Highly active products obtained based on antler stem cells, preparation methods and applications thereof

Abstract

The present invention proposes a highly active product obtained based on antler stem cells, a preparation method and its application, including preparing a serum-free culture medium, thawing the immortalized antler stem cells, centrifuging, inoculating and culturing, and passage, and finally resuspending and freeze-drying with water for injection to obtain freeze-dried powder. The culture medium consists of a basic culture medium and a growth additive, and each component is described in detail. The method can efficiently enrich the active products unique to antler wax flakes. It also involves the application of highly active products obtained based on antler stem cells in skin care products (such as water solutions, lotions, etc.) and health products (such as nutritional supplements, functional drinks), providing a new way for the development of related products, and has potential economic and social value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a highly active product obtained based on velvet antler stem cells, a preparation method and an application thereof. Background Art

[0002] As a biological material with important medicinal and health-care value, deer antler has received extensive attention in the fields of medicine and health care. It is composed of multiple components such as nerves, blood vessels, connective tissue, skin cartilage, etc., and is rich in a variety of bioactive ingredients such as proteins, peptides, amino acids, etc. Its medicinal and health-care value has been verified for a long time.

[0003] From the perspective of tissue structure, velvet antler can be divided into three sections: upper, middle and lower. Among them, the upper section contains skin and dense subcutaneous tissue, and the lower section is connected to the spherical antler tissue. This part is the area with more antler stem cells. The antler tissue can differentiate into the antler precursor cell layer, and the sliced ​​products of this part are commonly known as "wax slices". "Wax slices" have become the most valuable antler products at present because they contain rich bioactive ingredients and more significant medical and health care effects. Studies have found that there are more than 300 proteins in antler wax slice extracts that can promote the proliferation and migration of endothelial progenitor cells. However, the proportion of wax slices in the whole antler is extremely low, and when extracting the active ingredients in antler or antler wax slices, it is usually necessary to perform mechanical crushing, solution extraction, high pressure or ultrasonic treatment on the antler. Even so, the extraction yield of active ingredients is still not high, and there are a large number of extracellular matrix components, which to a certain extent limits the efficacy of antler.

[0004] In recent years, research on antler stem cells has made significant progress. For example, antler stem cells have shown many positive effects in regenerative medicine research. They can promote the proliferation and differentiation of human bone marrow mesenchymal stem cells, inhibit osteoclast formation, participate in cell repair and promote cell regeneration. At the same time, antler stem cell metabolites can reprogram the tumor microenvironment, enhance immune response, improve osteoarthritis symptoms, and promote chondrocyte growth. Similar to antler wax flakes, the efficacy of antler stem cells depends on a complex combination of multiple components. The abundance, isomers, modified products and protein-protein interactions of these components play a vital role in regulating cell growth and development. Numerous proteomic studies on antlers have shown that wax flakes have a different protein composition from other parts of antlers. These unique protein combinations are the key features that distinguish antler wax flakes from other antler products. In addition, preliminary laboratory studies have also found that antler stem cells have good proliferation potential and the ability to synthesize active substances.

[0005] In summary, although the prior art has achieved certain results in the research on antlers, it is still insufficient in efficiently obtaining the active ingredients specific to antler wax flakes. The present invention aims to provide a new approach to solve this problem. Summary of the invention

[0006] The embodiments of the present invention provide a highly active product obtained based on antler stem cells, a preparation method and its application, which are aimed at the defects of the current technology in extracting active ingredients from antler or antler wax flakes, such as low yield, many extracellular matrix components, influence of factors such as breeding conditions, and inability to efficiently obtain active ingredients specific to antler wax flakes.

[0007] The core technology of the present invention is mainly to enrich the unique active products of antler wax slices through large-scale cultivation of immortalized antler stem cells, using self-developed serum-free culture medium, and through the steps of culture medium preparation, cell recovery, inoculation and continuous passaging, as well as high-density antler cell pure water lysis and freeze-drying.

