WO2023217128A1 - Cellule de type précurseur épithélial de muqueuse gastrique, son procédé de préparation et son utilisation - Google Patents

Cellule de type précurseur épithélial de muqueuse gastrique, son procédé de préparation et son utilisation Download PDF

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WO2023217128A1
WO2023217128A1 PCT/CN2023/092963 CN2023092963W WO2023217128A1 WO 2023217128 A1 WO2023217128 A1 WO 2023217128A1 CN 2023092963 W CN2023092963 W CN 2023092963W WO 2023217128 A1 WO2023217128 A1 WO 2023217128A1
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cells
gastric mucosal
mucosal epithelial
epithelial precursor
gastric
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周丽
周伸奥
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上海赛立维生物科技有限公司
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a gastric mucosal epithelial precursor-like cell and its preparation method and application.
  • the self-renewing epithelium of the gastric body contains 4 types of terminally differentiated cells that are replaced at different rates: oxyntic cells (parietal cells), zymogen cells (chief cells), cervical mucus cells, and hormone-secreting enteroendocrine cells.
  • Neck mucus cells have secretory functions and serve as intermediate progenitors to chief cells.
  • stem cells Although the properties of stem cells differ in the gastric corpus and antrum, it is generally accepted that all gastric mucosal cells are derived from stem cells. Stem cells in adult tissues can regenerate all resident cell types in a lineage, and the starting point for studying stem cells is often to exploit their potential to regenerate lost or damaged tissue.
  • Peptic ulcer mainly refers to chronic ulcers that occur in the stomach and duodenum, namely gastric ulcer (GU) and duodenal ulcer (DU). It is a clinical Common chronic gastrointestinal diseases. Due to the use of gastric acid suppression drugs, gastric mucosal protection drugs, and Helicobacter pylori eradication drugs, the incidence of PU has shown a downward trend, and the effective cure rate has increased significantly. However, how to reduce the recurrence rate of PU is still a current issue. A big problem in the treatment of anterior PU.
  • MSCs mesenchymal stem cells
  • gastric mucosal epithelial precursor-like cells have the advantages of large number of cells, less invasiveness in sampling, ability to differentiate into gastric mucosal epithelial cells, and no immune rejection.
  • Using gastric mucosal epithelial precursor-like cell transplantation to treat GU may enhance the gastric mucosal barrier in patients with gastric ulcer, and is expected to become a novel treatment method.
  • gastric mucosal epithelial precursor-like cells can be transplanted to treat GU
  • people will have higher and higher requirements for gastric mucosal epithelial precursor-like cells.
  • Human primary cells will more closely represent gastric mucosal epithelial cells, but current technology only Limited to the isolation of differentiated gastric mucosal epithelial cells, these gastric mucosal epithelial cells cannot self-renew and therefore can only maintain activity for a few days in vitro or in an ex vivo environment.
  • the purpose of the present invention is to provide novel gastric mucosal epithelial precursor-like cells and their preparation methods and applications, so as to solve the problem that gastric mucosal epithelial precursor-like cells cannot be continuously passaged in vitro and cannot express gastric mucosal epithelial precursor-like cell markers. .
  • the preparation method of gastric mucosal epithelial precursor-like cells of the present invention includes the following steps:
  • S0 Provide gastric mucosal epithelial primary cells
  • S1 Place the gastric mucosal epithelial primary cells into a reprogramming medium for dedifferentiation culture until the confluence of the gastric mucosal epithelial precursor-like cells is not less than 80%, and use trypsin digestion solution to The gastric mucosal epithelial precursor-like cells are digested to obtain gastric mucosal epithelial precursor-like cells, wherein the reprogramming medium includes basal medium, gastrin and growth factors.
  • the beneficial effect of the method for preparing gastric mucosal epithelial precursor-like cells of the present invention is that by placing the gastric mucosal epithelial primary cells into a reprogramming medium for dedifferentiation and culture until the gastric mucosal epithelial precursor-like cells
  • the degree of confluence is not less than 80%, and the gastric mucosal epithelial precursor-like cells are digested using trypsin digestion solution to obtain gastric mucosal epithelial precursor-like cells
  • the reprogramming medium includes a basal medium , gastrin and growth factors to maintain the gastric mucosal epithelial precursor-like cells to continuously achieve self-renewal and proliferation in vitro culture
  • the preparation method of the gastric mucosal epithelial precursor-like cells also includes: S2: placing the gastric mucosal epithelial precursor-like cells into the reprogramming medium for expansion and culture until the gastric mucosal epithelium After the fusion degree of the precursor-like cells is not less than 80%, the gastric mucosal epithelial precursor-like cells are digested using the pancreatic digestion solution to obtain passaged gastric mucosal epithelial precursor-like cells.
