Pig stomach decellularized extracellular matrix hydrogel for protecting and repairing gastric mucosa and preparation method of pig stomach decellularized extracellular matrix hydrogel

Abstract

The invention discloses a pig stomach decellularized extracellular matrix hydrogel for protecting and repairing gastric mucosa and a preparation method thereof, the pig stomach decellularized extracellular matrix hydrogel is prepared by taking pig stomach tissue as a raw material, performing degreasing, decellularization and DNA removal on the pig stomach tissue to prepare a pig stomach decellularized extracellular matrix, and further performing digestion treatment by utilizing simulated gastric juice to prepare the pig stomach decellularized extracellular matrix hydrogel. The obtained pig stomach decellularized extracellular matrix hydrogel not only can form a physical barrier to protect the damaged part of the gastric mucosa, but also can play an anti-inflammatory role and promote the regeneration of the gastric mucosa, so that the protection and repair of the damaged part of the gastric mucosa can be realized at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a porcine stomach decellularized extracellular matrix hydrogel for protecting and repairing gastric mucosa and a preparation method thereof. Background Art

[0002] Gastric mucosa is located in the innermost layer of the stomach wall and has the function of secreting gastric acid and absorbing nutrients. Long-term use of drugs, drinking, strong tea or coffee, etc. may damage the gastric mucosal barrier, thereby causing gastritis, gastric ulcer and other gastric mucosa-related diseases. At present, the drugs for treating gastric mucosal damage clinically mainly include mucosal protective drugs and acid inhibitors, which are all achieved by reducing gastric acid stimulation. That is, existing treatment methods can only provide a protective effect that reduces gastric mucosa stimulation by gastric acid, and the damage and repair of gastric mucosa still need to rely on the regeneration ability of gastric mucosa itself, and the treatment effect is very limited. Therefore, it is extremely important to develop a treatment method that can protect gastric mucosa from gastric acid stimulation and promote the repair of ulcer sites, which is extremely important for accelerating gastric mucosal repair.

[0003] Decellularized extracellular matrix is ​​a natural biomaterial that is obtained by decellularizing human or animal organs / tissues using physical, chemical or biological methods. Decellularized extracellular matrix removes immunogenic substances such as DNA and retains effective ingredients such as collagen and glycosaminoglycans as much as possible, so it has excellent biocompatibility and is widely used in tissue regeneration and organ repair. Decellularized extracellular matrix can be further hydrolyzed to obtain decellularized extracellular matrix hydrogel, which can better simulate the microenvironment of human tissue and is conducive to cell proliferation and adhesion at the site of injury. However, the role of decellularized extracellular matrix hydrogel in the treatment of gastric mucosal injury has not yet been developed. Summary of the invention

[0004] The purpose of the present invention is to provide a porcine stomach decellularized extracellular matrix hydrogel for protecting and repairing gastric mucosa and a preparation method thereof. The porcine stomach decellularized extracellular matrix hydrogel prepared by the present invention can protect the damaged part of gastric mucosa by forming a physical barrier on the one hand, and on the other hand, it can play an anti-inflammatory role and promote gastric mucosal regeneration, thereby achieving a synergistic protection and repair effect on gastric mucosal damage.

[0005] To achieve the above object, the present invention adopts the following technical solution: A porcine stomach decellularized extracellular matrix hydrogel for protecting and repairing gastric mucosa, the preparation method of which comprises the following steps: 1) Soaking the pig stomach tissue in anhydrous ethanol, and then defatting it using supercritical carbon dioxide fluid to obtain defatted pig stomach tissue; 2) decellularizing the defatted porcine stomach tissue obtained in step 1) with a pancreatic enzyme solution and a Triton solution in sequence to obtain a decellularized porcine stomach tissue; 3) subjecting the decellularized porcine stomach tissue obtained in step 2) to DNA removal, sterilization, washing and drying in sequence to obtain porcine stomach decellularized extracellular matrix; 4) The porcine stomach decellularized extracellular matrix obtained in step 3) is further digested with simulated gastric juice, and the pH of the digestive juice is adjusted to neutral to obtain the porcine stomach decellularized extracellular matrix hydrogel.

[0006] Furthermore, the soaking time in step 1) is 12-48 hours.

[0007] Furthermore, in step 1), the pressure of the degreasing treatment using supercritical carbon dioxide fluid is 15-25 MPa (preferably 20 MPa); the treatment time is 3-5 h (preferably 4 h).

[0008] Furthermore, the mass concentration of the pancreatic enzyme solution in step 2) is 0.2% to 0.3% (preferably 0.25%).

[0009] Furthermore, the volume concentration of the Triton solution in step 2) is 0.25% to 0.75% (preferably 0.5%).

[0010] Furthermore, the amount of pancreatic enzyme solution and Triton solution in step 2) is calculated based on 5 mL per gram of defatted porcine stomach tissue.

