Hydrogel-coated human amniotic epithelial stem cells and exosomes thereof for use in the preparation of a medicament for improving intestinal inflammation

Abstract

The application discloses application of a hydrogel-coated human amniotic epithelial stem cell and exosomes thereof to preparation of a drug for improving intestinal inflammation, and intestinal inflammation symptoms of a mouse are significantly reduced through anal injection, which is manifested in faster recovery of body weight, lower disease activity score, and significant reduction of intestinal permeability increase caused by UC. Intestinal epithelial function reconstruction is promoted, which is manifested in intestinal permeability improvement, intestinal epithelial integrity improvement, inflammation reaction reduction, intestinal epithelial tissue regeneration promotion, and apoptosis reduction. According to the above results, the human amniotic epithelial stem cell and the exosomes thereof can be applied to UC treatment in combination with a biomaterial. The method has a wide prospect in clinical application of UC treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of hydrogel-coated human amniotic epithelial stem cells and their exosomes in the preparation of drugs to improve enteritis. Background Technology

[0002] Inflammatory bowel disease (IBD) is a recurrent chronic digestive tract disease. Research suggests that immune-mediated intestinal damage is a crucial pathological basis for its development, involving potential factors such as genetics, environment, and microorganisms, and exhibiting a complex pathophysiological mechanism. Based on different clinicopathological manifestations, IBD is mainly divided into two subtypes: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is confined to the colon, with superficial mucosal inflammation extending proximally continuously, potentially leading to ulcers, massive bleeding, toxic megacolon, and fulminant colitis. Crohn's disease, on the other hand, can affect any part of the digestive tract, with lesions typically discontinuous, characterized by transmural inflammation, and can lead to complications such as fibrotic strictures, fistulas, and abscesses. In addition, patients often suffer from extraintestinal complications, including skin diseases (such as erythema nodosum and pyoderma gangrenosa), eye diseases (such as conjunctivitis and uveitis), rheumatic diseases (such as ankylosing spondylitis and peripheral arthritis), hepatobiliary diseases (such as primary sclerosing cholangitis), urinary system diseases (such as kidney stones), and perianal complications (such as abscesses, fistulas, and strictures).

[0003] Treatment for IBD varies depending on the severity and extent of the disease, ranging from topical anti-inflammatory drugs to surgical resection of the affected bowel segment. Since immune dysfunction is a key factor in the pathogenesis of IBD, immunosuppression is a cornerstone of treatment. Commonly used drugs include non-targeted therapies such as aminosalicylate, corticosteroids, and immunomodulators. Although their effectiveness is recognized, some patients do not respond well to treatment. These therapies often increase the risk of side effects, including opportunistic infections, immune disorders (such as psoriasis-like rashes), and malignancies. Another effective treatment for IBD is targeted biological therapy, currently commonly used in clinical practice include anti-TNF antibodies, anti-IL12 and anti-IL23 antibodies, and JAK inhibitors. However, due to their high cost, lower safety profile, and high rate of secondary loss of response, these therapies often impose significant economic costs on patients and the healthcare system as a whole.

[0004] In recent years, studies have shown that stem cell transplantation can promote the regeneration of damaged tissue structure and function, and more and more scholars have discovered the role of stem cells in the treatment of intestinal diseases. Currently, the stem cell types used in IBD clinical research are mainly hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCT reduces the abnormal inflammatory immune response in IBD patients by resetting the immune system. However, the risk of serious adverse events after HSCT is high, and large-scale clinical studies on its safety and long-term efficacy are still lacking. MSCs used to treat IBD are usually derived from allogeneic umbilical cord blood, autologous bone marrow, or adipose tissue. Although many research teams have conducted numerous preclinical and clinical studies, their clinical application remains limited due to cell availability and poor targeting.

[0005] Given the increasing prevalence of IBD and the aforementioned limitations of current medical treatments, there is still a need to find safe and effective new therapies for IBD. These therapies should consider both safety and effectiveness, as well as accessibility and cost-effectiveness.