  • gastric mucosal epithelial precursor-like cells are further expanded and the obtained gastric mucosal epithelial precursor-like cells can express gastric mucosal epithelial precursor-like cell markers.
  • the reprogramming medium also includes ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF- ⁇ signaling inhibitors, nutritional supplements and buffers.
  • the content of gastrin is 0.1-5 nmol/L
  • the content of the growth factor is 190-340 ng/ml based on the volume of the basal culture medium.
  • the content of the ROCK kinase inhibitor is 5-15 ⁇ M
  • the content of the Wnt signaling pathway agonist is 1-10 ⁇ M
  • the content of the TGF- ⁇ signaling inhibitor is The content is 0.1-5 ⁇ M
  • the content of the nutritional supplement is 2-10%.
  • the growth factors include fibroblast growth factor 10, epidermal growth factor and basic fibroblast growth factor.
  • the content of the fibroblast growth factor 10 is 150-250ng/ml
  • the content of the epidermal growth factor is 10-30ng/mL
  • the alkaline component The content of fiber growth factor is 30-60ng/mL.
  • the gastric mucosal epithelial precursor-like cells positively express at least one molecule including LGR5 and CD44, at least one keratin including CK19, and at least one CD molecule including CD24.
  • the gastric mucosal epithelial precursor-like cells do not express HLA-DRPQ.
  • the present invention also provides a gastric mucosal epithelial precursor-like cell, which is prepared by the method for preparing gastric mucosal epithelial precursor-like cells.
  • the beneficial effect of the gastric mucosal epithelial precursor-like cells of the present invention is: on the gastric mucosa
  • the preparation method of epithelial precursor-like cells is simple, and the obtained gastric mucosal epithelial precursor-like cells can reduce the impact of gastric ulcer on the human body.
  • the present invention also provides an application of gastric mucosal epithelial precursor-like cells.
  • the gastric mucosal epithelial precursor-like cells are used to prepare a gastric mucosal epithelial precursor-like cell preparation for treating peptic ulcer.
  • Using the gastric mucosal epithelial precursor-like cells The effects of somatic cell preparations on peptic ulcer were examined after intervention in in vivo animal models.
  • the beneficial effect of the application of the gastric mucosal epithelial precursor-like cells of the present invention is that it can treat peptic ulcer and will not cause immune rejection after allogeneic transplantation.
  • the animal model includes a drug-induced gastric ulcer model.
  • Figure 1 is a photographic schematic diagram of the morphology of fourth-generation gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention
  • Figure 2 is a schematic diagram of the gene expression marker CD24 of gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of the gene expression marker LGR5 of gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention
  • Figure 4 is a schematic diagram of the gene expression marker CD44 of gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention
  • Figure 5 is a schematic diagram of the gene expression marker CK19 of gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention.
  • Figure 6 is a schematic diagram of the gene expression marker HLA-DRPQ of gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention.
  • Lgr5+ve cells could form micro-organisms similar to the stomach.
  • Structure namely gastric organoid
  • this liquid environment simulates the microenvironment of stem cells in the body
  • R-spondin Wnt signaling pathway activator
  • EGF is an important factor in maintaining the proliferation of Lgr5+ gastric stem cells in vitro.
  • Noggin can promote the increase in the number of gastric pits in organoids
  • laminin-rich Matrigel provides an environment that supports the growth of gastric organoids.
  • This gastric organoid model can undergo long-term proliferation and maintain stable genetic characteristics and biological behaviors in vitro.
  • progenitor cell population that can produce multiple cell subtypes, which can differentiate and form under in vitro culture induction conditions.
  • Gastric epithelial cells with different functions.
