Preparation method and application of cell lysate gel for treating oral ulcer

Abstract

The invention discloses a preparation method and application of a cell lysate gel for treating oral ulcers, and relates to the technical field of oral care. The cell lysate gel is prepared by mixing a functional solution and thrombin, the functional solution comprises a solvent and a solute, the solvent is a mesenchymal stem cell lysate, the solute comprises bovine fibrinogen, borneol, vitamins, polysaccharides, an immunopotentiator, chlorogenic acid, indomethacin, and ginkgo biloba, the vitamins comprise vitamin E and vitamin B2, the polysaccharides comprise astragalus polysaccharides and cuttlebone polysaccharides, the immunopotentiator comprises alpha-mannan peptide and total glucosides of white paeony. The invention avoids the side effects of mesenchymal stem cells, has the advantages of high fluidity, simple and convenient preparation, safety and environmental protection, good application treatment effect, and the like, and achieves remarkable results in the aspects of oral ulcer anti-inflammatory effect, oral ulcer angiogenesis, and enhancing rat immunity, thereby reflecting the great prospect of oral ulcer treatment.

Description

Technical Field

[0001] The invention relates to the technical field of oral care, and in particular to a preparation method and application of a cell lysate gel for treating oral ulcers. Background Art

[0002] Oral ulcers (OU) are the most common ulcerative disease in humans and a common complication of radiotherapy or chemotherapy in cancer patients. Impaired regenerative capacity of the oral mucosal epithelium compromises barrier function and evolves into destructive ulcers that fully penetrate the underlying connective tissue. As a result, oral ulcers are often accompanied by severe pain, increased risk of infection, impaired oral and pharyngeal function, and even decreased quality of life. Several causes of OU have been recognized, such as mucosal trauma, chemical irritation, autoimmune and inflammatory diseases. In order to relieve patients' pain and reduce the severity of the disease, a variety of treatment methods have been applied, including oral hygiene, anti-inflammatory drugs, local anesthetics, oral decontamination and mouthwash, however, most of them still believe that no effective treatment has been established.

[0003] Cell-free therapy is an emerging stem cell therapy technology. The benefits of cell-free therapy strategies include the ability to avoid the side effects of mesenchymal stem cells. Direct injection of stem cell lysate can cause embolism, tumorigenesis, immune response, etc. Traditionally, therapeutic macromolecules such as cytokines are administered by large-dose injection, which is a method with significant limitations. Studies have shown that mesenchymal stem cell lysate has anti-inflammatory effects, and extracellular vesicles of mesenchymal stem cells have also been shown to promote angiogenesis. In the process of oral ulcers in rats, inflammatory responses have always existed, and angiogenesis indicators also play an important role in the later repair process. Therefore, mesenchymal stem cell lysate was applied to the oral ulcer model. However, there are large differences in the collection methods and standards of stem cell lysates. The treatment plan for oral ulcers does not fully utilize stem cell therapy, the drug efficacy is narrow, and the treatment efficiency is low.

[0004] Hydrogel sealants can create a humid environment, absorb tissue exudates, and serve as a barrier to microbial invasion, and are widely used in various disease models. At present, most of the therapeutic drugs for oral ulcers are spray-type aqueous solutions or powders. The publication number CN110075146B discloses an oral ulcer spray and its preparation method, including the following components by volume: 75-85 parts of hyaluronic acid extract, 5-15 parts of stem cell extract, 3-8 parts of deionized water, 2-6 parts of preservatives and 0.1-0.5 parts of vitamin B2. It feels gentle and comfortable when used, and there is no scar after healing. The total effective rate for patients with oral ulcers is high, but it cannot play a longer role in the wound part. Hydrogels can fit well at the ulcer wound and increase the drug action time. However, most hydrogels have poor inherent forming adaptability, and it is easy to observe the undesirable fit between irregular damaged tissue and hydrogel at the interface. Therefore, most hydrogels lack spatial advantages and cannot dynamically adapt to wound defects, thereby guiding endogenous cells to assemble into a complete tissue structure. Summary of the invention