[0008] In a first aspect, the present invention provides a method for preparing a highly active product based on antler stem cells, the method comprising the following steps:

[0009] S00, preparing a serum-free stem cell culture medium without animal-derived components as a complete antler stem cell culture medium, wherein the serum-free stem cell culture medium consists of a antler stem cell basal culture medium and an antler stem cell growth additive;

[0010] S10, thawing the cell cryopreservation tube with immortalized antler stem cells, transferring the cell suspension in the cell cryopreservation tube to a centrifuge tube after thawing, adding antler stem cell complete culture medium, centrifuging and discarding the supernatant, then adding antler stem cell complete culture medium and blowing and mixing the cell precipitate;

[0011] S20, taking the mixed cell suspension to count and record, and then inoculating into a culture vessel for culture, and when the cell confluence reaches a set threshold, subculture can be performed;

[0012] S30. After subculturing a sufficient amount of cells, resuspend them with water for injection to rupture the cell membrane, and freeze-dry them to obtain freeze-dried powder.

[0013] Furthermore, in step S00, the antler stem cell basal culture medium consists of vitamins, inorganic salt ions, trace elements, sodium bicarbonate, glucose, ascorbic acid phosphate, non-essential amino acid supplements, alanine-glutamine, lipid mixture, γ-aminobutyric acid, trehalose and various amino acids.

[0014] Furthermore, in step S00, the antler stem cell growth additive consists of recombinant insulin, recombinant transferrin, pea protein hydrolysate, soy protein hydrolysate, biotin, superoxide dismutase, reduced glutathione, vitamin A, vitamin E, triiodothyronine, recombinant human fibroblast growth factor FGF2, recombinant human transforming growth factor TGF-b1, recombinant human heparin growth factor HGF, recombinant human collagen and trypsin inhibitor.

[0015] Furthermore, in step S00, the addition ratio of antler stem cell growth additive and antler stem cell basal culture medium is 1:20.

[0016] Furthermore, in step S20, the threshold is set to 90%.

[0017] In a second aspect, the present invention provides a highly active product obtained based on deer antler stem cells, which is prepared by the above-mentioned method for preparing a highly active product based on deer antler stem cells.

[0018] In a third aspect, the present invention provides an application of a highly active product obtained based on deer antler stem cells in skin care products.

[0019] Furthermore, the type of skin care products is any one of lotions, emulsions, essences, creams, eye creams, facial masks, gels, and cleansing products.

[0020] In a fourth aspect, the present invention provides an application of a highly active product obtained from deer antler stem cells in health care products.

[0021] Furthermore, the health product is a nutritional supplement or a functional drink.

[0022] The main contributions and innovations of the present invention are as follows:

[0023] 1. Culture medium innovation: We have developed a serum-free stem cell culture medium without animal-derived ingredients. It is composed of a specific basal culture medium and growth additives in proportion. The basal culture medium contains a variety of vitamins, inorganic salt ions and other ingredients, and the growth additives contain a variety of substances such as recombinant insulin. This culture medium can improve the growth efficiency and active substance production of antler stem cells, avoiding the potential risks brought by the introduction of animal-derived ingredients or other matrices, which is unprecedented in the existing technology.

[0024] 2. Improvement of cell culture and product enrichment methods: Immortalized antler stem cells were used for culture, and the detailed and optimized operation procedures from thawing of cell cryopreservation tubes, centrifugation, inoculation culture to subculture were clarified. For example, the critical threshold for subculture was determined when the cell confluence reached 90%. In the product enrichment stage, the method of resuspending the cells with water for injection to rupture the cell membrane and then freeze-drying to obtain freeze-dried powder retains the antler stem cells themselves and the intracellular proteome and metabolome components to the greatest extent, achieving efficient enrichment of the active products unique to antler wax flakes, overcoming the problems of low yield of active ingredients extracted from antlers and high extracellular matrix components in the existing technology.

[0025] 3. Diversified product applications: Not only limited to the application fields of traditional antler products, the highly active products obtained based on antler stem cells of the present invention can also be applied to the field of skin care products, including various types of products such as water-based solutions, emulsions, essences, etc., as well as nutritional supplements and functional drinks in the field of health care products, etc., opening up a broader market space for the commercial application of highly active products obtained based on antler stem cells, which is an innovative application direction not covered by the existing technology.

