubator for digestion for 8 minutes.

[0033] (7) Use moderate blowing to quickly pass the liquid through a 400-mesh sieve. Collect the filtered liquid and centrifuge it at 1200 rpm for 3 minutes.

[0034] (8) Discard the supernatant and resuspend the pellet in complete culture medium. Count the cells with a cell counter and calculate the number of cells to 5 × 10 5 / hole ~1×10 6 The cells were inoculated at a density of 100 / well in a six-well plate.

[0035] (9) After 12 hours, discard the culture medium, gently wash the wells with Wash Solution III, and add fresh complete culture medium. Change the medium every two days.

[0036] 3. Passaging of primary endothelial cells.

[0037] When primary PIMVECs grow to 90% confluence, wash twice with wash solution III. Add trypsin solution and let stand in the cell culture incubator for 5 minutes. Discard trypsin and add complete medium, collect cells and centrifuge, resuspend with complete medium and inoculate in a new cell culture plate. After 4 hours, aspirate the medium and replace with new complete medium.

[0038] 2. Culture of IPEC-J2 cells.

[0039] 1. Liquid preparation.

[0040] IPEC-J2 culture medium is a high-glucose DMEM solution containing fetal bovine serum, a penicillin-streptomycin mixture, and endothelial cell growth factor, wherein fetal bovine serum accounts for 10% of the volume of the high-glucose DMEM solution, the penicillin-streptomycin mixture accounts for 1% of the volume of the high-glucose DMEM solution, and the non-essential amino acid mixture accounts for 1% of the mass of the high-glucose DMEM solution. The culture medium is filtered through a 0.22 μm filter and stored at 4°C.

[0041] 0.1% pancreatin solution is a PBS buffer containing pancreatin, wherein the pancreatin accounts for 0.1% of the volume of PBS.

[0042] 2. Cell recovery.

[0043] IPEC-J2 cells were purchased from ATCC and stored in a liquid nitrogen tank in the laboratory. Before use, take out the cryotube, quickly place it in a 37°C water bath, and gently shake to thaw. After thawing, centrifuge at 1200rpm for 3 minutes, discard the supernatant, resuspend the cells in IPEC-J2 medium and inoculate them in a cell culture bottle. Change the medium after 24 hours.

[0044] 3. Cell passaging.

[0045] When the cells grow to 90%, discard the culture medium, wash twice with PBS, add 0.1% trypsin solution, and let it stand in the cell culture incubator for 3 minutes. After digestion, discard the trypsin solution, add IPEC-J2 culture medium, gently blow the cells, collect the liquid and centrifuge at 1200rpm for 3 minutes. Resuspend the cells and inoculate them in a new cell culture plate.

[0046] 3. Establishment of an in vitro multi-cell co-culture model for piglet weaning diarrhea.

[0047] IPEC-J2 cells of passage 20 were cultured at 2×10 5 The cells were inoculated at a density of 100 / well in the upper chamber of a 6-well Transwell plate, 1.6 ml of complete culture medium was added to the upper chamber, and 2.5 ml of complete culture medium was added to the lower chamber, and the medium was changed every two days.

[0048] On the 18th day of culture, primary cultured PIMVEC cells were cultured at 5 × 10 5 The cells were seeded at a density of 100 / well in the lower chamber of a 6-well Transwell plate, and the medium was changed every two days.

[0049] On the 21st day of culture, the upper epithelial cells formed a stable monolayer membrane structure, and the lower endothelial cells grew to 80%-90% fusion. At this time, lipopolysaccharide was added to the lower chamber, and 1 μg of lipopolysaccharide was added to each 1 ml of complete culture medium to establish an in vitro multicellular co-culture model of piglet weaning diarrhea.

[0050] 4. Testing of relevant indicators.

[0051] 1. TEER detection of transmembrane electrical resistance of monolayer cells.

[0052] After endothelial cells are inoculated, the transmembrane resistance (TEER) of epithelial cells is measured. Three points in different directions are taken as repeats during the measurement. The calculation formula is TEER = (measured resistance value - blank value) × monolayer surface area.

[0053] 2. Detection of tumor necrosis factor TNF-α concentration in the upper chamber.