  • Garcia et al. used a medium rich in EGF, HGF, fibroblast growth factor (FGF), etc. to conduct primary isolation and culture of adult gastric mucosal layer cells, and observed that in gastric cells growing in a single layer, Spherical colonies were formed on the surface of fibroblasts. Further identification of the cell clusters that grew into spheres revealed that these cells could express molecular markers unique to embryonic stem cells and gastric stem cells, and could spontaneously differentiate to form gastric epithelial progenitor cells under in vitro culture conditions.
  • EGF EGF
  • HGF fibroblast growth factor
  • Each gastric unit in the stomach contains a small population of monoclonal stem cells capable of lifelong epithelial self-renewal.
  • gastric stem cells are essentially similar to intestinal stem cells, little is known about them.
  • Gastric stem cells may be involved in the pathogenesis of gastric cancer, a global health problem.
  • gastric stem cells may reside in the isthmus, circulate slowly, produce progeny that migrate in both directions, differentiate into mature resident lineages, and have variable lifespans.
  • the lack of molecular markers is the biggest obstacle to gastric stem cell research. The identification of gastric stem cell markers in normal and disease states, as well as reliable methods for cultivating and expanding gastric stem cells in vitro, are the focus of research in this field.
  • antral stem cells are more Nearer the base of the gland, fewer types of progeny are produced that appear to have characteristics of hybridization between antral cells and intestinal stem cells.
  • At least a subset, if not all, of gastric antral stem cells carry the surface marker LGR5 and replicate rapidly (perhaps daily) in adult mice, where LGR5 can long-term promote all mature epithelial lineages.
  • LGR5 can long-term promote all mature epithelial lineages.
  • their growth requirements and signaling pathways should be further determined, as well as the determinants of whether these cells undergo sustained replication or lineage differentiation. Because stem cells throughout the stomach continuously respond to external signals and local tissue damage, they must occupy a complex niche that conveys homeostatic signals as well as information about infection and inflammation.
  • a combination of intrinsic and microenvironmental signals may transform gastric stem cells with proliferative potential into cells with abnormal, metaplastic differentiation patterns, leading to dysplasia and cancer. Because of our limited understanding of the stem cell microenvironment, few areas in basic gastroenterology research have generated as much interest or challenge. It will be important to develop methods to isolate and culture stem cells that express validated molecular markers. Infection and tissue damage can induce gastric metaplasia and cancer, and this development will advance the understanding of stem cell properties and responses to infection and tissue damage.
  • the present invention provides a method for preparing gastric mucosal epithelial precursor-like cells, which includes the following steps:
  • S0 Provide gastric mucosal epithelial primary cells
  • S1 Place the gastric mucosal epithelial primary cells into a reprogramming medium for dedifferentiation culture until the confluence of the gastric mucosal epithelial precursor-like cells is not less than 80%, and use trypsin digestion solution to Gastric mucosal epithelial precursor-like cells are digested to obtain gastric mucosa Epithelial precursor-like cells, wherein the reprogramming medium includes basal medium, gastrin and growth factors.
  • the gastric mucosal epithelial primary cells are put into a reprogramming medium for dedifferentiation culture until the confluence of the gastric mucosal epithelial precursor-like cells is not less than 80%, and trypsin digestion solution is used to
  • the gastric mucosal epithelial precursor-like cells are digested to obtain gastric mucosal epithelial precursor-like cells, wherein the reprogramming medium includes basal medium, gastrin and growth factors to maintain gastric mucosal epithelial precursors
  • the gastric mucosal epithelial precursor-like cells continuously realize self-renewal and proliferation in vitro culture, thus this application solves the problem that gastric mucosal epithelial precursor-like cells cannot be continuously passaged in vitro and cannot express gastric mucosal epithelial precursor-like cell markers.
  • the method for preparing gastric mucosal epithelial precursor-like cells further includes: S2: placing the gastric mucosal epithelial precursor-like cells into the reprogramming medium for expansion and culture until the After the fusion degree of the gastric mucosal epithelial precursor-like cells is not less than 80%, the gastric mucosal epithelial precursor-like cells are digested using the pancreatic digestion solution to obtain passaged gastric mucosal epithelial precursor-like cells. .
  • the gastric mucosal epithelial precursor-like cells can be continuously expanded in the reprogramming medium and the obtained gastric mucosal epithelial precursor-like cells can express gastric mucosal epithelial precursor-like cell markers.
