CN109864964B - Anti-aging composition containing stem cells and application thereof - Google Patents
Anti-aging composition containing stem cells and application thereof Download PDFInfo
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Abstract
The invention provides an anti-aging composition containing stem cells and application thereof, belonging to the technical field of biological medicines, wherein the composition specifically comprises the following components: a) stem cells; b) immune cells involved in the immune response; c) IPS cell extract; and d) a protective agent, wherein the protective agent is a mixture of the grape polyphenol and catalpol in a weight ratio of 1: 15.8-16.0. The stem cells, the immune cells and the IPS cell extracts in the composition can be used together to remove senescent dead and cancerated cells, clean the microenvironment of an organism, protect the proliferation and activation of human cells, further play the roles of regeneration and repair, delay senescence, reverse senescence injury and efficiently promote wound healing and bone growth.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an anti-aging composition containing stem cells and application thereof.
Background
China is rapidly stepping into aging society, and at present, China has about hundred million elderly people over the age of China. The elderly people aged over 60 in China in 2050 are expected to account for three years, and various troubles and problems caused by aging are more serious. Senescence and death are the ultimate fates that are difficult to avoid for individuals of any species, but to date our understanding of the mechanisms by which senescence occurs has been quite limited. In view of its importance, aging has been a focus of biological research, which is a direct manifestation of the slowing of human function, a natural process, and a complex and variable process. In the whole life process, the body is influenced and acted by various substances, energy and information in the external environment, and tissues and organs are inevitably damaged and have function decline. With the improvement of medical technology and living standard, the number of aging population increases, and with aging, physiological functions of human organs are affected, such as hypertension, hyperglycemia, hyperlipidemia, hyperuricemia, high weight, coronary atherosclerosis and other diseases increase. Delaying aging may reduce the occurrence of these diseases and improve the functional status of the body as a whole, thereby improving the quality of life of the body.
The research on aging resistance is a hot topic of the modern society and is also a target which is continuously pursued by people. At present, anti-aging methods are various, such as diet adjustment, proper exercise, folk prescription, and the like. Despite all the advantages, no means has been proven to be effective by systematic studies. The rise of regenerative medicine at the present stage has stimulated the research enthusiasm of people for various stem cells, tissue engineering scaffolds and cell growth factors. Stem Cells (SC) are a type of pluripotent cells with self-replicating ability, which can differentiate into various functional cells under certain conditions, and can perform regenerative repair on aging degenerative changes of various tissues, organs and systems, so that the structure and function of the cells are normalized and rejuvenated, and the cells regulate the body state as a whole, thereby being a systemic and fundamental health care. For example, patent application CN102486475A discloses a method for evaluating anti-aging effect of autologous adipose-derived stem cells, which can improve the metabolic function of body to various lipoproteins by applying adipose-derived stem cells for health care and treatment. For another example, patent application CN103387962A discloses an autologous stem cell anti-aging technology with anti-aging and life prolonging effects, which can achieve the purposes of improving organ functions, delaying aging and prolonging life by transplanting plants and protein-induced autologous hematopoietic stem cells (PPiHPS) periodically, for a long time, for many times, supplementing seed cells required for growth, regeneration and repair of organism tissues and organs of a living individual, and various organ functional cells generated thereby, and regeneratively repairing the aging structure and function of organism organs. However, simply using stem cells to achieve anti-aging of the body neglects the microenvironment of the stem cells, adjacent cells, secreted proteins, extracellular matrix, metabolic signals (oxygen, glucose, etc.), and mechanical parameters (shear stress, tissue hardness, etc.) … … all affect the behavior of the stem cells, and the actual anti-aging effect is not ideal.
Disclosure of Invention
The stem cells, the immune cells and the IPS cell extract in the composition are combined to remove senescent dead and cancerated cells, clean the microenvironment of an organism, protect the proliferation and activation of human cells, further play the roles of regeneration and repair, delay senescence, reverse senescence injury and efficiently promote wound healing and bone growth.
The technical scheme adopted by the invention for realizing the purpose is as follows:
[1] an anti-aging composition comprising stem cells, comprising:
a) stem cells;
b) immune cells involved in the immune response;
c) IPS cell extract; and
d) and (4) a protective agent. In the method of the invention, the stem cells are combined with the immune cells and the IPS cell (induced pluripotent stem cell) extract, which not only can play a role in removing aged dead and cancerated cells by the immune cells and ensure that the microenvironment of an organism is in a safe and clean state, but also can play unique roles in protecting the proliferation, activating, secreting antioxidant active substances and resisting aging of human cells by the IPS cell extract, meanwhile, the input of the stem cells can play roles in regeneration and repair, the recovery of the organism is accelerated, the aging process is further delayed, the respective curative effects can be mutually enhanced by the combination of the three, in addition, the protective agent can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, is favorable for maintaining the integrity, the regeneration and the bioavailability of the stem cells and the immune cells and plays an excellent anti-aging role of the composition, meanwhile, the traditional Chinese medicine composition has a certain effect of reversing aging injury, can efficiently promote wound healing and bone growth, has higher economic value and application prospect in the fields of beauty treatment, medical treatment, health care and the like, maintains the balance of the neuroendocrine system of an organism through various cytokines and hormones secreted by immune cells and stem cells, and can more effectively resist the aging of the organism without treating the disease first on the basis of scientifically preventing the occurrence of the disease.