[0054] The upper culture medium was collected before and 12 hours after the addition of lipopolysaccharide to the lower chamber of the lipopolysaccharide stimulation group, while the control group did not add lipopolysaccharide. The TNF-α concentration of the upper culture medium was detected using the Solebow ELISA kit. The specific steps are as follows: (1) Centrifuge the culture medium at 3000 rpm for 5 minutes and collect the supernatant for testing.

[0055] (2) Add 100 μL of the standard and the sample to be tested into a 96-well plate pre-coated with an anti-IgG antibody that binds to TNF-α. Seal the plate and place it in a 37°C incubator for 90 minutes.

[0056] (3) Wash the plate three times and tap it. Add 100 μL of secondary IgG antibody to each well, seal the plate and place it in a 37°C incubator for 60 minutes.

[0057] (4) Wash the plate three times and tap it. Add 100 μL of HRP working solution to each well, seal the plate and place it in a 37°C incubator for 30 minutes.

[0058] (5) Wash the plate three times and tap it. Add 100 μL of the colorimetric substrate TMB to each well, seal the plate and place it in a 37°C incubator for 15 minutes.

[0059] (6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm using an ELISA reader.

[0060] (7) Draw a standard curve and calculate the TNF-α concentration of each sample.

[0061] 5. Analysis of experimental results.

[0062] like Figure 1 As shown, the transmembrane resistance value of the cell layer in the in vitro multi-cell co-culture model group of piglet weaning diarrhea was significantly higher than that in the single-cell culture group, indicating that the stability of the IPEC-J2 epithelial cell membrane in the in vitro multi-cell co-culture model group of piglet weaning diarrhea was higher than that in the single-cell culture group.

[0063] like Figure 2 As shown, under normal conditions without lipopolysaccharide induction, there was no significant difference in the concentration of TNF-α between the single cell culture model and the in vitro multi-cell co-culture model of piglet weaning diarrhea. However, under lipopolysaccharide induction, the concentration of TNF-α in the in vitro multi-cell co-culture model of piglet weaning diarrhea was significantly increased, indicating that the in vitro multi-cell co-culture model of piglet weaning diarrhea is easier to successfully induce an inflammatory model.

[0064] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0065] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. An in vitro multi-cell co-culture model for piglet weaning diarrhea, characterized in that: The method for preparing the in vitro multi-cell co-culture model of piglet weaning diarrhea comprises the following steps: firstly inoculating and culturing IPEC-J2 cells in the upper chamber of a Transwell plate, and then inoculating and culturing primary porcine small intestinal endothelial cells in the lower chamber of the Transwell plate; when the upper IPEC-J2 cells form a stable monolayer membrane structure and the fusion degree of the lower porcine small intestinal endothelial cells reaches 80% to 90%, 0.1 μg / ml to 1 μg / ml of lipopolysaccharide is added to the complete culture medium of the lower chamber for induction for 10 h to 12 h, so as to obtain the in vitro multi-cell co-culture model of piglet weaning diarrhea.

2. The in vitro multi-cell co-culture model of piglet weaning diarrhea according to claim 1, characterized in that: The cell seeding density was 1×10 5 pcs / hole~5×10 5 Pieces / hole.

3. The in vitro multi-cell co-culture model of piglet weaning diarrhea according to claim 1, characterized in that: The IPEC-J2 cells are cultured for 15 to 19 days.

4. The in vitro multi-cell co-culture model of piglet weaning diarrhea according to claim 1, characterized in that: The time for culturing primary porcine small intestinal endothelial cells is 21 to 22 days.

5. The in vitro multi-cell co-culture model of piglet weaning diarrhea according to claim 1, characterized in that: The complete culture medium is a high-glucose DMEM solution containing fetal bovine serum, a penicillin-streptomycin mixture, and endothelial cell growth factor.

6. The in vitro multi-cell co-culture model of piglet weaning diarrhea according to claim 5, characterized in that: In the complete culture medium, serum accounts for 10% to 15% of the volume of the high-glucose DMEM solution, the penicillin-streptomycin mixture accounts for 1% to 2% of the volume of the high-glucose DMEM solution, and the endothelial cell growth factor accounts for 1% to 5% of the mass of the high-glucose DMEM solution.

7. Use of the in vitro multi-cell co-culture model for weaning diarrhea in piglets according to claim 1 in screening drugs for treating weaning diarrhea in piglets.

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