Preparation method and application of sheep-derived amniotic fluid exosome

Abstract

The invention relates to the technical field of biological materials, in particular to a preparation method and application of a sheep-derived amniotic fluid exosome. The preparation method specifically comprises the following steps: amniotic fluid extraction, centrifugal separation, batch treatment method ion exchange chromatography and batch treatment method hydrophobic chromatography. Wherein the batch treatment method ion exchange chromatography is to enrich proteins and nucleic acids with negative charges in exosomes and amniotic fluid by utilizing the property of negative charges carried by a cell membrane structure; according to the batch processing method hydrophobic chromatography, hydrophilic substances are separated by utilizing the hydrophobic property of a cell membrane structure. The purpose of the present application is to collect a trace amount of exosomes from a large amount of amniotic fluid, and to efficiently enrich exosomes by using a batch adsorption method. The method has the characteristic of mildness, damage to vesicles is extremely small, ultracentrifugation is not needed, and hundreds of liters of exosome stock solution can be treated at a time. The exosome is rich in growth factors, and the content of placenta growth factors is the highest.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to a method for preparing and applying sheep-derived amniotic fluid exosomes. Background Art

[0002] Exosomes are nano-scale membrane vesicles secreted by cells, and their diameter is usually 30-150nm. Exosomes contain a rich variety of bioactive molecules, including proteins, nucleic acids, and lipids. These bioactive molecules play an important role in information transmission between cells and can effectively regulate the physiological functions of cells. Amniotic fluid, as a specific environment for the growth and development of the fetus in the uterus, contains a large number of rich cells and a variety of bioactive molecules. In recent years, after many scientific studies, it has been found that exosomes also exist in amniotic fluid. These exosomes in amniotic fluid are very likely to play a vital role in the growth and development of the fetus and the health of the mother.

[0003] However, as of now, the preparation methods and practical applications of amniotic fluid exosomes are still relatively limited. This urgently requires further in-depth research and vigorous development. The source of amniotic fluid from sheep is wide, but the content of exosomes in amniotic fluid is negligible, and the volume of amniotic fluid is very large. Under normal circumstances, exosomes are collected by ultracentrifugation, but this method takes a very long time. When the ultrafiltration method is used for concentration, the long-term shear force will cause the vesicles to break, which will lead to poor uniformity of the product.

[0004] Therefore, it is necessary to find a gentle method to collect exosomes from amniotic fluid. Summary of the invention

[0005] The purpose of this application is to be able to collect trace amounts of exosomes from a large amount of amniotic fluid, and to efficiently collect exosomes by adopting a batch adsorption method. This method has mild characteristics, minimal damage to the vesicles, and does not require ultracentrifugation. It has a large processing capacity and can process hundreds of liters of exosome stock solution at a time. The present application provides a method for preparing and applying sheep-derived amniotic fluid exosomes. The exosomes are rich in growth factors, and the content of placental growth factor is the highest.

[0006] In the first aspect, the present application provides a method for preparing sheep-derived amniotic fluid exosomes, using the following technical scheme:

[0007] A method for preparing sheep-derived amniotic fluid exosomes, the preparation method specifically comprising the following steps: amniotic fluid extraction, centrifugal separation, batch ion exchange chromatography, batch hydrophobic chromatography;

[0008] The batch ion exchange chromatography utilizes the negative charge properties of the cell membrane structure to enrich the negatively charged proteins and nucleic acids in exosomes and amniotic fluid;

[0009] The batch hydrophobic chromatography utilizes the hydrophobic property of the cell membrane structure to separate the hydrophilic substances.

[0010] Optionally, the batch ion exchange chromatography comprises the following steps: diluting the supernatant after centrifugation with 20 mM phosphate buffer at pH 7.4 to reduce the conductivity to below 4 mS / cm; balancing DE52 with 20 mM phosphate buffer at pH 7.4, filtering and removing the balancing buffer; adding the supernatant after centrifugation at a ratio of 1 ml of DE52 cellulose to 100 ml of the supernatant after centrifugation, placing on a rotary mixer, and slowly mixing at 4° C. for 4-5 hours;

[0011] After the binding is completed, the DE52 filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity; washing is performed with a washing solution. The eluate is then mixed with the filler and slowly mixed and incubated on a rotary mixer for 5 minutes, and eluted three times. The three eluates are mixed together to obtain amniotic fluid exosomes.