In the summary of the invention and the preferred embodiments, the stem cells in the anti-aging composition comprising stem cells are one or a combination of at least two of optional but not limited to embryonic stem cells, adult stem cells, hematopoietic stem cells, neural stem cells, peripheral blood stem cells, adipose stem cells, mesenchymal stem cells, cardiac stem cells, placental sub-totipotent stem cells and amniotic membrane stem cells; more preferably embryonic stem cells, mesenchymal stem cells or hematopoietic stem cells; most preferably umbilical cord and placenta mesenchymal stem cells among the mesenchymal stem cells. The embryonic stem cell (ES stem cell) is a highly undifferentiated cell, has totipotency of development, can differentiate all tissues and organs of an adult animal, including germ cells, can be rapidly differentiated after treatment, and finally generates various cell lines, such as muscle cells, blood cells, nerve cells or develops into embryo bodies, so that the ES stem cell has wide prospects in the combined application of cell transplantation and other advanced biotechnology; adult stem cells have a critical ability to repair and regenerate in many tissues, organs, and epidermal and hematopoietic systems of animals, and under certain conditions, adult stem cells either produce new stem cells or differentiate according to a certain program to form new functional cells, thereby maintaining the dynamic balance of growth and deterioration of tissues and organs; hematopoietic stem cells are the only source of various blood cells in vivo, and are mainly present in bone marrow, peripheral blood and umbilical cord blood; transplantation of hematopoietic stem cells is the most effective method for treating hematological diseases, congenital genetic diseases, and multiple metastatic neoplastic diseases; the neural stem cells are expected to cure part of symptoms of patients with Parkinson's syndrome, so that the neural stem cells have potential application prospect on any central nervous system diseases; the adipose-derived stem cells have the potential of in vitro proliferation and multiple differentiation, and can be applied to the regeneration and repair of tissues and organs; mesenchymal stem cells are pluripotent stem cells that have all of the commonalities of stem cells, namely self-renewal and pluripotency; the clinical application is the most at present, and the combined application with hematopoietic stem cells can improve the success rate of transplantation and accelerate hematopoietic reconstruction; the placental sub-totipotent stem cells are close to embryonic stem cells in the development stage, can be differentiated to form more than 200 human tissue organ cells, but cannot form a complete human body; the amniotic membrane stem cells are derived from amniotic epithelium, express markers of various embryonic stem cells, have multi-differentiation capacity, have differentiation capacity exceeding that of bone marrow-derived mesenchymal stem cells, and have lower immunogenicity.
In the summary and preferred embodiment of the present invention, the stem cell is cultured by the following steps: and culturing the stem cells in a third culture medium, adding a second culture medium for subculture when the cultured cells are more than 92% confluent, and collecting fourth generation stem cells.
In the context of the present invention and in a preferred embodiment, the immune cells involved in the immune response in the anti-senescence composition comprising stem cells are in particular NK cells. NK cells (Natural killer cells) are important immune cells of the body, not only involved in the resistance to tumors, viral infections and immune regulation, but also in certain cases in the development of hypersensitivity reactions and autoimmune diseases.
In the summary of the invention and the preferred embodiment, the culture method of the NK cells is as follows: culturing NK cells in a first medium at a cell density of more than 4X 107After one/mL, a second culture medium is added for subculture, and fifth generation cells are collected.
In the summary and the preferred embodiment of the invention, the first culture medium consists of a clinical-grade serum-free culture medium and a first additive, and the components of the first additive and the concentration of the components in a DMEM culture solution are 2.2-2.5 mg/L sodium alginate, 10-11 mg/L potassium selenite, 11-14 mg/L sulfur-containing amino acid, 15-18 mg/L mannitol, 4-4.2 mg/L tender willow branch aqueous extract and 6-7 mg/L angelica dahurica leaf aqueous extract. The first culture medium can provide sufficient mass energy for the culture and development of the NK cells, the healthy and rapid formation of a cell line is ensured, and the tender willow branch aqueous extract and the angelica dahurica leaf aqueous extract can effectively accelerate the proliferation of the NK cells so as to quickly reach the predetermined cell density for transfer subculture.
In the invention content and the preferred embodiment, the second culture medium consists of a DMEM culture solution, fetal calf serum and a second additive, the total volume fraction of the fetal calf serum in the DMEM culture solution is 5-6%, and the components of the second additive and the concentration of the components in the DMEM culture solution are 6-10 mg/L glutathione, 12-14 mg/L ferric citrate, 5-6 mg/L fibroblast growth factor, 2-5 mg/L potassium selenite, 6-8 mg/L human albumin, 5-10 mg/L cinnamon water extract and 5-10 mg/L fingered citron water extract.
In the content and the preferable embodiment of the invention, the third culture medium consists of a clinical-grade serum-free culture medium and a third additive, and the concentration of each component of the third additive in a DMEM culture solution is 1-3 mg/L diethanolamine, 5-10 mg/L lauroylserine, 1-3 mg/L cysteine, 10-15 mg/L sorbitol, 5-10 mg/L proline, 1-3 mg/L vitamin C, 5-10 mg/L tender willow branch water extract and 5-10 mg/L kudzu root alcohol extract. The tender willow branch water extract and the kudzu root alcohol extract can activate related biological enzymes of stem cells on the genetic material level, effectively accelerate the differentiation and proliferation of the stem cells, and enable the stem cells to quickly reach the preset cell density for transfer subculture.
In the summary and preferred embodiment of the present invention, the preparation method of IPS cell extract in the anti-aging composition comprising stem cells specifically comprises:
(a) spreading 0.25-0.4 wt% gelatin in an aging container to cover the whole bottom, standing at 37 deg.C for 45-60 min, adding 5-7 times of gelatin volume of cell complete culture medium pre-incubated at 37 deg.C, adding cell with concentration not less than 2 × 105culturing cells/mL human embryonic fibroblasts with 6-8 times of gelatin volume until the growth concentration of feeder layer cells exceeds 92%;
(b) inoculating the cultured IPS cell suspension into the container in the step (a) at the cell amount of 1: 10-25, and performing CO treatment at 37 DEG C2Culturing for 3-10 days in an incubator;
(c) sucking out the cell culture solution in the step (b), washing with PBS for 2-5 times, adding cell lysate with the same volume of gelatin to dissolve cells, centrifuging to obtain supernatant, dialyzing with PBS at the temperature of 1-4 ℃ for 12-18 h, and freeze-drying the solution in a dialysis bag to obtain the cell culture solution. The IPS cell extract is rich in high biological active substances such as collagen, active peptide, growth factor, vitamin, mineral substances, antioxidant active enzyme and the like, has small antigenicity to a human body, is absorbed and utilized by the human body, has excellent anti-aging function, is organically compatible with stem cells and immune cells, protects the proliferation of the human body cells and the secretion of the antioxidant active substances, and can exert efficient cell activating and anti-aging functions.