[0005] The present invention is made in view of the above problems, and its purpose is to provide a mesenchymal stem cell lysate gel preparation for treating oral ulcers. The cell lysate gel preparation has the advantages of being simple and convenient to prepare, safe and environmentally friendly, having good application and treatment effects, and being suitable for the treatment of different stages of oral ulcers.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a mesenchymal stem cell lysate gel for treating oral ulcers. The stem cell lysate gel is prepared by mixing a functional solution and thrombin. The functional solution includes a solvent and a solute. The solvent is a mesenchymal stem cell lysate. The total protein concentration in the mesenchymal stem cell lysate is 30-40 μg / μl. The solute includes bovine fibrinogen, borneol, vitamins, polysaccharides, immunopotentiators, chlorogenic acid, indomethacin, and ginkgo biloba. The bovine fibrinogen and the thrombin are mixed to promote gelation.

[0008] Furthermore, the functional solution and the thrombin are mixed in a volume ratio of 1:1-1:2 to form a gel.

[0009] Furthermore, the vitamins include vitamin E and vitamin B2, the polysaccharides include astragalus polysaccharides and cuttlebone polysaccharides, and the immunopotentiators include α-mannan peptide and total glucosides of white peony.

[0010] Furthermore, the preparation method of the mesenchymal stem cell lysate is as follows:

[0011] A1. Separation: 1) Digest SHED with trypsin for two minutes, collect cells, and centrifuge at 1500-2000r / min for 10-12min. 2) Remove the supernatant, wash the cells with PBS, and centrifuge at 1500-2000r / min for 10-12min.

[0012] A2. Disruption: 1) Remove the supernatant and add deionized water to resuspend the cells to form a cell suspension. Let stand at room temperature for 30-50 minutes. 2) Place the cell suspension at ultra-low temperature for 30-40 minutes, then take it out and thaw it at room temperature. Repeat this process several times to obtain a freeze-thaw suspension.

[0013] A3. Determination and storage: 1) The freeze-thaw suspension is centrifuged at 2000-2500 r / min for 30-35 min, the precipitate is discarded, the supernatant is collected, filtered and packaged, and the protein concentration of the supernatant is measured by BCA protein quantitative method. 2) The supernatant after packaging is stored at ultra-low temperature.

[0014] Furthermore, the preparation method of the functional solution is as follows:

[0015] B1. Adding samples: Add the solutes into a beaker, which include the following components by weight: 5-50 parts of bovine fibrinogen, 1-20 parts of borneol, 1-8 parts of vitamin E, 1-8 parts of vitamin B2, 1-6 parts of astragalus polysaccharide, 1-6 parts of cuttlebone polysaccharide, 2-10 parts of α-mannan peptide, 2-10 parts of total glucosides of white paeony, 1-10 parts of chlorogenic acid, 1-6 parts of indomethacin, and 1-12 parts of ginkgo biloba.

[0016] B2. Dissolution: Take out the supernatant stored at ultra-low temperature in step A3, slowly thaw it under low temperature conditions, add the thawed supernatant into a beaker, and stir until all the solutes are completely dissolved.

[0017] Furthermore, the preferred solute composition in step B1 is made of the following raw materials in parts by weight: 30-42 parts of bovine fibrinogen, 1-5 parts of borneol, 3-6 parts of vitamin E, 5-8 parts of vitamin B2, 1-4 parts of astragalus polysaccharide, 4-6 parts of cuttlebone polysaccharide, 6-10 parts of α-mannan peptide, 6-10 parts of total glucosides of white peony, 3-7 parts of chlorogenic acid, 1-3 parts of indomethacin, and 8-12 parts of ginkgo.

[0018] Furthermore, the optimal solute composition in step B1 is made of the following raw materials in parts by weight: 36 parts of bovine fibrinogen, 3 parts of borneol, 6 parts of vitamin E, 8 parts of vitamin B2, 2 parts of astragalus polysaccharide, 5 parts of cuttlebone polysaccharide, 8 parts of α-mannan peptide, 8 parts of total glucosides of white peony, 5 parts of chlorogenic acid, 2 parts of indomethacin, and 12 parts of ginkgo.