[0026] The details of one or more embodiments of the invention are set forth in the following drawings and description so that other features, objects, and advantages of the invention are more readily apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0028] Figure 1 This is a diagram showing the gel electrophoresis analysis results of the antler stem cell extract and the antler wax slice and blood slice extraction samples according to an embodiment of the present invention;

[0029] Figure 2 This is a comparison chart of the types of proteins (a) and polypeptides (b) in the antler stem cell extract and the antler wax flake and blood flake extract according to an embodiment of the present invention;

[0030] Figure 3 This is a comparison chart of the content of the functional protein found in the antler growth center (the source of antler wax flakes) in three samples according to an embodiment of the present invention. DETAILED DESCRIPTION

[0031] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with one or more embodiments of this specification. Instead, they are merely examples of devices and methods consistent with some aspects of one or more embodiments of this specification as detailed in the appended claims.

[0032] It should be noted that: in other embodiments, the steps of the corresponding method are not necessarily performed in the order shown and described in this specification. In some other embodiments, the steps included in the method may be more or less than those described in this specification. In addition, a single step described in this specification may be decomposed into multiple steps for description in other embodiments; and multiple steps described in this specification may be combined into a single step for description in other embodiments.

[0033] Embodiment 1

[0034] The present invention aims to propose a preparation method for obtaining a highly active product based on antler stem cells. Figure 1 , the method comprises the following steps:

[0035] S00, medium preparation:

[0036] The currently reported antler stem cell culture medium is mainly composed of basal culture medium and fetal bovine serum. There are also reported serum-free culture media with animal-derived components such as collagen and bovine serum albumin. Some researchers have also tried to use deer serum instead of fetal bovine serum, but deer serum has a low yield and contains a large amount of antibodies and complement, and is not suitable for large-scale cell culture. The purpose of the present invention is to enrich the active products of antler stem cells. The use of animal-derived components and fetal bovine serum will introduce a large amount of xenogeneic proteins and heterologous metabolites, affecting the efficacy of the active components of antler stem cells. Therefore, based on the experience of developing serum-free stem cell culture media, the present invention specially customizes an antler stem cell serum-free culture medium without animal-derived components for immortalized antler stem cells;

[0037] This culture medium consists of two parts: antler stem cell basal culture medium and antler stem cell growth additive. Before cell culture, thaw the antler stem cell growth additive at 4 degrees or room temperature and add it to the antler stem cell basal culture medium at a ratio of 1:20 to prepare a complete culture medium, that is, an antler stem cell serum-free culture medium without animal-derived ingredients;

[0038] The basal medium for antler stem cells is composed of various amino acids, vitamins, inorganic salt ions, trace elements, sodium bicarbonate, glucose, ascorbic acid phosphate, non-essential amino acid supplements, alanine-glutamine, lipid mixture, γ-aminobutyric acid, and trehalose;

[0039] Deer antler stem cell growth supplement consists of recombinant insulin, recombinant transferrin, pea protein hydrolysate, soy protein hydrolysate, biotin (vitamin B7 or coenzyme R), superoxide dismutase, reduced glutathione, vitamin A, vitamin E, triiodothyronine, recombinant human fibroblast growth factor FGF2, recombinant human transforming growth factor TGF-b1, recombinant human heparin growth factor HGF, recombinant human collagen, and trypsin inhibitor.

[0040] The amino acids in the basal medium for antler stem cells can be:

[0041] 1. Essential amino acids: including lysine, tryptophan, phenylalanine, methionine (methionine), threonine, isoleucine, leucine, valine, etc. These amino acids cannot be synthesized by the cells themselves or the amount synthesized is not enough to meet the needs and must be obtained from the outside.

[0042] 2. Non-essential amino acids: such as alanine, glycine, proline, serine, tyrosine, cysteine, etc. Although cells can synthesize them by themselves, adding them to the culture medium can better meet the needs of cell growth.

[0043] Vitamins can be:

[0044] 1. Water-soluble vitamins: For example, B vitamins, including vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B6, vitamin B12, etc., play an important role in the energy metabolism of cells, nucleic acid synthesis, etc. There is also vitamin C (ascorbic acid), which has an antioxidant effect and helps maintain the normal physiological functions of cells.

