Preparation method and application of chicken small intestine epithelial cell apoptosis vesicles

Abstract

The invention discloses a preparation method of chicken small intestine epithelial cell apoptosis vesicles. The preparation method comprises the following steps: culturing chicken small intestine epithelial cells; inducing cell apoptosis; and preparing the apoptosis vesicles. The invention also discloses the chicken small intestine epithelial cell apoptosis vesicle prepared by the method and application thereof. The chicken small intestine epithelial cell apoptosis vesicle can load chicoric acid, and has the effects of resisting inflammation, enhancing cell oxidation resistance and promoting chicken small intestine epithelial cell proliferation and cell migration.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to apoptotic vesicles of chicken small intestinal epithelial cells that can be used as biological drugs, their preparation method, and their applications. Background Technology

[0002] Chicken small intestinal epithelial cells are the main cell type in the epithelial layer of the small intestinal mucosa and play a crucial role in the physiological processes of digestion, absorption, and immunity in chickens. Chicken small intestinal epithelial cells participate in physiological functions such as digestion, absorption, and secretion.

[0003] Cichoric acid is a naturally occurring organic compound, belonging to the polyphenol class, and is mainly found in plants such as chicory, echinacea, and dandelion. Cichoric acid possesses immunomodulatory, anti-inflammatory, hyaluronidase-inhibiting, and antiviral properties, and can be developed into drugs for use in the pharmaceutical field, or used as an antioxidant in the food and cosmetic industries.

[0004] The gut, as the primary site of nutrient digestion and absorption in chickens, and a crucial barrier against pathogenic microorganisms, plays a vital role in maintaining homeostasis. While chicoric acid possesses potential health benefits such as anti-inflammatory and antioxidant properties, its direct application is limited by its unstable chemical properties, including easy decomposition upon heating or exposure to light, poor membrane permeability, low oral bioavailability, easy degradation in the stomach, and poor intestinal absorption.

[0005] Apoptotic vesicles are small membrane-bound vesicles released during apoptosis, exhibiting good biocompatibility and tissue repair capabilities. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing a method for effectively preparing apoptotic vesicles from chicken small intestinal epithelial cells.

[0007] To achieve the above-mentioned objectives, this invention provides a method for preparing apoptotic vesicles from chicken small intestinal epithelial cells, comprising the following steps: S1. Culture chicken small intestinal epithelial cells; S2. Induces apoptosis; S3. Preparation of apoptotic vesicles.

[0008] Preferably, step S1 includes: tissue sampling and digestion, cell cluster enrichment, and 3D cell sphere culture.

[0009] Preferably, the tissue sampling and digestion steps are as follows: Healthy chicken embryos aged 18-20 days were selected, and small intestinal tissue was isolated under aseptic conditions. The intestines were thoroughly rinsed with pre-cooled phosphate-buffered saline to remove contents, and then the small intestinal tissue was minced into pieces of approximately 1-2 mm. 3 Small pieces; tissue fragments were placed in 1-10 mg / mL collagenase II and digested with shaking at 35-37℃ and 100-300 rpm for 20-50 minutes; Preferably, the cell cluster enrichment step is as follows: After digestion, the digestate was filtered through a sterile 100 µm pore size cell sieve to remove undigested large tissue fragments; the filtrate was collected and then filtered through a 40 µm pore size cell sieve; the cell components retained on the 40 µm sieve were retained and collected, and the collected 40-100 µm cell clusters were resuspended in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and washed by centrifugation at 300×g for 3-10 minutes; Preferably, the 3D cell spheroid culture steps are as follows: The centrifuged cell pellet was resuspended in complete culture medium; cell clusters were counted and the concentration was adjusted so that each cell cluster was equivalent to 2 × 10⁶ cells in the original isolate. 5 Single cells were seeded at a density of [number]; the culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 6 days, during which stable 3D small intestinal epithelial cell spheroids formed. Preferably, the complete culture medium was DMEM / F12 basal medium, supplemented with 2% B27, 10 ng / mL epidermal growth factor, and 10 ng / mL basic fibroblast growth factor.

