Application of ANKRD22 as target in preparation of gastrointestinal mucosa repair protective agent
阅读说明：本技术 以ankrd22为靶标在制备胃肠粘膜修复保护剂中的应用 (Application of ANKRD22 as target in preparation of gastrointestinal mucosa repair protective agent ) 是由 朱永良 柳景文 于 2019-09-17 设计创作，主要内容包括：本发明公开了一种以ANKRD22为靶标在制备胃肠粘膜修复保护剂中的应用,以ANKRD22为靶标是指包括采用基因敲除、基因敲减或者化学药物降低ANKRD22基因的表达。本发明为修复胃肠粘膜轻中度损伤提供了一种新的靶标,即通过抑制ANKRD22基因表达,不仅能够使胃组织LGR5+干细胞扩增,而且能够减轻胃组织炎症反应,增加胃粘液分泌。(The invention discloses application of ANKRD22 as a target in preparation of a gastrointestinal mucosa repair protective agent, wherein the ANKRD22 as the target is used for reducing the expression of an ANKRD22 gene by gene knockout, gene knockdown or chemical drugs. The invention provides a new target for repairing mild and moderate damage of gastrointestinal mucosa, namely, by inhibiting the expression of ANKRD22 gene, the invention not only can amplify the LGR5+ stem cells of stomach tissue, but also can relieve the inflammatory reaction of stomach tissue and increase the secretion of stomach mucus.)
1. The application of ANKRD22 in preparing gastrointestinal mucosa repair protective agent.
2. The use according to claim 1, wherein the gastrointestinal mucosa repair protectant is a drug that targets stomach tissue stem cells.
3. The use according to claim 1, wherein the gastrointestinal mucosa repair protectant targets ANKRD22, and expands LGR5+ stem cells of gastric tissue during repair of gastrointestinal mucosa injury.
4. The use of claim 1, wherein the gastrointestinal mucosa repair protectant targets ANKRD rd22, reduces the inflammatory response and increases mucus secretion from gastric tissue.
5. The use of any one of claims 1 to 4, wherein targeting ANKRD22 comprises reducing the expression of ANKRD22 gene by gene knock-out, gene knock-down, or chemical drug.
6. Use of macrophage of stomach tissue knocked out by ANKRD22 gene in preparation of gastrointestinal mucosa repair protective agent.
7. The use of claim 6, wherein the gastric tissue macrophage knockout with ANKRD22 is prepared by a method comprising the steps of:
s1, stimulating mild and moderate gastrointestinal mucosa injury of an ANKRD22 gene-deleted mouse by adopting an intragastric perfusion mode;
s2, during the 24-48h period of gastrointestinal mucosa repair, taking off the stomach tissue block from the mouse, and digesting the stomach tissue block by adopting enzyme mixed liquor, wherein the enzyme mixed liquor comprises collagenase IV, hyaluronidase and deoxyribonuclease;
s3, stopping digestion by adopting fetal calf serum after digestion is finished, adding the product into a 50ml centrifuge tube, centrifuging at the temperature of 4 ℃ at 800rmp/min, and re-suspending cell sediment by using an RPMI1640 complete culture medium;
s4, separating and purifying the stomach tissue macrophage by using a Percoll equal density gradient precipitation method;
s5.2500rmp/min, centrifuging at 4 ℃, and sucking out a cloudy middle-layer cell band by using a suction pipe;
s6, adding 3 times of Percoll liquid into RPMI1640 complete culture medium for dilution, centrifuging at 800rmp/min and 4 ℃, and suspending cell sediment by using the RPMI1640 complete culture medium, wherein the cell suspension is the gastric tissue macrophage knocked out by ANKRD22 gene.
8. The use of claim 7, wherein the amount of HCl/EtOH intragastric administration is 5mL/kg body weight in the mild-to-moderate irritation of gastrointestinal mucosa at S1.