[0011] Furthermore, in step 2), the temperature for treatment with pancreatic enzyme solution is 37° C. and the time is 2 to 6 hours (preferably 4 hours).

[0012] Furthermore, in step 2), the treatment time with the Triton solution is 2 to 4 hours (preferably 3 hours).

[0013] Furthermore, the pig stomach decellularized extracellular matrix obtained in step 3) is white in appearance and has a DNA content of less than 50 ng / mg.

[0014] Furthermore, the simulated gastric fluid in step 4) is a hydrochloric acid solution containing pepsin, wherein the concentration of pepsin is 10 mg / mL and the concentration of the hydrochloric acid solution is 0.01 mol / mL.

[0015] Furthermore, the amount of simulated gastric fluid in step 4) is converted to 30-100 mL per gram of porcine gastric decellularized extracellular matrix.

[0016] Furthermore, in step 4), the digestion time using simulated gastric fluid is 6 to 24 hours (preferably 12 hours).

[0017] The obtained porcine stomach decellularized extracellular matrix hydrogel can not only effectively reduce the area of ​​gastric mucosal damage, but also effectively reduce the content of inflammatory factors (TNF-α, IL-6, IL-1β) in gastric mucosal tissue. Therefore, it can be used to prepare drugs, functional health products and other related products for protecting and repairing gastric mucosal damage.

[0018] Compared with the prior art, the main advantages of the present invention are: The porcine stomach decellularized extracellular matrix hydrogel prepared by the present invention has a low DNA content, while effectively retaining active substances such as collagen, glycosaminoglycans and elastin, which can effectively reduce the damaged area of ​​gastric mucosa and reduce the content of inflammatory factors in gastric mucosa, play a dual role of protecting and repairing the gastric mucosa, and achieve synergistic treatment of gastric mucosal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the DNA content of the pig stomach decellularized extracellular matrix prepared in Examples 1 to 3 and the original pig stomach.

[0020] Figure 2 This is a comparison chart of the total protein content of the pig stomach decellularized extracellular matrix prepared in Example 1 and the original pig stomach.

[0021] Figure 3 This is a histological staining comparison of the pig stomach decellularized extracellular matrix prepared in Example 1 and the original pig stomach.

[0022] Figure 4 This is a sample comparison diagram of the porcine stomach decellularized extracellular matrix powder and the porcine stomach decellularized extracellular matrix hydrogel in Example 4.

[0023] Figure 5 These are images of stomach specimens of mice in each group in Example 5.

[0024] Figure 6 This is a comparison of the relative ulcer areas of each group of mice in Example 5.

[0025] Figure 7 This is a comparison of TNF-α content in gastric tissue of each group of mice in Example 5.

[0026] Figure 8 This is a comparison of the IL-1β content in the gastric tissue of each group of mice in Example 5.

[0027] Fig. 9 This is a comparison of the IL-6 content in the gastric tissue of each group of mice in Example 5. DETAILED DESCRIPTION

[0028] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0029] Embodiment 1: 1) Wash and chop fresh pig stomach tissue, soak it in anhydrous ethanol for 24 hours, and then use supercritical carbon dioxide fluid to defat it at a pressure of 20 MPa for 4 hours to obtain defatted pig stomach tissue; 2) Take 20 g of the defatted porcine stomach tissue, first treat it with 100 mL of a 0.25% pancreatic enzyme solution at 37°C for 4 h, and then treat it with 100 mL of a 0.5% Triton solution for 3 h to obtain decellularized porcine stomach tissue; 3) The obtained decellularized porcine stomach tissue was treated in 100 mL, 300 U / mL DNA enzyme solution for 48 hours, then sterilized by soaking in 75% ethanol solution for 2 hours, washed with sterile pure water, and freeze-dried to obtain porcine stomach decellularized extracellular matrix.

[0030] Example 2 1) Wash and chop fresh pig stomach tissue, soak it in anhydrous ethanol for 24 hours, and then use supercritical carbon dioxide fluid to defat it at a pressure of 15MPa for 5 hours to obtain defatted pig stomach tissue; 2) Take 20 g of the defatted porcine stomach tissue, first treat it with 100 mL of a 0.25% pancreatic enzyme solution at 37°C for 3 h, then treat it with 100 mL of a 0.75% Triton solution for 2 h to obtain decellularized porcine stomach tissue; 3) The obtained decellularized porcine stomach tissue was treated in 100 mL, 300 U / mL DNA enzyme solution for 48 hours, then sterilized by soaking in 75% ethanol solution for 2 hours, washed with sterile pure water, and freeze-dried to obtain porcine stomach decellularized extracellular matrix.