[0010] Step S2 includes: after culturing for 6 days, adding chicoric acid aqueous solution to the cells in the 3D spheroid culture to make the final concentration of chicoric acid in the culture medium 100-300 µM, and continuing to culture for 18-24 hours. Preferably, step S3 includes: sample pretreatment and vesicle release; fractional centrifugation to remove impurities; vesicle purification and concentration; and density gradient fine separation.

[0011] Preferably, the sample pretreatment and vesicle release include: After chicoric acid-induced apoptosis, the culture supernatant containing 3D cell spheroids and the cell spheroids were collected and placed on ice. Pulsed sonication was performed under ice bath conditions at 80-100 W power: 2 seconds on, 1 second off, for 3-5 minutes to fully lyse the cells and release intracellular vesicles.

[0012] Preferably, the step-by-step centrifugation to remove impurities includes: The ultrasonic lysis buffer was first centrifuged at 300×g for 5-10 minutes at 4℃ to remove intact cells and large fragments from the precipitate. The supernatant was collected, transferred to a new tube, and centrifuged again at 2000×g for 5-10 minutes at 4℃ to remove the precipitate containing cell debris, retaining the vesicle-rich supernatant.

[0013] Preferably, the vesicle purification and concentration includes: The collected supernatant was injected into a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed in PBS at a volume ratio greater than 1:1000 at 4°C for 12-36 hours to remove small molecule contaminants and salt ions. After dialysis, the sample was transferred to a 100 kDa ultrafiltration centrifuge tube and concentrated to the final volume at 4000×g at 4°C.

[0014] Preferably, the fine separation of the density gradient includes: Add 1-2 mL of vesicle separation reagent, slowly stack 1-2 mL of concentrated vesicle sample to avoid interface disturbance, centrifuge at 16000×g for 30-60 minutes at 4℃; after centrifugation, a milky white vesicle layer is seen at the gradient interface, which is the apoptotic vesicle suspension.

[0015] On the other hand, the present invention also provides apoptotic vesicles of chicken small intestinal epithelial cells prepared according to the above method.

[0016] Preferably, the apoptotic vesicles of the chicken small intestinal epithelial cells are rich in chicoric acid. More preferably, the chicoric acid content in the apoptotic vesicles is 13.7-16.7 μg / mg protein.

[0017] Preferably, the diameter of the apoptotic vesicles in the chicken small intestinal epithelial cells is 50 to 500 nm.

[0018] On the other hand, the present invention also provides the application of the apoptotic vesicles of chicken small intestinal epithelial cells in the preparation of anti-inflammatory drugs.

[0019] On the other hand, the present invention also provides the application of the aforementioned apoptotic vesicles of chicken small intestinal epithelial cells in the preparation of drugs that enhance the antioxidant capacity of chicken small intestinal epithelial cells.

[0020] On the other hand, the present invention also provides the application of the aforementioned apoptotic vesicles of chicken small intestinal epithelial cells in the preparation of a drug that promotes the proliferation of chicken small intestinal epithelial cells.

[0021] On the other hand, the present invention also provides the application of the aforementioned chicken small intestinal epithelial cell apoptotic vesicles in the preparation of a drug that promotes the migration of chicken small intestinal epithelial cells.

[0022] Compared with existing technologies, the method of this invention is simple and easy to operate. The apoptotic vesicles of chicken small intestinal epithelial cells prepared are rich in chicoric acid, which has anti-inflammatory effects, enhances the antioxidant capacity of cells, and promotes the proliferation and migration of chicken small intestinal epithelial cells. The apoptotic vesicles of this invention loaded with chicoric acid have good biocompatibility, significantly improve the bioavailability and drug stability of chicoric acid, and promote the absorption of chicoric acid. This overcomes the limitations of chicoric acid in existing technologies and provides a promising solution for its effective delivery to intestinal targets. It can be used as a biological drug to promote the health of the chicken digestive tract and promote the breeding of superior chickens. Attached Figure Description

[0023] Figure 1 The morphology of chicken small intestinal epithelial cells is shown. (A) Chicken small intestinal epithelial cells in spheroidal culture; scale bar: 100 μm; (B) Morphology of chicken small intestinal epithelial cells induced with apoptosis; scale bar: 100 μm.