9. The use of claim 7, wherein in step S2, prior to digesting the stomach tissue mass with the enzyme cocktail, the method further comprises rinsing the blood from the tissue mass thoroughly with 4 ℃ saline, removing visible fat and connective tissue with sterile scissors, and cutting the tissue mass to a thickness of 1-3 mm3Small pieces of size, and then digested.
10. A gastrointestinal mucosa repair protective agent, which is prepared from macrophages of gastric tissue in which ANKRD22 gene is knocked out.
Technical Field
The invention relates to the field of biomedicine, in particular to application of ANKRD22 as a target in preparation of a gastrointestinal mucosa repair protective agent.
Background
The gastrointestinal tract is an important digestive and absorptive organ of a human body, and food can be fully ground by the stomach and uniformly mixed with gastric acid to form pasty chyme for the absorption of the small intestine. The gastrointestinal mucosa can protect the stomach from being damaged, but is very easy to be damaged by diet, medicines, bad mood and the like. Gastrointestinal mucosal injury is a common and frequently occurring disease worldwide, with almost all suffering from it throughout life. With the increase of social activities of people, the incidence of diseases related to gastrointestinal mucosa injury caused by exogenous injury factors such as alcohol and irritant food is increasing year by year, and particularly, the gastrointestinal mucosa injury caused by alcohol is increasing day by day. Ethanol damages gastrointestinal mucosa barrier through direct or indirect action, which results in increased mucosa permeability, neutrophil infiltration and increased free radicals, thereby causing symptoms of inflammation, pain and the like. At present, scholars at home and abroad carry out a great deal of research on gastrointestinal mucosa injury repair, discuss the gastrointestinal mucosa injury mechanism caused by ethanol and research on medicaments for preventing and treating alcoholic gastrointestinal mucosa injury, but no internationally recognized effective medicament is published up to now.
The key means for protecting gastrointestinal mucosa clinically is to inhibit gastric acid and resist helicobacter pylori, and the main medicaments are alkaline antacids, proton pump inhibitors, H2 receptor antagonists, gastrointestinal mucosa protective agents and the like. In recent years, the importance of strengthening the defense function of the gastrointestinal mucosa has been recognized, and it is considered that strengthening the protection of the gastrointestinal mucosa can play a positive role in repairing the damage of the gastrointestinal mucosa. Currently, the main representatives of gastrointestinal mucosa protective agents are: prostaglandins, teprenone, sucralfate, bismuth potassium citrate, etc. have different mechanisms of action. Although these drugs have good therapeutic effects, there are increasing reports on adverse reactions. Therefore, the search for safer and more effective gastrointestinal mucosa protective agent medicaments is of great significance for overcoming the global disease.
Stem cells exist in gastrointestinal tissues, and have a damage repair function. With the proposal of the concept that the improvement of the gastrointestinal mucosa repair quality is the key to the mucosal injury treatment, the inventor hopes to find a targeting molecule through continuous exploration, which can specifically target the gastric tissue stem cells and enhance the stem cell amplification capability so as to repair the gastrointestinal mucosa injury without damaging the normal tissue cells. Therefore, providing a novel target for effectively improving the quality of repairing gastrointestinal mucosa injury is a problem which needs to be solved urgently at present.
Disclosure of Invention
ANKRD22 is a small molecule protein with 4 ankyrin repeat motifs (ANKs). Each ANK contained 2 inverse-a-screws and 1
In view of the above, the main objective of the present invention is to find a new target for repairing mild-moderate damage of gastrointestinal mucosa, especially targeting gastric tissue stem cells, so as to better prevent and/or treat diseases related to mild-moderate gastrointestinal mucosa damage.
The invention provides a novel stem cell-targeted potential target of a gastrointestinal mucosa repair protective agent, namely application of ANKRD22 gene as a target spot in preparation of the gastrointestinal mucosa protective agent.
ANKRD22(Ankyrin repeat domain-containing protein 22) is a small molecule protein with 4 Ankyrin repeat motifs (ANK) and has high expression levels in human gastric tissue and macrophages.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides the use of ANKRD22 as a target for the preparation of a gastrointestinal mucosal repair protectant.