[0045] 2. Fat-soluble vitamins: Like vitamin A, they affect cell differentiation and proliferation; vitamin D participates in calcium and phosphorus metabolism and may play a role in processes such as cell mineralization; vitamin E is an antioxidant that can protect cells from free radical damage; vitamin K may play a certain role in cell activities related to physiological processes such as blood coagulation.

[0046] Inorganic salt ions can be:

[0047] 1. Cations: including potassium ions (K + ), which is essential for maintaining the membrane potential of cells, participating in the regulation of cell osmotic pressure and the activity regulation of various enzymes; sodium ion (Na +) also participates in the regulation of cell osmotic pressure and some physiological processes; calcium ions (Ca 2+ ) plays a key role in cell signal transduction, cell adhesion, muscle contraction, etc.; magnesium ions (Mg 2+ ) is a cofactor of many enzymes and is involved in energy metabolism, nucleic acid and protein synthesis within cells.

[0048] 2. Anion: Chloride ion (Cl - ) is the main anion in the extracellular fluid and is involved in maintaining the osmotic pressure and acid-base balance of the cell; phosphate ion (PO4 3- ) is a component of biological macromolecules such as nucleic acids and phospholipids, and plays an important role in the formation and maintenance of cell genetic material and membrane structure; sulfate ion (SO4 2- ) may be involved in the modification of some proteins and polysaccharides.

[0049] Trace elements can be:

[0050] 1. Iron (Fe): It is a component of many enzymes in cells, such as cytochrome oxidase, and is involved in cell respiration and energy metabolism.

[0051] 2. Zinc (Zn): It regulates the activity of various enzymes and participates in processes such as cell proliferation, differentiation and DNA synthesis.

[0052] 3. Copper (Cu): It is a component of some oxidases and participates in intracellular redox reactions and iron metabolism.

[0053] 4. Manganese (Mn): It affects the activity of some enzymes and participates in processes such as cellular sugar metabolism and protein synthesis.

[0054] 5. Selenium (Se): It is a component of some antioxidant enzymes, such as glutathione peroxidase, which has antioxidant effects and protects cells from oxidative damage.

[0055] 6. Lithium (Li): It is considered an essential nutrient that can promote cell growth and metabolism, cell proliferation and self-renewal. Secondly, lithium has a neuroprotective effect, can improve synaptic plasticity, and inhibit cell apoptosis. Lithium ions can also regulate the potential of cell membranes, affect the electrophysiological characteristics of cells, and thus affect cell function.

[0056] 7. Molybdenum (Mo): It is a trace element necessary for cell metabolism and enzyme activity, and is involved in the active sites of enzymes involved in key reactions in carbon, nitrogen and sulfur metabolism.

[0057] The specific ingredients are shown in Table 1 and Table 2 below:

[0058] Table 1 Composition of basal culture medium for antler stem cells

[0059]

[0060] Table 2 Composition of Deer Antler Stem Cell Growth Supplement

[0061]

[0062] S10, Deer antler stem cell culture:

[0063] The immortalized antler stem cells used in the present invention have a cell line establishment process that has been disclosed in the patent (CN202311164955.6). This patent is to solve the problems of slow proliferation and low viability of antler stem cells in in vitro culture and amplification applications. It uses a self-developed culture medium and adds a variety of plant-derived ingredients to replace the components in conventional culture medium. It can enhance the viability of antler stem cells, accelerate the proliferation rate, and extend the number of cell passages, thereby achieving cell immortalization. At the same time, the biological characteristics of immortalized antler stem cells can be identified by a variety of methods. Therefore, the present invention uses the immortalized antler stem cells obtained by culture according to this patent.