[0024] Figure 2 The results of apoptotic vesicle identification are shown. (A) Transmission electron microscopy observation results, scale bar: 100 μm; (B) Particle size determination results; (C) Western blot results.

[0025] Figure 3 The anti-inflammatory effects of apoptotic vesicles were demonstrated. (A) Anti-inflammatory effect of TNF-α; (B) Anti-inflammatory effect of IL-6.

[0026] Figure 4 The antioxidant effect of apoptotic vesicles was demonstrated.

[0027] Figure 5 The cell proliferation-promoting effect of apoptotic vesicles was demonstrated.

[0028] Figure 6 The cell migration-promoting effect of apoptotic vesicles is shown. (A) Crystal violet staining results, scale bar: 100 μm; (B) Number of migrating cells. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] 1. Research Methods and Materials 1.1 Experimental Materials 1.1.1 Laboratory Animals Chicken embryos aged 18-20 days were selected and purchased from the Nanhai Poultry Farm. They were raised in an environment that met the standards for the management of laboratory animals and underwent acclimatization before the experiment.

[0031] 1.1.2 Main Reagents DMEM / F12 medium (Gibco), fetal bovine serum (FBS, BI), 0.25% trypsin-EDTA solution (Hyclone), chicoric acid (purity ≥98%, Sigma), phosphate-buffered saline (PBS, pH 7.4, Solarbio), lipopolysaccharide (LPS, Sigma), hydrogen peroxide (H2O2, Aladdin), superoxide dismutase (SOD) assay kit, glutathione peroxidase (GSH-Px) assay kit, malondialdehyde (MDA) assay kit (Nanjing Jiancheng Biotechnology Institute), CCK-8 assay kit (Dojindo), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) ELISA assay kit (R&DSystems), TSG101 antibody, Alix antibody, β-Tubulin antibody (Cell Signaling Technology), goat anti-mouse / rabbit secondary antibody (Jackson ImmunoResearch).

[0032] 1.1.3 Main Instruments CO2 cell culture incubator (Thermo Fisher Scientific), benchtop high-speed centrifuge (Eppendorf), transmission electron microscope (TEM, Hitachi), nanoparticle size and potential analyzer (Malvern), multi-functional microplate reader (BioTek), inverted phase contrast microscope (Olympus), Western blot electrophoresis and membrane transfer equipment (Bio-Rad).

[0033] 1.2 Experimental Methods 1.2.1 Isolation, Culture and Processing of Chicken Small Intestinal Epithelial Cell Clusters Tissue sampling and digestion: Healthy chicken embryos aged 18-20 days were selected, and small intestinal tissue was isolated under aseptic conditions. The intestine was thoroughly rinsed with pre-cooled phosphate-buffered saline (PBS) to remove contents, and then the small intestinal tissue was minced into pieces of approximately 1-2 mm. 3 Small pieces of tissue were placed in 1 mg / mL collagenase II (dissolved in PBS or Hanks balanced salt solution containing calcium and magnesium ions) and digested by shaking at 37°C and 200 rpm for 35 minutes.

[0034] Cell cluster enrichment: After digestion, the digestate was filtered through a sterile 100 µm pore size cell sieve to remove undigested large tissue fragments. The filtrate was collected and then filtered through a 40 µm pore size cell sieve. The cellular components retained on the 40 µm sieve were retained and collected; this portion mainly consisted of cell clusters (crypt structures) with a diameter ranging from 40 to 100 µm. The collected 40–100 µm cell clusters were resuspended in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and washed by centrifugation at 300 × g for 5 minutes.