In the context of the present invention and preferred embodiments, the IPS cell extract is prepared by a method wherein the cell complete medium in step (a) comprises: DMEM + 15-20 g/L fetal bovine serum + 3-5 mg/L sodium alginate + 4-10 g/L + 10-12 mg/L sulfur-containing amino acid + 5-6 mg/L sodium pyruvate + 0.2-0.5 mg/L beta-carotene + 0.12-0.15 mg/L red sage root extract. The cell complete culture medium can promote the rapid differentiation and propagation of human embryonic fibroblasts, is beneficial to the synthesis and secretion of active substances by IPS cells, promotes the cell culture solution cultured for a short time to be rich in the active substances of the IPS cells, and greatly enhances the medical care effect of the composition.
In the invention and the preferred embodiment, the protective agent in the anti-aging composition containing stem cells is a mixture of grape polyphenol and catalpol in a weight ratio of 1: 15.8-16.0. The applicant finds that the mixture of the grape polyphenol and the catalpol in a specific weight ratio is added to the anti-aging composition containing the stem cells, so that the biological function of the composition is maintained, the mutual synergistic effect of the grape polyphenol and the catalpol can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, the integrity, the reproducibility and the bioavailability of the stem cells and the immune cells are maintained, and the excellent anti-aging effect of the composition is exerted.
In the summary and the preferred embodiment of the invention, the extraction method of the glucan polyphenol in the protective agent comprises the following steps: crushing grape skins and grape seeds under the protection of nitrogen flow, adding petroleum ether for degreasing, crushing, vacuum-drying, adding 75% ethanol solution according to the solid-to-liquid ratio of 1: 2-5, leaching at the constant temperature of 45-55 ℃ for 2-5 h, taking clear liquid, evaporating to dryness at low temperature, and crushing to obtain the grape skin and grape seeds. The grape polyphenol, namely grape procyanidin, is a natural plant polyphenol, has various biological activities such as oxidation resistance, free radical damage resistance, radiation resistance, cardiovascular protection and the like, can also play a synergistic role when being matched with catalpol, can retain the inherent biological activity of the grape polyphenol to a greater extent by extracting the grape polyphenol by the method, and has short extraction time and easy operation.
In the summary and preferred embodiments of the present invention, the components of the anti-aging composition comprising stem cells occupy the composition in the following weight percentages:
a) 35-98 wt% of dry cells;
b) 0.1-35% by weight of immune cells involved in the immune response;
c) 0.1-30 wt% IPS cell extract; and
d)1.8 to 5 wt% of a protective agent.
In the context of the present invention and in preferred embodiments, the anti-aging composition comprising stem cells may be formulated for oral administration.
In the summary and preferred embodiments of the invention, the forms of the anti-aging composition comprising stem cells suitable for oral administration include the form of tablets and/or capsules prepared by conventional methods with pharmaceutically acceptable excipients including, but not limited to:
1) a binder, preferably pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose;
2) fillers, preferably lactose, galactose, corn starch, microcrystalline cellulose or calcium hydrogen phosphate;
3) a lubricant, preferably magnesium stearate, talc or silica;
4) disintegrating agent, preferably potato starch, sodium starch glycolate;
5) wetting agents, preferably sodium lauryl sulfate.
In the context of the present invention and in preferred embodiments, the stem cell-containing anti-aging composition is suitable for oral administration in a form including a tablet and/or capsule that can be coated by methods well known in the art.
In the context of the present invention and in preferred embodiments, the forms of the anti-senescence stem cell-containing composition suitable for oral administration include liquid formulations which may take the form of solutions, syrups or suspensions, or they may be provided in anhydrous form for reconstitution with water or other suitable vehicle prior to use. The liquid formulation may be prepared by conventional methods with pharmaceutically acceptable additives including, but not limited to:
1) a suspending agent, preferably sorbitol syrup, a cellulose derivative or hydrogenated edible lipid;
2) an emulsifier, preferably lecithin or acacia;
3) a non-aqueous vehicle, preferably almond oil, oily esters, glycerin or fractionated vegetable oils;
4) preservatives, preferably methyl hydroxybenzoate, propyl hydroxybenzoate or sorbic acid.
In the context of the present invention and in the preferred embodiments, the anti-aging composition comprising stem cells may be formulated for oral administration and may also contain buffer salts, flavoring agents, coloring agents and sweetening agents as desired.
[2] The present invention also provides the method for preparing the anti-aging composition comprising stem cells as described in item [1], specifically: and (3) under sterile conditions and at the temperature of 1-2 ℃, sequentially adding the protective agent, the immune cells participating in immune response and the IPS cell extract into the stem cells, uniformly mixing, and freezing to obtain the composition.
[3] The present invention also provides the use of the stem cell-containing antiaging composition as described in items [1] and [2], particularly the use of the composition in the preparation of an antiaging preparation.
