WO2021052049A1 - Use of ankrd22 as target in preparation of gastrointestinal mucosa repair protectant - Google Patents

Abstract

Disclosed is the use of ANKRD22 as a target in the preparation of a gastrointestinal mucosa repair protectant. Using ANKRD22 as a target refers to reducing the expression of an ANKRD22 gene by means of using gene knockout, gene knockdown or chemical drugs. The present invention provides a new target for repairing mild and moderate injuries to gastrointestinal mucosa, that is, by means of inhibiting the expression of the ANKRD22 gene, stomach tissue LGR5+ stem cells can be amplified, the inflammatory response in the stomach tissue can be reduced, and the amount of gastric mucus secretion can be increased.

Description

Application of ANKRD22 as a target in the preparation of gastrointestinal mucosal repair and protective agent Technical field

The invention relates to the field of biomedicine, in particular to the application of ANKRD22 as a target in the preparation of a gastrointestinal mucosal repair protective agent.

Background technique

The gastrointestinal tract is an important digestion and absorption organ of the human body. Food needs to be fully grinded by the stomach and mixed with gastric acid to form a paste-like chyme that can be absorbed by the small intestine. Although the gastrointestinal mucosa can protect the stomach from damage, it is extremely vulnerable to damage from diet, drugs, and bad moods. Gastrointestinal mucosal injury is a common and frequently-occurring disease worldwide, and almost everyone suffers from this disease in their lifetime. With the increase of people’s social activities nowadays, the incidence of gastrointestinal mucosal damage-related diseases caused by exogenous injury factors such as alcohol and irritating food is also increasing year by year, especially the mild to moderate gastrointestinal mucosal damage caused by alcohol is increasing. . Ethanol directly or indirectly damages the gastrointestinal mucosal barrier, leading to increased mucosal permeability, neutrophil infiltration, and free radicals, resulting in symptoms such as inflammation and pain. At present, scholars at home and abroad have conducted a large number of studies on gastrointestinal mucosal injury repair, explore the mechanism of gastrointestinal mucosal injury caused by alcohol, and research drugs to prevent and treat alcoholic gastrointestinal mucosal injury. However, no internationally recognized effective drugs have been published.

Clinically, the key means to protect the gastrointestinal mucosa is to inhibit gastric acid and anti-Helicobacter pylori. The main drugs are basic antacids, proton pump inhibitors, H2 receptor antagonists and gastrointestinal mucosal protective agents. In recent years, people have realized the importance of strengthening the gastrointestinal mucosal defense function, and believe that adding emphasis on gastrointestinal mucosal protection can play a positive role in the repair of gastrointestinal mucosal damage. At present, the main representatives of gastrointestinal mucosal protective agents are: prostaglandin drugs, teprenone, sucralfate, bismuth potassium citrate, etc., and their mechanisms of action are not the same. Although these drugs have good therapeutic effects, reports about their adverse reactions are also increasing. Therefore, finding safer and more effective gastrointestinal mucosal protective drugs is of great significance to overcome this global disease.

There are stem cells in the gastrointestinal tissues, and stem cells have damage repair functions. With the concept that “improving the quality of gastrointestinal mucosal repair is the key to the treatment of mucosal injury”, the inventor hopes to find a targeted molecule that can specifically target gastric tissue stem cells and enhance stem cell expansion through continuous exploration. The ability to repair gastrointestinal mucosal damage without damaging normal tissue cells. Therefore, it is an urgent problem to provide a new type of target that can effectively improve the repair quality of gastrointestinal mucosal damage.

Summary of the invention

ANKRD22 is a small molecule protein with 4 ankyrin repeat motifs (ANK). Each ANK contains 2 reverse á spirals and 1

The hairpin-like L-shaped structure, about 30-34 amino acid residues in size, forms a high-affinity molecular connection gallows structure. There are many proteins in the body that contain ANK motifs and have a wide range of functions. ANKRD22 has a high level of expression in human stomach tissues and macrophages. It is a molecule that links cellular metabolic reprogramming and nuclear reprogramming, suggesting the target of ANKRD22 It is closely related to the repair function of gastric stem cells. Through experiments, it is found that ANKRD22 is associated with the repair of mild to moderate gastrointestinal mucosal damage, and may be used as a target for the treatment of gastrointestinal mucosal damage. Therefore, using ANKRD22 as the target to screen a new type of gastrointestinal mucosal repair and protective agent targeting gastric tissue stem cells has important social significance and a broad economic market.