[0031] Example 3 1) Wash and chop fresh pig stomach tissue, soak it in anhydrous ethanol for 24 hours, and then use supercritical carbon dioxide fluid to defat it at a pressure of 20 MPa for 4 hours to obtain defatted pig stomach tissue; 2) Take 20 g of the defatted porcine stomach tissue, first treat it with 100 mL of a 0.2% pancreatic enzyme solution at 37°C for 6 h, and then treat it with 100 mL of a 0.25% Triton solution for 2 h to obtain decellularized porcine stomach tissue; 3) The obtained decellularized porcine stomach tissue was treated in 100 mL, 300 U / mL DNA enzyme solution for 48 hours, then sterilized by soaking in 75% ethanol solution for 2 hours, washed with sterile pure water, and freeze-dried to obtain porcine stomach decellularized extracellular matrix.

[0032] Performance Testing (1) Using the original pig stomach as a control, the DNA content of the pig stomach decellularized extracellular matrix prepared in Examples 1 to 3 was tested. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that the DNA content of the pig stomach decellularized extracellular matrix prepared in the examples is less than 50 ng / mg, which is significantly reduced compared with the original pig stomach.

[0033] (2) Using the original pig stomach as a control, the total protein content of the pig stomach decellularized extracellular matrix prepared in Example 1 was tested. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the total protein content of the pig stomach decellularized extracellular matrix is ​​90.97%, which is slightly higher than that of the original pig stomach.

[0034] (3) Using the original pig stomach as a control, the pig stomach decellularized extracellular matrix prepared in Example 1 was characterized by histological staining. The results are as follows: Figure 3 As shown in the figure. The H&E staining image shows that the cell nuclei of the original pig stomach are clearly visible, and the cell edges are clear and complete; while the cell nuclei are almost not observed in the pig stomach decellularized extracellular matrix prepared in Example 1, but the tissue structure remains intact, indicating that the cells have been destroyed and the cell nuclei have been cleanly removed. The Masson staining results and PAS staining results show that the pig stomach decellularized extracellular matrix prepared in Example 1 still contains a large amount of collagen fibers and carbohydrate substances, indicating that the decellularization process did not cause obvious damage to the extracellular matrix.

[0035] Example 4 2 g of the porcine stomach decellularized extracellular matrix prepared in Example 1 was digested in 100 mL of simulated gastric fluid (wherein the concentration of pepsin was 10 mg / mL and the concentration of hydrochloric acid solution was 0.01 mol / mL) for 12 h, and then the pH of the digestive fluid was adjusted to neutral to obtain a porcine stomach decellularized extracellular matrix hydrogel.

[0036] Example 5 The protective and repairing effects of porcine gastric decellularized extracellular matrix hydrogel on gastric mucosa were evaluated through the ethanol acute gastric injury model experiment.

[0037] 1. Experimental Materials (1) Test samples and reagents Original pig stomach powder, pig stomach decellularized extracellular matrix prepared in Example 1, pig stomach decellularized extracellular matrix hydrogel prepared in Example 4, pig skin decellularized extracellular matrix hydrogel, colloidal pectin bismuth capsule, anhydrous ethanol, 4% paraformaldehyde, TNF-α test kit, IL-6 test kit, IL-1β test kit.

[0038] The porcine skin decellularized extracellular matrix hydrogel used was prepared using porcine skin tissue as raw material according to the method described in Examples 1 and 4. (2) Experimental instruments Electronic analytical balance, tissue grinder, constant temperature incubator, electric constant temperature water bath, and microplate reader.

[0039] 2. Experimental methods and results (1) Experimental groups There were blank control group, model group, positive control group, original porcine stomach powder group, porcine stomach decellularized extracellular matrix powder group, porcine skin decellularized extracellular matrix hydrogel group, and porcine stomach decellularized extracellular matrix hydrogel group, with 10 mice in each group.

[0040] (2) Dosage Blank control group (pure water); model group (ethanol injury); positive control group (ethanol injury + colloidal bismuth pectin 0.5g / kg); original porcine stomach powder group (ethanol injury + original porcine stomach powder 0.2g / kg); porcine stomach decellularized extracellular matrix powder group (ethanol injury + porcine stomach decellularized extracellular matrix powder 0.2g / kg); porcine skin decellularized extracellular matrix hydrogel group (ethanol injury + porcine skin decellularized extracellular matrix hydrogel 0.2g / kg); porcine stomach decellularized extracellular matrix hydrogel group (ethanol injury + porcine stomach decellularized extracellular matrix hydrogel 0.2g / kg); all test samples used pure water as the dispersion medium, and were administered at 0.1mL / 10g, and were continuously administered by gavage for 7 days.