[0064] Recovery of immortalized antler stem cells: Take out the cell cryopreservation tube (containing immortalized antler stem cells) from liquid nitrogen, place it in a 37°C water bath or cell recovery instrument to thaw, shake the cryopreservation tube from time to time to make it melt rapidly within 2 minutes, and be careful not to immerse the mouth of the cryopreservation tube in the water bath to avoid contamination; after the cell cryopreservation solution is completely melted, wipe the outer wall of the cryopreservation tube with 75% alcohol, open the lid, transfer the cell suspension to a 15mL centrifuge tube, add 5ml of antler stem cell complete culture medium, centrifuge at 1000rpm for 3-5min, and discard the supernatant. Add 1-2mL of complete culture medium and gently blow away the mixed cell pellet.

[0065] Inoculation: Take a certain amount of cell suspension for counting, record key parameters such as cell number and viability, and inoculate cells into culture dishes or culture bottles. After culturing in a 37°C, 5% CO2 incubator for 48 hours (the specific time depends on the cell state and inoculation density), the cells can be passaged when the confluence reaches 90%.

[0066] It is worth mentioning that the confluence of antler stem cells should not reach 100%, especially when the inoculation is uneven, and timely digestion and subculture are required, otherwise it is easy to cause cell stacking growth, or even aggregation into balls, which will seriously cause cell aging and differentiation. In the clean bench, discard the culture medium in the culture bottle and add PBS to wash the cells 1-2 times. Add 1-2mL of trypsin (0.05%) to digest the cells and digest them at 37℃ for 1-2min. After digestion, gently tap the culture bottle. When most of the cells shrink and become round under the microscope and a small number of cells begin to flow, add more than twice the amount of trypsin to terminate digestion. Use a pipette to gently blow the cells that have not completely fallen off the wall of the bottle, and gently blow and mix the cell suspension to disperse the cells completely. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 400g for 3min. Discard the supernatant, add 1mL of culture medium or PBS solution to resuspend the cells, and count them. Add 15mL of culture medium to the T75 bottle and inoculate antler stem cells according to the number of 1.5E6. The culture flask was placed at 37°C, 5% CO2, and saturated humidity.

[0067] S20, Enrichment of highly active products obtained from antler stem cells:

[0068] After a sufficient amount of antler stem cells is cultured through subculture, the cells obtained by culture can be collected by digestion and centrifugation. Resuspend them with water for injection at a density of 10 million cells per milliliter, and use the low osmotic pressure of the water for injection to rupture the membrane of the antler stem cells and release the cell contents. Finally, put the cell suspension into a -80℃ refrigerator.

[0069] After the cell suspension is completely frozen, the frozen antler stem cell suspension is placed in a vacuum freeze dryer to complete the freeze-drying process of the cell suspension at -80°C to obtain a complete, highly active product freeze-dried powder based on antler stem cells.

[0070] In this way, the active ingredients of antler stem cells enriched by the method of the present invention retain the antler stem cells themselves and the intracellular proteome and metabolome components, such as skeletal proteins, transport proteins, signal transduction proteins, enzymes, peptides, lipids and polyamine compounds, to the greatest extent. The use of immortalized antler stem cells as production raw materials can stably produce high-value active products contained in antlers without being affected by factors such as seasons, climate changes, and breeding conditions. After large-scale continuous cultivation, the production cycle and production cost of enriched active products will be greatly reduced, promoting the commercialization of antler stem cell-related products. This invention can not only provide a new raw material for functional foods and medical beauty markets, but antler stem cells may also expand a wider application space for the research and treatment of diseases such as Alzheimer's disease, hyperglycemia, and hypertension, and have long-term social and economic value.

[0071] Embodiment 2

[0072] Based on the same concept, the present invention also proposes the steps of continuous passage of antler stem cells under serum-free culture conditions and enrichment of active products:

[0073] Step 1: Preparation of serum-free medium for antler stem cells. The antler stem cell growth additive is stored at -20 degrees. After thawing at 4 degrees or room temperature, it is added to the antler stem cell basal medium at a ratio of 1:20 to prepare a complete antler stem cell medium. At the same time, prepare a conventional antler stem cell medium consisting of alpha-MEM basal medium plus 10% fetal bovine serum.