[0012] Optionally, the washing solution is 20 mM phosphate, 10 mM sodium chloride at pH 7.4.

[0013] Optionally, the eluent is 20 mM phosphate, 50 mM sodium chloride at pH 7.4.

[0014] Optionally, the batch hydrophobic chromatography comprises the following steps: adding 2M ammonium sulfate to the supernatant after centrifugation; balancing the phenyl hydrophobic filler with a balancing buffer solution of 20mM phosphate and 2M ammonium sulfate at pH 7.4, and filtering to remove the balancing buffer; adding the supernatant after centrifugation at a ratio of 1ml of phenyl hydrophobic filler to 100ml of the supernatant after centrifugation, placing on a rotating mixer, and slowly mixing at 4°C for 4-5h;

[0015] After the binding is completed, the phenyl hydrophobic filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity. Wash with washing solution. Sequentially use eluent 1 and eluent 2 to mix with the filler, slowly mix and incubate for 5 minutes on a rotating mixer, and elute three times each; mix the three eluents obtained by eluting with eluent 2 together to obtain amniotic fluid exosomes.

[0016] Optionally, the washing solution is 20 mM phosphate, 1 M sodium chloride at pH 7.4.

[0017] Optionally, the eluent 1 is 20 mM phosphate, 500 mM sodium chloride at pH 7.4.

[0018] Optionally, the eluent 2 is 20 mM phosphate, 150 mM sodium chloride at pH 7.4.

[0019] Optionally, the ovine source is sheep.

[0020] In a second aspect, the present application provides the use of sheep-derived amniotic fluid exosomes prepared by the above-mentioned preparation method in preparing a composition for tissue repair, regeneration and / or disease treatment.

[0021] Optionally, the composition is a cosmetic.

[0022] In summary, the present application includes at least one of the following beneficial technical effects:

[0023] 1. This application provides for the first time a method for preparing sheep-derived amniotic fluid exosomes. The method is simple, efficient, low-cost, harmless to ewes and lambs, and can be used to prepare sheep-derived amniotic fluid exosomes on a large scale.

[0024] 2. This application discovered that sheep amniotic fluid exosomes are rich in growth factors, among which placental growth factor is the highest, providing a new direction and basis for the application of sheep amniotic fluid exosomes.

[0025] 3. The preparation method of the present application can prepare sheep-derived amniotic fluid exosomes on a large scale, providing sufficient raw material guarantee for its application.

[0026] 4. Sheep-derived amniotic fluid exosomes have broad application prospects and can provide new tools and raw materials for tissue repair and regeneration, disease treatment, cosmetics and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the TEM result of amniotic fluid exosomes obtained in Example 4 of the present application. DETAILED DESCRIPTION

[0028] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0030] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0031] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0032] The present application provides a method for preparing sheep-derived amniotic fluid exosomes. The preparation method specifically comprises the following steps:

[0033] (1) Sheep amniotic fluid collection

[0034] We select healthy sheep in the late stage of pregnancy and collect amniotic fluid during delivery. The collection process strictly follows clean operation specifications to ensure the quality and safety of the amniotic fluid.

[0035] The collected sheep amniotic fluid was immediately placed in a sterile centrifuge tube, stored at 4°C, and processed as soon as possible.

[0036] (2) Centrifugal separation

[0037] The collected sheep amniotic fluid was centrifuged at 3000 rpm for 15 min at 4°C to remove cells and debris.

[0038] The supernatant was taken and centrifuged at 10000 rpm for 30 min at 4°C to further remove tiny particles and impurities.

[0039] The supernatant was obtained by further filtration and sterilization using a 0.22 um polyethersulfone filter membrane.

[0040] (3) Batch ion exchange chromatography

[0041] By utilizing the negatively charged properties of the cell membrane structure, exosomes and negatively charged proteins and nucleic acids in the amniotic fluid can be enriched, thereby removing other impurities in the amniotic fluid and greatly reducing the volume.