[0019] Furthermore, the weight unit in step B1 is mg, the volume of the thawed supernatant added in step B2 is 5-70 ml, and the bovine fibrinogen concentration of the functional solution is in the range of 1-1.4 mg / ml.

[0020] Furthermore, the weight unit is mg, and the preferred functional solution compositions are dissolved in 30 ml of the mesenchymal stem cell lysate, and mixed with the thrombin in a ratio of 1:1.5 to prepare a gel.

[0021] Furthermore, the concentration of thrombin is 21 U / ml.

[0022] In a second aspect, the present invention further provides an application of a mesenchymal stem cell lysate gel for treating oral ulcers.

[0023] Furthermore, the mesenchymal stem cell lysate gel for treating oral ulcers was applied to a rat oral ulcer model, and achieved significant results in terms of anti-inflammatory effect, oral ulcer angiogenesis, and enhancement of the rat immune system in the rat oral ulcer model.

[0024] The beneficial effects of the present invention are:

[0025] (1) The cell lysate gel for treating oral ulcers prepared by the present invention has good fluidity and can have an ideal fit with the irregular damaged tissue on the surface of oral ulcers, thereby improving the treatment effect.

[0026] (2) The cell lysate gel for treating oral ulcers prepared by the present invention can significantly promote the relative content of CD206, CD31, and VEGF, and significantly reduce the relative content of iNOS, that is, enhance the anti-inflammatory effect of oral ulcers and the angiogenesis of oral ulcers.

[0027] (3) The cell lysate gel for treating oral ulcers prepared by the present invention is simple and convenient to prepare, safe and environmentally friendly.

[0028] (4) The cell lysate gel for treating oral ulcers prepared by the present invention not only targets oral ulcers, but also adds an immunopotentiator, aiming to treat oral ulcers from the outside to the inside. After the gel is decomposed, the immunopotentiator enters the body along with saliva, significantly promoting the relative content of CD14 and IgG, thereby enhancing individual immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Figure 2 shows the effect of different combinations of gels on the relative content of CD206 in the rat oral ulcer model. The lowercase letters in the figure indicate that the data of groups a, b, and c were subjected to one-way analysis of variance under the conditions of the control group, solution group, gel group, and gel group. The * in the figure indicates that the control group, solution group, and gel group were subjected to significant analysis with the gel, respectively. p <0.05,** p <0.01,*** p <0.001, **** p <0.0001, n=5;

[0030] Figure 2 The effect of different combination gels on the relative content of iNOS in the rat oral ulcer model. Figure 1 The same, n=5;

[0031] Figure 3 The effect of different combination gels on the relative content of CD31 in the rat oral ulcer model. Figure 1 The same, n=5;

[0032] Figure 4 The effect of different combination gels on the relative content of VEGF in the rat oral ulcer model. Figure 1 The same, n=5;

[0033] Figure 5 The effect of different combination gels on the relative content of CD14 in the rat oral ulcer model. Figure 1 The same, n=5;

[0034] Figure 6 The effect of different combination gels on the relative content of IgG in the rat oral ulcer model. Figure 1 The same, n=5;

[0035] Figure 7 The effect of the optimal combination gel on HUVEC cell angiogenesis in vitro;

[0036] Figure 8 This is a direct view of the mucosal pathological sections showing the repairing effect of the optimal combination gel in the rat oral ulcer model. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0038] A preparation method and application of a mesenchymal stem cell lysate gel for treating oral ulcers, including a preparation method of a mesenchymal stem cell lysate gel for treating oral ulcers, as shown in Examples 1-4 for details, and also including an application of a mesenchymal stem cell lysate gel for treating oral ulcers, as shown in Examples 5-7 for details.