In view of this, the main purpose of the present invention is to find new targets for repairing mild to moderate gastrointestinal mucosal damage, especially targeting stem cells of gastric tissue, so as to better prevent and/or treat mild to moderate gastrointestinal mucosal damage related disease.

The present invention provides a novel potential target of a gastrointestinal mucosal repair protective agent targeting stem cells, that is, the application of the ANKRD22 gene as a target in the preparation of a gastrointestinal mucosal protective agent.

ANKRD22 (Ankyrin repeat domain-containing protein 22) is a small molecule protein with 4 ankyrin repeat motifs (ANK), which has a high level of expression in human stomach tissues and macrophages.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

In the first aspect, the present invention provides the application of ANKRD22 as a target in the preparation of a protective agent for gastrointestinal mucosal repair.

Further, the gastrointestinal mucosal repair and protective agent is a drug that targets gastric tissue stem cells.

Further, the gastrointestinal mucosal repair protective agent uses ANKRD22 as a target, and expands the gastric tissue LGR5+ stem cells when the gastrointestinal mucosal damage is repaired.

Further, the gastrointestinal mucosal repair and protective agent targets ANKRD22 to reduce the inflammatory reaction of the gastric tissue and increase the secretion of mucus in the gastric tissue.

Further, targeting ANKRD22 refers to the use of gene knockout, gene knockdown, or chemical drugs to reduce the expression of the ANKRD22 gene.

In the second aspect, the present invention provides the application of gastric tissue macrophages knocked out with ANKRD22 gene in the preparation of gastrointestinal mucosal repair protective agents.

Further, the preparation method of gastric macrophages knocked out with ANKRD22 gene includes the following steps:

S1. Gavage mice with ANKRD22 gene deletion to stimulate the gastrointestinal mucosa with mild to moderate damage;

S2. During the 24-48h period of gastrointestinal mucosal repair, remove the gastric tissue block from the mouse, and use the enzyme mixture to digest the gastric tissue block. The enzyme mixture includes collagenase IV, hyaluronidase and deoxyribonuclease;

S3. After the digestion is completed, use fetal bovine serum to terminate the digestion, add the product to a 50ml centrifuge tube, centrifuge at 800 rpm, 4°C, and resuspend the cell pellet in RPMI 1640 complete medium;

S4. Separation and purification of gastric macrophages by Percoll isocratic gradient precipitation method;

After centrifugation at S5.2500rmp/min at 4°C, suck out the cloud-like middle layer cell band with a pipette;

S6. Dilute with 3 times volume of RPMI 1640 complete medium of Percoll solution, centrifuge at 800 rpm at 4°C, and resuspend the cell pellet in RPMI 1640 complete medium. The cell suspension is the ANKRD22 knockout gastric tissue macrophages cell.

Furthermore, in S1, the gastrointestinal mucosa was mild to moderately damaged and stimulated, and the amount of HCl/EtOH gavage was 5 mL/kg body weight.

Further, in S2, during 24-48 hours of gastrointestinal mucosal repair, the gastric tissue block is removed from the mouse, and the cut gastric tissue block is digested with an enzyme mixture preheated to 37°C.

Further, in S2, removing the gastric tissue block from the mouse, specifically including immersing the mouse killed by the neck in 75% alcohol, and then placing the mouse on a sterile clean table, using surgical scissors and tweezers Remove the block of stomach tissue.

Furthermore, in S2, before digesting the gastric tissue mass with the enzyme mixture, it also includes thoroughly flushing the blood of the tissue mass with 4℃ normal saline, removing visible fat and connective tissue with sterile scissors, and cutting the tissue mass into 1～3㎜ ³ size small pieces, and then digest. It can be understood that cutting the gastric tissue into small pieces can better digestion and improve the quality and efficiency of digestion.