[0006] The placenta is obstetric medical waste generated after the delivery of the fetus. As a vital organ for the exchange of substances between the fetus and mother, it consists of the amnion, chorion, and decidua. At the innermost part of the amnion is a layer of cells facing the amniotic cavity and surrounding the amniotic fluid, called human amniotic epithelial stem cells (hAESCs). hAESCs are homologous to embryonic stem cells, differentiating from the inner cell mass of the blastocyst on day 8 of fertilization, and possess strong differentiation capacity and plasticity. Studies have shown that hAESCs can secrete various immunomodulatory factors, anti-angiogenic proteins, and anti-inflammatory factors, exhibiting immunomodulatory capabilities. Furthermore, hAESCs are widely available, easily obtained, and have no ethical issues, exhibiting low immunogenicity (expressing HLA-E and HLA-G, but not β2-microglobulin or co-stimulatory factors). Based on these facts, we have innovatively developed a new approach to applying hAESCs in IBD treatment, realizing the reuse of waste biological resources. Meanwhile, because this treatment method is clearly targeted, safe, effective, and has no toxic side effects, it is closer to clinical translation and application. Summary of the Invention

[0007] The technical problem this invention aims to solve is as follows: This invention provides the application of hydrogel-coated human amniotic epithelial stem cells and their exosomes in the preparation of drugs to improve enteritis. This method uses human hydrogel-coated amniotic epithelial stem cells and their exosomes as a treatment method, administered via rectal injection to treat colitis. This addresses the following shortcomings: current drug treatments for colitis have limited efficacy, significant side effects, or are expensive; current cell therapies, administered intravenously, pose risks of pulmonary embolism and difficulty in targeting cells to the colon, thus affecting treatment efficacy.

[0008] To address the aforementioned technical challenges, hydrogel-coated human amniotic epithelial stem cells and their exosomes were prepared to improve their application in enteritis drugs.

[0009] The method for preparing hydrogel-coated human amniotic epithelial stem cells and their exosomes includes the following steps:

[0010] Primary human amniotic epithelial stem cells were isolated and expanded in a complete culture medium to obtain a complete culture medium containing human amniotic epithelial stem cells. The complete culture medium containing human amniotic epithelial stem cells was mixed with hydrogel material to prepare a hydrogel material cell mixture suspension. Hydrogel-coated human amniotic epithelial stem cells were obtained from the hydrogel material cell mixture suspension.

[0011] Alternatively, exosomes of human amniotic epithelial stem cells can be extracted from the culture supernatant of human amniotic epithelial stem cells.

[0012] Primary human amniotic epithelial stem cells were isolated, specifically including:

[0013] After washing the amniotic membrane tissue multiple times with a phosphate-balanced solution containing antibiotics, it was then subjected to enzymatic digestion and centrifugation to harvest primary human amniotic epithelial stem cells.

[0014] The human amniotic epithelial stem cells in the whole culture medium containing human amniotic epithelial stem cells are human amniotic epithelial stem cells of passage number less than P3.

[0015] The hydrogel material is a hydrogel modified with RGD cell adhesion peptides.

[0016] The hydrogel material described uses RGD hydrogel, model number TWG003, consists of hydrogel and diluent, with a volume ratio of hydrogel to diluent of 1:3.1 to 4.9.

[0017] The human amniotic epithelial stem cells in the hydrogel material cell suspension were (0.1–1.5) × 10⁻⁶. 7 / ml.

[0018] Furthermore, one of the technical solutions adopted by the present invention is: a method for isolating hAESCs from amniotic tissue of discarded placenta, the method comprising the following steps:

[0019] Step 1: Obtaining the amniotic membrane; After obtaining informed consent from the healthy mother undergoing cesarean section, the amniotic membrane is mechanically detached from the inner surface of the placenta and transferred to a biosafety cabinet;

[0020] Step 2: Isolation of hAESCs; wash away blood and other impurities from the amnion surface with washing solution, cut into small pieces, digest with trypsin, centrifuge to obtain cell pellet. Resuspend in culture medium and pass through a cell sieve;

[0021] Step 3: Culture of hAESCs; After cell counting, approximately 1×10⁷ cells were seeded into a 15cm culture dish and placed in a 37°C, 5% CO₂ cell culture incubator. After the cells adhered to the dish, the culture medium was changed every two days.

[0022] Step 4: Cryopreservation of hAESCs; After the cells have grown to confluence, digest them with trypsin, centrifuge and collect them, place them in cryovials, put them in a cryopreservation box and store them at -80 degrees Celsius, and transfer them to liquid nitrogen after 12 hours.