The invention has the beneficial effects that:
1) the stem cells are combined with the immune cells and the IPS cell extract, so that the removing effect of the immune cells on aging dead and cancerated cells can be exerted, the microenvironment of an organism is in a safe and clean state, the IPS cell extract can also exert the unique effects of protecting the proliferation and the activation of human cells, secreting antioxidant active substances and resisting aging, meanwhile, the input of the stem cells can exert the regeneration and repair effects, the organism rehabilitation is accelerated, the aging process is further delayed, and the respective curative effects can be mutually enhanced by the combination of the stem cells, the immune cells and the IPS cell extract;
2) the mixture of the grape polyphenol and the catalpol in a special weight ratio is added into the anti-aging composition containing the stem cells, so that the biological function of the composition is kept, the mutual synergistic effect of the grape polyphenol and the catalpol can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, the integrity, the regeneration and the bioavailability of the stem cells and the immune cells are kept, and the excellent anti-aging effect of the composition is exerted;
3) the composition has a certain effect of reversing aging injury, can efficiently promote wound healing and bone growth, and has higher economic value and application prospect in the fields of beauty treatment, medical treatment, health care and the like;
4) the balance of the neuroendocrine system of the organism is maintained through various cell factors and hormones secreted by immune cells and stem cells, and the organism aging is more effectively resisted without treating the disease first on the basis of scientifically preventing the disease.
The dye and adhesive composition provided by the invention makes up for the defects of the prior art, and is reasonable in design and convenient to operate.
Detailed Description
Although the following text sets forth a detailed description of numerous different embodiments, it should be understood that the legal scope of the invention is defined by the words of the claims set forth at the end of this patent. The detailed description is to be construed as exemplary only and does not describe every possible embodiment since describing every possible embodiment would be impractical, if not impossible. Numerous alternative embodiments could be implemented, using either current technology or technology developed after the filing date of this patent, which would still fall within the scope of the claims.
Unless a term is defined herein to mean … … "or a similar statement is expressly defined unless the term is used in this patent in the statement" as used herein, the term '____' is not intended to expressly or implicitly limit the meaning of the term beyond its plain or ordinary meaning and the term should not be construed as being limited in scope based on any statement made in any part of this patent (other than the language of the claims). To the extent that any term recited in the claims at the end of this patent is referred to in this patent in a manner consistent with a single meaning, that is done for sake of clarity only so as to not confuse the reader, and it is not intended that such claim term by limited, by implication or otherwise, to that single meaning.
The technical solutions in the preferred embodiments of the present invention are clearly and completely described, and it is obvious that the described preferred embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Of course, and in no way intended to limit the scope of the invention, it is intended that any product which performs the function of the invention not necessarily achieve all of the advantages described herein, nor do they necessarily achieve all of the objectives described above.
The present invention is further described in detail with reference to the following examples:
example 1:
the present embodiment provides an anti-aging composition containing stem cells, wherein the anti-aging composition containing stem cells comprises the following components by weight:
a) 50% by weight of dry cells;
b) 25% by weight of immune cells involved in the immune response;
c)22 wt% IPS cell extract; and
d) 3% by weight of protective agent.
In the method of the invention, the stem cells are combined with the immune cells and the IPS cell (induced pluripotent stem cell) extract, which not only can play a role in removing aged dead and cancerated cells by the immune cells and ensure that the microenvironment of an organism is in a safe and clean state, but also can play unique roles in protecting the proliferation, activating, secreting antioxidant active substances and resisting aging of human cells by the IPS cell extract, meanwhile, the input of the stem cells can play roles in regeneration and repair, the recovery of the organism is accelerated, the aging process is further delayed, the respective curative effects can be mutually enhanced by the combination of the three, in addition, the protective agent can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, is favorable for maintaining the integrity, the regeneration and the bioavailability of the stem cells and the immune cells and plays an excellent anti-aging role of the composition, meanwhile, the traditional Chinese medicine composition has a certain effect of reversing aging injury, can efficiently promote wound healing and bone growth, has higher economic value and application prospect in the fields of beauty treatment, medical treatment, health care and the like, maintains the balance of the neuroendocrine system of an organism through various cytokines and hormones secreted by immune cells and stem cells, and can more effectively resist the aging of the organism without treating the disease first on the basis of scientifically preventing the occurrence of the disease.
In this embodiment, the stem cells in the antiaging composition containing stem cells are adipose-derived stem cells, and the stem cells are cultured by: and (3) culturing the adipose-derived stem cells in a third culture medium, adding a second culture medium for subculture when the cultured cells are up to 92% confluent, and collecting fourth generation stem cells.
In this embodiment, the immune cells involved in immune response in the anti-aging composition comprising stem cells are specifically NK cells, and the culture method of the NK cells is as follows: culturing NK cells in a first medium to a cell density of 4 × 107After one/mL, a second culture medium is added for subculture, and fifth generation cells are collected.
The first culture medium consists of a clinical-grade serum-free culture medium and a first additive, wherein the concentrations of the components of the first additive in a DMEM culture solution are 2.2mg/L sodium alginate, 10mg/L potassium selenite, 11mg/L sulfur-containing amino acid, 15mg/L mannitol, 4mg/L tender willow branch aqueous extract and 6mg/L dahurian angelica leaf aqueous extract. The first culture medium can provide sufficient mass energy for the culture and development of the NK cells, the healthy and rapid formation of a cell line is ensured, and the tender willow branch aqueous extract and the angelica dahurica leaf aqueous extract can effectively accelerate the proliferation of the NK cells so as to quickly reach the predetermined cell density for transfer subculture.
The second culture medium consists of a DMEM culture solution, fetal calf serum and a second additive, the total volume fraction of the fetal calf serum in the DMEM culture solution is 5%, and each component of the second additive and the concentration of the second additive in the DMEM culture solution are 6mg/L glutathione, 12mg/L ferric citrate, 5mg/L fibroblast growth factor, 2mg/L potassium selenite, 6mg/L human albumin, 5mg/L cinnamon water extraction and 5mg/L fingered citron water extraction.
