Panda-derived exosome gel as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of panda-derived exosome gel. The preparation method comprises the following steps: step 1, culturing panda umbilical cord-derived mesenchymal stem cells by using a culture medium; 2, culturing in an air-permeable culture bottle, and then collecting cell supernate; 3, centrifuging for five times, and continuing to resuspend with the PBS buffer solution to obtain an exosome solution; and 4, adding carbomer into a proper amount of distilled water, continuously stirring, sterilizing after the carbomer is swelled into a semitransparent solution, then adding the exosome solution and triethanolamine, uniformly stirring, gradually adding the ethylparaben solution, triethanolamine and glycerol, then adding distilled water, uniformly stirring, and storing to obtain the exosome gel. The invention further provides panda-derived exosome gel and an effect of the panda-derived exosome gel in promoting wound healing of pandas. The invention provides a panda-derived new medicine which can be used for promoting wound healing of pandas and has a relatively good anti-inflammatory effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, more particularly to an application of an exosome gel derived from giant panda umbilical cord mesenchymal stem cells in skin wound repair of giant pandas. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are known for their excellent self-renewal and differentiation capabilities, and play an increasingly important role in wound healing research and treatment in human and veterinary clinics. However, there are still limitations in the clinical application of MSCs in skin wound treatment, such as cell source diversity, allogeneic and xenogeneic immune rejection, chromosomal variation, poor implantation and survival, malignant transformation and mutation, and risk of carcinogenic / tumor support. In recent years, a large number of studies have shown that stem cell transplantation promotes wound healing through the paracrine mechanism of exosome release. Compared with stem cell therapy, exosomes have very low tumorigenicity, weak immune response, and are easily recognized and endocytosed by recipient cells. Exosomes are small extracellular vesicles, usually 30-150 nanometers in diameter, produced by various cell types and tissues, and exist in biological fluids such as plasma, urine, saliva, and breast milk. Studies have shown that exosomes from umbilical cord mesenchymal stem cells (UC-MSCs-Exos) promote angiogenesis, promote granulation tissue formation, accelerate re-epithelialization, and help wound healing in excisional wound and burn models, showing important prospects and practical value in wound healing.

[0003] Captive giant pandas will experience various diseases, including trauma, fractures, craniocerebral injury, tumors and other diseases requiring surgical intervention. Although the body's damaged tissues will initiate the corresponding regeneration and repair program after injury, this regeneration and repair ability will gradually decrease with age. Elderly giant pandas also have poor wound healing ability after surgical treatment, and are prone to wound infection. However, the commonly used broad-spectrum antibiotics are prone to drug resistance and unstable treatment effect, and there is currently no good solution. Therefore, it is of great significance to develop new drugs to promote wound healing in giant pandas and reduce the incidence of wound infection, improve the health and postoperative welfare of giant pandas, etc. SUMMARY

[0004] The present application is aimed at the problem that the wound healing ability of elderly captive giant pandas is impaired, and there is currently no giant panda-derived drug for wound healing treatment. A safe and reliable giant panda-derived exosome gel based on giant panda umbilical cord mesenchymal stem cells is provided.

[0005] The present application is implemented as follows:

[0006] A preparation method of a giant panda-derived exosome gel, comprising the following steps:

[0007] Step one, culture the umbilical cord-derived mesenchymal stem cells (UC-MSCs) of giant panda with the culture medium, when the confluence reaches 80%, discard the complete culture medium, and wash the adherent cells with PBS buffer;

[0008] Step two, add sufficient low-sugar DMEM solution to the gas-permeable culture flask, and place it in a 37℃, 5% CO2 incubator for culture, collect the cell supernatant after 48 hours.

[0009] Step three, centrifuge the collected cell supernatant for the first time to remove cells;

[0010] Take the supernatant after the first centrifugation and move it to a new centrifuge tube, centrifuge it for the second time to remove cell debris;

[0011] Take the supernatant after the second centrifugation and move it to a new centrifuge tube, centrifuge it for the third time to remove larger vesicles;

[0012] Take the supernatant after the third centrifugation, filter it through a filter membrane to remove large particle impurities, collect the filtrate, and resuspend it with 4℃ pre-cooled PBS buffer after the fourth centrifugation to remove the supernatant;

[0013] Centrifuge the above resuspension for the fifth time to remove the supernatant, continue to resuspend it with PBS buffer, and obtain the exosome solution.