  • the reprogramming medium further includes ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF- ⁇ signaling inhibitors, nutritional supplements and buffers.
  • the ROCK kinase inhibitor Y-27632 is derived from Taoshu Biotech
  • the Wnt signaling pathway agonist CHIR99021 is derived from Taoshu Biotech
  • the TGF- ⁇ signaling inhibitor A8301 is derived from Taoshu Biotech.
  • the nutritional supplement includes N2 Nutritional supplements and B27 nutritional supplements, the N2 nutritional supplement is from Yisheng Bio, and the B27 nutritional supplement is from Yisheng Bio.
  • the content of gastrin is 0.1-5 nmol/L
  • the content of the growth factor is 190-340 ng/ml based on the volume of the basal culture medium.
  • the content of gastrin is any one of 0.5nmol/L, 1nmol/L, 2nmol/L, 3nmol/L and 4nmol/L
  • the content of the growth factor is any one of 200ng/ml, 230ng/ml, 250ng/ml, 270ng/ml, 300ng/ml and 320ng/ml.
  • the content of the ROCK kinase inhibitor is 5-15 ⁇ M
  • the content of the Wnt signaling pathway agonist is 1-10 ⁇ M
  • the TGF- ⁇ The content of the signal inhibitor is 0.1-5 ⁇ M
  • the content of the nutritional supplement is 2-10%.
  • the content of the ROCK kinase inhibitor is any one of 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, and 14 ⁇ M.
  • the content of the Wnt signaling pathway agonist is 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M and 9 ⁇ M
  • the content of the TGF- ⁇ signal inhibitor is 0.5 ⁇ M, 1 ⁇ M, 1.5 ⁇ M, 2 ⁇ M, 2.5 ⁇ M, 3 ⁇ M, 3.5 ⁇ M and 4 ⁇ M
  • the content of the nutritional supplement is any one of 3%, 4%, 5%, 6%, 7%, 8% and 9%.
  • the growth factors include fibroblast growth factor 10, epidermal growth factor and basic fibroblast growth factor.
  • the fibroblast growth factor 10 (English Named Fibroblast Growth Factors 10, English abbreviation FGF10) is derived from Shanghai Xinyu Biotechnology, the epidermal growth factor (English name is Epidermal growth factor, English abbreviation EGF) is derived from nearshore organisms, and the alkaline fibroblast growth factor (The English name is basic fibrobast growth factor, the English abbreviation is bFGF) is derived from offshore organisms; the gastrin (English name is gastrin) is derived from Beijing Boermai Biotechnology.
  • the epidermal growth factor English name is Epidermal growth factor, English abbreviation EGF
  • EGF epidermal growth factor
  • alkaline fibroblast growth factor The English name is basic fibrobast growth factor, the English abbreviation is bFGF
  • the gastrin (English name is gastrin) is derived from Beijing Boermai Biotechnology.
  • the content of the fibroblast growth factor 10 is 150-250ng/ml, and the content of the epidermal growth factor is 10-30ng/mL, based on the volume of the basal culture medium, so The content of the basic fibroblast growth factor is 30-60ng/mL.
  • the content of the fibroblast growth factor 10 is 160ng/ml, 170ng/ml, 180ng/ml, 190ng/ml, 200ng/ml, 210ng/ml , any one of 220ng/ml, 230ng/ml and 240ng/ml
  • the content of the epidermal growth factor is any one of 15ng/mL, 20ng/mL, 25ng/mL
  • the content of the factor is any one of 35ng/mL, 40ng/mL, 45ng/mL, 50ng/mL and 55ng/mL.
  • the gastric mucosal epithelial precursor-like cells positively express at least one molecule including LGR5 and CD44, at least one keratin including CK19, and at least one CD molecule including CD24.
  • the gastric mucosal epithelial precursor-like cells do not express HLA-DRPQ.
  • the HLA-DRPQ is HLA-DR/DP/DQ.
  • the present invention also provides a gastric mucosal epithelial precursor-like cell
  • Somatic-like cells are prepared by the method for preparing gastric mucosal epithelial precursor-like cells.
