A preparation method of a gel patch for repairing gastric tissue

Abstract

The invention discloses a preparation method of a gel patch for repairing gastric tissue, which is characterized in that the gastric tissue of a mammal is first taken, and then decellularized after washing to obtain a decellularized gastric patch matrix; then a viscous hyaluronic acid aqueous solution containing omeprazole is prepared; then the viscous hyaluronic acid aqueous solution is placed in a porous cell culture plate, glacial acetic acid is added for soaking, and after soaking, the viscous hyaluronic acid is taken out from the porous cell culture plate and attached to the gastric patch matrix, and freeze-dried to obtain a gel patch for repairing gastric tissue; the advantage is that the gel patch has good biocompatibility, contains a small amount of natural cytokines, can be slowly degraded and absorbed, and the degradation products can participate in the remodeling of extracellular matrix components, and have suitable mechanical properties and good plasticity. And the gel patch can effectively block the persistent damage of external harmful factors to the stomach, induce the self-healing and repair of gastric mucosa and muscle layer tissue, promote the regeneration of gastric wall tissue, and has no toxic side effects on the human body.

Description

Technical Field

[0001] The invention relates to a therapeutic article for gastric diseases, and in particular to a preparation method of a gel patch for repairing gastric tissue. Background Art

[0002] Since the digestive tract of the human body is directly connected to the external environment, coupled with some unreasonable eating habits that stimulate and wear the walls of the digestive tract, as well as bacteria and microbial invasion, the mucosal layer of the digestive tract epithelium is easily damaged, leading to gastric mucosal-related diseases, such as chronic inflammation, atrophic gastritis, atrophic gastritis with intestinal metaplasia and dysplasia, and chronic peptic ulcers even lead to acute gastric perforation, threatening the patient's life. At present, there are almost no other treatments for chronic inflammation of the stomach except for drug treatment. For more serious gastric diseases, such as gastric cancer, surgical resection combined with radiotherapy and chemotherapy is the main treatment method, which is more damaging to the human body and has obvious side effects. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing a gel patch for repairing gastric tissue. The prepared gel patch can induce self-healing and repair of gastric tissue, has significant therapeutic effect and few side effects.

[0004] The technical solution adopted by the present invention to solve the above technical problem is: a method for preparing a gel patch for repairing gastric tissue, comprising the following specific steps:

[0005] (1) obtaining a mammalian stomach tissue, washing it, and performing a decellularization treatment to obtain a decellularized stomach patch matrix;

[0006] (2) dissolving omeprazole in pure water to obtain an omeprazole aqueous solution with a concentration of 1 to 20 mg / mL, and then dissolving high molecular weight hyaluronic acid in the omeprazole aqueous solution to obtain a viscous hyaluronic acid aqueous solution containing omeprazole with a hyaluronic acid concentration of 1 to 20 mg / mL;

[0007] (3) The viscous hyaluronic acid aqueous solution obtained in step (2) is placed in a porous cell culture plate, and glacial acetic acid with a mass concentration of 0.1 to 2% is added to completely immerse the viscous hyaluronic acid in the porous cell culture plate, and the viscous hyaluronic acid is soaked for 2 to 24 hours. Then, the viscous hyaluronic acid is taken out from the porous cell culture plate and attached to the gastric patch substrate obtained in step (1). After being rinsed with PBS buffer with a concentration of 0.1 M, it is placed in a freeze dryer for freeze drying to obtain a gel patch for repairing gastric tissue.

[0008] Furthermore, the specific method of step (1) is as follows: the stomach tissue of a mammal is rinsed with clean water to remove impurities, and is soaked in 75% medical alcohol for 30 minutes, and is washed three times with a 0.1M PBS buffer solution, each time for 5 minutes, and then the stomach tissue is placed in a high osmotic pressure solution to disrupt the membrane for 24 hours, and then rinsed with a PBS solution containing double antibodies (streptomycin and penicillin) and placed in a mixture of 10000-50000U / mL of DNA enzyme and 1000-2000U / mL of RNA enzyme in a volume ratio of 10:1, and treated in a 37°C water bath for 2 hours; the above membrane disruption and nuclease treatment are repeated 2-3 times to obtain a decellularized gastric patch matrix.

