A method and application for detecting goblet cell-associated antigen channels in the piglet intestine.

Abstract

This invention discloses a method and application for detecting goblet cell-related antigen channels in the piglet intestine. It relates to the field of antigen detection technology. The method includes the following steps: taking a portion of the mid-jejunum from a piglet; processing it; cutting the processed mid-jejunum into small pieces; incubating the pieces in a culture medium containing a marker; fixing; dehydrating and clearing the fixed pieces; embedding them in paraffin; preparing tissue sections; staining the tissue sections with a rabbit antibody against the goblet cell marker protein mucin 2 and a goat anti-rabbit fluorescent secondary antibody; mounting with the nuclear dye DAPI; and observing and analyzing under a fluorescence microscope. The method provided by this invention is simple to operate and can successfully detect goblet cell-related antigen channels in weaned piglets, laying the foundation for subsequent pig nutrition formulations.

Description

Technical Field

[0001] This invention relates to the field of antigen detection technology, and more specifically to a method and application for detecting goblet cell-associated antigen channels in the piglet intestine. Background Technology

[0002] Goblet cells are secretory cells in the intestinal mucosal epithelium. They secrete mucins to form a mucus layer, constituting the host's first line of defense against pathogens and toxins. Recent studies have found that goblet cells can present intestinal contents (such as food antigens and microorganisms) to lamina propria immune cells via goblet cell-associated antigen channels, directly participating in mucosal immune regulation. This phenomenon plays a crucial role in maintaining intestinal immune homeostasis and inducing immune tolerance or immune responses, and is essential for maintaining intestinal homeostasis. Furthermore, during lactation, beneficial bacteria in the gut can translocate to extraintestinal tissues via goblet cell-associated antigen channels, also playing a protective role in the body's health.

[0003] Weaned piglets often suffer from intestinal diseases such as diarrhea due to their underdeveloped intestinal immune system, which greatly endangers the healthy development of the pig industry. Identifying the existence of goblet cell-associated antigen (GAT) channels in piglets could provide an important target for protecting their intestinal health. However, current detection methods for GAT channels are limited to human and mouse intestinal tissues; a detection method for GAT channels in piglets has not yet been established.

[0004] Therefore, the detection method and application of goblet cell-associated antigen channels in the piglet intestine is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method and application for detecting goblet cell-associated antigen channels in the piglet intestine.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for detecting goblet cell-associated antigen channels in the piglet intestine, comprising the following steps:

[0008] (1) Take the middle section of the jejunum of piglets;

[0009] (2) Process the middle section;

[0010] (3) Cut the processed middle section into small pieces;

[0011] (4) Incubate small pieces in DMEM low-glucose medium containing the marker;

[0012] (5) After the small pieces have been incubated, fix them in place;

[0013] (6) Dehydration and transparency of the fixed small pieces;

[0014] (7) Paraffin embedding treatment;

[0015] (8) Prepare tissue sections;

[0016] (9) Tissue sections were stained with rabbit-derived antibody against goblet cell marker protein mucin 2 and goat anti-rabbit fluorescent secondary antibody;

[0017] (10) Mounting with the nuclear dye DAPI;

[0018] (11) Observe and analyze under a fluorescence microscope.

[0019] Preferred: Step (2) treatment: Remove the mesentery, cut the intestinal segment longitudinally, and rinse with sterile PBS;

[0020] Step (3) Small piece: 4-5mm × 4-5mm;

[0021] Step (4) The markers were 10% fetal bovine serum and 10 mg / mL tetramethylrhodamine; incubation conditions: 37°C cell culture incubator, 1 hour;

[0022] Step (5) uses a 4% paraformaldehyde solution for fixation, and the fixation time is 24 hours;

[0023] Step (6) Dehydration process: Soak in 75%, 85%, 95% and 100% alcohol in sequence; Transparency process: Soak in xylene twice, 10 minutes each time.

[0024] Step (8) The slices are 4 μm thick and dried at 37 °C.

[0025] Preferred: Step (11) Observation and analysis:

[0026] If mucin 2 and dextran staining are present in the same cell, it indicates that the goblet cell-associated antigen channel in the piglet intestine has been successfully detected;

[0027] If, during the uptake phase, dextran fluorescence is mainly located in the upper part of the cell; during the completed uptake phase, dextran fluorescence is mainly located in the lower part of the cell; and during the presentation phase, dextran fluorescence is mainly located in the lamina propria immune cells adjacent to goblet cells, then it indicates that the goblet cell-associated antigen channel in the piglet intestine mediates the process of goblet cell uptake and presentation of intestinal antigens.

[0028] The present invention also provides the application of any of the above methods in the preparation of swine nutrient products.

[0029] Preferred: Nutrient: Regulates goblet cell-associated antigen pathways in piglets, improving intestinal health in piglets during pig production.

[0030] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method and application for detecting goblet cell-associated antigen channels in the piglet intestine, and the technical effects achieved are as follows:

[0031] 1. The method provided by this invention is simple to operate;

[0032] 2. The method of the present invention can successfully detect goblet cell-associated antigen channels in weaned piglets.