[0074] Step 2: Resuscitate an immortalized antler stem cell: Take the cell cryopreservation tube out of liquid nitrogen, place it in a 37°C water bath to thaw, shake the cryopreservation tube from time to time to make it melt rapidly within 2 minutes; after the cell cryopreservation solution is completely melted, wipe the outer wall of the cryopreservation tube with 75% alcohol, open the lid, transfer the cell suspension to a 15mL centrifuge tube, and add 5mL PBS solution. Use a centrifuge at 1000rpm, room temperature, centrifuge for 3-5 minutes, and discard the supernatant. Add 1mL PBS solution and gently blow away the mixed cell pellet. Take 20µL of cell suspension and count using AO / PI staining.

[0075] Step 3. Take 6 T25 cell flasks and divide them into two groups. Add the serum-free medium for antler stem cells and fetal bovine serum medium prepared in step 1 respectively. According to the cell counting results, 5E5 antler stem cells were inoculated into each cell culture flask. The cells were mixed and placed in a 37°C, 5% CO2 incubator for 48 hours. The cell confluence reached about 90%. In the clean bench, discard the culture medium in the culture flask and add PBS to wash the cells 1-2 times. Add 0.5mL of trypsin (0.05%) to digest for 1-2min. After digestion, gently tap the culture flask. When most of the cells shrink and become round under the microscope and a small number of cells begin to flow, 4mL of culture medium can be added to terminate the digestion. Use a pipette to gently blow the cells that have not completely fallen off the wall of the flask, and gently blow and mix the cell suspension to make the cells completely dispersed. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 400g for 3min. The supernatant was discarded, 1 mL of culture medium was added to resuspend the cells, and the next generation of cells was inoculated again after counting. The excess cells were collected, resuspended with injection water at a concentration of 1E7 cells / mL, and stored in a -80°C refrigerator.

[0076] Step 4. After 10 consecutive passages, the antler stem cell suspensions enriched by the two culture media were placed in a vacuum freeze dryer and freeze-dried overnight at -80°C to obtain complete lyophilized powders of highly active products based on antler stem cells. The powders were reconstituted with water for injection, and 10 µL samples were taken to determine the protein concentrations in the extracts obtained from the two culture media using the Bradford method, as shown in Table 3 below:

[0077] Table 3 - Differences in expansion times and protein concentrations of continuously passaged antler stem cells in two culture media

[0078]

[0079] It can be seen from the above results that the serum-free culture medium developed for immortalized antler stem cells in the present invention can maintain stable antler stem cell expansion, and can avoid the problems of cell aging and decreased proliferation rate compared to conventional fetal bovine serum culture medium. In addition, at the same cell concentration, due to the higher state and metabolic activity of antler stem cells cultured in serum-free culture medium, the protein concentration is higher than that of conventional fetal bovine serum culture medium. The antler stem cell culture medium in the present invention is a serum-free, animal-free culture medium developed for an immortalized antler stem cell. Compared with the culture medium using fetal bovine serum or deer serum, it has an extremely low protein content, more stable culture performance, and no xenobiotic contamination, and is more suitable for the enrichment of active substances in antler stem cells. Benefiting from the current gradually decreasing cost of cell culture medium raw materials such as recombinant proteins, this antler stem cell culture medium can support large-scale antler stem cell culture and enrichment of active products.

[0080] Embodiment 3

[0081] Based on the same concept, the present invention also proposes the following steps for comparing the active products enriched with antler stem cells with the extracts from antler wax slices and antler blood slices:

[0082] Step 1: Cultivation of antler stem cells and enrichment of active products. Resuscitation of an immortalized antler stem cell: remove the cell cryovial from liquid nitrogen, place it in a 37°C water bath to thaw, shake the cryovial from time to time to make it melt rapidly within 2 minutes; after the cell cryopreservation solution is completely melted, wipe the outer wall of the cryovial with 75% alcohol, open the lid, transfer the cell suspension to a 15mL centrifuge tube, and add 5ml of antler stem cell complete medium. Use a centrifuge at 1000rpm, room temperature, centrifuge for 4 minutes, and discard the supernatant. Add 1mL of antler stem cell complete medium and gently blow to mix the cell pellet. Count the cells in the cell suspension. The number of frozen antler stem cells is about 3.0E6. Inoculate one antler stem cell into 2 T75 cell culture flasks, each containing 15ml of antler stem cell medium and 1.5E6 seed cells. The cell culture flask was placed in a 37°C, 5% CO2 incubator for 48 hours. When the cell confluence reached 90%, approximately 6.0E6 antler stem cells could be harvested from each T75 flask.