[0014] Step four, add carbomer to an appropriate amount of distilled water, continuously stir until it swells into a translucent solution, then sterilize it in a high-pressure steam sterilization pot, then add the exosome solution obtained in step three, triethanolamine, stir uniformly, gradually add nipagin ethyl ester solution and triethanolamine, glycerol, then add distilled water, stir uniformly, and store in a-80℃ refrigerator for standby, which is the exosome gel.

[0015] In step two, the gas-permeable culture flask can be 175 cm 2 Gas-permeable culture flask. The so-called sufficient low-sugar DMEM solution is relative to the amount of mesenchymal stem cells, as long as it meets the culture needs of mesenchymal stem cells, for example, 35 mL-50 mL low-sugar DMEM solution can be added.

[0016] A further solution is:

[0017] In step one, the umbilical cord-derived mesenchymal stem cells of giant panda are the 4th generation of umbilical cord-derived mesenchymal stem cells of giant panda.

[0018] The reason for limiting the 4th generation of umbilical cord-derived mesenchymal stem cells of giant panda is generally to obtain sufficient amount of cells for extracting exosomes. Because the umbilical cord-derived mesenchymal stem cells of giant panda are very rare and precious, through multiple subculture, sufficient amount of cells can be obtained.

[0019] Many studies have been conducted on the subculture of the mesenchymal stem cells from the umbilical cord of the giant panda, and those skilled in the art can select the corresponding culture method according to the prior art. The culture can also be performed in the following manner:

[0020] The umbilical cord mesenchymal stem cells are isolated, inoculated into a culture dish with the corresponding culture medium, and placed in a 37℃, 5% CO2 incubator for culture, which is recorded as the primary cells (P0). When the confluence of the cells reaches 70% to 90%, the cells are subcultured: the culture medium is discarded, the cells are washed twice with PBS, and the cells are digested with 0.25% trypsin for 2 min to 5 min, and then the digestion is terminated. The cells are collected by centrifugation at 800 g for 5 min and cultured with the corresponding culture medium, which is recorded as P1. When the confluence of the P1 cells reaches 70% to 90%, the cells are subcultured again, which is recorded as P2, and the process is repeated in sequence until P4, i.e., the fourth generation of the umbilical cord-derived mesenchymal stem cells of the giant panda.

[0021] A further scheme is:

[0022] The culture medium in step one is: low-sugar DMEM solution + 10% volume FBS + 1% volume penicillin-streptomycin solution + 10 ng / mL bFGF.

[0023] The low-sugar DMEM solution is a conventional standard product, and for example, a low-sugar DMEM solution produced by the Gibco company can be used. The 10% volume FBS means that 10% of the volume of the low-sugar DMEM solution is FBS.

[0024] The 1% volume penicillin-streptomycin solution means that 1% of the volume of the low-sugar DMEM solution is penicillin-streptomycin solution.

[0025] The 10 ng / mL bFGF means that the final concentration of bFGF in the culture medium is 10 ng / mL, i.e., 10 ng of bFGF is added to 1 mL of the culture medium.

[0026] A further scheme is:

[0027] The penicillin-streptomycin solution contains 10,000 units / mL of penicillin and 10,000 μg / mL of streptomycin.

[0028] A further scheme is:

[0029] In step three, the first centrifugation condition is: 4℃, 300 g centrifugation for 30 min.

[0030] The second centrifugation condition is: 4℃, 2000 g centrifugation for 30 min.

[0031] The third centrifugation condition is: 4℃, 10000 g centrifugation for 45 min;

[0032] The fourth centrifugation condition is: 4℃, 100000 g centrifugation for 70 min;

[0033] The fifth centrifugation condition is: 4℃, 100000 g centrifugation for 70 min.

[0034] The purpose of the fourth and fifth centrifugations is to precipitate the exosomes.

[0035] Further, the scheme is:

[0036] In step three, the filter membrane is a 0.22 μm filter membrane.

[0037] The application further provides a giant panda-derived exosome gel prepared by the preparation method of the aforementioned giant panda-derived exosome gel.

[0038] The application further provides an application of the giant panda-derived exosome gel, which is used for promoting wound healing of giant pandas.

[0039] Further, the scheme is:

[0040] The giant panda-derived exosome gel contains 0.4 mg / mL to 1.0 mg / mL of exosomes.