[0035] 3D cell spheroid culture: The centrifuged cell pellet was resuspended in complete culture medium (DMEM / F12 basal medium supplemented with 2% B27, 10 ng / mL epidermal growth factor EGF, and 10 ng / mL basic fibroblast growth factor bFGF). Cell clusters were counted and the concentration was adjusted so that each cell cluster was equivalent to 2 × 10⁶ cells in the original isolate. 5 Single cells were seeded at a density in ultra-low adsorption 96-well plates. The culture plates were incubated at 37°C, 5% CO2, and saturated humidity for 6 days, during which stable 3D small intestinal epithelial cell spheroids were formed.

[0036] Apoptosis induction treatment: After 6 days of culture, chicoric acid aqueous solution was added to the 3D spheroidized cells to bring the final concentration of chicoric acid in the culture medium to 300 µM. The cells were cultured for another 24 hours, while an untreated control group (with an equal volume of PBS) was also included. The culture plates were returned to the incubator and cultured for another 24 hours. Cells and supernatant were then collected for subsequent analysis.

[0037] 1.2.2 Preparation of apoptotic vesicles Sample pretreatment and vesicle release: After chicoric acid-induced apoptosis (300 µM, 24 h), the culture supernatant containing 3D cell spheroids and the cell spheroids were collected and placed on ice. The cells were thoroughly lysed and intracellular vesicles were released using a pulsed ultrasonic lysing device (titanium alloy probe) under ice bath conditions (100 W power: 2 s on, 1 s off, for 5 minutes).

[0038] Graded centrifugation to remove impurities: The ultrasonic lysis buffer was first centrifuged at 300×g for 10 minutes (4℃) to remove intact cells and large fragments from the precipitate. The supernatant was collected, transferred to a new tube, and centrifuged again at 2000×g for 10 minutes (4℃) to remove the precipitate containing cell debris and retain the supernatant rich in vesicles.

[0039] Vesicle purification and concentration: The collected supernatant (approximately 5 mL) was injected into a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed in PBS at a ratio of ≥1:1000 (v / v) for 24 hours at 4°C to remove small molecule contaminants and salt ions. After dialysis, the sample was transferred to a 100 kDa ultrafiltration centrifuge tube and concentrated to a final volume of 0.5 mL at 4,000 × g at 4°C.

[0040] Fine separation of density gradients: Add 2 mL of vesicle separation reagent (Takara, Capturem EV Kit) to the bottom of a 15 mL ultracentrifuge tube, and slowly stack 1 mL of concentrated vesicle sample on top to avoid interfacial disturbance. Centrifuge at the lowest acceleration setting (setting 1) for 60 minutes (4℃). After centrifugation, a milky white vesicle layer will be visible at the gradient interface. Use an 18G blunt-tipped needle and syringe to accurately aspirate this layer.

[0041] Vesicle collection and preservation: The collected apoptotic vesicle suspension can be used directly in downstream experiments or stored for a short period in PBS at 4°C; for long-term storage, it needs to be aliquoted to -80°C.

[0042] 1.2.3 Detection and identification of apoptotic vesicles Transmission electron microscopy (TEM) observation: 20 μL of apoptotic vesicle suspension was dropped onto a copper grid, left to stand at room temperature for 10 minutes, negatively stained with 2% phosphotungstic acid for 5 minutes, and allowed to air dry naturally. The morphology, size and structural characteristics of the apoptotic vesicles were then observed and photographed under a transmission electron microscope to record the morphology, size and structural characteristics of the apoptotic vesicles.

[0043] Particle size analysis: Apoptotic vesicles were diluted 200 times with PBS, and their particle size distribution and zeta potential were determined using a nanoparticle size and potential analyzer. Each sample was measured three times, and the average value was taken.