[0041] (3) Experimental process The mice were fasted but not watered for 24 hours before the last oral administration. After waiting for 1 hour, all groups except the blank control group were gavaged with anhydrous ethanol at a dose of 0.1 mL / 10 g to induce acute gastric injury. After waiting for 1.5 hours, the animals were killed by cervical dislocation, the stomach tissue was quickly removed by opening the abdomen, cut open along the greater curvature of the stomach, and the contents were gently rinsed with 4°C saline. The stomach tissue was immediately collected to observe the damage to the gastric mucosa and measure various indicators.

[0042] (4) Evaluation of gastric mucosal damage in mice The stomach tissues of each group were photographed and recorded. Figure 5 Then, the damaged area of ​​gastric mucosa was counted using Image J software, and the ratio of the damaged area to the total stomach area was used as the relative ulcer area. The results are shown in Figure 6 As shown. Combined Figure 5 and Figure 6 The results analysis showed that both the positive control group and porcine stomach decellularized extracellular matrix hydrogel had obvious protective and repairing effects on gastric mucosa, among which the porcine stomach decellularized extracellular matrix hydrogel group had the smallest relative ulcer area, and was significantly better than the original porcine stomach powder group, porcine stomach decellularized extracellular matrix powder group and porcine skin decellularized extracellular matrix hydrogel group, indicating that porcine stomach decellularized extracellular matrix hydrogel had the best protective and repairing effects on ethanol-induced gastric mucosal damage, and using hydrogel as a dosage form was an important prerequisite for its effect.

[0043] (5) Determination of TNF-α, IL-6, and IL-1β Contents in Mouse Gastric Tissue The gastric tissues of each group were homogenized, and the contents of TNF-α, IL-6, and IL-1β were determined according to the instructions of the kit. The results are shown in Figures 7-9 As shown in the figure, compared with the blank control group, the levels of TNF-α, IL-6, and IL-1β in the gastric tissue of the mice in the model group were significantly increased, and the levels of TNF-α, IL-6, and IL-1β in the original porcine stomach powder group, porcine stomach decellularized extracellular matrix powder group, and porcine skin decellularized extracellular matrix hydrogel group were basically equivalent to those in the model group, while the levels of TNF-α, IL-6, and IL-1β in the positive control group and porcine stomach decellularized extracellular matrix hydrogel group were lower than those in the model group, indicating that porcine stomach decellularized extracellular matrix hydrogel has a significant anti-inflammatory effect on the gastric mucosa, which is beneficial to promote the repair of gastric mucosal damage.

[0044] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing a porcine gastric decellularized extracellular matrix hydrogel for protecting and repairing gastric mucosa, characterized in that: The following steps are involved: 1) Soaking the pig stomach tissue in anhydrous ethanol, and then defatting it using supercritical carbon dioxide fluid to obtain defatted pig stomach tissue; 2) decellularizing the defatted porcine stomach tissue obtained in step 1) with a pancreatic enzyme solution and a Triton solution in sequence to obtain a decellularized porcine stomach tissue; 3) subjecting the decellularized porcine stomach tissue obtained in step 2) to DNA removal, sterilization, washing and drying in sequence to obtain porcine stomach decellularized extracellular matrix; 4) The porcine stomach decellularized extracellular matrix obtained in step 3) is further digested with simulated gastric juice, and the pH of the digestive juice is adjusted to neutral to obtain the porcine stomach decellularized extracellular matrix hydrogel.

2. The preparation method according to claim 1, characterized in that: The soaking time in step 1) is 12-48 hours.

3. The preparation method according to claim 1, characterized in that: In step 1), the degreasing treatment is performed using supercritical carbon dioxide fluid at a pressure of 15-25 MPa for 3-5 hours.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the pancreatic enzyme solution in step 2) is 0.2% to 0.3%, and the amount used is 5 mL per gram of defatted pig stomach tissue; The temperature for treatment with pancreatic enzyme solution is 37°C and the time is 2 to 6 hours.

5. The preparation method according to claim 1, characterized in that: The volume concentration of the Triton solution in step 2) is 0.25% to 0.75%, and the amount used is 5 mL for each gram of defatted pig stomach tissue; the treatment time with the Triton solution is 2 to 4 hours.

6. The preparation method according to claim 1, characterized in that: Step 3) The obtained pig stomach decellularized extracellular matrix is ​​white in appearance, and the DNA content is less than 50 ng / mg.

7. The preparation method according to claim 1, characterized in that: The simulated gastric fluid in step 4) is a hydrochloric acid solution containing pepsin, wherein the concentration of pepsin is 10 mg / mL and the concentration of the hydrochloric acid solution is 0.01 mol / mL; the amount used is 30-100 mL per gram of pig stomach decellularized extracellular matrix; and the digestion treatment time is 6-24 hours.

8. A porcine stomach decellularized extracellular matrix hydrogel prepared by the method of claim 1.

9. Use of the porcine stomach decellularized extracellular matrix hydrogel as claimed in claim 8 in the preparation of products for protecting and repairing gastric mucosal damage.

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