[0042] The specific operation method is as follows: Dilute the supernatant after centrifugation with 20mM phosphate buffer at pH 7.4 to reduce the conductivity to below 4mS / cm. Equilibrate DEAE cellulose (DE52) with 20mM phosphate buffer at pH 7.4, and filter to remove the equilibration buffer. Add the supernatant after centrifugation at a ratio of 1ml DE52 cellulose to 100ml supernatant after centrifugation, place on a rotating mixer, and slowly mix at 4°C for 4-5h. Avoid generating a lot of foam during mixing.

[0043] After the binding is completed, the DE52 filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity. Washing is performed using a washing solution (20 mM phosphate, 10 mM sodium chloride at pH 7.4). The eluent (20 mM phosphate, 50 mM sodium chloride at pH 7.4) is then mixed with the filler and slowly mixed and incubated for 5 minutes on a rotating mixer, and eluted three times. The three eluents are mixed together to obtain an eluent for ion exchange chromatography.

[0044] (4) Batch hydrophobic chromatography

[0045] Taking advantage of the hydrophobic nature of the cell membrane structure, some hydrophilic substances can be separated in one step using the hydrophobic chromatography method.

[0046] The specific operation method is as follows: add 2M ammonium sulfate to the eluent of ion exchange chromatography. Equilibrate the phenyl hydrophobic filler with an equilibrium buffer solution (20mM phosphate, 2M ammonium sulfate at pH 7.4), and filter to remove the equilibrium buffer. Add the eluent of ion exchange chromatography in a ratio of 1ml of phenyl hydrophobic filler to 100ml of the eluent of ion exchange chromatography, place on a rotary mixer, and slowly mix for 4-5h at 4°C. Avoid generating a lot of foam during mixing.

[0047] After the binding is completed, the phenyl hydrophobic filler is introduced into the chromatography column, and the eluate of the ion exchange chromatography is filtered by gravity. Washing is performed using a washing solution (20 mM phosphate at pH 7.4, 1 M sodium chloride). Sequentially, eluent 1 (20 mM phosphate at pH 7.4, 500 mM sodium chloride) and eluent 2 (20 mM phosphate at pH 7.4, 150 mM sodium chloride) are mixed with the filler and slowly mixed and incubated on a rotating mixer for 5 minutes, and each is eluted three times. The three eluates obtained by eluting with eluent 2 are mixed together to obtain amniotic fluid exosomes.

[0048] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0049] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0050] The present application is further described in detail below in conjunction with the embodiments and test results.

[0051] Example 1

[0052] This embodiment provides a method for preparing amniotic fluid exosomes, wherein the subjects are healthy sheep in the late stage of pregnancy.

[0053] The above preparation method specifically comprises the following steps:

[0054] (1) Sheep amniotic fluid collection

[0055] We select healthy sheep in the late stage of pregnancy and collect amniotic fluid during delivery. The collection process strictly follows clean operation specifications to ensure the quality and safety of the amniotic fluid.

[0056] The collected sheep amniotic fluid was immediately placed in a sterile centrifuge tube, stored at 4°C, and processed as soon as possible.

[0057] (2) Centrifugal separation

[0058] The collected sheep amniotic fluid was centrifuged at 3000 rpm for 15 min at 4°C to remove cells and debris.

[0059] The supernatant was taken and centrifuged at 10000 rpm for 30 min at 4°C to further remove tiny particles and impurities.

[0060] The supernatant was obtained by further filtration and sterilization using a 0.22 um polyethersulfone filter membrane.

[0061] (3) Ultrafiltration concentration

[0062] The supernatant after centrifugation is ultrafiltered and concentrated through an external pressure ultrafiltration membrane (low shear force), and the molecular weight cutoff of the ultrafiltration membrane is 100kDa. The ultrafiltration process is carried out at 4°C, the pressure is 0.1MPa, the flow rate is 0.5L / min, and the volume of the liquid after ultrafiltration concentration is 1 / 20 of the original.

[0063] (4) Exosome extraction

[0064] Ultracentrifugation was used to extract sheep amniotic fluid exosomes. The ultrafiltrated and concentrated liquid was ultracentrifuged at 100,000 rpm for 2 h at 4°C, and the resulting precipitate was the amniotic fluid exosomes. The precipitate was washed three times with PBS buffer to remove residual impurities.