Furthermore, in S2, the enzyme mixture contains 50 mg/mL collagenase I, 50 mg/mL collagenase IV, 5 mg/mL hyaluronidase, and 1 U/mL deoxyribonuclease.

Furthermore, in S3, after the digestion is completed, fetal bovine serum is used to terminate the digestion, and the product is added to a 50ml centrifuge tube, centrifuged at 800 rpm and 4°C, and the cell pellet is resuspended in RPMI 1640 complete medium; if the supernatant is sticky after digestion It can be digested by adding deoxyribonuclease again, stop the reaction with fetal bovine serum, centrifuge again at 800rmp/min, 4℃, resuspend the cell pellet with RPMI 1640 complete medium, and temporarily put it in the 37℃ incubator; The suspended cell suspension was mixed, centrifuged at 800 rpm at 4°C, and resuspended in RPMI1640 complete medium;

Furthermore, in S4, the Percoll isocratic gradient precipitation method is used to separate and purify gastric tissue macrophages. Specifically, use a glass syringe with a 14-gauge needle to add 45% and 35% Percoll in descending order of density. Slowly add the solution along the side wall of the 15ml centrifuge tube, add 3ml for each gradient, be careful not to generate bubbles during the operation; use a pipette to slowly add the 3ml cell suspension to the centrifuge tube along the wall of the tube, and spread it on the Percoll density gradient liquid Surface; In an implementation of the present invention, 9 parts of Percoll solution and 1 part of 10*D-Hanks' solution are used to prepare 90% Percoll mother liquor, and Percoll mother liquor and D Hanks' solution are used to prepare 35% and 45% Percoll Solution

Further, in S6, add 3 times the volume of Percoll solution RPMI 1640 complete medium diluted, after centrifugation at 800 rpm, 4 ℃, resuspend the cell pellet with a small amount of RPMI 1640 complete medium, the cell suspension is the ANKRD22 of the present invention Knockout gastric macrophages.

In the present invention, the centrifugation at 800rmp/min and 4°C, where the centrifugal speed of 800rmp/min is to make the cells precipitate without breaking; the centrifugation temperature of 4°C mainly keeps the cells at a lower temperature and slows down Growth and metabolism, so that it can exist more stably.

In the third aspect, the present invention provides a gastrointestinal mucosal repair and protection agent, which is prepared by using ANKRD22 gene knockout gastric tissue macrophages.

It should be noted that the present invention has discovered through research that the deletion of the ANKRD22 gene can not only repair mild to moderate damage to the gastrointestinal mucosa, but also amplify LGR5+ stem cells in gastric tissue, reduce gastric tissue inflammation, increase gastric mucus secretion, and can be used for repair Mild to moderate gastrointestinal mucosal damage. Therefore, ANKRD22 gene can be used as a target to prepare gastrointestinal mucosal repair protective agents; as long as the expression of ANKRD22 gene can be reduced, it can be used to treat mild to moderate gastrointestinal mucosal damage.

It should also be noted that the present invention has been researched and found that the deletion of the ANKRD22 gene has no significant impact on the growth and development of the biological individual itself, and has no significant impact on the undamaged gastric tissue. Therefore, the ANKRD22 gene can be used as a target to prepare a gastrointestinal mucosal repair protective agent. .

The inventors found in the research that ANKRD22 is a new target related to the expansion of LGR5+ stem cells after mild to moderate damage to gastric tissue, and the use of CRISPR/Cas9 technology for gene knockout or shRNA interference with ANKRD22 gene expression can effectively alleviate Symptoms of mild to moderate gastrointestinal mucosal damage in experimental mice.

In the specific experiment of the present invention, the gene expression of ANKRD22 is interfered by gene knockout technology, which can effectively alleviate the mild to moderate stomach caused by hydrochloric acid ethanol solution (150mM HCl/60% absolute ethanol solution, HCl/EtOH). Symptoms of intestinal mucosal damage. Furthermore, in experiments to detect ANKRD22 knockout or overexpressing cells in vivo and in vitro, we found that the ANKRD22 target is closely related to the Wnt-Ca2+ signaling pathway.