  • the preparation method of the gastric mucosal epithelial precursor-like cells is simple, and the obtained gastric mucosal epithelial precursor-like cells can reduce the impact of gastric ulcer on the human body.
  • the present invention also provides an application of gastric mucosal epithelial precursor-like cells.
  • the gastric mucosal epithelial precursor-like cells are used to prepare a gastric mucosal epithelial precursor-like cell preparation for treating peptic ulcer.
  • Using the gastric mucosal epithelial precursor-like cells The effects of somatic cell preparations on peptic ulcer were examined after intervention in in vivo animal models. It can treat peptic ulcers and will not cause immune rejection after allogeneic transplantation.
  • the animal model includes a drug-induced gastric ulcer model.
  • Gastric mucosal epithelial tissue was used as the starting material to obtain human gastric mucosal epithelial precursor-like cells that positively express CD24, LGR5, CD44 and CK19.
  • gastric mucosal epithelial tissue was normal gastric mucosal epithelial tissue.
  • the normal gastric mucosal epithelial tissue is a surgical sample derived from a patient who is no more than 70 years old.
  • the patient has no infectious viral infection after medical examination, and the patient has not used steroid hormone drugs within 6 months before surgery.
  • the patient was fully informed about the purpose of obtaining surgical samples before surgery and signed an informed consent form.
  • sterile PBS buffer to clean and sterilize the gastric mucosal epithelial tissue.
  • 3 ml of cell digestion solution is composed of type I collagenase and sterile PBS buffer.
  • the volume of sterile PBS buffer and trypsin digestion solution is the same, and the volume percentage of type I collagenase in the cell digestion solution is 1%;
  • the components of the reprogramming medium include: epithelial cell growth factor EGF with a content of 20 ng/ml based on the volume of the basal medium DMEM/F12 (from Wuhan Pronosai Life Technology Co., Ltd.) and 50 ng/ml.
  • fibroblast growth factor 10 g/ml basic fibroblast growth factor bFGF, 1% nutritional supplement N2 (1X), 1% nutritional supplement B27 (1X), 10 ⁇ M ROCK kinase inhibitor Y-27632 , Wnt signaling pathway agonist CHIR99021 at 3 ⁇ M, TGF- ⁇ signaling inhibitor at 1 ⁇ M A8301, gastrin at 1 nanomol/L and fibroblast growth factor 10 (FGF10) at 200 ng/ml in 5% fetal bovine serum (FGF10)
  • FBS from Beijing Baiolaibo Technology Co., Ltd.
  • Figure 1 is a photographic schematic diagram of the morphology of fourth-generation gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention.
  • Figure 2 is a schematic diagram of the gene expression marker CD24 of gastric mucosal epithelial precursor-like cells in an embodiment of the present invention
  • Figure 3 is a schematic diagram of the gene expression marker LGR5 of gastric mucosal epithelial precursor-like cells in an embodiment of the present invention
  • Figure 4 is a schematic diagram of the gene expression marker CD44 of gastric mucosal epithelial precursor-like cells according to an embodiment of the present invention
  • Figure 5 is A schematic diagram of the gene expression marker CK19 of gastric mucosal epithelial precursor-like cells in an embodiment of the present invention
  • Figure 6 is a schematic diagram of the gene expression marker HLA-DRPQ of gastric mucosal epithelial precursor-like cells in an embodiment of the present invention.
  • the fourth-generation gastric mucosal epithelial precursor-like cells are rinsed with sterile PBS buffer, and then the fourth-generation gastric mucosal precursor-like cells are treated with trypsin digestion solution.
  • the epithelial precursor-like cells are digested and then centrifuged. After the centrifugation, the cell pellet is collected. The centrifugation speed is 300g and the centrifugation time is 5 minutes; 100 ⁇ l of dye is added to the cell pellet. Resuspend the cells in the buffer into the flow tube, then add 5 ⁇ l of the flow cytometry antibody to be tested and incubate for 20 minutes. Resuspend the cells in each flow tube with 400 ⁇ l of staining buffer to obtain a cell suspension. The cell suspension is subjected to flow cytometry.
  • the specific steps of flow cytometry include:
  • the cell pellet was resuspended in liquid and then transferred to a flow tube.
  • the resuspended cell mixture was subjected to flow cytometry detection of surface markers.