[0023] In one aspect of the present invention, the serological reactions of the parturient in step 1 of the above preparation method are all negative for HIV, syphilis, hepatitis A, hepatitis B and hepatitis C.

[0024] In one aspect of the present invention, step 1 of the above preparation method requires placing the amnion in a conical flask containing 50 ml of culture medium and transporting it at 4 degrees Celsius.

[0025] In one aspect of the present invention, in step 2 of the above preparation method, the washing solution is formulated as PBS buffer with added penicillin and streptomycin, and the entire amnion is rinsed three times with the washing solution.

[0026] In one aspect of the present invention, step 2 of the above preparation method includes a pre-digestion: adding 0.25% pancreatic enzyme and digesting in a 37°C water bath for 10 minutes; formal digestion: adding 0.25% pancreatic enzyme and digesting in a 37°C water bath for 30 minutes, shaking up and down 10 times every 10 minutes during the process; and terminating digestion: adding a digestion termination solution in a 1:1 ratio.

[0027] In one aspect of the present invention, the conditions for centrifuging and collecting cells in step 2 of the above preparation method are: at room temperature, centrifuge at 1000 rpm for 10 min, and then discard the supernatant.

[0028] To solve the above-mentioned technical problems, the second technical solution adopted by the present invention is: a method for treating colitis by using hydrogel-coated human amniotic epithelial stem cells, the method comprising the following steps:

[0029] Step 1: Resuscitation of hAESCs; Remove the cell cryopreservation tubes from liquid nitrogen, quickly place them in a 37°C water bath to thaw, and then centrifuge. Resuspend the cells in hAESC culture medium.

[0030] Step 2: Hydrogel coating of hAESCs; after cell counting, the hydrogel material was mixed with hAESC culture medium containing cells at a ratio of 4:1 to prepare a hydrogel-coated cell suspension, ultimately achieving a cell density of 1×10⁻⁶. 7 / ml.

[0031] Step 3: Anal injection of hAESCs; After anesthetizing the mice, approximately 200 μl of hydrogel-coated cell suspension was injected into the colon via the anus.

[0032] In one aspect of the present invention, step 2 of the above preparation method requires that the hydrogel material be placed at room temperature for equilibration beforehand.

[0033] In one aspect of the present invention, step 2 of the above preparation method requires mixing the hydrogel material with the cell-containing hAESCs culture medium and then gently blowing it 5-10 times.

[0034] In one aspect of the invention, step 3 of the above preparation method requires maintaining mouse anesthesia with 2% isopentane inhalation.

[0035] In one aspect of the present invention, step 3 of the above preparation method requires inserting a specially designed anal syringe 4 cm deep into the colon and slowly injecting the solution.

[0036] In one aspect of the present invention, after the anal injection in step 3 of the above preparation method, the anus needs to be glued with skin glue and broken open after 6 hours.

[0037] To solve the above-mentioned technical problems, the third technical solution adopted by the present invention is: a method for treating colitis using human amniotic epithelial stem cell exosomes, the method comprising the following steps:

[0038] Step 1: Extraction of exosomes from hAESCs culture supernatant; collect hAESCs culture supernatant and obtain exosomes by gradient centrifugation.

[0039] Step 2: Anal injection of hAESCs exosomes; after anesthetizing the mice, PBS containing exosomes was injected into the colon via the anus.

[0040] In one aspect of the present invention, step 1 of the above preparation method requires taking hAESCs in the logarithmic growth phase.

[0041] In one aspect of the present invention, the hAESCs culture system in step 1 of the above preparation method does not contain serum.

[0042] In one aspect of the present invention, step 1 of the above preparation method requires collecting the culture supernatant of hAESCs after 48 hours of culture.

[0043] In one aspect of the invention, step 2 of the above preparation method requires maintaining mouse anesthesia with 2% isopentane inhalation.

[0044] In one aspect of the present invention, step 2 of the above preparation method requires inserting a specially designed anal syringe 4 cm deep into the colon and slowly injecting the solution.

[0045] In one aspect of the present invention, after the anal injection in step 2 of the above preparation method, the anus needs to be glued with skin glue and broken open after 6 hours.