[0065] Example 2

[0066] This embodiment provides a method for preparing amniotic fluid exosomes, wherein the subjects are healthy sheep in the late stage of pregnancy.

[0067] The above preparation method specifically comprises the following steps:

[0068] (1) Sheep amniotic fluid collection: follow the corresponding steps of Example 1.

[0069] (2) Centrifugal separation: Follow the corresponding steps in Example 1.

[0070] (3) Batch ion exchange chromatography

[0071] By utilizing the negatively charged properties of the cell membrane structure, exosomes and negatively charged proteins and nucleic acids in the amniotic fluid can be enriched, thereby removing other impurities in the amniotic fluid and greatly reducing the volume.

[0072] The specific operation method is as follows: Dilute the supernatant after centrifugation with 20mM phosphate buffer at pH 7.4 to reduce the conductivity to below 4mS / cm. Equilibrate DEAE cellulose (DE52) with 20mM phosphate buffer at pH 7.4, and filter to remove the equilibration buffer. Add the supernatant after centrifugation at a ratio of 1ml DE52 cellulose to 100ml supernatant after centrifugation, place on a rotating mixer, and slowly mix at 4°C for 4-5h. Avoid generating a lot of foam during mixing.

[0073] After the binding is completed, the DE52 filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity. Wash with a washing solution (20mM phosphate at pH 7.4, 10mM sodium chloride). Then, the eluent (20mM phosphate at pH 7.4, 50mM sodium chloride) is mixed with the filler and slowly mixed and incubated for 5 minutes on a rotating mixer, and eluted three times. The three eluents are mixed together to obtain amniotic fluid exosomes.

[0074] Example 3

[0075] This embodiment provides a method for preparing amniotic fluid exosomes, wherein the subjects are healthy sheep in the late stage of pregnancy.

[0076] The above preparation method specifically comprises the following steps:

[0077] (1) Sheep amniotic fluid collection: follow the corresponding steps of Example 1.

[0078] (2) Centrifugal separation: Follow the corresponding steps in Example 1.

[0079] (3) Batch hydrophobic chromatography

[0080] Taking advantage of the hydrophobic nature of the cell membrane structure, some hydrophilic substances can be separated in one step using the hydrophobic chromatography method.

[0081] The specific operation method is as follows: Add 2M ammonium sulfate to the supernatant after centrifugation. Equilibrate the phenyl hydrophobic filler with an equilibrium buffer solution (20mM phosphate, 2M ammonium sulfate at pH 7.4), and filter to remove the equilibrium buffer. Add the supernatant after centrifugation at a ratio of 1ml of phenyl hydrophobic filler to 100ml of the supernatant after centrifugation, place on a rotating mixer, and slowly mix for 4-5h at 4°C. Avoid generating a lot of foam during mixing.

[0082] After the binding is completed, the phenyl hydrophobic filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity. Wash with a washing solution (20mM phosphate at pH 7.4, 1M sodium chloride). Sequentially, eluent 1 (20mM phosphate at pH 7.4, 500mM sodium chloride) and eluent 2 (20mM phosphate at pH 7.4, 150mM sodium chloride) are mixed with the filler and slowly mixed and incubated on a rotating mixer for 5 minutes, eluting three times each. The three eluates obtained by eluting with eluent 2 are mixed together to obtain amniotic fluid exosomes.

[0083] Example 4

[0084] This embodiment provides a method for preparing amniotic fluid exosomes, wherein the subjects are healthy sheep in the late stage of pregnancy.

[0085] The above preparation method specifically comprises the following steps:

[0086] (1) Sheep amniotic fluid collection: follow the corresponding steps of Example 1.

[0087] (2) Centrifugal separation: Follow the corresponding steps in Example 1.

[0088] (3) Batch ion exchange chromatography: Follow the corresponding steps of Example 2.

[0089] (4) Batch hydrophobic chromatography: The amniotic fluid exosomes obtained by the “batch ion exchange chromatography” were subjected to “batch hydrophobic chromatography” according to the corresponding steps of Example 3 to obtain an amniotic fluid exosome solution.

[0090] Example 5

[0091] This embodiment provides a method for preparing amniotic fluid exosomes, wherein the subjects are healthy sheep in the late stage of pregnancy.