  • the antibody names and product numbers are: CD24 (abcam, ab290730); CD44 (abcam, ab254530); HLA-DRPQ (abcam, ab7856),
  • the positivity rate of the positive peak of marker CD24 is 96.3%
  • the positivity rate of the positive peak of marker LGR5 is 96.7%
  • the positivity rate of the positive peak of marker CD44 is 99.9 %
  • the positive rate of the positive peak of marker CK19 is 93.2%.
  • the positive rate of the positive peak is greater than 70%, indicating that the gastric mucosal epithelial precursor-like cells positively express the marker. Therefore, the gastric mucosal epithelial precursor-like cells of the present invention are positive.
  • the P4 generation gastric mucosal epithelial precursor cells show the characteristics of gastric mucosal epithelial precursor cells; referring to Figure 6, the positivity rate of the positive peak of the marker HLA-DRPQ is 0.31%, and the positivity rate of the positive peak is 0.31%. A rate greater than 70% indicates that the gastric mucosal epithelial precursor-like cells positively express the marker. Therefore, the gastric mucosal epithelial precursor-like cells of the present invention negatively express HLA-DRPQ, indicating that the gastric mucosal epithelial precursor-like cells of the present invention do not express MHC. - Class II antigen. After the patient transplants the cells, the body cannot recognize the cells through the MHC class II antigen of the immune system. The patient's immune system will not launch an immune attack on them, so the patient's body is less likely to produce a rejection reaction. .
  • Gastric mucosal epithelial precursor-like cells are inoculated on sodium alginate gel to prepare a gastric mucosal epithelial precursor-like cell preparation.
  • the gastric mucosal epithelial precursor-like cell preparation is used to repair ulcerated gastric mucosal tissue.
  • the gastric mucosal epithelial precursor-like cell preparation The cell-like preparation is a patch of gastric mucosal epithelial precursor-like cells, in which each square centimeter of gastric mucosal epithelial precursor-like cell patch contains 1 ⁇ 10 6 gastric mucosal Epithelial precursor-like cells.
  • precursor cells or stem cells especially those derived from adult gastric mucosal epithelium, to repair damaged gastric tissue is a promising new therapy for the treatment of gastric diseases.
  • the gastric mucosal barrier is a complex system involving physical, chemical, and biological defense mechanisms that protect the stomach from irritating foods, hydrochloric acid, and pepsin activity.
  • Several conditions such as gastritis, gastric erosions, and ulcers, can disrupt the gastric mucosal barrier leading to its destruction.
  • Gastric mucosal injury is a common disease in veterinary medicine. Due to many frequently used medications such as NSAIDs or corticosteroids.
  • There are several diseases that can disrupt mucosal defense mechanisms including Helicobacter pylori infection, liver or kidney disease, adrenocortical insufficiency, shock, spinal cord disease, autoimmune disease, primary gastrointestinal disease, and tumors.
  • gastric or duodenal ulcers are usually caused by defects in the gastric mucosa or duodenal epithelial barrier function.
  • a gastric ulcer has been described as a large "moon crater" defect in the stomach lining. Endoscopic studies revealed that 48.5% of dogs had ulcers in the stomach or proximal duodenum.
  • Gastric cancer is also more common in gastric cancer than in other livestock animals because tumor resection results in deep gastric wounds requiring tissue reconstruction. Most gastric malignancies are carcinomas, accounting for 50-90%. This is followed by sarcoma and malignant lymphoma. Some studies have attempted to transplant gastric mucosal defects.
  • Gastric mucosal defects can be caused by a variety of factors, including the use of nonsteroidal anti-inflammatory drugs, Helicobacter pylori infection, gastrointestinal and spinal cord diseases, and tumors, causing great pain to patients.
  • the animal model is the beagle gastric ulcer model, and the indication is gastric ulcer;
  • Each male beagle was treated with ketamine hydrochloride for induction and maintenance of anesthesia.
  • gastric mucosal epithelial precursor cell patch After surgery, the effect of the gastric mucosal epithelial precursor cell patch on gastric mucosal repair was detected through endoscopic and blood biochemical methods.
  • the gastric mucosal epithelium can continuously regenerate and repair to cope with the internal environment, pathogens and foreign substances, and gastric precursor cells play a key role in this.