[0039] Example 1

[0040] A method for preparing a mesenchymal stem cell lysate gel for treating oral ulcers, comprising the following steps performed in sequence: A. preparation of a mesenchymal stem cell lysate, B. preparation of a functional solution, and C. preparation of a gel. The preparation process is completed in an ultra-clean environment, and the drug raw materials, experimental equipment, etc. are strictly sterilized or disinfected. The specific steps are as follows:

[0041] Preparation of mesenchymal stem cell lysate: 1) Digest SHED with trypsin for two minutes, collect cells, and centrifuge at 1500r / min for 10min. 2) Remove the supernatant, wash the cells with PBS, and centrifuge at 1500r / min for 10min, repeat 3 times. 3) Remove the supernatant, add deionized water to resuspend the cells to form a cell suspension. And let it stand at room temperature for 30min. 4) Place the cell suspension at -80℃ for 30min, then take it out and thaw it at room temperature, repeat 4 times to obtain a freeze-thaw suspension. 5) After the freeze-thaw suspension is centrifuged at 2000r / min for 30min, discard the precipitate, take the supernatant, filter and aliquot, and measure the protein concentration of the supernatant by BCA protein quantification method. 6) The aliquoted supernatant is stored at -80℃.

[0042] Preparation of functional solution: 1) Adding samples: Add drugs to a beaker, including the following components by weight: 5-50 parts of bovine fibrinogen, 1-20 parts of borneol, 1-8 parts of vitamin E, 1-8 parts of vitamin B2, 1-6 parts of astragalus polysaccharide, 1-6 parts of sea cucumber polysaccharide, 2-10 parts of α-mannan peptide, 2-10 parts of total glycosides of white peony, 1-10 parts of chlorogenic acid, 1-6 parts of indomethacin, and 1-12 parts of ginkgo. 2) Dissolving: Take out the prepared mesenchymal stem cell lysate, take the weight unit in mg, and the added volume of the mesenchymal stem cell lysate is 5-70 ml, slowly add it to the beaker, stir while adding, and the stirring method includes but is not limited to manual glass rod stirring, automatic stirring with a magnetic stirrer, etc., stirring until all solutes are completely dissolved.

[0043] Preparation of gel: 1) Prepare thrombin, dissolve lyophilized thrombin powder in 25mmol / l calcium chloride solution to prepare a thrombin solution with a concentration of 21U / ml. 2) Use a dual-drug mixing system to install two syringes containing fibrinogen solution and thrombin solution respectively on the dual injection stand, and mix the two solutions evenly by pushing the linkage push rod. The volume ratio of the functional solution to the thrombin solution is designed to be within the range of 1:1-1:2 to prepare the gel.

[0044] Example 2

[0045] A composition of a mesenchymal stem cell lysate gel for treating oral ulcers, having but not limited to the following effects:

[0046] 1) Mesenchymal stem cell lysate, which has anti-inflammatory effects, and the extracellular vesicles of mesenchymal stem cells have also been shown to promote angiogenesis; 2) Bovine fibrinogen, which is a gelling agent. Fibrinogen and thrombin can form a gel after mixing. Specifically, thrombin cleaves the fibrinogen molecule into fibrinopeptide A and peptide B, leading to the formation of fibrin monomers. At the same time, thrombin also activates factor XIII, which participates in the cross-linking of fibrin, allowing fibrin to form a stable, non-brittle clot. In this process, the concentration of fibrinogen determines the adhesion strength of the gel, while the concentration of thrombin determines the speed of gel formation; 3) Borneol, which can eliminate inflammation and has a good analgesic effect. It can also promote exfoliation of the oral mucosa and heal ulcers and erosions; 4) Vitamin E, which can dilate peripheral blood vessels, improve the patient's blood circulation, and promote ulcer healing; 5) Vitamin B2. Vitamin B2 itself does not directly break down into small molecules to provide energy for cells, but it is a coenzyme component of various enzymatic reactions in the body, especially enzymes related to energy metabolism, such as succinate dehydrogenase involved in the tricarboxylic acid cycle and complex II (succinate-Q reductase) in the mitochondrial respiratory chain. These enzymes play a key role in the production of ATP, so an adequate supply of vitamin B2 is essential for energy metabolism in the process of cell proliferation; 6) Astragalus polysaccharide promotes the proliferation of oral mucosal fibroblasts, showing the phenomenon of accelerated healing of oral ulcers, and Astragalus polysaccharide can also improve the body's immune abnormalities; 7) Cuttlebone polysaccharide can reduce the levels of IL-6 and TNF-α in patients with oral ulcers, effectively accelerating the healing of oral ulcers; 8) α-Mannan peptide can enhance the phagocytic function of macrophages, enhance the function of bone marrow hematopoietic stem cells, and accelerate the growth of peripheral blood cells; 8 ) Total glucosides of white paeony are a group of monoterpenoids extracted from white paeony. They have the effects of regulating immunity, protecting the kidneys, protecting the liver, anti-inflammatory, anti-depression and affecting cell proliferation, and have no obvious toxic side effects; 9) Chlorogenic acid is the main active ingredient in many Chinese herbal medicines and Chinese herbal compound preparations. It has anti-inflammatory, antioxidant, antibacterial and healing effects; 10) Indomethacin, as a non-steroidal anti-inflammatory drug, exerts its anti-inflammatory effect by inhibiting prostaglandins. Compared with corticosteroids, it also has analgesic effects, and has fewer side effects, making it safer and more effective; 11) Ginkgo biloba promotes local material exchange and removes harmful metabolites, thereby enhancing the body's immunity.