[0033] 3. This lays the foundation for subsequent in vitro screening of nutrients that may affect porcine goblet cell-related antigen channels, and utilizes these nutrients to regulate porcine goblet cell-related antigen channels, thereby improving the intestinal health of piglets during pig production. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1 The attached figure is a fluorescence detection diagram of the goblet cell-associated antigen channel in piglets provided by the present invention, in which cells showing positive results for both dextran and mucin 2 are observed; white * indicates goblet cells containing the goblet cell-associated antigen channel.

[0036] Figure 2 The attached figure shows the fluorescence detection of goblet cell-related antigen channels in piglets at different stages provided by the present invention. In the uptake stage, dextran staining is mainly located in the upper part of the goblet cells; in the uptake completion stage, dextran staining is mainly located in the lower part of the goblet cells; and in the presentation stage, dextran staining is mainly located in the lamina propria immune cells adjacent to the goblet cells.

[0037] Figure 3 The attached figure is a fluorescence detection diagram of the goblet cell-associated antigen channel in piglets provided by the present invention. In the figure, the green fluorescence of the goblet cells has completed the uptake of dextran in the intestinal lumen and presented it to the immune cells below. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] This invention discloses a method and application for detecting goblet cell-associated antigen channels in the piglet intestine.

[0040] In this example, the DMEM low-glucose culture medium was obtained from Sigma-Aldrich, fetal bovine serum was obtained from Gibco, the rabbit antibody for mucin 2 (immunogen amino acid sequence: MYLEAGDVVVRQEERCVCRDGRLHCRQIRLIGQSCTAPKIHMDCSNLTALATSKPRALSCQTLAAGYYHTECVSGCVCPDGLMDDGRGGCVVEKECPCVHNNDLYSSGAKIKVDCNTCTCKRGRWV, as shown in SEQ ID No. 1) was obtained from Proteintech, and the goat anti-rabbit fluorescent secondary antibody, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, was used. TM 488 and tetramethylrhodamine-labeled dextran were obtained from Invitrogen, and the DAPI-containing mounting solution was obtained from Beyotime Biotechnology. All other unmentioned raw materials were commercially available; all unmentioned methods were conventional methods.

[0041] Example 1

[0042] A method for detecting goblet cell-associated antigen channels in the piglet intestine, comprising the following steps:

[0043] (1) (Select 7kg weaned piglets, fast for 10 hours, euthanize and open the abdominal cavity) Take 5cm of the middle part of the jejunum of the weaned piglets, place it in sterile PBS at 37℃, and immediately transfer it to the ultra-clean workbench.

[0044] (2) Treatment of the middle section: cut off the mesentery, cut the intestinal segment longitudinally, and gently rinse the intestinal segment with 37°C sterile PBS;

[0045] (3) Cut the intestinal segment into 5mm×5mm pieces and place them in a 48-well cell culture plate;

[0046] (4) Add 500 μl of DMEM low-glucose medium (containing 10% fetal bovine serum and 10 mg / mL tetramethylrhodamine-labeled dextran) to the culture wells (in the culture plate) containing intestinal tissue blocks, and place in a 37°C cell culture incubator;

[0047] (5) After 1 hour, the intestinal tissue block was removed and fixed by soaking in 4% paraformaldehyde;

[0048] (6) After fixing for 24 hours, the intestinal tissue blocks were soaked in 75%, 85%, 95%, and 100% alcohol in sequence to dehydrate them. Then, the intestinal tissue was soaked in xylene twice for 10 minutes each time to make it transparent.

[0049] (7) Place the intestinal tissue block in melted paraffin. After the paraffin has completely soaked the intestinal tissue block, clamp the intestinal tissue block into the embedding frame for cooling and embedding.

[0050] (8) Place the embedded wax block on a microtome, slice it to a thickness of 4μm, flatten the slice on a glass slide, and dry it in an oven at 37℃.

[0051] (9) Using rabbit-derived antibodies against goblet cell marker protein mucin 2 and goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488) Stain the sections;

[0052] (10) Place 100 μL of mounting solution containing the nuclear dye DAPI on a glass slide and mount it;

[0053] (11) Observe the blue fluorescence (DAPI), green fluorescence (mucin 2), and red fluorescence (dextran) under a fluorescence microscope and take pictures;

[0054] The results are as follows Figure 1 As shown, transepithelial dextran staining was observed in the small intestinal epithelium of piglets, indicating that the intestinal epithelium can absorb dextran from the intestinal lumen. Further analysis using mucin 2 staining revealed that some mucin 2 and dextran staining occurred within the same cell (white *), indicating successful detection of the goblet cell-associated antigen channel in the piglet intestine. Some dextran staining also occurred independently of mucin 2 (white arrows), suggesting the presence of other cells in the piglet intestinal epithelium that also absorb intestinal dextran.