It should also be noted that the inventors have discovered through research that gastric macrophages lacking the ANKRD22 gene can reduce the inflammatory response in gastric tissue after mild to moderate injury and increase gastric mucus secretion. Therefore, the ANKRD22 gene can also be used as a target. Gastric tissue macrophages prepare gastrointestinal mucosal repair protective agent.

It can be understood that the ANKRD22 gene knockout gastric tissue macrophages of the present invention have a good effect of repairing gastrointestinal mucosal damage and can reduce gastric tissue inflammation. Therefore, they can be prepared as a gastrointestinal mucosal repair protective agent for repair Slight to moderate damage to the gastrointestinal mucosa. Among them, the medicine may also include other pharmaceutically acceptable auxiliary or active ingredients, which are not specifically limited here.

In summary, the present invention proves that ANKRD22 is a new target of gastrointestinal mucosal repair and protective agent targeting LGR5+ stem cells, which can reduce gastric tissue inflammation, increase gastric mucus secretion, and is closely related to the Wnt-Ca2+ signaling pathway, and uses any technology Interfering with the expression of ANKRD22 gene can effectively repair the symptoms of mild to moderate damage to the gastrointestinal mucosa.

In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

The present invention provides a new target for repairing mild to moderate damage of gastrointestinal mucosa, that is, by inhibiting the expression of ANKRD22 gene, not only can the gastric tissue LGR5+ stem cells be expanded, but also gastric tissue inflammation can be reduced, and gastric mucus secretion can be increased. The ANKRD22 gene knockout gastric tissue macrophages of the present invention can be used to develop new gastrointestinal mucosal repair and protective agents that target stem cells.

Description of the drawings

Figure 1: The effect of ANKRD22 gene deletion on mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH. Among them, WT, wild

type: wild type mouse. Ankrd22-/-: ANKRD22 gene deletion mice. Control (Ctrl): control group. HCl/EtOH, hydrochloric acid ethanol solution: the experimental group of hydrochloric acid ethanol gavage.

Figure 2: The effect of deletion of the ANKRD22 gene on the level of LGR5 in mouse gastric tissue 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH. Among them, Lgr5: G protein coupled receptor 5.

Figure 3: The effect of deletion of ANKRD22 gene on the levels of Mucin2, Mucin5AC and Mucin6 in mouse gastric tissue 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH. Among them, Muc2: Mucin 2; Muc5ac: Mucin 5AC; Muc6: Mucin 6;

Figure 4: The effect of deletion of the ANKRD22 gene on the level of Ca2+ in mouse gastric tissue cells 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH.

Figure 5: The effect of deletion of ANKRD22 gene on the levels of inflammatory factors IL-1β and IL-18 of macrophages in mouse gastric tissue 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH. Among them, IL-1β: Interleukin 1β; IL-18: Interleukin 18.

detailed description

The present invention discovers a new therapeutic target during the study of gastrointestinal mucosal damage, namely the ANKRD22 gene. Research has confirmed that knocking out the ANKRD22 gene reduces the expression of the ANKRD22 gene and can effectively repair the gastrointestinal mucosa in mild to moderate conditions. Injury, expand the gastric tissue LGR5+ stem cells, reduce gastric tissue inflammation, increase gastric mucus secretion, and protect the gastrointestinal mucosa. It is understandable that as long as the expression of ANKRD22 gene can be reduced, the effect of repairing mild to moderate gastrointestinal mucosal damage can be achieved; therefore, in addition to gene knockout, gene knockdown or chemical drugs can also reduce the expression of ANKRD22 gene. The effect of protecting the gastrointestinal mucosa.

In addition, the study of the present invention has confirmed that the extraction of ANKRD22 gene knockout gastric tissue macrophages can repair mild to moderate damage to the gastrointestinal mucosa, reduce the inflammation of the gastric tissue, and protect the gastrointestinal mucosa; therefore, ANKRD22 Knockout gastric macrophages can be used to develop new targeted drugs for gastrointestinal mucosal repair and protection agents.