[0083] In the clean bench, discard the culture medium in the culture flask and add PBS to wash the cells twice. Add 2mL of trypsin (0.25%) for digestion for 2min. After digestion is completed, gently tap the culture flask. When most of the cells shrink and become round under the microscope and a small number of cells begin to flow, 6mL of culture medium can be added to terminate digestion. Use a pipette to gently blow the cells that have not completely fallen off the wall of the flask, and gently blow and mix the cell suspension to completely disperse the cells. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 400g for 3min. Discard the supernatant, add 1mL of culture medium to resuspend the cells, and count. After one round of cell culture, about 12.0E6 antler stem cells were harvested from two T75 cell culture flasks. If the cells are collected at this time, the harvested antler stem cells can be resuspended with 1.2mL of water for injection to obtain a cell suspension with a density of about 10.0E6 cells / mL.

[0084] The antler stem cell suspension was placed in a -80°C refrigerator for freezing. After the antler stem cell suspension was frozen, the frozen cell suspension was placed in a vacuum freeze dryer and freeze-dried overnight at -80°C to obtain a complete antler stem cell active product freeze-dried powder, which was then placed in a -80°C refrigerator for storage.

[0085] Step 2: Extraction of commercial antler wax slice and blood slice protein. The whole wax slice and blood slice of antler were purchased from the official flagship store of a antler specialty store on Taobao platform. The price of the whole wax slice is 100 yuan / g, and the price of the antler blood slice is 30 yuan / g. Grind the whole wax slice and blood slice into powder with a grinder, collect 5g of whole wax slice powder of antler and 5g of blood slice powder of antler into two 50ml centrifuge tubes respectively, add 40ml of water for injection, place the centrifuge tube on a water bath shaker, shake at 200rpm, 25℃ for 2 hours, and then put the two centrifuge tubes into a 4℃ refrigerator and let it stand overnight for extraction. The obtained extract was filtered through 0.2µm and stored in a -80℃ refrigerator;

[0086] Step 3. Comparison of active proteins and peptides in antler stem cells, antler wax flakes, and blood flakes. The antler stem cell freeze-dried powder collected in step 1 and the antler wax flake and blood flake extract freeze-dried powder collected in step 2 were reconstituted with the original volume of water for injection and filtered with a 0.2µm filter membrane. The reconstituted antler stem cell active product was an aqueous solution of 10.0E6 cells / mL, and the commercial antler extract was an aqueous solution of 0.125g wax flake or blood flake / mL. Take 10µL of the sample and use the Bradford method to determine the protein concentration of the extract. The results are shown in Table 4 below:

[0087] Table 4 - Comparison of protein content between antler stem cell freeze-dried powder solution and antler wax flake and blood flake extracts

[0088]

[0089] At the same time, gel electrophoresis analysis (SDS-PAGE) was performed on the three samples. The results showed that although the protein content in the antler wax slices was high, the bands were fuzzy, which may be due to protein degradation; the components in the antler blood slices were concentrated around a molecular weight of 55kDa, with relatively fewer types; while in the antler stem cell extract samples, the protein bands were clear and the molecular weight distribution range was the widest, showing more product diversity ( Figure 1 ).

[0090] The aqueous solutions of active ingredients from three sources of velvet antler were analyzed using an Orbitrap Eclipse mass spectrometer. The data was analyzed using data-independent proteomics and compared with the Cervidae-Cervus protein database in the Uniport database.

[0091] The results showed that nearly 6,000 proteins were identified from antler stem cell freeze-dried powder solution and antler samples, of which antler stem cells had the most abundant protein types (5,832 types), of which 66% were only found in antler stem cell samples. The number of protein types in antler wax flake extract was second (1,948 types), and the number of protein types in antler blood flake was the lowest ( Figure 2 a).