[0055] In addition, such as Figure 2 As shown, different stages of goblet cell uptake of dextran via the goblet cell-associated antigen (GAA) channel were observed in the small intestine of piglets. During the uptake phase, dextran fluorescence was mainly located in the upper part of the cell; during the completion of uptake, dextran fluorescence was mainly located in the lower part of the cell; and during the presentation phase, dextran fluorescence was mainly located in the lamina propria immune cells adjacent to the goblet cells. These findings indicate that the goblet cell-associated antigen channel in the piglet intestine mediates the uptake and presentation of intestinal antigens by goblet cells.

[0056] Example 2

[0057] A method for detecting goblet cell-associated antigen channels in the piglet intestine, comprising the following steps:

[0058] (1) Take a 2cm section of the middle part of the jejunum of weaned piglets, place it in sterile PBS at 37℃, and immediately transfer it to a clean bench.

[0059] (2) Treatment of the middle section: cut off the mesentery, cut the intestinal segment longitudinally, and gently rinse the intestinal segment with 37°C sterile PBS;

[0060] (3) Cut the intestinal segment into 4mm×4mm pieces and place them in a 48-well cell culture plate;

[0061] (4) Add 500 μl of DMEM low-glucose medium (containing 10% fetal bovine serum and 10 mg / mL tetramethylrhodamine-labeled dextran) to the culture well containing the intestinal tissue block, and place it in a 37°C cell culture incubator;

[0062] (5) After 1 hour, the intestinal tissue block was removed and fixed by soaking in 4% paraformaldehyde;

[0063] (6) After fixing for 24 hours, the intestinal tissue blocks were soaked in 75%, 85%, 95%, and 100% alcohol in sequence to dehydrate them. Then, the intestinal tissue was soaked in xylene twice for 10 minutes each time to make it transparent.

[0064] (7) Place the intestinal tissue block in melted paraffin. After the paraffin has completely soaked the intestinal tissue block, clamp the intestinal tissue block into the embedding frame for cooling and embedding.

[0065] (8) Place the embedded wax block on a microtome, slice it to a thickness of 4μm, flatten the slice on a glass slide, and dry it in an oven at 37℃.

[0066] (9) Using rabbit-derived antibodies against goblet cell marker protein mucin 2 and goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488) Stain the sections;

[0067] (10) Place 100 μL of mounting solution containing the nuclear dye DAPI on a glass slide and mount it;

[0068] (11) Observe the blue fluorescence (DAPI), green fluorescence (mucin 2) and red fluorescence (dextran) under a fluorescence microscope and take pictures.

[0069] The results are as follows Figure 3 As shown, goblet cells labeled with mucin 2 (green fluorescence) were found in the small intestinal epithelium of piglets to take up dextran (red fluorescence) and present it to two adjacent lamina propria immune cells (two red fluorescence sites below).

[0070] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0071] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting goblet cell-associated antigen channels in the piglet intestine, characterized in that, Includes the following steps: (1) Take the middle section of the jejunum of piglets; (2) Process the middle section; (3) Cut the processed middle section into small pieces; (4) Incubate the small pieces in DMEM low-glucose medium containing the marker; (5) After the small pieces have been incubated, fix them in place; (6) Dehydration and transparency of the fixed small pieces; (7) Paraffin embedding treatment; (8) Prepare tissue sections; (9) Tissue sections were stained with rabbit-derived antibody against goblet cell marker protein mucin 2 and goat anti-rabbit fluorescent secondary antibody; (10) Mounting with the nuclear dye DAPI; (11) Observe and analyze under a fluorescence microscope; The markers in step (4) are 10% fetal bovine serum and 10 mg / mL tetramethylrhodamine-labeled dextran; Step (11) Observation and analysis: If mucin 2 and dextran staining are present in the same cell, it indicates that the goblet cell-associated antigen channel in the piglet intestine has been successfully detected; If, during the uptake phase, dextran fluorescence is mainly located in the upper part of the cell; during the completed uptake phase, dextran fluorescence is mainly located in the lower part of the cell; and during the presentation phase, dextran fluorescence is mainly located in the lamina propria immune cells adjacent to goblet cells, then it indicates that the goblet cell-associated antigen channel in the piglet intestine mediates the process of goblet cell uptake and presentation of intestinal antigens.

2. The method as described in claim 1, characterized in that: The treatment described in step (2) is as follows: remove the mesentery, cut the intestinal segment longitudinally, and rinse with sterile PBS; The small piece mentioned in step (3) is 4~5mm × 4~5mm; The incubation conditions described in step (4) are: 37°C cell culture incubator, 1 hour; The fixation in step (5) uses a 4% paraformaldehyde solution and the fixation time is 24 hours; The dehydration process in step (6) involves soaking the food in 75%, 85%, 95%, and 100% alcohol in sequence; the transparency process involves soaking the food in xylene twice, for 10 minutes each time. Step (8) The slices are 4 μm thick and dried at 37 °C.

3. The application of the method according to any one of claims 1 to 2 in the preparation of swine nutrient solutions.

4. The application as described in claim 3, characterized in that, The nutrient: regulates the goblet cell-associated antigen pathway in piglets, improving the intestinal health of piglets during pig production.

Images