[0041] The application provides an exosome gel prepared by using giant panda mesenchymal stem cells, a preparation method and an application, which is a new drug of giant panda origin and can be used for promoting wound healing of giant pandas and has good anti-inflammatory effect. The application effectively solves the problem that the wound healing ability of old captive giant pandas is impaired, and there is no drug of giant panda origin for wound healing treatment at present. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 It is an application effect schematic diagram of an embodiment of the application. DETAILED DESCRIPTION

[0043] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0044] Embodiment one

[0045] The embodiment provides a preparation method of a giant panda-derived exosome gel, which comprises the following steps:

[0046] Step one, 175cm 3In the air-permeable culture bottle, the medium is used in an amount of 45-50 mL, and the medium is: low-sugar DMEM solution + 10% volume FBS + 1% (penicillin-streptomycin solution + 10 ng / mL bFGF, the culture of the fourth generation of giant panda umbilical cord-derived mesenchymal stem cells (UC-MSCs), and the inoculation of the UC-MSCs is in an amount of 8×10 5 -10×10 5 / bottle, and when the confluence reaches 80%, the complete culture medium is discarded, and the adherent cells are washed with PBS buffer.

[0047] Step two, continue to culture at 175 cm 3 In the air-permeable culture bottle, 35 mL-50 mL low-sugar DMEM solution is added, and the culture is placed in a 37°C, 5% CO2 incubator for culture, and the cell supernatant is collected after 48 h.

[0048] Step three, the collected supernatant is centrifuged at 4°C, 300 g for 30 min to remove cells. The supernatant is moved to a new centrifuge tube, and centrifuged at 4°C, 2000 g for 30 min to remove cell debris; the supernatant is moved to a new centrifuge tube, and centrifuged at 4°C, 10000 g for 45 min to remove larger vesicles; filtered through a 0.22 μm filter membrane, and the filtrate is collected, and centrifuged at 4°C, 100000 g for 70 min; the supernatant is removed, resuspended with 10 mL pre-cooled PBS buffer, and then centrifuged at 4°C, 100000 g for 70 min again; the supernatant is removed and resuspended with PBS buffer to obtain an exosome solution;

[0049] Step four, 0.1 g carbomer 940 is added to an appropriate amount of distilled water, and stirred constantly. After it swells into a translucent solution, it is sterilized in a high-pressure steam sterilization pot, and then different volumes of exosome solution, 0.005 g triethanolamine, 0.2 g nipagin ethyl solution, and 0.005 g triethanolamine, 0.5 g glycerol are added as needed, and then distilled water is added to 10 mL. After stirring uniformly, it is stored in a-80°C refrigerator for standby, and different concentrations of exosome gels are obtained.

[0050] Example two

[0051] Using the giant panda-derived exosome gel prepared in Example one, the effect of the exosome gel on wound healing is studied by taking mice as experimental objects. The specific process is as follows:

[0052] Mice were anesthetized with chloral hydrate at a dose of 0.3 mg / kg body weight, and then the back of the mice was shaved. The back of the mice was disinfected with 75% alcohol solution, and a full-thickness wound about 1.0 cm x 1.0 cm rectangular incision was made on the skin of the mice with a surgical knife. All mice were randomly divided into a control group, a blank gel group without drug loading, a low-dose Exo-gel group (the amount of exosomes in the gel was 0.4 mg / mL), and a high-dose Exo-gel group (the amount of exosomes in the gel was 1.0 mg / mL), with 3 mice in each group. The same dose of gel was given to each group every day. Image J software was used to measure and calculate the wound area at 0 d, 3 d, and 7 d after the wound, to evaluate the quality of wound healing. The results are shown in FIG. 8. Figure 1

[0053] No obvious redness was observed in the wound of the model mice, and the diet and mental state were normal. On the third day of treatment, more yellow thick inflammatory products appeared in the wounds of the control group and the blank gel group, while no obvious inflammatory reaction was observed in the wounds of the exosome group. The wound healing effect of the exosome group was higher than that of the control group and the blank gel group, and the healing effect of the high-dose exosome group (1.0 mg / mL) was higher than that of the low-dose exosome group (0.4 mg / mL). On the seventh day of treatment, the wounds of the exosome gel group healed significantly, with a healing rate of 87.63% in the low-dose group and 90.42% in the high-dose group, which was significantly higher than that in the low-dose group and showed good full-thickness damage treatment effect. The blank gel group without drug loading had no obvious effect on wound healing.