[0047] Example 3

[0048] A method for preparing a mesenchymal stem cell lysate gel for treating oral ulcers, wherein the BCA protein quantification method comprises the following steps: 1) preparing a BCA working solution, mixing BCA reagents A and B at a ratio of 50:1, and shaking them thoroughly for use; 2) preparing a standard protein solution, using bovine serum albumin (BSA) as a standard, dissolving it with an appropriate PBS buffer and preparing a standard protein solution with a certain concentration gradient; 3) gradient diluting the standard into 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, and 10 mg / ml; 4) adding samples, taking 500 ul of each concentration of the standard and the supernatant and adding them to a 96-well plate respectively, setting 3 replicate wells for each concentration to improve accuracy, and then adding 500 ul to each well. The BCA working solution was gently shaken to mix; 5) Incubate and culture, place the 96-well plate in an incubator at 37°C for 30 minutes to allow the protein to be fully mixed with the BCA working solution; 6) Determine the protein concentration, after the incubation, cool to room temperature, and use an ELISA reader to measure the absorbance value of each well of the 96-well plate at a wavelength of 562nm. 7) Calculate the protein concentration, with the standard protein concentration as the horizontal axis and the corresponding absorbance value as the vertical axis, and draw a standard curve. The protein concentration and the absorbance value are linearly related, and the R of the standard curve 2 >0.99, the absorbance value of the supernatant to be measured is between the absorbance values ​​corresponding to the 0 mg / ml standard and the 10 mg / ml standard, and the protein concentration of the supernatant is obtained by substituting the absorbance value of the supernatant into the standard curve.

[0049] Example 4

[0050] A method for preparing a mesenchymal stem cell lysate gel for treating oral ulcers, wherein the specific steps of culturing the SHED are as follows: 1) using sterile PBS buffer to repeatedly rinse the deciduous teeth in a clean bench, extracting the pulp tissue with a pulp extraction needle, cutting it into pieces, and placing it in a sterile small conical bottle, adding 1 ml of 3 mg / ml collagenase type I and 4 mg / ml dispase to the small conical bottle, mixing them in a ratio of 1:1, and digesting the pulp tissue in a 37°C water bath for 1 h, shaking it several times during the period until the formed pulp tissue is no longer visible. After dispersion, transfer it into a centrifuge tube, wash it with PBS buffer 3 times, centrifuge it at 1000 r / min for 2 minutes each time, discard the supernatant, add the culture medium to mix, repeatedly blow and beat the discrete cell clumps, pass through a cell sieve (with an inner diameter of 70 μm) sterilized by high pressure, filter to obtain single discrete cells, count the cells with a hemocytometer, and count them at 1×10 4 ~1×10 5The cells were inoculated into a 25 mL plastic culture flask at a density of 100 μg / mL, and 2-3 mL of DMEM medium containing 20% ​​FBS and high glucose was added. The cells were then incubated at 37°C and 5% CO 2 Culture in an incubator. After 3 days, completely replace the culture medium, discard the non-adherent cells, and change the medium once every 2-3 days. After the cells fuse into a monolayer (80-90% confluence), digest with 0.25% trypsin at room temperature for 3-5 minutes, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, and pass the precipitate at a ratio of 1:2. 2) Take the first generation of deciduous dental pulp cells in the logarithmic growth period, dilute them in multiples with high-glucose DMEM culture medium containing 20% ​​fetal bovine serum, adjust the cell density to 10-15 cells / mL, mix them thoroughly, and inoculate them in a 96-well plate at 100μL / well. After culturing for 24 hours, mark the single cell wells and replenish the liquid to 200μL / well, and change the medium every 4 days. When the number of cells increases, change the medium every 2-3 days.