[0046] Compared with the prior art, the present invention has the following advantages:

[0047] (1) This invention is the first to use hydrogel-coated human amniotic epithelial stem cells and their exosomes for rectal injection to treat UC. This product has the following two advantages: ① Rectal injection avoids the risks of pulmonary embolism and immune rejection faced in the clinical application of cell therapy, thus improving the therapeutic effect; ② Hydrogel coating of human amniotic epithelial stem cells can help cells resist the harsh environment in the intestine, allowing them to reside in the intestine in a more active form and enhancing their therapeutic potential.

[0048] (2) Compared with oral administration and intravenous injection, colonic administration allows the active ingredients to act directly on intestinal epithelial cells, avoiding the low delivery efficiency caused by physiological processes such as gastrointestinal absorption and the first-pass effect of the liver, and reducing immune rejection. Compared with intravenous injection, rectal instillation requires less medical technology, and patients can perform it at home after simple training, making treatment convenient, saving medical resources, and showing good prospects for clinical translation. Attached Figure Description

[0049] Figure 1 : The binding morphology of hydrogel materials with hAESC and the characterization of hAESC exosomes

[0050] (A) Morphology of hAESC cytoskeleton coated with hydrogel;

[0051] (B) hAESC electron micrograph of the hydrogel coating;

[0052] (C) Proliferation of hAESCs coated with hydrogel;

[0053] (D) Electron micrograph of hAESC exosomes;

[0054] (E)hAESC exosome particle size analysis;

[0055] (F)hAESC exosome marker protein.

[0056] Figure 2 Hydrogel-coated hAESC anal injection promotes DSS-induced colonic epithelial remodeling.

[0057] (A) Flowchart of anal injection of hydrogel-coated hAESC for the treatment of DSS-induced UC in mice.

[0058] (B) Changes in body weight of mice in the treatment group and the control group;

[0059] (C) Clinical activity scores of mice in the treatment group and the control group;

[0060] (D) Colon morphology of mice in the treatment group and the control group;

[0061] (E) The ratio of colon length to body weight in mice in the treatment group and the control group;

[0062] (F) H&E staining of colon tissues from mice in the treatment and control groups;

[0063] (G) Pathological scores of colon tissues in mice in the treatment and control groups;

[0064] (H) By detecting the concentration of FITC-Dextran in serum, the intestinal permeability of mice in the treatment group and the control group was compared.

[0065] (I) mRNA expression levels of genes related to the tightness of intercellular connections between epithelial cells in the colonic epithelial tissue of mice in the treatment and control groups.

[0066] Figure 3 Hydrogel-coated hAESC rectal injection reduces DSS-induced colitis.

[0067] (A) Proliferation of intestinal epithelium in mice in the treatment group and the control group;

[0068] (B) mRNA expression levels of proliferation-related genes in the intestinal epithelial tissue of mice in the treatment and control groups;

[0069] (C) CD45 inflammatory cell infiltration in the epithelial tissues of mice in the treatment and control groups;

[0070] (D) mRNA expression levels of inflammation-related genes in the colon tissues of mice in the treatment and control groups;

[0071] (E) Expression of cleaved-caspase 3, an apoptosis-related gene, in the colon tissues of mice in the treatment and control groups;

[0072] (F) Apoptosis in the colon tissue of mice in the treatment and control groups (TUNEL staining);

[0073] (G) mRNA expression levels of apoptosis-related genes in the colonic epithelial tissue of mice in the treatment and control groups.

[0074] Figure 4 Rectal injection of human amniotic epithelial stem cell exosomes promotes recovery from DSS-induced colitis.

[0075] (A) Flowchart of anal injection of human amniotic epithelial stem cell exosomes for the treatment of DSS-induced UC in mice;

[0076] (B) Changes in body weight of mice in the treatment group and the control group;

[0077] (C) Survival rate of mice in the treatment group and the control group;

[0078] (D) Colon morphology of mice in the treatment group and the control group;

[0079] (E) The ratio of colon length to body weight in mice in the treatment group and the control group;

[0080] (F) H&E staining of colon tissues from mice in the treatment and control groups;

[0081] (G) Pathological scores of colon tissues in mice in the treatment and control groups;

[0082] (H) Proliferation of intestinal epithelium in mice in the treatment group and the control group;

[0083] (I) CD45 inflammatory cell infiltration in the epithelial tissues of mice in the treatment group and the control group. Detailed Implementation

[0084] Example 1: Preparation of hAESCs-related solutions

[0085] 1. Preparation of hAESCs culture medium: Add 15% (v / v) KSR, 1× non-essential amino acids, 1× L-glutamine, 1× sodium pyruvate, 1× antibiotics to DMEM / F12 medium, and add 10 ng / ml EGF before use.