[0044] Western blot detection of specific proteins: Total protein was extracted from apoptotic vesicles, and protein concentration was determined using the BCA method. 30 μg of protein sample was subjected to SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. TSG101 antibody (1:1000) and Cleaved Caspase-3 antibody (1:1000) were added, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed three times with TBST for 10 minutes each time, and the corresponding goat anti-mouse / rabbit secondary antibody (1:5000) was added, followed by incubation at room temperature for 1 hour. After washing the membrane three more times with TBST, chemiluminescence was used for color development, and the results were observed and analyzed using a gel imaging system.

[0045] Chicoric acid content determination: The chicoric acid content in apoptotic vesicles was determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (25:75, v / v); flow rate: 1.0 mL / min; detection wavelength: 326 nm; column temperature: 30℃; injection volume: 10 μL. Standard curves were plotted using chicoric acid standards of different concentrations, and the chicoric acid content in apoptotic vesicles was calculated based on the peak area of ​​the samples.

[0046] 1.2.4 Study on the function of apoptotic vesicles Anti-inflammatory experiment: An inflammation model was established using LPS stimulation of chicken small intestinal epithelial cells. Cells were divided into a control group, an LPS model group, and an apoptotic vesicle treatment group (with added prepared apoptotic vesicles). The LPS model group and the apoptotic vesicle treatment group were stimulated with 1 μg / mL LPS for 6 hours, followed by further culture of the apoptotic vesicle treatment group with added apoptotic vesicles for 24 hours. Cell culture supernatants were collected, and the levels of TNF-α and IL-6 were detected using an ELISA kit.

[0047] Antioxidant experiment: An oxidative stress model was constructed by treating cells with 100 μM H2O2. Cells were divided into a control group, an H2O2 model group, and an apoptotic vesicle treatment group. Cells in the H2O2 model group and the apoptotic vesicle treatment group were stimulated with H2O2 for 2 hours, followed by the addition of apoptotic vesicles to the apoptotic vesicle treatment group for 24 hours of further culture. Cells were collected, and SOD and GSH-Px activities were measured according to the kit instructions.

[0048] Cell proliferation experiment: Cell proliferation was detected using the CCK-8 assay. Chicken small intestinal epithelial cells were seeded into 96-well plates and divided into a control group and treatment groups with different concentrations of apoptotic vesicles (0.1, 1, and 10 μg / mL), with 6 replicates per group. After culturing for 24, 48, and 72 hours, 10 μL of CCK-8 solution was added to each well, and the cells were cultured for another 2 hours. The absorbance (OD) at 450 nm was measured using a microplate reader, and the cell proliferation rate was calculated.

[0049] Cell migration experiment: Cell migration assays were performed using Transwell chambers (8.0 μm pore size). The lower chamber was filled with culture medium containing 10% FBS and apoptotic vesicles (1 μg / mL), and the upper chamber was seeded with 5 × 10⁶ cells / mL. 4 Chicken small intestinal epithelial cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The chambers were removed, unmigrated cells in the upper chamber were wiped away, and the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Five fields of view were randomly selected under a microscope to count the number of migrating cells.

[0050] 2. Experimental Results 2.1 Observation of chicken small intestinal epithelial cell morphology In the primary culture stage, cells grow in a polygonal, adherent manner with uniform morphology. After passage and 3D spheroidization culture, cells aggregate into a tightly packed spherical structure. After induction with 300 μM chicoric acid for 24 hours, the 3D cell spheroid structure appears loose under a microscope, with some cells becoming rounded and shrunken, and cell membrane budding and apoptotic body formation observed. Figure 1 (A and B in the text).

[0051] 2.2 Identification of Apoptotic Vesicles Transmission electron microscopy (TEM) observation revealed that the prepared apoptotic vesicles were round or oval, relatively uniform in size, with a diameter ranging from approximately 50 to 500 nm, and exhibited a typical double-membrane structure. Figure 2 (A in the middle).

[0052] Particle size analysis: Using a nanoparticle size and potential analyzer, the average particle size of apoptotic vesicles was determined to be 180.5 ± 20.3 nm, the polydispersity index (PDI) was 0.25 ± 0.03, and the zeta potential was -18.5 ± 3.2 mV, indicating that the vesicle particle size distribution was relatively concentrated and had a certain degree of stability. Figure 2 (B in the middle).