Example 1. Interference with gene expression of ANKRD22 can effectively alleviate the mild to moderate gastrointestinal mucosal injury symptoms of experimental mice caused by hydrochloric acid ethanol solution (150mM HCl/60% absolute ethanol solution, HCl/EtOH).

In this example, it was verified that interference with the gene expression of ANKRD22 can effectively repair the mild to moderate gastrointestinal mucosal injury symptoms caused by HCl/EtOH in experimental mice. Among them, an experimental mouse model of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH was established. However, compared with wild-type mice, the symptoms of gastrointestinal mucosal damage in ANKRD22 knockout mice were significantly alleviated, including: reduction of gastrointestinal mucosal bleeding points and reduction of uneven mucosal particles.

details as follows:

HCl/EtOH experimental mouse model of mild to moderate gastrointestinal mucosal injury

The ANKRD22 knockout mouse with C57BL/6J background was commissioned by Saiye (Guangzhou) Biotechnology Co., Ltd. to construct. C57BL/6J wild-type female mice were from the Experimental Animal Center of Zhejiang University of Traditional Chinese Medicine.

The animals were kept in a constant temperature and humidity, 12-hour light-dark cycle breeding room. Feed and water can be eaten freely. All animal procedures were performed in accordance with the procedures approved by the Animal Welfare Ethics Committee.

Wild-type mice: Female mice are randomly divided into control group and HCl/EtOH group on the 42nd day (D42) after birth, with at least 6 mice in each group.

ANKRD22 gene-deficient mice: Female mice were randomly divided into control group and HCl/EtOH group on the 42nd day (D42) after birth, with at least 6 mice in each group.

HCl/EtOH group: The mice were fasted with water for 24 hours and then gavage HCl/EtOH, 5mL/kg body weight, 150mM HCl and 100% ethanol, dissolved in PBS at a volume ratio of 4:6.

Control group: mice were given the same amount of PBS by gavage.

The mice were sacrificed 24 hours after modeling, and blood samples and tissues were collected. The mice were anesthetized with isoflurane, and blood was collected from the eyeballs. The gastric tissue was separated and fixed in 4% paraformaldehyde solution overnight. In addition, the isolated tissues are quickly frozen in liquid nitrogen until further processing.

Figure 1 shows the effect of deletion of ANKRD22 gene on mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH.

As shown in Figure 1, the gastrointestinal mucosa of wild-type mice (WT-control) was pale pink in general and smooth and smooth; the gastrointestinal mucosa of ANKRD22 gene-deficient mice (Ankrd22-/-control) and the gastrointestinal mucosa of the WT-control group There is no significant difference in the mucosa; the gastrointestinal mucosa of wild-type mice (WT-HCl/EtOH) treated with HCl/EtOH for 24 hours has many red strip-shaped bleeding points, and the mucosa is uneven and appears to be fine granular. HCl/EtOH caused mild to moderate gastrointestinal mucosal damage in mice; when ANKRD22 gene-deficient mice were treated with HCl/EtOH (Ankrd22-/-HCl/EtOH), the gastrointestinal mucosal red striped bleeding spots were more than Ankrd22-/- The control group was significantly reduced and the mucosal granular shape was reduced, indicating that the mice in this group had a stronger role in repairing gastrointestinal mucosa damage.

Example 2. Interfering with the gene expression of ANKRD22 can significantly increase the number of LGR5+ stem cells amplified in the gastric tissue of experimental mice with mild to moderate gastrointestinal mucosal damage.

Figure 2 shows the effect of deletion of the ANKRD22 gene on the level of LGR5 in mouse gastric tissue 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH.

After adding the mouse stomach tissue to Trizol and grinding, RNA was extracted and reverse transcribed into cDNA. Real-time PCR was used to detect the relative expression of LGR5: HCl/EtOH 24h after mild to moderate gastrointestinal mucosal injury. Ankrd22-/- mouse stomach The level of LGR5 in tissues (Ankrd22-/-HCl/EtOH) was significantly higher than that of wild-type mice (Ankrd22-/-control). P<0.05 represents statistically significant difference.