[0051] Example 5

[0052] An application of a mesenchymal stem cell lysate gel for treating oral ulcers. The effect of solute on gelation is shown in Table 1. There is no substance in the solute that has similar functions to the gelation factors bovine fibrinogen and thrombin, but chlorogenic acid will affect the gelation by affecting the pH value of the functional solution. When the final concentration of chlorogenic acid exceeds 0.133 mg / ml, that is, when the volume ratio of the functional solution to the thrombin solution is 1:1, 1:1.5, and 1:2, the concentration of chlorogenic acid in the functional solution exceeds 0.26 mg / ml, 0.33 mg / ml, and 0.39 mg / ml, respectively, no gelation occurs. When the final concentration of chlorogenic acid is between 0.06 and 0.12, the gelation speed becomes significantly faster.

[0053] Table 1 Experimental study on the effect of chlorogenic acid concentration on gel formation

[0054]

[0055] Note: Groups 1-16 in Table 1 are based on the preparation of the gel in Example 1. 16 groups of data were selected from a limited number of experiments to illustrate the effect of the concentration range of chlorogenic acid on gelation, of which Groups 1-12 are data when the volume ratio of the functional solution to the thrombin solution is 1:1.5, Groups 13-14 are data when the volume ratio of the functional solution to the thrombin solution is 1:1, and Groups 15-16 are data when the volume ratio of the functional solution to the thrombin solution is 1:2. The addition of the mass of the remaining solutes, as long as they are within the weight fraction range, has no significant effect on gelation.

[0056] Example 6

[0057] An application of a mesenchymal stem cell lysate gel for treating oral ulcers, wherein the prepared gel is subjected to a variety of experimental tests, including but not limited to experiments on the fluidity and adhesion of the gel, experiments on the anti-inflammatory effects of the gel in a rat oral ulcer model, experiments on the effects on angiogenesis in a rat oral ulcer model, experiments on the effects on rat immunity, etc., and the best gel component composition is selected from a series of gels prepared with different component proportions having better effects.

[0058] 1. Some gel combinations are as follows:

[0059] a. Bovine fibrinogen 30 mg, borneol 1 mg, vitamin E 3 mg, vitamin B2 5 mg, astragalus polysaccharide 1 mg, cuttlebone polysaccharide 4 mg, α-mannan peptide 6 mg, total glycosides of white peony 6 mg, chlorogenic acid 3 mg, indomethacin 1 mg, ginkgo biloba 8 mg, mesenchymal stem cell lysate 30 ml, 21 U / ml thrombin solution 45 ml.

[0060] b. Bovine fibrinogen 36 mg, borneol 3 mg, vitamin E 6 mg, vitamin B2 8 mg, astragalus polysaccharide 2 mg, cuttlebone polysaccharide 5 mg, α-mannan peptide 8 mg, total glycosides of white peony 8 mg, chlorogenic acid 5 mg, indomethacin 2 mg, ginkgo biloba 12 mg, mesenchymal stem cell lysate 30 ml, 21 U / ml thrombin solution 45 ml.

[0061] c. Bovine fibrinogen 42 mg, borneol 5 mg, vitamin E 8 mg, vitamin B2 10 mg, astragalus polysaccharide 3 mg, cuttlebone polysaccharide 6 mg, α-mannan peptide 10 mg, total glycosides of white peony 10 mg, chlorogenic acid 6 mg, indomethacin 3 mg, ginkgo biloba 12 mg, mesenchymal stem cell lysate 30 ml, 21 U / ml thrombin solution 45 ml.