[0086] 2. Preparation of cryopreservation solution for hAESCs: 90% FBS + 10% DMSO;

[0087] Example 2: Isolation and cryopreservation of hAESCs

[0088] 1. Obtaining the human amniotic membrane: With the authorization and consent of the mother undergoing cesarean section, placental tissue was taken from healthy mothers (serological tests for HIV, syphilis, hepatitis A, hepatitis B, and hepatitis C were all negative). The placenta was cross-cut, and the entire amniotic membrane was obtained through mechanical separation. The human amniotic membrane was placed in an Erlenmeyer flask containing 50 ml of culture medium and transported to the laboratory at 4 degrees Celsius.

[0089] 2. Isolation of hAESCs: Add antibiotics to 1×PSB buffer to obtain a washing buffer. Wash the amnion membrane three times with this buffer to remove blood and other impurities. Cut the amnion membrane into small pieces and transfer them to 50ml centrifuge tubes. Add 15ml of pre-incubated 0.25% trypsin, incubate at 37°C for 10 minutes, discard the digestion solution, and transfer the amnion membrane to another new 50ml centrifuge tube. Add 20ml of 0.25% trypsin to the new tube and digest at 37°C for 30 minutes, shaking up and down 5 times every 5 minutes. Stop the digestion with serum-free digestion stop solution, centrifuge at 1000rpm for 10 minutes, discard the supernatant, and resuspend the cells in 4ml of culture medium. Add 16ml of culture medium, mix well, and pass through 200-mesh and 400-mesh cell sieves to obtain hAESCs cell suspensions.

[0090] 3. Culture of hAESCs: After counting with a cell counting chamber, approximately 1×10⁶ cells were cultured. 7 One cell was seeded into a 15cm culture dish and placed in a 37°C, 5.5% CO2 cell culture incubator. After the cells adhered, the culture medium was changed. The culture medium was changed every two days.

[0091] 4. Cryopreservation of hAESCs: After the cells have grown to confluence, add 5 ml of 0.25% trypsin to each 15 cm culture dish and incubate at 37°C for about 7 minutes. When the cells become rounded and detach like quicksand when the culture dish is shaken, add serum-free digestion stop solution. Pipette the cells to form a single-cell suspension and transfer to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes at room temperature and discard the supernatant. Resuspend the cells in cryopreservation solution, collect them in cryovials, place them in a cryopreservation box, and store at -80°C. After 12 hours, transfer to liquid nitrogen for cryopreservation.

[0092] Example 3: Extraction of exosomes from human amniotic epithelial cell culture supernatant

[0093] 1. Take hAESCs in the logarithmic growth phase, seed them in 15cm culture dishes, and incubate them in a 37°C, 5.5% CO2 cell culture incubator.

[0094] 2. Once the cell density reaches 70%, discard the supernatant, wash the cells twice with 1×PBS buffer, add serum-free amniotic epithelial stem cell culture medium, and incubate in a 37°C, 5.5% CO2 cell culture incubator.

[0095] 3. After culturing for 48 hours, collect the cell culture supernatant, collect hAESCs exosomes by gradient centrifugation, resuspend in PBS, aliquot, and store at -80℃ for later use.

[0096] Example 4: Construction of a mouse ulcerative colitis disease model and anal injection of hAESCs

[0097] 1.6-8 week old C57BL / 6 male rats were acclimatized for one week and then randomly divided into a treatment group and a sham-operated group. They were given sterilized tap water containing 3% DSS for days 1 to 5. On day 6, they were given normal drinking water.

[0098] 2. On day 6, hAESCs were resuscitated, counted, and then prepared into a hydrogel cell suspension.

[0099] 3. Mice were anesthetized with 2% isoflurane inhalation. The mice in the treatment group were placed face down on the operating table, their tails were lifted to expose the anus to the surgeon's line of sight, and the anus was gently clamped with forceps.