[0092] The above preparation method specifically comprises the following steps:

[0093] (1) Sheep amniotic fluid collection: follow the corresponding steps of Example 1.

[0094] (2) Centrifugal separation: Follow the corresponding steps in Example 1.

[0095] (3) Flow-through ion exchange chromatography

[0096] The supernatant after centrifugation was diluted with 20 mM phosphate buffer at pH 7.4 to reduce the conductivity of the supernatant after centrifugation to below 4 mS / cm. DEAE cellulose (DE52) was loaded into a 1 ml chromatography column and connected to an Akta medium pressure chromatography device, and balanced with 20 mM phosphate buffer at pH 7.4 at a flow rate of 1 ml / min. 100 ml of the supernatant after centrifugation was passed through the chromatography column, and the loading was stopped when the column pressure reached 0.3 MPa.

[0097] After binding, wash with a washing solution (20 mM phosphate, 10 mM sodium chloride at pH 7.4) and then elute with an eluting solution (20 mM phosphate, 50 mM sodium chloride at pH 7.4). The eluted solution is the amniotic fluid exosome solution.

[0098] Performance test results

[0099] The performance of the amniotic fluid exosomes prepared in the above Examples 1-5 was tested respectively.

[0100] (I) Processing capacity analysis

[0101] The processing capacity analysis results of Examples 1-5 are shown in Table 1.

[0102] Table 1 Processing capacity of Examples 1-5

[0103] Example No. Processed amniotic fluid volume (ml) Preparation time (h) Limiting Factors 1 50 10 Ultracentrifugation capacity is small and centrifugation time is long 2 200-300 0.5 - 3 200-300 0.5 - 4 200-300 0.5 - 5 50 0.5 Column blockage

[0104] As shown in Table 1, Example 1 uses the traditional centrifugation method to extract exosomes. Since the ultracentrifuge is usually an angle rotor desktop centrifuge with a rotor capacity of 1-5 ml, the maximum processing volume per batch is 10 ml, and the processing time per batch is at least 2 hours. Therefore, it takes 10 hours to process 50 ml, which is difficult to achieve industrialization.

[0105] In addition, Example 5 adopts the flow-through adsorption method. Since enrichment is easily caused at the head of the chromatography column, which causes the chromatography column to be blocked, the column force of 1 ml of filler increases significantly when treating 50 ml of amniotic fluid, and the treatment cannot be continued. Therefore, compared with the batch method, the flow-through adsorption method has certain limitations when it comes to industrial scale-up.

[0106] Based on the above, Examples 2-4 use a batch adsorption method for extraction and purification, which has a large processing volume, a short processing time, and is easy to scale up industrially.

[0107] (II) Particle size analysis

[0108] Dynamic light scattering (DLS) was used to analyze the particle size of the amniotic fluid exosomes obtained in each example. The analysis results are shown in Table 2.

[0109] Table 2 Particle size analysis and total protein analysis results

[0110]

[0111] As shown in Table 2, the particle size of sheep amniotic fluid exosomes is mainly distributed between 30-150nm, with an average particle size of 80nm.

[0112] (III) Analysis of total exosome protein

[0113] Exosomes were extracted by ultracentrifugation, and the total protein of the amniotic fluid exosomes obtained in each example was analyzed.

[0114] Take 1 ml of amniotic fluid exosomes obtained in each example, ultracentrifuge at 100000 rpm for 2 h at 4 ° C, collect the supernatant and precipitate after ultracentrifugation, resuspend the precipitate with 100 ul cell lysis buffer, and use the BCA method to determine the protein content released by the exosomes and the protein content in the ultracentrifugation supernatant. The calculation method is as follows: protein content in exosomes / (protein content in exosomes + protein content in supernatant) is the purity.

[0115] The test results are shown in Table 2.

[0116] As shown in Table 2, the protein content in sheep amniotic fluid exosomes is rich, with an average of 500 μg of protein per milliliter of sheep amniotic fluid exosomes. Among them, Example 1 uses ultracentrifugation to better remove free protein, so the purity is higher. Examples 2, 3 and 5 contain more impurities, mainly because the chromatography column has a strong adsorption capacity for some free protein impurities, and the removal effect is not good at one time. Example 4 is purified in two steps, and the purity of the protein in the amniotic fluid exosomes is close to that of Example 1.