Further, the gastrointestinal mucosa repair protective agent is a drug targeting the stem cells of the gastric tissue.
Further, the gastrointestinal mucosa repair protective agent targets ANKRD22, and expands the LGR5+ stem cells of the stomach tissue during repair of gastrointestinal mucosa injury.
Further, the gastrointestinal mucosa repair protective agent takes ANKRD22 as a target, reduces the inflammatory reaction of the stomach tissue and increases the mucus secretion of the stomach tissue.
Further, targeting ANKRD22 is meant to include decreasing expression of the ANKRD22 gene using gene knock-out, gene knock-down, or chemical drugs.
In a second aspect, the invention provides the use of a gastric tissue macrophage knocked out by ANKRD22 gene in the preparation of a gastrointestinal mucosa repair protective agent.
Further, a method for preparing macrophage of stomach tissue with ANKRD22 gene knockout, comprising the following steps:
s1, stimulating mild and moderate gastrointestinal mucosa injury of an ANKRD22 gene-deleted mouse by adopting an intragastric perfusion mode;
s2, during the 24-48h period of gastrointestinal mucosa repair, taking off the stomach tissue block from the mouse, and digesting the stomach tissue block by adopting enzyme mixed liquor, wherein the enzyme mixed liquor comprises collagenase IV, hyaluronidase and deoxyribonuclease;
s3, stopping digestion by adopting fetal calf serum after digestion is finished, adding the product into a 50ml centrifuge tube, centrifuging at the temperature of 4 ℃ at 800rmp/min, and re-suspending cell sediment by using an RPMI1640 complete culture medium;
s4, separating and purifying the stomach tissue macrophage by using a Percoll equal density gradient precipitation method;
s5.2500rmp/min, centrifuging at 4 deg.C, and sucking out the cloud-like middle layer cell band with a suction tube;
s6, adding 3 times of Percoll liquid into RPMI1640 complete culture medium for dilution, centrifuging at 800rmp/min and 4 ℃, and suspending cell sediment by using the RPMI1640 complete culture medium, wherein the cell suspension is the gastric tissue macrophage knocked out by ANKRD22 gene.
Further, in S1, the amount of HCl/EtOH lavage was 5mL/kg body weight in mild to moderate injury stimulation of gastrointestinal mucosa.
Further, in S2, during the gastrointestinal mucosa repair period of 24-48h, the gastric tissue mass was removed from the mouse and the minced gastric tissue mass was digested with an enzyme mixture preheated to 37 ℃.
Further, in S2, the stomach tissue mass is removed from the mouse, specifically, the decapitated mouse is soaked in 75% alcohol, and then the mouse is placed on a sterile super clean bench, and the stomach tissue mass is removed with surgical scissors and forceps.
Further, in S2, before the digestion of the stomach tissue mass with the enzyme mixture, the digestion process further includes the step of adding 4 ℃ physiological saltThoroughly flushing blood of the tissue blocks with water, removing visible fat and connective tissues with sterile scissors, and shearing the tissue blocks into 1-3 mm3Small pieces of size, and then digested. It will be appreciated that cutting the pieces of stomach tissue into smaller pieces provides better digestion, improving the quality and efficiency of digestion.
Further, in S2, the enzyme mixture contained 50mg/mL of collagenase I, 50mg/mL of collagenase IV, 5mg/mL of hyaluronidase, and 1U/mL of DNase.