[0100] 4. Connect the 1ml syringe containing the hydrogel cell suspension to the infusion set tubing, gently insert the tubing into the anus to a depth of 4cm, and slowly inject the hydrogel cell suspension.

[0101] 5. After the injection is complete, leave the tubing in the anus for 15 seconds. Remove the tubing, clamp the anus closed with tweezers, and seal the anus with skin glue.

[0102] After 6.6 hours, the mouse anus was examined and the adhesions were broken.

[0103] 7. On the 9th day, administer another rectal injection, following the same procedure as above.

[0104] 8. Mice in the sham-operated group underwent the same surgical procedure as mice in the treatment group, and the injected substance was a 4:1 mixture of hydrogel and PBS.

[0105] Example 5: Construction of a mouse model of ulcerative colitis and anal injection of hAESC exosomes

[0106] 1. The mouse UC model was constructed in the same manner as above.

[0107] 2. Starting from day 5, hAESC exosomes are collected every other day, resuspended in PBS, and injected rectally. The surgical procedure is the same as above.

[0108] Illustration ( Figure 1 The results showed that the hAESCs coated with the hydrogel material were spherical and suspended within the hydrogel material. Figure 1 A, B). hAESC exosomes exhibit a typical goblet-shaped membranous morphology. Figure 1 C), with a particle size of approximately 100 micrometers. Figure 1D), expresses exosome marker proteins CD9, CD63, and CD81, but does not express the organelle marker protein Calnexin. Figure 1 E).

[0109] Example 6: Observation of disease phenotypes and sample collection in mice

[0110] 1. Weigh each group of mice at the same time every day, observe the mice's condition, collect fecal samples for occult blood testing and record the results, and score them according to the Disease Activity Index (DAI) scale.

[0111] 2. On the day of the experiment's endpoint, the mice were anesthetized and euthanized by cervical dislocation. They were then placed in a supine position on the operating table, and the abdominal skin and muscles were cut open to expose the colon.

[0112] 3. Remove the entire colon, including the cecum and anus, take a picture, measure the length of the colon, cut off the cecum, and rinse the colon contents with pre-cooled PBS.

[0113] Illustration ( Figure 2 , 4 The results showed that hydrogel-coated hAESC and rectal injection of hAESC exosomes improved DSS-induced colitis, including promoting weight recovery. Figure 2 B, 4B), reduce disease activity score ( Figure 2 C) Improve survival rate Figure 4 C) Improves colon length shortening ( Figure 2 D, E; 4D, E).

[0114] Example 7 Intestinal permeability detection

[0115] 1. Mice were fasted for 4 hours, and a FITC-dextran (40,000 kDa) solution with a concentration of 80 mg / ml was prepared.

[0116] 2. Mice were administered the drug via gavage at a dose of 60 mg / 100 g body weight.

[0117] 3.4 hours later, mice were anesthetized with isoflurane inhalation, and blood was collected from the inner canthal venous plexus behind the eyeball by inserting an anticoagulant capillary. The blood was collected into a 1.5ml EP tube, centrifuged at 350g at room temperature for 10 minutes, and the clear supernatant was aspirated.

[0118] 4. Add different concentrations of FITC-dextran working solution and mouse serum samples to the microplate, with two replicates for each sample.

[0119] 5. Test the fluorescence intensity of each well using a fluorescence microplate reader. Set the wavelength of the fluorescence microplate reader to 490 nm. Repeat the measurement three times.

[0120] Illustration ( Figure 2The results showed that anal injection of hydrogel-coated hAESC improved DSS-induced increased intestinal epithelial permeability. Figure 2 H).

[0121] Example 8: Colonic Histology Examination

[0122] 1. Open the colon longitudinally, with the mucosa facing outward, and roll the entire colon into a Swiss roll.

[0123] 2. After fixing the colon tissue in 4% PFA overnight, it was routinely dehydrated with alcohol, cleared with xylene, impregnated with paraffin, and then embedded.

[0124] 3. Prepare paraffin sections with a thickness of 5 μm.

[0125] 4. Perform HE staining on the cut lung pathological sections.