From the results of Figure 1 and Figure 2, it can be seen that ANKRD22 gene deletion can repair mild to moderate gastrointestinal mucosal damage, affect the level of LGR5 in gastric tissue, increase LGR5+ cell expansion, and thus exhibit a gastrointestinal mucosal protective effect targeting LGR5+ stem cells.

Example 3. Interfering with the gene expression of ANKRD22 can significantly increase the gastric mucus secretion level of experimental mice with mild to moderate gastrointestinal mucosal damage.

Figure 3 shows the effect of deletion of the ANKRD22 gene on the levels of Mucin2, Mucin5AC and Mucin6 in mouse gastric tissue 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH.

After the mouse gastric tissue was added to Trizol and ground, RNA was extracted and reverse transcribed into cDNA. Real-time PCR was used to detect the relative expression of Mucin2, Mucin5AC and Mucin6: HCl/EtOH 24h Ankrd22-/ after mild to moderate gastrointestinal mucosal injury -Mouse stomach tissue (Ankrd22-/-HCl/EtOH) Mucin2, Mucin5AC and Mucin6 levels were significantly increased compared with wild-type mice (Ankrd22-/-control). P<0.05 represents statistically significant difference.

Figure 3 shows that ANKRD22 gene deletion can inhibit the Wnt-Ca2+ signaling pathway, reduce the Ca2+ concentration of gastric tissue cells, indirectly increase the activation level of the classic Wnt pathway, and increase the expansion of LGR5+ stem cells.

Example 4. ANKRD22 regulates the Ca2+ concentration of gastric tissue cells and the activation level of Wnt pathway through the Wnt-Ca2+ signaling pathway.

Figure 4 shows the effect of deletion of the ANKRD22 gene on the level of Ca2+ in mouse gastric tissue cells 24 after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH.

After digesting mouse stomach tissue into single cells, add 10μmol/L fluo-4 (Invitrogen, USA) and 0.02%

F-127 (Sigma), incubated at 37°C in the dark for 1 h, FACSCanto II flow cytometer was used to detect the FITC fluorescence intensity. The average FITC fluorescence intensity represents the level of Ca2+ in the cell.

As shown in Figure 4, ANKRD22 gene-deficient mice (Ankrd22-/-control) had no significant difference in the concentration of Ca2+ in gastric tissue cells from the WT-control group; while HCl/EtOH 24h after mild to moderate gastrointestinal mucosal injury Compared with wild-type mice (WT-HCl/EtOH), the concentration of Ca2+ in gastric tissue cells of mice (Ankrd22-/-HCl/EtOH) was significantly reduced. P<0.05 represents statistically significant difference.

Figure 4 shows that ANKRD22 gene deletion can inhibit the Wnt-Ca2+ signaling pathway, reduce the Ca2+ concentration of gastric tissue cells, indirectly increase the activation level of the canonical Wnt pathway, and increase the expansion of LGR5+ stem cells.

Example 5. Gastric macrophages with deletion of the ANKRD22 gene can significantly reduce the level of inflammation in the gastric tissue of experimental mice with mild to moderate gastrointestinal mucosal damage.

Figure 5 shows the effect of deletion of the ANKRD22 gene on the levels of IL-1β and IL-18 in macrophages of mouse gastric tissue after the repair of mild to moderate gastrointestinal mucosal damage caused by HCl/EtOH.

The ANKRD22 knockout mouse gastric tissue macrophages and wild-type mouse gastric tissue macrophages after HCl/EtOH stimulation of the gastrointestinal mucosa for 24 hours were extracted, and the ANKRD22 knockout mice and wild-type mouse gastric tissue macrophages were extracted after intragastric administration with PBS for 24 hours. Type mouse gastric macrophages were used as controls. The mouse gastric tissue was added to RIPA strong lysate and ground to extract the protein, and the absorbance was detected by a microplate reader using IL-1β and IL-18 ELISA kit (Abcam).