The third culture medium consists of a clinical-grade serum-free culture medium and a third additive, wherein the concentrations of the components of the third additive in a DMEM culture solution are 1mg/L diethanolamine, 5mg/L lauroylserine, 1mg/L cysteine, 10mg/L sorbitol, 5mg/L proline, 1mg/L vitamin C, 5mg/L tender willow branch water extract and 5mg/L kudzu root alcohol extract. The tender willow branch water extract and the kudzu root alcohol extract can activate related biological enzymes of stem cells on the genetic material level, effectively accelerate the differentiation and proliferation of the stem cells, and enable the stem cells to quickly reach the preset cell density for transfer subculture.
In this embodiment, the method for preparing the IPS cell extract in the anti-aging composition comprising stem cells specifically comprises:
(a) spreading 0.25 wt% gelatin in a container to cover the whole bottom, standing at 37 deg.C for 45min, adding 5 times of gelatin volume of cell complete culture medium pre-incubated at 37 deg.C, adding cell with concentration of 2 × 105cells/mL, 6 times gelatin volume of human embryo fibroblasts, cultured until the growth concentration of feeder layer cells reaches 92%;
(b) the IPS cell suspension obtained by the cultivation is inoculated in the container of step (a) in a cell amount of 1:10 and CO is added at 37 DEG C2Culturing in an incubator for 3 d;
(c) sucking out the cell culture solution in the step (b), washing with PBS for 2 times, adding cell lysate with the same volume of gelatin to dissolve cells, centrifuging to obtain supernatant, dialyzing with PBS solution at the temperature of 1 ℃ for 12 hours, and freeze-drying the solution in the dialysis bag to obtain the cell culture solution. The IPS cell extract is rich in high biological active substances such as collagen, active peptide, growth factor, vitamin, mineral substances, antioxidant active enzyme and the like, has small antigenicity to a human body, is absorbed and utilized by the human body, has excellent anti-aging function, is organically compatible with stem cells and immune cells, protects the proliferation of the human body cells and the secretion of the antioxidant active substances, and can exert efficient cell activating and anti-aging functions.
In the preparation method of the IPS cell extract, the complete cell culture medium in the step (a) comprises the following components: DMEM +15g/L fetal bovine serum +3mg/L sodium alginate +4g/L +10mg/L sulfur-containing amino acid +5mg/L sodium pyruvate +0.2mg/L beta-carotene +0.12mg/L Salvia miltiorrhiza root extract. The cell complete culture medium can promote the rapid differentiation and propagation of human embryonic fibroblasts, is beneficial to the synthesis and secretion of active substances by IPS cells, promotes the cell culture solution cultured for a short time to be rich in the active substances of the IPS cells, and greatly enhances the medical care effect of the composition.
In this example, the protective agent in the anti-aging composition containing stem cells is specifically a mixture of grape polyphenol and catalpol in a weight ratio of 1: 15.8. The applicant finds that the mixture of the grape polyphenol and the catalpol in a specific weight ratio is added to the anti-aging composition containing the stem cells, so that the biological function of the composition is maintained, the mutual synergistic effect of the grape polyphenol and the catalpol can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, the integrity, the reproducibility and the bioavailability of the stem cells and the immune cells are maintained, and the excellent anti-aging effect of the composition is exerted.
The extraction method of the grape polyphenol in the protective agent comprises the following steps: crushing grape skin and grape seed under the protection of nitrogen flow, adding petroleum ether for degreasing, crushing, vacuum-drying, adding 75% ethanol solution according to the solid-to-liquid ratio of 1:2, leaching at the constant temperature of 45 ℃ for 2h, taking clear liquid, evaporating to dryness at low temperature, and crushing to obtain the grape skin and grape seed. The grape polyphenol, namely grape procyanidin, is a natural plant polyphenol, has various biological activities such as oxidation resistance, free radical damage resistance, radiation resistance, cardiovascular protection and the like, can also play a synergistic role when being matched with catalpol, can retain the inherent biological activity of the grape polyphenol to a greater extent by extracting the grape polyphenol by the method, and has short extraction time and easy operation.
Example 2:
this example provides [1] an anti-aging composition comprising stem cells, comprising:
a) stem cells;
b) immune cells involved in the immune response;
c) IPS cell extract; and
d) and (4) a protective agent. In the method of the invention, the stem cells are combined with the immune cells and the IPS cell (induced pluripotent stem cell) extract, which not only can play a role in removing aged dead and cancerated cells by the immune cells and ensure that the microenvironment of an organism is in a safe and clean state, but also can play unique roles in protecting the proliferation, activating, secreting antioxidant active substances and resisting aging of human cells by the IPS cell extract, meanwhile, the input of the stem cells can play roles in regeneration and repair, the recovery of the organism is accelerated, the aging process is further delayed, the respective curative effects can be mutually enhanced by the combination of the three, in addition, the protective agent can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, is favorable for maintaining the integrity, the regeneration and the bioavailability of the stem cells and the immune cells and plays an excellent anti-aging role of the composition, meanwhile, the traditional Chinese medicine composition has a certain effect of reversing aging injury, can efficiently promote wound healing and bone growth, has higher economic value and application prospect in the fields of beauty treatment, medical treatment, health care and the like, maintains the balance of the neuroendocrine system of an organism through various cytokines and hormones secreted by immune cells and stem cells, and can more effectively resist the aging of the organism without treating the disease first on the basis of scientifically preventing the occurrence of the disease.
In this embodiment, the stem cells in the anti-aging composition comprising stem cells are umbilical cord, placental mesenchymal stem cells. Mesenchymal stem cells are pluripotent stem cells that have all of the commonalities of stem cells, namely self-renewal and pluripotency; at present, the application is most in clinical application, and the combined application with hematopoietic stem cells can improve the success rate of transplantation and accelerate hematopoietic reconstruction.