[0126] Illustration ( Figure 2 , 4 The results showed that hydrogel-coated hAESC and hAESC exosomes, when injected anally, promoted DSS-induced colonic epithelial remodeling and restored colonic tissue structure. Figure 2 F, G; 4F, G).

[0127] Example 9 Apoptosis Detection (Paraffin Section)

[0128] 1. Dewaxing and hydration: Place the paraffin slices into the following reaction solutions in sequence: xylene I for 10 minutes → xylene II for 10 minutes → xylene III for 5 minutes → 100% ethanol I for 5 minutes → 100% ethanol II for 5 minutes → 95% ethanol for 2 minutes → rinse with running water for 5 minutes.

[0129] 2. Follow the instructions of the Yisheng TUNEL Apoptosis Detection Kit (Alexa Fluor 488) to detect intestinal epithelial apoptosis.

[0130] 3. Observe and photograph using a laser scanning confocal microscope (OLYMPUS, FV3000).

[0131] Example 10 Immunohistochemical staining (paraffin sections)

[0132] 1. The dewaxing and hydration processes are the same as above.

[0133] 2. Antigen retrieval: Boil 0.01M sodium citrate buffer (pH=6.0) beforehand, place the paraffin tissue slide in the retrieval solution, microwave on high for 20 minutes, and then let it cool naturally.

[0134] 3. Blocking: Immerse paraffin tissue slides in 3% hydrogen peroxide solution and incubate at room temperature for 15 minutes. Rinse with PBS for 5 minutes each time, 3 times. Label the tissue with a histochemical pen, add 5% BSA blocking solution to the center of the tissue, and incubate at room temperature for 1 hour.

[0135] 4. Antibody incubation and DAB color development: Follow the instructions for the Xinbosheng immunohistochemistry kit.

[0136] 5. Hematoxylin counterstaining: Place the slide in hematoxylin for 30 seconds, then rinse with tap water for 10 minutes.

[0137] 6. After dehydration and mounting, observe under a microscope.

[0138] Illustration ( Figure 3 The results showed that anal injection of hydrogel-coated hAESC improved DSS-induced apoptosis of intestinal epithelium and reduced TUNEL-positive cells. Figure 3 F), decreased expression of cleaved-caspase 3 (F) Figure 3 E).

[0139] Example 11 Immunofluorescence staining (paraffin sections)

[0140] 1. The dewaxing, hydration, and antigen retrieval steps are the same as above.

[0141] 2. Blocking and antibody incubation: Label the tissue with a histochemical pen, add 5% BSA blocking solution to the center of the tissue, and incubate at room temperature for 1 hour. Remove the blocking solution, add primary antibody diluted with the blocking solution (1:200) to the center of the tissue, and incubate overnight at 4 degrees Celsius in a humidified chamber, protected from light.

[0142] 3. Secondary antibody incubation: Remove the primary antibody and rinse the tissue with PBS for 5 minutes each time (3 times). Add the secondary antibody diluted in PBS (1:500) to the center of the tissue, and incubate in a humidified chamber at room temperature for 1 hour, protected from light.

[0143] 4. Mounting: Aspirate the secondary antibody and rinse the tissue with PBS for 5 minutes each time (3 times). Add a drop of mounting solution containing DAPI to the center of the tissue and incubate for 2 minutes. Cover the tissue with a coverslip.

[0144] 5. Observation and photography: Observe and photograph under a fluorescence microscope.

[0145] Illustration ( Figure 3 , 4 The results showed that hydrogel-coated hAESC and exosomes injected anally improved the DSS-induced inhibition of intestinal epithelial proliferation. Figure 3 A, 4H), reduce inflammatory cell infiltration ( Figure 3 C、4I)

[0146] Example 12 Detection of relative mRNA expression levels in colonic epithelial tissue

[0147] 1. Turn the entire colon tissue inside out to expose the colonic epithelium.

[0148] 2. Immerse in D-Hank's solution containing 5% EDTA and digest on ice for 30 minutes.

[0149] 3. Scrape intestinal epithelial tissue with a blade, rinse twice with PBS, and then freeze at -80°C.

[0150] 4. Total RNA extraction: Follow the instructions for the Aikerui AG21017 RNA extraction kit.

[0151] 5. Reverse transcription of cDNA: Follow the instructions for the AG11706 reverse transcription reagent premix.