[0062] 2. The fluidity and adhesion of some better gel combinations were evaluated, and the results are shown in Table 2 and Table 3:

[0063] Table 2 Viscosity of different combination gels at 37°C

[0064]

[0065] Table 3 Adhesion of different combination gels at 37°C

[0066]

[0067] 3. The anti-inflammatory effects of some superior gel combinations in the rat oral ulcer model were evaluated. The results are as follows: Figure 1 and Figure 2 As shown, the gel group used mesenchymal stem cell lysate gel, the solution group replaced the gel in the gel group with the corresponding solution components, the gel group only retained the corresponding bovine fibrin, mesenchymal stem cell lysate and thrombin components in the gel group, and the control group replaced the gel in the gel group with sterile water. The results showed that compared with the control group, the solution group, the gel group and the gel group could significantly increase the relative level of CD206 and significantly reduce the relative level of iNOS. Compared with the single solution group and the single gel group, the gel group had a more significant effect on the relative levels of CD206 and iNOS. In the rat oral ulcer model, macrophages are an important immune cell. When M0 macrophages are stimulated by activators, they will differentiate into different phenotypes, namely M1 macrophages and M2 macrophages. M1 macrophages play a pro-inflammatory role, and M2 macrophages play an anti-inflammatory role. CD204 is a common marker of mouse M2 macrophages, and inducible nitric oxide synthase (iNOS) is used to represent M1 macrophages. It is concluded that the mesenchymal stem cell lysate gel for treating oral ulcers of the present invention can better promote the anti-inflammatory effect in the rat oral ulcer model, among which the gel combination b has the best promoting effect.

[0068] 4. The effects of some superior gel combinations on angiogenesis in a rat oral ulcer model were evaluated. The results are as follows: Figure 3 and Figure 4 As shown, the gel group used mesenchymal stem cell lysate gel, the solution group replaced the gel in the gel group with the corresponding solution components, the gel group retained only the corresponding bovine fibrin, mesenchymal stem cell lysate and thrombin components in the gel group, and the control group replaced the gel in the gel group with sterile water. The results showed that compared with the control group, the solution group, the gel group and the gel group could significantly increase the relative levels of CD31 and VEGF. Compared with the single solution group and the single gel group, the gel group had a more significant effect on the relative levels of CD31 and VEGF. In the rat oral ulcer model, the protein marker CD31 (platelet and endothelial cell adhesion molecule) is often used to mark the vascular epithelium, and vascular endothelial growth factor (VEGF) can induce angiogenesis in vivo. It is concluded that the mesenchymal stem cell lysate gel for treating oral ulcers of the present invention can better promote angiogenesis in the rat oral ulcer model, among which the gel combination b has the best promotion effect.

[0069] 5. The effects of some better gel combinations on the immunity of rats were evaluated. The results are as follows: Figure 5and Figure 6 As shown in the figure, the gel group used mesenchymal stem cell lysate gel, the solution group replaced the gel in the gel group with the corresponding solution components, the gel group retained only the corresponding bovine fibrin, mesenchymal stem cell lysate and thrombin components in the gel group, and the control group replaced the gel in the gel group with sterile water. The results showed that compared with the control group, the solution group, the gel group and the gel group could significantly increase the relative levels of CD14 and IgG. Compared with the single solution group and the single gel group, the gel group had a more significant effect on the relative levels of CD14 and IgG. In the rat oral ulcer model, CD14, as an important surface protein of immune cells, It is an important molecule that plays the role of recognition, binding and signal transmission in these processes. IgG is a type of immunoglobulin and the most important type of antibody in the humoral immune response. As a part of the immune response, IgG participates in the defense and repair process of oral ulcers. It is concluded that the mesenchymal stem cell lysate gel for treating oral ulcers of the present invention is added with immunopotentiators such as α-mannan peptide and total glucosides of white peony. When the gel is gradually decomposed at the oral ulcer, the α-mannan peptide and total glucosides of white peony enter the body with saliva and are absorbed by the intestines, stomach and other organs. In the rat oral ulcer model, it can better promote the body's immune ability, among which the gel combination b has the best promoting effect.

[0070] Example 7

[0071] An application of mesenchymal stem cell lysate gel for treating oral ulcers. The gel combination B with the best overall performance was selected to continue the in vitro experiment on the effect on HUVEC cell angiogenesis and observe the mucosal pathological sections of the gel combination B in the rat oral ulcer model to evaluate the repair effect.