  • This kind of specific precursor cells that exist in the gastric mucosal epithelium are adult stem cells. They always maintain the ability of self-renewal. After the aging or damaged cells are shed, they can divide and differentiate to produce new gastric mucosal epithelial cells for supplementation, thereby maintaining The homeostasis of epithelial cells promotes the regeneration and repair of epithelial cells in the gastric mucosa, and even the development of cancer.
  • Gastric precursor cells play a key role in the dynamic renewal and post-injury repair of gastric mucosal epithelium. With the development of technologies such as lineage tracing and molecular markers, breakthroughs have been made in understanding the physiological characteristics of gastric precursor cells. Molecular markers such as Lgr5 and SOX2 have also been identified, and in vitro research models have been established. Since the dynamic balance of proliferation and differentiation of gastric precursor cells is the key to maintaining the homeostasis of the stem cell pool, sustained inflammatory response can induce accelerated proliferation of precursor cells and cause instability in the gastric stem cell pool, thereby participating in the occurrence of gastric mucosal lesions.
  • H.pylori infection induces resting-proliferation differentiation imbalance and cell phenotype transformation of gastric precursor cells, which may be one of the mechanisms of gastric cancer.
  • gastric precursor cells may also serve as the cells of origin of gastric tumors and participate in multiple steps of the occurrence and development of gastric cancer. It is crucial to further explore the mechanism of gastric precursor cells in the repair of gastric mucosal damage and their role in the occurrence of intestinal metaplasia and dysplasia in the gastric mucosa, especially how to integrate gastric precursor cells with the pathogenic mechanism of H. pylori , the molecular mechanism and origin of gastric cancer may become a research focus in the future. With the development and deepening of precursor cell research technology and theory, the study of gastric precursor-like cells has played an important role in gastric diseases. It has broad prospects in disease prevention and treatment.
  • gastric mucosal epithelial precursor-like cells into the damaged gastric mucosa, in addition to the gastric mucosal epithelial precursor-like cells themselves may differentiate into gastric mucosal epithelial cells, the cytokines they secrete may also interact with gastric mucosal epithelial precursor cells.
  • somatic cells not only promotes the differentiation of gastric mucosal epithelial precursor-like cells into gastric mucosal epithelial cells, but also stimulates the migration and proliferation of vascular endothelial cells to form new blood vessels, increase mucosal blood supply, inhibit inflammatory reactions and reduce fibrous tissue. Proliferate and promote ulcer healing, thereby achieving the purpose of wound repair and submucosal tissue reconstruction.

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Abstract

La présente invention concerne une cellule de type précurseur épithélial de muqueuse gastrique, son procédé de préparation et son utilisation. Le procédé de préparation de la cellule de type précurseur épithélial de muqueuse gastrique comprend les étapes suivantes : fournir des cellules épithéliales de muqueuse gastrique primaires ; placer les cellules épithéliales de muqueuse gastrique primaires dans un milieu de reprogrammation pour une culture de dédifférenciation jusqu'à ce que la confluence des cellules de type précurseur épithélial de muqueuse gastrique ne soit pas inférieure à 80 %, et effectuer un traitement de digestion sur les cellules de type précurseur épithélial de muqueuse gastrique à l'aide d'une solution de trypsine pour obtenir les cellules de type précurseur épithélial de muqueuse gastrique, le milieu de reprogrammation comprenant un milieu basique, de la gastrine et un facteur de croissance. La présente invention résout le problème selon lequel des cellules de type précurseur épithélial de muqueuse gastrique ne peuvent pas être transmises en continu in vitro et ne peuvent pas exprimer des marqueurs de cellules de type précurseur épithélial de muqueuse gastrique.
PCT/CN2023/092963 2022-05-10 2023-05-09 Cellule de type précurseur épithélial de muqueuse gastrique, son procédé de préparation et son utilisation WO2023217128A1 (fr)

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CN117025508A (zh) 2023-11-10
WO2023217134A1 (fr) 2023-11-16
CN117018033A (zh) 2023-11-10
CN117025507A (zh) 2023-11-10
WO2023217125A1 (fr) 2023-11-16
CN117025504A (zh) 2023-11-10
WO2023217121A1 (fr) 2023-11-16

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