In this embodiment, the stem cell culture method comprises: and culturing the stem cells in a third culture medium, adding a second culture medium for subculture when the cultured cells reach 93% confluence, and collecting fourth generation stem cells.
In an embodiment, the immune cells involved in the immune response in the anti-aging composition comprising stem cells are specifically NK cells. NK cells (Natural killer cells) are important immune cells of the body, not only involved in the resistance to tumors, viral infections and immune regulation, but also in certain cases in the development of hypersensitivity reactions and autoimmune diseases.
In this embodiment, the NK cell culture method is: culturing NK cells in a first medium until the cultured cell density reaches 5 × 107After one/mL, a second culture medium is added for subculture, and fifth generation cells are collected.
In this embodiment, the first culture medium is composed of a clinical-grade serum-free culture medium and a first additive, and the components of the first additive and the concentrations thereof in the DMEM culture solution are 2.4mg/L of sodium alginate, 11mg/L of potassium selenite, 12mg/L of sulfur-containing amino acid, 17mg/L of mannitol, 4.1mg/L of an aqueous extract of tender willow branches and 6.8mg/L of an aqueous extract of dahurian angelica leaves. The first culture medium can provide sufficient mass energy for the culture and development of the NK cells, the healthy and rapid formation of a cell line is ensured, and the tender willow branch aqueous extract and the angelica dahurica leaf aqueous extract can effectively accelerate the proliferation of the NK cells so as to quickly reach the predetermined cell density for transfer subculture.
In this embodiment, the second culture medium is composed of DMEM culture solution, fetal bovine serum, and a second additive, wherein the total volume fraction of the fetal bovine serum in the DMEM culture solution is 5.5%, and the components of the second additive and the concentrations thereof in the DMEM culture solution are 8mg/L glutathione, 13mg/L ferric citrate, 5.5mg/L fibroblast growth factor, 4mg/L potassium selenite, 7mg/L human albumin, 8mg/L cinnamon water extract and 8mg/L fingered citron water extract.
In this embodiment, the third medium is composed of a clinical-grade serum-free medium and a third additive, and the respective components of the third additive and the concentrations thereof in the DMEM medium are 2mg/L diethanolamine, 10mg/L lauroyl serine, 2mg/L cysteine, 14mg/L sorbitol, 6mg/L proline, 3mg/L vitamin C, 8mg/L tender willow branch aqueous extract and 8mg/L kudzu root alcohol extract. The tender willow branch water extract and the kudzu root alcohol extract can activate related biological enzymes of stem cells on the genetic material level, effectively accelerate the differentiation and proliferation of the stem cells, and enable the stem cells to quickly reach the preset cell density for transfer subculture.
In this embodiment, the method for preparing the IPS cell extract in the anti-aging composition comprising stem cells specifically comprises:
(a) spreading 0.4 wt% gelatin in a container to cover the whole bottom, standing at 37 deg.C for 60min, adding 6 times of gelatin volume of cell complete culture medium pre-incubated at 37 deg.C, adding cell with concentration of 2.5 × 105cells/mL, human embryonic fibroblasts 8 times the gelatin volume, cultured until the growth concentration of feeder layer cells reaches 95%;
(b) the IPS cell suspension obtained by the cultivation is inoculated in the container of the step (a) with the cell amount of 1:15 and CO at 37 DEG C2Culturing for 7d in an incubator;
(c) sucking out the cell culture solution in the step (b), washing with PBS for 5 times, adding cell lysate with the same volume of gelatin to dissolve cells, centrifuging to obtain supernatant, dialyzing with PBS at 4 ℃ for 16h, and freeze-drying the solution in the dialysis bag. The IPS cell extract is rich in high biological active substances such as collagen, active peptide, growth factor, vitamin, mineral substances, antioxidant active enzyme and the like, has small antigenicity to a human body, is absorbed and utilized by the human body, has excellent anti-aging function, is organically compatible with stem cells and immune cells, protects the proliferation of the human body cells and the secretion of the antioxidant active substances, and can exert efficient cell activating and anti-aging functions.
In this example, the IPS cell extract was prepared by using the cell complete medium of step (a) as follows: DMEM +18g/L fetal bovine serum +4mg/L sodium alginate +10g/L +11mg/L sulfur-containing amino acid +5.5mg/L sodium pyruvate +0.4mg/L beta-carotene +0.15mg/L Salvia miltiorrhiza root extract. The cell complete culture medium can promote the rapid differentiation and propagation of human embryonic fibroblasts, is beneficial to the synthesis and secretion of active substances by IPS cells, promotes the cell culture solution cultured for a short time to be rich in the active substances of the IPS cells, and greatly enhances the medical care effect of the composition.
In this example, the protective agent in the anti-aging composition comprising stem cells is specifically a mixture of grape polyphenol and catalpol in a weight ratio of 1: 16. The applicant finds that the mixture of the grape polyphenol and the catalpol in a specific weight ratio is added to the anti-aging composition containing the stem cells, so that the biological function of the composition is maintained, the mutual synergistic effect of the grape polyphenol and the catalpol can avoid the damage of enzyme substances in the IPS extract to the stem cells and the immune cells, the integrity, the reproducibility and the bioavailability of the stem cells and the immune cells are maintained, and the excellent anti-aging effect of the composition is exerted.