[0117] (IV) Nucleic acid content determination

[0118] The nucleic acid content in the amniotic fluid exosomes obtained in each example was measured by fluorescent quantitative PCR. The measurement results are shown in Table 3.

[0119] Table 3 Analysis of nucleic acid content and cytokine content

[0120] Example No. miRNA content (ng / ml) mRNA content (ng / ml) PIGF (μg / ml) IGF (μg / ml) 1 12 8 140 20 2 15 10 190 35 3 13 9 184 38 4 14 9.5 180 35 5 11 9.5 195 40

[0121] As can be seen from Table 3, the sheep amniotic fluid exosomes obtained in each example contain a variety of nucleic acid molecules, such as miRNA, mRNA, etc.

[0122] (V) Cytokine content analysis

[0123] The content of placental growth factor and insulin-like growth factor in the amniotic fluid exosomes obtained in each example was determined by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 3.

[0124] As shown in Table 3, the sheep amniotic fluid exosomes obtained in each example contain rich growth factors. The cytokine content in the sheep amniotic fluid exosomes obtained in Examples 2-5 is basically the same, and significantly higher than that in Example 1, which may be because Examples 2-5 also extracted a small amount of free cytokines in the amniotic fluid when purifying the amniotic fluid exosomes.

[0125] (VI) Morphological observation

[0126] The morphology of the amniotic fluid exosomes obtained in Example 4 was observed using a transmission electron microscope (TEM). Figure 1 shown.

[0127] Depend on Figure 1 It can be seen that sheep amniotic fluid exosomes are round or oval membrane vesicle structures with complete membrane structures and clear internal structures.

[0128] Example 6

[0129] This example provides the use of the amniotic fluid exosomes obtained in Example 4 in tissue repair and regeneration.

[0130] The specific method is as follows: 20 mice were selected and randomly divided into four groups, 5 mice in each group. Superficial second-degree and deep second-degree skin wound models were made on the back of mice. The sheep amniotic fluid exosomes prepared in Example 4 were applied to the wound of the experimental group mice, and the normal saline was applied to the control group mice. The healing of the wound of the mice was observed every day, the wound area was measured, and the healing rate and healing time at 7d and 14d were calculated.

[0131] The results are shown in Table 4.

[0132] Table 4 Application of amniotic fluid exosomes obtained in Example 4 in tissue repair and regeneration

[0133]

[0134] As shown in Table 4, the healing speed of skin wounds in the experimental group mice was significantly accelerated.

[0135] Example 7

[0136] This example provides the application of the amniotic fluid exosomes obtained in Example 4 in the field of cosmetics.

[0137] The specific method is as follows: prepare a cosmetic emulsion containing the amniotic fluid exosomes obtained in Example 4.

[0138] Formula 1 is as follows: amniotic fluid exosomes 0.01%, hyaluronic acid 0.1%, vitamin E 0.5%, glycerol 3%, emulsifier 0.1%, preservative 0.5%, and deionized water balance.

[0139] Formula 2 is as follows: collagen (type I, Zhejiang Chongshan Biological Products Co., Ltd.) 0.01%, hyaluronic acid 0.1%, vitamin E 0.5%, glycerol 3%, emulsifier 0.1%, preservative 0.5%, and deionized water balance.

[0140] 30 volunteers were recruited for the trial experiment and randomly divided into three groups, with 10 people in each group. The experimental group used the cosmetic lotion of formula 1, and the control group used the cosmetic lotion of formula 2. Use it once a day in the morning and evening for 8 weeks. Before use, 4 weeks of use, and 8 weeks of use, the volunteers' skin was tested, including the degree of skin wrinkles, skin texture, pore fineness, skin moisture content, and skin elasticity recovery rate. Among them, the degree of skin wrinkles, skin texture and pore fineness were evaluated using the Visia skin detector, and the skin moisture content and skin elasticity recovery rate were evaluated using the CK skin detector.

[0141] The test results after 8 weeks of use are shown in Table 5.