[0009] Furthermore, the high osmotic pressure solution used in the step (1) is a mixture of a TritonX-100 solution with a mass concentration of 1 to 5% and a NaCl solution with a molar concentration of 1 to 5 M in a volume ratio of 1:1.

[0010] Furthermore, the mammal is a pig, a sheep or a rabbit.

[0011] Furthermore, the gastric tissue is gastric mucosa and submucosal tissue, or the entire gastric wall.

[0012] Furthermore, in the step (2), the molecular weight of the hyaluronic acid is greater than or equal to 1,200,000 Da.

[0013] Compared with the prior art, the advantages of the present invention are that compared with traditional drug therapy, the gel patch prepared by the present invention has good biocompatibility, contains a small amount of natural cytokines, can be slowly degraded and absorbed, and the degradation products can participate in the remodeling of extracellular matrix components, and has suitable mechanical properties and good plasticity. Moreover, the gel patch can effectively block the persistent damage to the stomach by external harmful factors, induce the self-healing and repair of gastric mucosa and muscle layer tissue, promote the regeneration of gastric wall tissue, and has no toxic side effects on the human body. In addition, the gel patch can make the omeprazole drug therein achieve the effect of slow release, reduce the frequency of administration, and omeprazole can inhibit the secretion of gastric acid, slow down the degradation rate of the patch, and also has appropriate anti-inflammatory effect, helping the gastric tissue to repair better. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic diagram of a finished product of the gel patch of the present invention;

[0015] Figure 2 These are pictures showing the effects of the gel patch of the present invention in different stages when used for gastric tissue repair in mice;

[0016] Figure 3 Comparison of pathological sections of mouse stomach tissue before and after repair. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the accompanying drawings.

[0018] Embodiment 1: A method for preparing a gel patch for repairing gastric tissue, comprising the following specific steps:

[0019] (1) Take the gastric mucosa and submucosal tissue of the pig, rinse with clean water to remove blood and other impurities, soak with 75% medical alcohol for 30 minutes, wash three times with 0.1M PBS buffer, each time for 5 minutes, and then place the gastric mucosa and submucosal tissue in a high osmotic pressure solution obtained by mixing a 2% TritonX-100 solution with a molar concentration of 2M NaCl solution in a volume ratio of 1:1 to disrupt the membrane for 24 hours, then rinse with a PBS solution containing double antibodies and place in a mixed solution of 10000U / mL DNase and 2000U / mL RNase in a volume ratio of 10:1, and treat in a 37°C water bath for 2 hours; repeat the above membrane disruption and nuclease treatment 3 times to obtain a decellularized gastric patch matrix;

[0020] (2) dissolving omeprazole in pure water to obtain an omeprazole aqueous solution with a concentration of 2 mg / mL, and then dissolving high molecular weight hyaluronic acid with a molecular weight greater than or equal to 1,200,000 Da in the omeprazole aqueous solution to obtain a viscous hyaluronic acid aqueous solution containing omeprazole with a hyaluronic acid concentration of 10 mg / mL;

[0021] (3) Place the viscous hyaluronic acid aqueous solution obtained in step (2) in a porous cell culture plate, add glacial acetic acid with a mass concentration of 2% to completely immerse the viscous hyaluronic acid in the porous cell culture plate, and soak for 12 hours. Then, take the viscous hyaluronic acid out of the porous cell culture plate and attach it to the gastric patch substrate obtained in step (1). After rinsing with 0.1M phosphate buffered saline (PBS), place it in a freeze dryer for freeze drying to obtain a gel patch for repairing gastric tissue.