Further, in S3, after digestion is completed, fetal calf serum is used for stopping digestion, the product is added into a 50ml centrifuge tube, centrifugation is carried out at 800rmp/min and 4 ℃, and cell precipitation is suspended by RPMI1640 complete culture medium; if the supernatant is viscous after digestion, adding deoxyribonuclease for digestion, stopping reaction with fetal calf serum, centrifuging at 4 deg.C at 800rmp/min, suspending cell precipitate with RPMI1640 complete culture medium, and temporarily placing in 37 deg.C incubator; mixing all the resuspended cell suspensions, centrifuging at 800rmp/min and 4 ℃, and then resuspending the mixture in RPMI1640 complete medium;
further, in S4, the method for separating and purifying macrophages in gastric tissue by Percoll isopycnic gradient precipitation is specifically that 45% and 35% Percoll solutions are slowly added along the side wall of a 15ml centrifuge tube from large to small according to the density by using a glass syringe with a 14-gauge long needle head, 3ml is added for each gradient, and no air bubbles are generated in the operation process; slowly adding 3ml of cell suspension into a centrifuge tube along the tube wall by using a straw, and paving the cell suspension on the liquid surface with Percoll density gradient; in one implementation of the present invention, 90% of Percoll mother liquor is prepared from 9 parts of Percoll solution and 1 part of 10 × D-Hanks 'solution, and 35% and 45% of Percoll solution is prepared from Percoll mother liquor and DHanks' solution;
further, in S6, RPMI1640 complete medium with 3 times volume of Percoll solution was added for dilution, and after centrifugation at 800rmp/min and 4 ℃, a small amount of RPMI1640 complete medium was used to resuspend cell pellets, which were the ANKRD22 gene knockout gastric tissue macrophages of the present invention.
In the invention, the cell is centrifuged at 800rmp/min and 4 ℃, wherein the centrifugation speed of 800rmp/min is used for precipitating the cells without breaking; the centrifugation temperature of 4 ℃ is mainly to enable the cells to be at a lower temperature, slow down the growth and metabolism of the cells and enable the cells to exist more stably.
In a third aspect, the present invention provides a gastrointestinal mucosa repair protective agent prepared from macrophages from gastric tissue having the ANKRD22 gene knocked out.
The research shows that the deletion of the ANKRD22 gene can repair mild and moderate damage of gastrointestinal mucosa, can expand LGR5+ stem cells in stomach tissue, relieve inflammation of the stomach tissue and increase secretion of gastric mucus, and can be used for repairing mild and moderate damage of the gastrointestinal mucosa. Therefore, the ANKRD22 gene can be used as a target point to prepare a gastrointestinal mucosa repair protective agent; if the expression of ANKRD22 gene can be reduced, the effect of treating mild and moderate gastrointestinal mucosa injury can be achieved.
It should be noted that, through research, the deletion of the ANKRD22 gene has no obvious influence on the growth and development of biological individuals and no obvious influence on non-damaged stomach tissues, so that the ANKRD22 gene can be used as a target point to prepare the gastrointestinal mucosa repair protective agent.
The inventor finds in research that ANKRD22 is a novel target spot related to LGR5+ stem cell expansion after mild and moderate damage of gastric tissue, and gene knockout or shRNA mode interference of ANKRD22 is carried out by using CRISPR/Cas9 technology, so that the symptom of mild and moderate gastrointestinal mucosal damage of experimental mice can be effectively relieved.
In the specific experiment of the invention, the gene expression of ANKRD22 is interfered by a gene knockout technology, so that the symptom of mild and moderate gastrointestinal mucosal injury of an experimental mouse caused by a hydrochloric acid ethanol solution (150mM HCl/60% absolute ethanol solution, HCl/EtOH) can be effectively relieved. Further, in vitro and in vivo experiments for detecting ANKRD22 knockout or over-expression cells, we found that the ANKRD22 target is closely related to Wnt-Ca2+ signaling pathway.
It should be noted that, through research, the inventors found that macrophages in gastric tissue with deletion of ANKRD22 gene can reduce inflammatory reaction of gastric tissue after mild and moderate injury, and increase secretion of gastric mucus, so that the gastrointestinal mucosa repair protective agent can be prepared from macrophages in gastric tissue with ANKRD22 gene as target.