[0142] Table 5 Application of amniotic fluid exosomes obtained in Example 4 in the field of cosmetics

[0143] Experimental equipment index Recipe 1 Recipe 2 Visia Skin wrinkle improvement (%) 25±3 13±2 Visia Skin texture improvement (%) 20±2 16±4 Visia Pore ​​fineness (%) 17±2 9±5 CK Skin moisture content (%) 32±5 21±3 CK Skin elasticity recovery rate (%) 28±5 16±4

[0144] As shown in Table 5, the use of cosmetic lotions containing amniotic fluid exosomes can significantly improve skin wrinkles, sagging, dryness and other problems, increase skin elasticity and firmness, and make the skin smoother and more delicate.

[0145] In summary, the sheep amniotic fluid exosomes provided by the present application contain rich growth factors, among which the placental growth factor has the highest content. At the same time, the preparation method of the present application is simple, efficient, and low-cost, and sheep amniotic fluid exosomes can be prepared on a large scale. Sheep amniotic fluid exosomes have broad application prospects and can provide new tools and raw materials for the fields of tissue repair and regeneration, disease treatment, cosmetics, etc.

[0146] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing sheep-derived amniotic fluid exosomes, characterized in that: The preparation method specifically comprises the following steps: amniotic fluid extraction, centrifugal separation, batch ion exchange chromatography, batch hydrophobic chromatography; The batch ion exchange chromatography utilizes the negative charge properties of the cell membrane structure to enrich the negatively charged proteins and nucleic acids in exosomes and amniotic fluid; The batch hydrophobic chromatography utilizes the hydrophobic property of the cell membrane structure to separate the hydrophilic substances.

2. The preparation method according to claim 1, characterized in that The batch ion exchange chromatography comprises the following steps: diluting the supernatant after centrifugation with a 20 mM phosphate buffer at pH 7.4 to reduce the conductivity to below 4 mS / cm; balancing DE52 with a 20 mM phosphate buffer at pH 7.4, filtering and removing the balancing buffer; adding the supernatant after centrifugation at a ratio of 1 ml of DE52 cellulose to 100 ml of the supernatant after centrifugation, placing on a rotary mixer, and slowly mixing at 4° C. for 4-5 hours; After the binding is completed, the DE52 filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity; washing is performed using a washing solution; the eluate is then mixed with the filler and slowly mixed and incubated on a rotary mixer for 5 minutes, and eluted three times; the three eluates are mixed together to obtain amniotic fluid exosomes.

3. The preparation method according to claim 2, characterized in that The wash solution was 20 mM phosphate, 10 mM sodium chloride at pH 7.

4.

4. The preparation method according to claim 2, characterized in that The eluent was 20 mM phosphate, 50 mM sodium chloride, pH 7.

4.

5. The preparation method according to claim 1, characterized in that The batch hydrophobic chromatography comprises the following steps: adding 2M ammonium sulfate to the supernatant after centrifugation; balancing the phenyl hydrophobic filler with a balancing buffer solution of 20mM phosphate and 2M ammonium sulfate at pH 7.4, filtering and removing the balancing buffer solution; adding the supernatant after centrifugation in a ratio of 1ml of phenyl hydrophobic filler to 100ml of the supernatant after centrifugation, placing on a rotating mixer, and slowly mixing at 4°C for 4-5h; After the binding is completed, the phenyl hydrophobic filler is introduced into the chromatography column, and the supernatant after centrifugation is filtered by gravity; washing is performed with a washing solution; eluent 1 and eluent 2 are mixed with the filler in turn, and then slowly mixed and incubated on a rotating mixer for 5 minutes, and eluted three times each; the eluents obtained from the three elutions with eluent 2 are mixed together to obtain amniotic fluid exosomes.

6. The preparation method according to claim 5, characterized in that The wash solution was 20 mM phosphate, 1 M sodium chloride at pH 7.

4.

7. The preparation method according to claim 5, characterized in that The eluent 1 is 20 mM phosphate, 500 mM sodium chloride at pH 7.

4.

8. The preparation method according to claim 5, characterized in that The eluent 2 is 20 mM phosphate, 150 mM sodium chloride at pH 7.

4.

9. The preparation method according to claim 1, characterized in that: The sheep source is sheep.

10. Use of sheep amniotic fluid exosomes obtained by the preparation method according to any one of claims 1 to 9 in preparing a composition for tissue repair, regeneration and / or disease treatment; Optionally, the composition is a cosmetic.

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