[0022] Embodiment 2: A method for preparing a gel patch for repairing gastric tissue, comprising the following specific steps:

[0023] (1) Take the whole layer of stomach wall of the pig, rinse it with clean water to remove blood and other impurities, soak it in 75% medical alcohol for 30 minutes, wash it three times with 0.1M PBS buffer, each time for 5 minutes, and then place the whole layer of stomach wall in a high osmotic pressure solution obtained by mixing a 5% TritonX-100 solution with a molar concentration of 3M NaCl solution in a volume ratio of 1:1 to disrupt the membrane for 24 hours, then rinse it with a PBS solution containing double antibodies and place it in a mixture of 50000U / mL DNA enzyme and 2000U / mL RNA enzyme in a volume ratio of 10:1, and treat it in a 37℃ water bath for 2 hours; repeat the above membrane disruption and nuclease treatment 3 times to obtain a decellularized gastric patch matrix;

[0024] (2) dissolving omeprazole in pure water to obtain an omeprazole aqueous solution with a concentration of 20 mg / mL, and then dissolving high molecular weight hyaluronic acid with a molecular weight greater than or equal to 1,200,000 Da in the omeprazole aqueous solution to obtain a viscous hyaluronic acid aqueous solution containing omeprazole with a hyaluronic acid concentration of 20 mg / mL;

[0025] (3) Place the viscous hyaluronic acid aqueous solution obtained in step (2) in a porous cell culture plate, add glacial acetic acid with a mass concentration of 1% to completely immerse the viscous hyaluronic acid in the porous cell culture plate, and soak for 24 hours. Then, take the viscous hyaluronic acid out of the porous cell culture plate and attach it to the gastric patch substrate obtained in step (1). After rinsing with 0.1M phosphate buffered saline (PBS), place it in a freeze dryer for freeze drying to obtain a gel patch for repairing gastric tissue.

[0026] Embodiment 3: A method for preparing a gel patch for repairing gastric tissue, comprising the following specific steps:

[0027] (1) The gastric mucosa and submucosal tissue of the rabbit were taken, rinsed with clean water to remove blood and other impurities, and soaked in 75% medical alcohol for 30 minutes, and washed three times with 0.1M PBS buffer, each time for 5 minutes, and then the gastric mucosa and submucosal tissue were placed in a high osmotic pressure solution obtained by mixing a 3% TritonX-100 solution with a molar concentration of 5M NaCl solution in a volume ratio of 1:1 for 24 hours, and then rinsed with a PBS solution containing double antibodies and placed in a mixture of 30000U / mL DNA enzyme and 1000U / mL RNA enzyme in a volume ratio of 10:1, and treated in a 37°C water bath for 2 hours; the above membrane disruption and nuclease treatment were repeated twice to obtain a decellularized gastric patch matrix;

[0028] (2) dissolving omeprazole in pure water to obtain an omeprazole aqueous solution with a concentration of 10 mg / mL, and then dissolving high molecular weight hyaluronic acid with a molecular weight greater than or equal to 1,200,000 Da in the omeprazole aqueous solution to obtain a viscous hyaluronic acid aqueous solution containing omeprazole with a hyaluronic acid concentration of 5 mg / mL;

[0029] (3) The viscous hyaluronic acid aqueous solution obtained in step (2) is placed in a porous cell culture plate, and glacial acetic acid with a mass concentration of 0.5% is added to completely immerse the viscous hyaluronic acid in the porous cell culture plate, and the viscous hyaluronic acid is soaked for 10 hours. Then, the viscous hyaluronic acid is taken out from the porous cell culture plate and attached to the gastric patch substrate obtained in step (1). After being rinsed with 0.1M phosphate buffered saline (PBS), it is placed in a freeze dryer for freeze drying to obtain a gel patch for repairing gastric tissue.

[0030] In the above embodiments, the stomach tissues used were all taken from freshly dead pigs, rabbits and other mammals. The PBS solution containing double antibodies used in the decellularization treatment of step (1) is penicillin and streptomycin, and penicillin, streptomycin and phosphate buffer (PBS) are prepared by the existing conventional preparation method to obtain the PBS solution containing double antibodies. In addition, the multi-porous cell culture plate can be selected according to the size of the required patch, such as a 6-well, 12-well or 24-well cell culture plate.