[0053] Biomarker detection: Western blot results showed that TSG101 and Cleaved Caspase-3 were specifically expressed in apoptotic vesicles, proving that the prepared vesicles were of apoptotic origin. Figure 2 (C in the middle).

[0054] Chicoric acid content determination: High performance liquid chromatography analysis showed that the chicoric acid content in apoptotic vesicles was 15.2±1.5 μg / mg protein, confirming that apoptotic vesicles carried chicoric acid.

[0055] 2.3 Results of apoptotic vesicle function study Anti-inflammatory effects: Compared with the control group, the levels of TNF-α and IL-6 in the cell culture supernatant of the LPS model group were significantly increased (P<0.01); compared with the LPS model group, the TNF-α level in the apoptotic vesicle treatment group (LPS+CA-apoV) decreased from 125.3±10.2 pg / mL to 45.6±5.8 pg / mL. Figure 3 In A), the IL-6 level decreased from 98.7±8.5 pg / mL to 32.1±4.2 pg / mL. Figure 3 The difference between the two values ​​(B) was statistically significant (P<0.01), indicating that apoptotic vesicles can effectively inhibit the inflammatory response.

[0056] Antioxidant effect: Compared with the control group, the intracellular SOD activity in the H2O2 model group decreased from 120.5±12.3 U / mg prot to 45.6±6.7 U / mg prot, and the GSH-Px activity decreased from 85.2±9.1 U / mg prot to 28.3±4.5 U / mg prot (P<0.01). Compared with the H2O2 model group, the SOD activity in the apoptotic vesicle treatment group (H2O2+CA-apoV) increased to 98.7±10.3 U / mg prot, and the GSH-Px activity increased to 65.4±8.2 U / mg prot, with significant differences (P<0.01), indicating that apoptotic vesicles can enhance the antioxidant capacity of cells. Figure 4 ).

[0057] Cell proliferation promotion effect: The CCK-8 assay results showed that, compared with the control group, the OD values ​​of cells in the 10 μg / mL and 50 μg / mL apoptotic vesicle treatment groups were significantly increased at 48 and 72 hours (P<0.05). At 72 hours, the cell proliferation rate of the 10 μg / mL treatment group increased by 28%, and the cell proliferation rate of the 50 μg / mL treatment group increased by 42%, indicating that apoptotic vesicles can promote the proliferation of chicken small intestinal epithelial cells. Figure 5 ).

[0058] Promotes cell migration: Cell migration assays showed that, compared with the control group (120.3±15.6 migrating cells), the apoptotic vesicle treatment group (CA-apoV) had 276.5±22.3 migrating cells, a significant increase (P<0.01), approximately 2.3 times that of the control group, indicating that apoptotic vesicles can effectively promote cell migration. Figure 6 (A and B in the text).

[0059] The apoptotic vesicles loaded with chicoric acid of the present invention have good biocompatibility, significantly improve the bioavailability and drug stability of chicoric acid, and promote the absorption of chicoric acid. This overcomes the limitations of chicoric acid in the prior art and provides a very promising solution for its effective delivery to intestinal targets.