In this embodiment, the method for extracting the glucan polyphenol in the protective agent comprises: crushing grape skin and grape seed under the protection of nitrogen flow, adding petroleum ether for degreasing, crushing, vacuum-drying, adding 75% ethanol solution according to the solid-to-liquid ratio of 1:4, leaching at the constant temperature of 48 ℃ for 4h, taking clear liquid, evaporating to dryness at low temperature, and crushing to obtain the grape skin and grape seed. The grape polyphenol, namely grape procyanidin, is a natural plant polyphenol, has various biological activities such as oxidation resistance, free radical damage resistance, radiation resistance, cardiovascular protection and the like, can also play a synergistic role when being matched with catalpol, can retain the inherent biological activity of the grape polyphenol to a greater extent by extracting the grape polyphenol by the method, and has short extraction time and easy operation.
In this embodiment, the components of the anti-aging composition comprising stem cells account for the following weight percentages of the composition by weight:
a) 56% by weight dry cells;
b) 24% by weight of immune cells involved in the immune response;
c)15 wt% IPS cell extract; and
d) 5% by weight of a protective agent.
In this embodiment, the anti-aging composition comprising stem cells can be formulated for oral administration.
In this embodiment, the forms of the anti-aging composition comprising stem cells suitable for oral administration include the form of tablets and/or capsules prepared by conventional methods with pharmaceutically acceptable excipients including, but not limited to:
1) a binder, preferably pregelatinized corn starch;
2) a bulking agent, preferably corn starch;
3) a lubricant, preferably magnesium stearate;
4) a disintegrant, preferably sodium starch glycolate;
5) wetting agents, preferably sodium lauryl sulfate.
In this embodiment, the form of the anti-aging composition comprising stem cells suitable for oral administration includes the form of tablets and/or capsules that may be coated by methods well known in the art.
In this embodiment, the forms of the anti-aging composition comprising stem cells suitable for oral administration include liquid formulations which may take the form of solutions, syrups or suspensions, or they may be presented as an anhydrous product for reconstitution with water or other suitable vehicle prior to use. The liquid formulation may be prepared by conventional methods with pharmaceutically acceptable additives including, but not limited to:
1) a suspending agent, preferably sorbitol syrup;
2) an emulsifier, preferably gum arabic;
3) a non-aqueous vehicle, preferably glycerol;
4) the preservative is preferably methyl hydroxybenzoate.
In this embodiment, the anti-aging composition comprising stem cells may be formulated for oral administration and may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents as needed.
The present embodiment also provides the item [2 ]: the method for preparing the anti-aging composition comprising stem cells according to item [1], which comprises: aseptically and sequentially adding the protective agent, immune cells participating in immune response and IPS cell extract into stem cells at 1 ℃, uniformly mixing and freezing to obtain the product.
The present embodiment also provides the item [3 ]: use of the stem cell-containing antiaging composition described in the items [1] and [2], particularly use of the composition for the production of a antiaging preparation.
Comparative example V3:
comparative example V3 is essentially the same as example 2 except that in comparative example V3, the composition contains no stem cells and consists of: b) 80% by weight of immune cells involved in the immune response; c)15 wt% IPS cell extract; and d) 5% by weight of a protective agent.
Comparative example V4:
comparative example V4 is essentially the same as example 2 except that in comparative example V4, the composition does not contain immune cells involved in the immune response and consists of: a) 56% by weight dry cells; c)39 wt% IPS cell extract; and d) 5% by weight of a protective agent.
Comparative example V5:
comparative example V5 is essentially the same as example 2, except that in comparative example V5, the composition does not contain an IPS cell extract and comprises the following components: a)71 wt% dry cells; b) 24% by weight of immune cells involved in the immune response; and d) 5% by weight of a protective agent.
Comparative example V6:
comparative example V6 is essentially the same as example 2 except that in comparative example V6, the composition does not contain a protectant and has the following composition: a)61 wt% dry cells; b) 24% by weight of immune cells involved in the immune response; c)15 wt% IPS cell extract.
Comparative example V7:
comparative example V7 is essentially the same as example 2 except that in comparative example V7 the protective agent of the composition does not contain catalpol.
Comparative example V8:
comparative example V8 is essentially the same as example 2 except that in comparative example V8 the composition does not contain any glucan polyphenol in the protectant.
Comparative example V9:
comparative example V9 is substantially the same as example 2 except that in comparative example V9, the first medium was supplemented with an aqueous extract of tender willow branches and an aqueous extract of dahurian angelica leaves.
Comparative example V10:
comparative example V10 is substantially the same as example 2 except that in comparative example V10, the second supplement of the second medium is not supplemented with aqueous extracts of cinnamon and fingered citron
Comparative example V11:
comparative example V11 is substantially the same as example 2 except that in comparative example V11, the third supplement of the third medium is not supplemented with an aqueous extract of tender willow branches and an alcohol extract of kudzu root.
Comparative example V12:
comparative example V12 is essentially the same as example 2 except that in comparative example V12, the IPS cell extract is prepared without the addition of beta-carotene and a Salvia miltiorrhiza root extract to the cell-complete medium.
Test example:
a, establishing a natural aging animal model:
balbc mice, 260, female, 10 months of age, weighing 25 ± 5 g. Carrying out adaptive feeding for 7d before the experiment begins, and carrying out the experiment after the quarantine observation is qualified.
All mice are fed and managed according to the standard, and other measures are not taken to establish an animal model of the natural aging of the mice.
Animal management criteria are as follows:
animal house: the SPF level is managed strictly according to national standards;
animal feed: sterilizing by high-pressure irradiation;
animal drinking: double distilled purified water (independent water purification system);
animal bedding: sterilizing the sawdust at high temperature and high pressure;
the animal room is entered and the special experimental clothes are replaced, and all the articles entered into the animal room are sterilized at high temperature and high pressure (except special instruments and medicines).
Grouping and administration of animals:
balbc mice were randomly grouped into 20 mice each. Each group of mice was injected with the compositions of examples 1-2 and comparative examples V3-V12 or medical saline, respectively, in tail vein at regular intervals every month.