As shown in Figure 5, the levels of IL-1β and IL-18 in gastric macrophages of mice treated with HCl/EtOH for 24 hours were significantly higher than those in the PBS control group, indicating that mice with mild to moderate gastrointestinal mucosal injury Gastric tissue inflammation was enhanced; and the levels of IL-1β and IL-18 in gastric macrophages of mice with ANKRD22 gene deletion after HCl/EtOH treatment were significantly lower than those of wild-type mouse gastric macrophages. P<0.05 represents statistically significant difference.

Figure 5 shows that gastric macrophages with ANKRD22 gene deletion can significantly reduce gastric tissue inflammation caused by gastrointestinal mucosal damage, and play a protective role in gastrointestinal mucosa.

The above content is a further detailed description of the present invention in conjunction with specific implementations, and it cannot be considered that the specific implementations of the present invention are limited to these descriptions. More precisely, the present invention relates to the antibodies, devices, kits, etc. and their uses disclosed herein, as well as the control of ANKRD22 levels, and various modifications can be made according to the details, and these modifications are within the scope of the claims. The scope of equivalent claims does not deviate from the spirit of the present invention.

Claims (10)

1. The application of ANKRD22 as the target in the preparation of gastrointestinal mucosal repair protective agent.
2. The application according to claim 1, wherein the gastrointestinal mucosal repair and protective agent is a drug targeting gastric tissue stem cells.
3. The application according to claim 1, wherein the gastrointestinal mucosal repair protective agent targets ANKRD22, and expands gastric tissue LGR5+ stem cells when the gastrointestinal mucosal damage is repaired.
4. The application according to claim 1, characterized in that the gastrointestinal mucosal repair and protective agent targets ANKRD22 to reduce gastric tissue inflammation and increase gastric tissue mucus secretion.
5. The application according to any one of claims 1 to 4, wherein the targeting of ANKRD22 refers to the use of gene knockout, gene knockdown, or chemical drugs to reduce the expression of the ANKRD22 gene.
6. Application of gastric tissue macrophages knocked out with ANKRD22 gene in preparation of gastrointestinal mucosal repair protective agent.
7. The application according to claim 6, wherein the preparation method of gastric macrophages knocked out with ANKRD22 gene comprises the following steps:

   S1. Gavage mice with ANKRD22 gene deletion to stimulate the gastrointestinal mucosa with mild to moderate damage;

   S2. During the 24-48h period of gastrointestinal mucosal repair, remove the gastric tissue block from the mouse, and use the enzyme mixture to digest the gastric tissue block. The enzyme mixture includes collagenase IV, hyaluronidase and deoxyribonuclease;

   S3. After the digestion is completed, use fetal bovine serum to terminate the digestion, add the product to a 50ml centrifuge tube, centrifuge at 800 rpm, 4°C, and resuspend the cell pellet in RPMI 1640 complete medium;

   S4. Separation and purification of gastric macrophages by Percoll isocratic gradient precipitation method;

   S5. After centrifugation at 2500rmp/min and 4℃, suck out the cloud-like middle layer cell band with a pipette;

   S6. Dilute with 3 times volume of RPMI 1640 complete medium of Percoll solution, centrifuge at 800 rpm at 4°C, and resuspend the cell pellet in RPMI 1640 complete medium. The cell suspension is the ANKRD22 knockout gastric tissue macrophages cell.
8. The application according to claim 7, characterized in that, in S1, the amount of HCl/EtOH gavage is 5 mL/kg body weight during mild to moderate damage to the gastrointestinal mucosa.
9. The application according to claim 7, characterized in that, in S2, before digesting the gastric tissue mass with the enzyme mixture, it also includes thoroughly flushing the blood of the tissue mass with 4℃ normal saline, and removing the visible tissue with sterile scissors fat, connective tissue, the tissue cut into blocks of size 1 to 3 mm ³ pieces, and then digested.
10. A gastrointestinal mucosal repair and protective agent, which is characterized in that it is prepared by using ANKRD22 gene knockout gastric tissue macrophages.

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