[0054]

[0055] Note: n number of mice; *P < 0.05, **P < 0.01 compared with the control group.

[0056] Summary of the effects of the examples:

[0057] The wounds of the exosome gel group healed significantly, and the healing rate of the high-dose group was significantly higher than that of the low-dose group, showing good full-thickness damage treatment effect. The blank gel group without drug loading had no obvious effect on wound healing. This indicates that the exosome gel derived from giant pandas has a promoting effect on wound healing.

[0058] Although the present application has been described with reference to the explanatory examples thereof, the above examples are only the preferred embodiments of the present application, and the embodiments of the present application are not limited to the above examples. It should be understood that those skilled in the art can design many other modifications and embodiments, which will fall within the scope and spirit of the principles disclosed in the present application.​

Claims

1. A method for preparing giant panda-derived exosome gel, characterized in that The steps include: Step 1: Cultivate giant panda umbilical cord-derived mesenchymal stem cells with culture medium. When the confluency reaches 80%, discard the complete culture medium and wash the adherent cells with PBS buffer. Step 2: Add sufficient low-glucose DMEM solution to the breathable culture bottle, place it in the incubator for culture, and collect the cell supernatant after 48 hours; Step 3: Centrifuge the collected cell supernatant for the first time to remove cells; The supernatant after the first centrifugation was transferred to a new centrifuge tube and centrifuged a second time to remove cell debris; The supernatant after the second centrifugation was transferred to a new centrifuge tube and centrifuged a third time to remove larger vesicles; The supernatant after the third centrifugation was taken and filtered through a filter membrane to remove large particles of impurities. The filtrate was collected and the supernatant was removed after the fourth centrifugation and resuspended in PBS buffer pre-cooled at 4°C. After the resuspension was centrifuged for the fifth time to remove the supernatant, it was resuspended in PBS buffer to obtain an exosome solution; Step 4: Add carbomer to an appropriate amount of distilled water and stir continuously. After it swells into a translucent solution, place it in a high-pressure steam sterilizer for sterilization. Then add the exosome solution and triethanolamine obtained in step 3, stir evenly, then gradually add ethyl paraben solution, triethanolamine, and glycerol, then add distilled water, stir evenly, and store in a -80°C refrigerator for use. This is the exosome gel.

2. The method for preparing the giant panda-derived exosome gel according to claim 1, characterized in that: In step 1, the giant panda umbilical cord-derived mesenchymal stem cells are the fourth generation giant panda umbilical cord-derived mesenchymal stem cells.

3. The method for preparing the giant panda-derived exosome gel according to claim 1, characterized in that: The culture medium in step 1 is: low-glucose DMEM solution + 10% volume FBS + 1% volume penicillin-streptomycin solution + 10 ng / mL bFGF.

4. The method for preparing the giant panda-derived exosome gel according to claim 3, wherein: The penicillin-streptomycin solution contains 10,000 units / mL of penicillin and 10,000 μg / mL of streptomycin.

5. The method for preparing the giant panda-derived exosome gel according to claim 1, characterized in that: In step 2, the culture conditions are: culture in an incubator at 37°C and 5% CO2.

6. The method for preparing the giant panda-derived exosome gel according to claim 1, characterized in that: In step 3, the first centrifugation conditions were: 4°C, 300 g for 30 min; The second centrifugation conditions were: 4°C, 2000 g for 30 min; The third centrifugation conditions were: 4°C, 10,000 g for 45 min; The fourth centrifugation conditions were: 4°C, 100,000 g for 70 min; The fifth centrifugation conditions were: 4°C, 100,000 g for 70 min.

7. The method for preparing giant panda-derived exosome gel according to claim 1, characterized in that: In step 3, the filter membrane is a 0.22 μm filter membrane.

8. A giant panda-derived exosome gel, characterized by: It is prepared by the preparation method of giant panda-derived exosome gel according to any one of claims 1 to 7.

9. The use of the giant panda-derived exosome gel according to claim 8, characterized in that: Used to promote wound healing in giant pandas.

10. The use of the giant panda-derived exosome gel according to claim 9, characterized in that: The giant panda-derived exosome gel contains 0.4 mg / mL to 1.0 mg / mL of exosomes.

Images