The ANKRD22 gene knockout gastric tissue macrophage disclosed by the invention has a good effect of repairing gastrointestinal mucosa injury, and can reduce the inflammatory reaction of gastric tissue, so that the macrophage can be completely prepared into a gastrointestinal mucosa repair protective agent for repairing the mild and moderate gastrointestinal mucosa injury. The medicament may further include other pharmaceutically acceptable auxiliary components or active components, which are not specifically limited herein.
In summary, the invention proves that ANKRD22 is a new target of a gastrointestinal mucosa repair protective agent targeting LGR5+ stem cells, can reduce inflammatory reaction of gastric tissues, increases secretion of gastric mucus, is closely related to a Wnt-Ca2+ signal pathway, and can effectively repair mild and moderate damage symptoms of gastrointestinal mucosa by interfering with ANKRD22 gene expression by any technology.
In conclusion, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the invention provides a new target for repairing mild and moderate damage of gastrointestinal mucosa, namely, by inhibiting the expression of ANKRD22 gene, the invention not only can amplify the LGR5+ stem cells of stomach tissue, but also can relieve the inflammatory reaction of stomach tissue and increase the secretion of stomach mucus. The macrophage of the gastric tissue with the knockout ANKRD22 gene can be used for developing a novel gastrointestinal mucosa repair protective agent targeting stem cells.
Drawings
FIG. 1: effect of deletion of ANKRD22 Gene on HCl/EtOH-induced mild to moderate gastrointestinal mucosal injury. Wherein, WT, world
type: wild type mice. Ankrd 22-/-: mice with deletion of ANKRD22 gene. Control (ctrl): and (4) a control group. HCl/EtOH, ethanolic HCl: and (3) an experimental group for intragastric administration of hydrochloric acid and ethanol.
FIG. 2: effect of deletion of ANKRD22 gene on LGR5 levels in mouse gastric tissue after HCl/EtOH-induced mild-to-moderate gastrointestinal mucosal injury repair 24. Wherein, Lgr 5: g protein coupled receptor 5.
FIG. 3: effect of deletion of ANKRD22 Gene on Mucin2, Mucin5AC and Mucin6 levels in mouse gastric tissue after HCl/EtOH induced mild to moderate gastrointestinal mucosal injury repair 24. Wherein, Muc 2:
FIG. 4: effect of deletion of ANKRD22 gene on Ca2+ levels in mouse gastric tissue cells after HCl/EtOH-induced repair of mild to moderate gastrointestinal mucosal injury 24.
FIG. 5: effect of deletion of ANKRD22 Gene on the levels of mouse gastric tissue macrophage inflammatory factors IL-1 β and IL-18 after HCl/EtOH-induced mild-moderate gastrointestinal mucosal injury repair 24. Wherein, IL-1 β: interleukin 1 β; IL-18: interleukin 18.
Detailed Description
According to the invention, a new therapeutic target, namely ANKRD22 gene, is found in the process of researching gastrointestinal mucosa injury, and researches prove that the ANKRD22 gene is knocked out to reduce the expression of ANKRD22 gene, the mild and moderate gastrointestinal mucosa injury can be effectively repaired, LGR5+ stem cells of a stomach tissue are amplified, the inflammatory reaction of the stomach tissue is relieved, the mucus secretion of the stomach tissue is increased, and the effect of protecting the gastrointestinal mucosa is achieved. It can be understood that the effect of repairing mild and moderate gastrointestinal mucosal injury can be achieved as long as the expression of ANKRD22 gene can be reduced; thus, in addition to gene knock-out, gene knockdown or chemical reduction of expression of the ANKRD22 gene may also serve the same protective effect on the gastrointestinal mucosa.
In addition, the research of the invention proves that the macrophage of the gastric tissue knocked out by the ANKRD22 gene can repair the mild and moderate damage of the gastrointestinal mucosa after being extracted, and alleviate the inflammatory reaction of the gastric tissue, thereby playing the role of protecting the gastrointestinal mucosa; therefore, the macrophage of the gastric tissue with the ANKRD22 gene knocked out can be used for developing a novel targeted drug of the gastrointestinal mucosa repair protective agent.