The composition for injection is adjusted to 2 × 10 cell density with physiological saline6Per mL, 0.2mL of cell sap was injected per mouse into the tail vein.
Saline was used as a negative control, and each mouse was injected with 0.2 mL.
TABLE 1 animal grouping and dosing Table
The survival state of the mice is continuously observed and recorded, the survival condition of the mice is checked every day, and the survival number of the mice in each group is counted at the bottom of every month.
TABLE 2 survival of groups of mice at 23 months
| Example one another | Quantity (only) | Survival number at 23 months (only) | Survival rate (%) |
| 1 | 20 | 18 | 90 |
| 2 | 20 | 19 | 95 |
| V3 | 20 | 10 | 50 |
| V4 | 20 | 11 | 55 |
| V5 | 20 | 8 | 40 |
| V6 | 20 | 14 | 70 |
| V7 | 20 | 15 | 75 |
| V8 | 20 | 14 | 70 |
| V9 | 20 | 15 | 75 |
| V10 | 20 | 16 | 80 |
| V11 | 20 | 14 | 70 |
| V12 | 20 | 15 | 75 |
| Physiological saline control group | 20 | 1 | 5 |
As can be seen from table 2, the effective components of the anti-aging composition containing mesenchymal stem cells of the present invention are effective when used alone (mouse survival rate is 40-55%), the effect of the components when used together (mouse survival rate is 90-95%) is significant, and in addition, from the survival state of the mouse, after 13 months of age, each group of mice gradually shows different aging signs, such as emaciation, hunched back, shivering, reaction retardation, periocular lesions, and the like, and the aging phenomenon after 18 months of age is very significant, and from the overall view, the anti-aging composition containing mesenchymal stem cells of the present invention preferably shows the best mental state for the mice of examples 1 and 2.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (7)
1. An anti-aging composition comprising stem cells, characterized by: the components of the anti-aging composition containing stem cells occupy the composition by weight percent:
a) 35-98 wt% of dry cells;
b) 0.1-35% by weight of immune cells involved in the immune response;
c) 0.1-30 wt% IPS cell extract; and
d)1.8 to 5 wt% of a protective agent;
wherein the protective agent is a mixture of grape polyphenol and catalpol in a weight ratio of 1: 15.8-16.0;
the immune cells participating in immune response specifically refer to NK cells, and the culture method comprises the following steps: culturing NK cells in a first medium at a cell density of more than 4X 107After the cells are cultured per mL, adding a second culture medium for subculture, and collecting fifth generation cells;
the first culture medium consists of a clinical-grade serum-free culture medium and a first additive, wherein the concentrations of the components of the first additive in a DMEM culture solution are 2.2-2.5 mg/L sodium alginate, 10-11 mg/L potassium selenite, 11-14 mg/L sulfur-containing amino acid, 15-18 mg/L mannitol, 4-4.2 mg/L tender willow branch aqueous extract and 6-7 mg/L angelica dahurica leaf aqueous extract;
the second culture medium consists of a DMEM culture solution, fetal calf serum and a second additive, the total volume fraction of the fetal calf serum in the DMEM culture solution is 5-6%, and the components of the second additive and the concentration of the components in the DMEM culture solution are 6-10 mg/L glutathione, 12-14 mg/L ferric citrate, 5-6 mg/L fibroblast growth factor, 2-5 mg/L potassium selenite, 6-8 mg/L human albumin, 5-10 mg/L cinnamon water extract and 5-10 mg/L fingered citron water extract.
2. The composition of claim 1, wherein:
the stem cell is one or a composition of at least two of adult stem cells, hematopoietic stem cells, neural stem cells, peripheral blood stem cells, adipose-derived stem cells, mesenchymal stem cells and amniotic membrane stem cells.
3. The composition of claim 2, wherein:
the culture method of the stem cells comprises the following steps: and culturing the stem cells in a third culture medium, adding a second culture medium for subculture when the cultured cells are more than 92% confluent, and collecting fourth generation stem cells.
4. The composition of claim 3, wherein: the third culture medium consists of a clinical-grade serum-free culture medium and a third additive, wherein the concentrations of the components of the third additive in a DMEM culture solution are 1-3 mg/L diethanolamine, 5-10 mg/L lauroyl serine, 1-3 mg/L cysteine, 10-15 mg/L sorbitol, 5-10 mg/L proline, 1-3 mg/L vitamin C, 5-10 mg/L tender willow branch water extract and 5-10 mg/L kudzu root alcohol extract.
5. The composition of claim 1, wherein: the preparation method of the IPS cell extract comprises the following steps:
(a) spreading gelatin in a container, covering the bottom, standing at 37 deg.C, adding cell complete culture medium pre-incubated at 37 deg.C, adding human embryo fibroblast, and culturing until the growth concentration of feeder layer cell exceeds 92%;
(b) inoculating the cultured IPS cell suspension into the container in the step (a) at the cell amount of 1: 10-25, and performing CO treatment at 37 DEG C2Culturing for 3-10 days in an incubator;
(c) sucking out the cell culture solution in the step (b), washing with PBS, adding cell lysate to dissolve cells, centrifuging to obtain supernatant, dialyzing with PBS at the temperature of 1-4 ℃, and freeze-drying the solution in a dialysis bag to obtain the cell culture solution.
6. A method for preparing an anti-aging composition comprising stem cells according to any one of claims 1 to 5, which is characterized by comprising: and (3) under sterile conditions and at the temperature of 1-2 ℃, sequentially adding the protective agent, the immune cells participating in immune response and the IPS cell extract into the stem cells, uniformly mixing, and freezing to obtain the composition.
7. Use of the anti-aging composition comprising stem cells according to any one of claims 1 to 6, in particular to use of the composition in the preparation of an anti-aging formulation.
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