[0092] Peptide analysis showed that more than 30,000 peptides were identified from antler stem cell extracts and antler samples, of which antler stem cells had the most abundant protein types (28,929), and 83% of the peptide types were only found in antler stem cell samples. The number of peptides in antler wax flake extracts was second (5,150), and the number of peptides in antler blood flakes was the least ( Figure 2 b).

[0093] According to existing studies, some functional proteins that are only found in the antler growth center (the source of antler wax flakes) can be found among these identified proteins. These proteins include important transcriptional regulators, functional enzymes and transport proteins, etc. They play a key role in inhibiting cell apoptosis, promoting cell proliferation and differentiation, neural development and repair, etc. ( Figure 3 a). Proteomic analysis showed that these proteins were present in large quantities in antler stem cells, and their abundance was much higher than that in antler wax slices, but were almost absent in blood samples ( Figure 3 b). In addition, some extracellular matrix-related proteins, such as MANF, HAPLN and VCAN, only exist in antler wax slices or blood slices, and their proportion is relatively high.

[0094] Based on the concentration of antler stem cells in this example, the protein content of 10.0E6 cells / mL antler stem cell extract is 27.8% higher than that of 0.125g / mL antler wax flake extract (Table 1). At the same time, the content of antler wax flake-specific proteins and peptides is also higher ( Figure 3 b). Cultivating 10.0E6 antler stem cells only requires no more than 30 mL of complete medium and 150 cm 2 The culture surface is 48 hours. Through large-scale expansion of antler stem cells, the proteins, peptides and other active substances unique to these antler wax flakes can be efficiently harvested, and the cost is equivalent to or lower than that of obtaining antler wax flakes through cultivation.

[0095] Those skilled in the art should understand that the technical features of the above embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a highly active product based on velvet antler stem cells, characterized in that: The following steps are involved: S00, preparing a serum-free stem cell culture medium without animal-derived components as a complete antler stem cell culture medium, wherein the serum-free stem cell culture medium consists of a antler stem cell basal culture medium and an antler stem cell growth additive; The addition ratio of the antler stem cell growth additive to the antler stem cell basal culture medium is 1:20; the antler stem cell basal culture medium consists of amino acids, vitamins, inorganic salt ions, trace elements, sodium bicarbonate, glucose, ascorbic acid phosphate, non-essential amino acid supplements, alanine-glutamine, lipid mixture, γ-aminobutyric acid, and trehalose; the antler stem cell growth additive consists of recombinant insulin, recombinant transferrin, pea protein hydrolysate, soy protein hydrolysate, superoxide dismutase, reduced glutathione, vitamin A, vitamin E, triiodothyronine, recombinant human fibroblast growth factor FGF2, recombinant human transforming growth factor TGF-b1, recombinant human heparin growth factor HGF, recombinant human collagen, and trypsin inhibitor; S10, thawing the cell cryopreservation tube with immortalized antler stem cells, transferring the cell suspension in the cell cryopreservation tube to a centrifuge tube after thawing, adding antler stem cell complete culture medium, centrifuging and discarding the supernatant, then adding antler stem cell complete culture medium and blowing and mixing the cell precipitate; S20, taking the mixed cell suspension, counting and recording, and then inoculating into a culture vessel for culture, and when the cell confluence reaches 90%, subculture can be performed; S30. After subculturing a sufficient amount of cells, resuspend them with water for injection to rupture the cell membrane, and freeze-dry them to obtain freeze-dried powder.

2. A highly active product obtained from velvet antler stem cells, characterized in that: The product is prepared by the method for preparing a highly active product based on antler stem cells as described in claim 1.

3. A skin care product, characterized in that: Including the highly active product obtained based on deer antler stem cells as described in claim 2.

4. A skin care product according to claim 3, characterized in that: The skin care product is any one of an aqueous solution, emulsion, essence, facial cream, eye cream, facial mask, gel, and facial cleanser.

5. A health product, characterized in that: Including the highly active product obtained based on deer antler stem cells as described in claim 2.

6. A health care product according to claim 5, characterized in that: The health care product is a nutritional supplement or a functional drink.

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