[0072] 1. Effects on HUVEC cell angiogenesis in vitro (cultured for 24 hours). The culture results are as follows: Figure 7 As shown, the left picture is the control group (CN), and the right picture is the experimental group with the addition of gel combination b (CL / FT). After the two were co-cultured under the same environmental conditions for 24 hours, the cultured cells were observed under a fluorescence microscope. The results showed that after 24 hours of in vitro culture, the HUVEC cells in the experimental group formed more blood vessels compared with the control group. It can be seen that CL / FT promotes the angiogenesis of HUVEC cells, indicating that CL / FT has a good potential for treating oral ulcers.

[0073] 2. Observe the mucosal pathological sections of gel combination b in the rat oral ulcer model. The results are as follows: Figure 8As shown, the picture is an enlarged view of the black box part from top to bottom. The results show that the oral mucosa of the control group (CN), solution group (CL) and gel group (FT) had local irregular depressions and ulcers, but the oral mucosa of the gel group (CL / FT) was repaired and smoothed, and the red area was more obvious, which intuitively reflects the good therapeutic effect of CL / FT on oral ulcers.

Claims

1. A method for preparing a cell lysate gel for treating oral ulcers, characterized in that: The functional solution is prepared by mixing a functional solution and thrombin, wherein the functional solution comprises a solvent and a solute, wherein the solvent is a mesenchymal stem cell lysate, and the solute comprises, by weight: 5-50 parts of bovine fibrinogen, 1-20 parts of borneol, 1-8 parts of vitamin E, 1-8 parts of vitamin B2, 1-6 parts of astragalus polysaccharide, 1-6 parts of cuttlebone polysaccharide, 2-10 parts of α-mannan peptide, 2-10 parts of total glucosides of white paeony, 1-10 parts of chlorogenic acid, 1-6 parts of indomethacin, and 1-12 parts of ginkgo biloba; The preparation method of the mesenchymal stem cell lysate is as follows: A1. Separation: 1) Digest SHED with trypsin for two minutes, collect cells, and centrifuge at 1500-2000r / min for 10-12min; 2) Remove the supernatant, wash the cells with PBS, and centrifuge at 1500-2000r / min for 10-12min; A2. Disruption: 1) Remove the supernatant, add deionized water to resuspend the cells to form a cell suspension, and let it stand at room temperature for 30-50 minutes; 2) Place the cell suspension at ultra-low temperature for 30-40 minutes, then take it out and thaw it at room temperature, repeat several times to obtain a freeze-thaw suspension; A3. Determination and storage: 1) The freeze-thaw suspension is centrifuged at 2000-2500 r / min for 30-35 min, the precipitate is discarded, the supernatant is collected, filtered and packaged, and the protein concentration of the supernatant is measured by BCA protein quantification method; 2) The packaged supernatant is stored at ultra-low temperature.

2. The method for preparing a cell lysate gel for treating oral ulcers according to claim 1, characterized in that: The preparation method of the functional solution is: B1. Add sample: add the solute into the beaker; B2. Dissolution: Take out the supernatant stored at ultra-low temperature in step A3, slowly thaw it under low temperature conditions, add the thawed supernatant into a beaker, and stir until all the solutes are completely dissolved.

3. The method for preparing a cell lysate gel for treating oral ulcers according to claim 2, characterized in that: The unit of weight is mg, the volume of the thawed supernatant added in step B2 is 5-70 ml, and the concentration of bovine fibrinogen in the functional solution is in the range of 1-1.4 mg / ml.

4. The method for preparing a cell lysate gel for treating oral ulcers according to claim 3, characterized in that: The functional solution and the thrombin are mixed in a volume ratio of 1:1-1:2 to prepare a gel.

5. The method for preparing a cell lysate gel for treating oral ulcers according to claim 4, characterized in that: The concentration of thrombin was 21 U / ml.

6. Use of the gel prepared by the method for preparing a cell lysate gel for treating oral ulcers as claimed in any one of claims 1 to 5 in preparing a drug for treating oral ulcers.

Images