[0152] 6. Real-time quantitative PCR: Using the AG11701 premixed qPCR kit, dilute the reverse-transcribed cDNA to an appropriate concentration and perform real-time quantitative PCR according to the following system.

[0153]

[0154] 7. The reaction procedure is as follows: 95℃ for 10 minutes, (95℃ for 10 seconds, 60℃ for 20 seconds, 72℃ for 15 seconds) × 40.

[0155] 8. After the reaction is complete, confirm the amplification curve and the melting curve.

[0156] Illustration ( Figure 2 The results showed that anal injection of hydrogel-coated hAESC promoted DSS-induced remodeling of colonic epithelial function and upregulated the expression of genes related to intestinal integrity, including Z0-1, Muc2, and Mptx1. Figure 2 H); the expression of genes related to intestinal epithelial cell proliferation, such as Pcna, Lama3, Mcm6, and Ly6a, is upregulated. Figure 3 B) The expression of genes Fas, Bas, and Apaf1, which are associated with apoptosis of intestinal epithelial cells, decreased.

[0157] The results showed that anal injection of hydrogel-coated hAESC reduced DSS-induced intestinal inflammation and decreased the expression of genes related to intestinal inflammation, including Cxcl1, Mip1, Il1b, Il6, Il17a, Il12a, Ifnγ, and Tnfα. Figure 3 D).

[0158] This invention provides a novel method for treating ulcerative colitis (UC) in mice using hydrogel-coated human amniotic epithelial stem cells and their exosomes. Intrarectal injection significantly reduced enteritis symptoms in mice, manifested as faster weight recovery, lower disease activity scores, and a significant reduction in UC-induced increased intestinal permeability. It promoted intestinal epithelial function reconstruction, resulting in improved intestinal permeability, enhanced intestinal epithelial integrity, reduced inflammatory response, promoted intestinal epithelial tissue regeneration, and reduced apoptosis. Based on these results, human amniotic epithelial stem cells and their exosomes, combined with biomaterials, can be applied to the treatment of UC. This method has broad prospects for clinical application in the treatment of UC.

[0159] In this specification, the present invention has been described with reference to specific embodiments. These embodiments are provided merely to aid in understanding the method and core ideas of the invention. The description of the invention is illustrative and not restrictive. Those skilled in the art can easily make improvements and modifications without departing from the principles of the invention, and such improvements and modifications also fall within the scope of protection of the claims.

Claims

1. The application of hydrogel-coated human amniotic epithelial stem cells and their exosomes in the preparation of drugs for improving ulcerative colitis, characterized in that, The method for preparing hydrogel-coated human amniotic epithelial stem cells and exosomes of human amniotic epithelial stem cells includes the following steps: Primary human amniotic epithelial stem cells were isolated and expanded in a complete culture medium to obtain a complete culture medium containing human amniotic epithelial stem cells. The complete culture medium containing human amniotic epithelial stem cells was mixed with hydrogel material to prepare a hydrogel material cell mixture suspension. Hydrogel-coated human amniotic epithelial stem cells were obtained from the hydrogel material cell mixture suspension. Alternatively, exosomes of human amniotic epithelial stem cells can be extracted from the culture supernatant of human amniotic epithelial stem cells.

2. The application according to claim 1, characterized in that, Primary human amniotic epithelial stem cells were isolated, specifically including: After washing the amniotic membrane tissue multiple times with a phosphate-balanced solution containing antibiotics, it was then subjected to enzymatic digestion and centrifugation to harvest primary human amniotic epithelial stem cells.

3. The application according to claim 1, characterized in that, The human amniotic epithelial stem cells in the whole culture medium containing human amniotic epithelial stem cells are human amniotic epithelial stem cells of passage number less than P3.

4. The application according to claim 1, characterized in that, The hydrogel material is a hydrogel modified with RGD cell adhesion peptides.

5. The application according to claim 1, characterized in that, The hydrogel material used is VitroGel® RGD hydrogel, model TWG003. The hydrogel of model TWG003 includes hydrogel and diluent, and the volume ratio of hydrogel to diluent is 1:3.1~4.

9.

6. The application according to claim 1, characterized in that, The human amniotic epithelial stem cells in the hydrogel material cell suspension were (0.1~1.5)×10⁻⁶. 7 / ml.

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