Claims

1. A method for preparing apoptotic vesicles from chicken small intestinal epithelial cells, comprising the following steps: S1. Culture chicken small intestinal epithelial cells; S2. Induces apoptosis; S3. Preparation of apoptotic vesicles; Step S1 includes: tissue sampling and digestion, cell cluster enrichment, and 3D cell sphere culture. The tissue sampling and digestion steps are as follows: Healthy chicken embryos aged 18-20 days were selected, and small intestinal tissue was isolated under aseptic conditions. The intestines were thoroughly rinsed with pre-cooled phosphate-buffered saline to remove contents, and then the small intestinal tissue was minced into pieces of approximately 1-2 mm. 3 Small pieces; tissue fragments were placed in 1-10 mg / mL collagenase II and digested with shaking at 35-37℃ and 100-300 rpm for 20-50 minutes; The cell cluster enrichment steps are as follows: After digestion, the digestate was filtered through a sterile 100 µm pore size cell sieve to remove undigested large tissue fragments; the filtrate was collected and then filtered through a 40 µm pore size cell sieve; the cell components retained on the 40 µm sieve were retained and collected, and the collected 40-100 µm cell clusters were resuspended in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and washed by centrifugation at 300×g for 3-10 minutes; The 3D cell spheroid culture steps are as follows: The centrifuged cell pellet was resuspended in complete culture medium; cell clusters were counted and the concentration was adjusted so that each cell cluster was equivalent to 2 × 10⁶ cells in the original isolate. 5 Single cells were seeded at a density of 1000 mg / mL; the culture plate was placed in an incubator at 37°C, 5% CO2, and saturated humidity for 6 days, during which stable 3D small intestinal epithelial cell spheroids were formed; the complete culture medium was DMEM / F12 basal medium, supplemented with 2% B27, 10 ng / mL epidermal growth factor and 10 ng / mL basic fibroblast growth factor. Step S2 includes: after culturing for 6 days, adding chicoric acid aqueous solution to the cells in the 3D spheroid culture to make the final concentration of chicoric acid in the culture medium 100-300 µM, and continuing to culture for 18-24 hours. Step S3 includes: sample pretreatment and vesicle release; fractional centrifugation to remove impurities; vesicle purification and concentration; and density gradient fine separation.

2. The method according to claim 1, characterized in that, The sample pretreatment and vesicle release include: After chicoric acid-induced apoptosis, the culture supernatant containing 3D cell spheroids and the cell spheroids were collected and placed on ice. Pulsed sonication was performed under ice bath conditions at 80-100 W power: 2 seconds on, 1 second off, for 3-5 minutes to fully lyse the cells and release intracellular vesicles.

3. The method according to claim 1, characterized in that, The step-by-step centrifugation process for removing impurities includes: The ultrasonic lysis buffer was first centrifuged at 300×g for 5-10 minutes at 4℃ to remove intact cells and large fragments from the precipitate. The supernatant was collected, transferred to a new tube, and centrifuged again at 2000×g for 5-10 minutes at 4℃ to remove the precipitate containing cell debris, retaining the vesicle-rich supernatant.

4. The method according to claim 1, characterized in that, The vesicle purification and concentration include: The collected supernatant was injected into a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed in PBS at a volume ratio greater than 1:1000 at 4°C for 12-36 hours to remove small molecule contaminants and salt ions. After dialysis, the sample was transferred to a 100 kDa ultrafiltration centrifuge tube and concentrated to the final volume at 4000×g at 4°C.

5. The method according to claim 1, characterized in that, The fine separation of the density gradient includes: Add 1-2 mL of vesicle separation reagent, slowly stack 1-2 mL of concentrated vesicle sample to avoid interface disturbance, centrifuge at 16000×g for 30-60 minutes at 4℃; after centrifugation, a milky white vesicle layer is seen at the gradient interface, which is the apoptotic vesicle suspension.

6. Apoptotic vesicles of chicken small intestinal epithelial cells prepared by the method according to any one of claims 1 to 5.

7. The apoptotic vesicles of chicken small intestinal epithelial cells according to claim 6, characterized in that, The apoptotic vesicles of the chicken small intestinal epithelial cells were rich in chicoric acid.

8. The apoptotic vesicles of chicken small intestinal epithelial cells according to claim 6, wherein the diameter of the apoptotic vesicles of chicken small intestinal epithelial cells is 50 to 500 nm.

9. The use of apoptotic vesicles of chicken small intestinal epithelial cells as described in any one of claims 6 to 8 in the preparation of any one of the following drugs: anti-inflammatory drugs; drugs that enhance the antioxidant capacity of chicken small intestinal epithelial cells; drugs that promote the proliferation of chicken small intestinal epithelial cells; drugs that promote the migration of chicken small intestinal epithelial cells.

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