CN110475534A

CN110475534A - It is a kind of based on the regenerated cosmetic formulation for delaying body aging of pluripotent stem cell differentiation

Info

Publication number: CN110475534A
Application number: CN201880004904.8A
Authority: CN
Inventors: 高扬; 杨芷
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-02-23
Filing date: 2018-02-23
Publication date: 2019-11-19
Also published as: WO2019161540A1

Abstract

It is a kind of based on the regenerated cosmetic formulation for delaying body aging of pluripotent stem cell differentiation, autologous fat derived stem cells 5 × 10 are contained in every milliliter⁵~10 × 10⁵It is a；Fibronectin FN5 microgram~10 micrograms；Electrolyte solution and cytokine profiles and auxiliary reagent, while also disclosing the preparation method of cosmetic formulation.The cosmetic formulation includes the effective components such as self versatile stem cell and cytokine profiles, the fast breeding regeneration of cell is able to carry out after injection or implanting to human body, by stem cell body respective organization directed differentiation, body can be damaged or the cell tissue of aging is replaced and repairs, keep activity and continuous release cell factor, great injury repair and therapeutic effect are played, so that rejuvenation state is presented in body such as skin.

Description

It is a kind of based on the regenerated cosmetic formulation for delaying body aging of pluripotent stem cell differentiation

A skin caring preparation for delaying aging based on differentiation and regeneration of pluripotent stem cells

Technical Field

[0001] The invention relates to the technical field of biological medicines, in particular to a cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells.

Background

[0002] The stem cell is a seed cell which is not differentiated and developed, has unlimited self-renewal and replication capacity, and can be differentiated into specific tissues, which is the basis of the stem cell for skin care and beauty and even tissue regeneration. The stem cells are divided into embryonic stem cells and adult stem cells, the embryonic stem cells existing in blastula can self-renew and replicate and differentiate into 220 cells with different functional types, and the cells with different types form a human body; adult stem cells exist in many tissues and organs of adults and differentiate into only specific types of cells that are, or are associated with, themselves. They not only serve as self-repair lines in humans, but are also responsible for maintaining the regeneration of certain human organs/tissues, such as blood, skin and small intestine tissues.

[0003] Scientific research shows that two kinds of adult stem cells are confirmed to exist in human skin, one is epidermal stem cell which exists in the basal layer of epidermis and has the main functions of supplementing epidermal cells, maintaining the balance of skin cells and repairing damaged tissues; the other is the dermal stem cells existing in the dermal papilla dermis, has strong self-renewal capacity, can induce the formation of hair follicles and migrate into the dermal layer of the cross hair follicles, proliferate and differentiate into fibroblasts (the fibroblasts can secrete components of extracellular matrix), and has the main functions of filling the skin with elasticity and reducing wrinkles. However, with age, skin turnover becomes slow and the activity and number of stem cells gradually decrease; ultraviolet light produces toxins and reactive oxygen species on the skin, which all damage the most sensitive stem cells; at the same time, low temperature, low humidity and sudden changes in the environment impair the barrier function of the skin. The above-mentioned intrinsic or extrinsic environmental factors affect the function of stem cells.

[0004] The stem cell growth factor is a multifunctional strong cytokine, is combined with a specific receptor of a target cell in a human body microenvironment, plays biological functions of stimulating differentiation and proliferation of the target cell, promoting synthesis and secretion of the target cell, chemotactic induction of inflammatory cells and the like, and starts a metabolic process in the cell and substance and information transmission between the cells. Many cytokine receptors have kinase activity, particularly tyrosine kinase activity (e.g. PDGF receptor, EGF receptor). The combination of the cell factors and the corresponding receptors activates the receptors, promotes the formation of subcutaneous blood vessels, repairs the subcutaneous blood microcirculation system, improves the microenvironment for cell growth, induces the proliferation and differentiation of various cells, accelerates the process of skin new cells replacing aging cells, and achieves the effect of delaying aging.