[0031] The gel patch prepared by the present invention was used to repair the stomach tissue of mice to verify the effect, specifically:

[0032] (1) First, gastric perforation model was created in mice: inhalation gas anesthesia was used, and multiple rats were fixed in a supine position on a constant temperature operating table. The surgical area was disinfected and covered with a drape. Starting from the xiphoid process, a midline incision of about 3 cm was made along the white line of the abdomen. The skin and subcutaneous tissue were cut layer by layer, the greater omentum and the lesser omentum were bluntly separated, the stomach was gently pulled out, and a disposable punch was used to create a gastric wall defect. At the same time, lipopolysaccharide LPS (5 mg / kg animal) and interferon γ-IFN (5 ug / kg animal) were injected into the ulcer site to cause inflammation in the defective part, simulating clinical ulcerative gastric perforation.

[0033] (2) The full-thickness gastric wall gel patch prepared in Example 2 of the present invention was placed in the gastric wall defect area of ​​the rat, and the patch was sutured with the surrounding gastric wall tissue using surgical thread, and then sutured layer by layer, and finally sutured the skin, and sterile dressing was applied. The rat was placed in a lateral position on a constant temperature resuscitation table, and monitored by a dedicated person until it woke up, and then transferred to a cage. It was fasted and deprived of water within 24 hours after surgery, and was fed with whole milk powder within 72 hours, and then raised normally in the animal room. By naked eye observation and histological detection and analysis, the gastric wall was completely repaired on the 21st day.

[0034] The protection scope of the present invention includes but is not limited to the above embodiments, and its protection scope is subject to the claims. Any replacement, deformation, and improvement of the technology that can be easily thought of by technicians in this field shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a gel patch for repairing gastric tissue, characterized in that The specific steps include: (1) obtaining a mammalian stomach tissue, washing it, and performing a decellularization treatment to obtain a decellularized stomach patch matrix; (2) dissolving omeprazole in pure water to obtain an omeprazole aqueous solution with a concentration of 1 to 20 mg / mL, and then dissolving high molecular weight hyaluronic acid in the omeprazole aqueous solution to obtain a viscous hyaluronic acid aqueous solution containing omeprazole with a hyaluronic acid concentration of 1 to 20 mg / mL; (3) placing the viscous hyaluronic acid aqueous solution obtained in step (2) in a porous cell culture plate, adding glacial acetic acid with a mass concentration of 0.1 to 2% to completely immerse the viscous hyaluronic acid in the porous cell culture plate, and soaking for 2 to 24 hours, then taking the viscous hyaluronic acid out of the porous cell culture plate and attaching it to the gastric patch substrate obtained in step (1), rinsing it with PBS buffer with a concentration of 0.1 M, and then placing it in a freeze dryer for freeze drying to obtain a gel patch for repairing gastric tissue.

2. The method for preparing a gel patch for repairing gastric tissue according to claim 1, characterized in that: The specific method of step (1) is as follows: the stomach tissue of a mammal is rinsed with clean water to remove impurities, and then soaked in 75% medical alcohol for 30 minutes, washed three times with 0.1M PBS buffer, each time for 5 minutes, and then the stomach tissue is placed in a high osmotic pressure solution to break the membrane for 24 hours, and then rinsed with a PBS solution containing penicillin and streptomycin and placed in a mixed solution of 10000-50000U / mL DNA enzyme and 1000-2000U / mL RNA enzyme in a volume ratio of 10:1, and treated in a 37°C water bath for 2 hours; the above membrane breaking and enzyme treatment are repeated 2-3 times to obtain a decellularized gastric patch matrix.

3. The method for preparing a gel patch for repairing gastric tissue according to claim 2, characterized in that: The high osmotic pressure solution used in the step (1) is a mixture of a TritonX-100 solution with a mass concentration of 1 to 5% and a NaCl solution with a molar concentration of 1 to 5M in a volume ratio of 1:

1.

4. The method for preparing a gel patch for repairing gastric tissue according to claim 1, characterized in that: The mammal is a pig, a sheep or a rabbit.

5. The method for preparing a gel patch for repairing gastric tissue according to claim 1, characterized in that: The stomach tissue is the gastric mucosa and submucosal tissue, or the whole layer of the stomach wall.

6. The method for preparing a gel patch for repairing gastric tissue according to claim 1, characterized in that: In the step (2), the molecular weight of the hyaluronic acid is greater than or equal to 1,200,000 Da.

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