[0005] The stem cells produce and secrete a large amount of bioactive factors, and the bioactive factors of the stem cells can effectively regulate and control the cell signal conduction of organisms and activate the stem cells of human bodies, so that the damaged, diseased and aged cells of the organisms are physiologically repaired or replaced. For example, stem cells can produce stem cell growth factor (SCF), Nerve Growth Factor (NGF)

Stromal cell derived growth factor (SDF), Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), Epidermal Growth Factor (EGF), interleukin-6 and interleukin-7 (IL-6 and IL-7), megakaryocyte colony stimulating factor (M-CSF), Tumor Necrosis Factor (TNF), Interferon (IFN) and other factors, and these cytokines have the functions of promoting cell proliferation, differentiation, resisting apoptosis and the like; stem cells can also produce natural immune proteins such as IgG, IgA, IgM, IgD, IgE, etc., which can counteract or repair the damage caused to the cells of the body by themselves and external factors.

[0006] The bioactive factor generated by the secretion of the stem cell is utilized to activate the stem cell of the organism, and then the cells of the organism with injury, pathological changes and aging are physiologically repaired or replaced by the stem cell, so that the biological activating agent has wide application prospect in the fields of disease prevention and treatment, health care and beauty.

[0007] The skin, which is the largest organ constituting the human body organ, occupies about 16% of the body volume, has an area of 1.8m2, a thickness of 2 ~ 4 mm, and a weight of about 3kg, and is composed of epidermis, dermis, and subcutaneous fat layer, and is in direct contact with the external environment, plays a role of protecting the human body from external stimuli such as various harmful factors including harmful microorganisms and ultraviolet rays, and has an important role of protecting the skin, and is an organ essential for maintaining biochemical functions required for the whole body metabolism.

[0008] Skin aging is a very complex process. The method is mainly characterized in that the telomere of the chromosome is shortened, the DNA methylation level is reduced, the expression level of various cytokines is changed, the number and the functions of growth factors such as EGF and the like and receptors thereof are changed, one important reason is subcutaneous vascular atrophy, so that the skin blood supply is insufficient, the physiological effect is generated, the cells lack nutrition, the external reactivity is gradually reduced, the proliferation and differentiation capacity of the skin cells is weakened, the metabolism is slowed down, and the skin has aging symptoms such as wrinkles, color spots and the like.

[0009] With the expansion of beauty and plastic market, more and more people choose to apply methods such as laser spot removal, photon skin tendering and the like to carry out facial beauty. Laser skin cosmetology is a treatment of the skin by photothermal action, which achieves the goal of cosmetology by generating high energy, accurately focusing, locally generating high energy in the skin tissue of the human, and removing and destroying the tissue. However, the number of people with postoperative complications caused by improper treatment selection and improper operation has increased year by year. The adverse effects of excessive tissue damage, severe inflammatory reaction, capillary irritation, etc. can be caused by improper operation. Major side effects include edema, blisters, pigmentation, hypopigmentation, telangiectasia, increased skin sensitivity, and the like.

[0010] Therefore, in the current beauty industry, the traditional chemical beauty and physical beauty cannot meet the requirements, natural plant extract components are prosperous in beauty treatment, and the stem cell beauty treatment is more and more concerned with remarkable efficacy and lasting effect. The stem cells act on beauty treatment, the traditional constraints of chemical beauty treatment and physical beauty treatment are eliminated, brand new experience is brought to people, and beauty and youth are realized from the interior of a human body.

[0011] Regenerative medicine has been a field of much attention and research and exploration as a medical cosmetic which is mainly plastic surgery for repair and reconstruction and medical cosmetic which is mainly young, and adipose-derived mesenchymal stem cells have solved many medical cosmetic problems.

[0012] Firstly, the convenience of obtaining the source materials is not only the greatest advantage of the fat-derived ADSCs, but also the advantage of plastic surgery. On one hand, the adipose tissues are widely distributed in the body and the reserves are rich; liposuction, on the other hand, is a well-established routine procedure for orthopedic surgery with little surgical risk and is readily available as a routine "waste byproduct". For patients, the fat-sucking sculptures can enjoy the rejuvenation and miraculous effects of stem cells at the same time, so that the fat-sucking sculptures can achieve a win-win life remodeling, the pain and fear are less than those of bone marrow extraction, and the risk of blood source pollution and immunological rejection is avoided, thereby forming the advantage of clinical application.

[0013] It was reported abroad that the death rate was 0 and the medical accident rate was only 0.68% in 66570 liposuction procedures performed in the early stage of liposuction performed in 1994 ~ 2000, it was found that liposuction is safe and reliable, and that the yields of MSCs in bone marrow were relatively low, accounting for only 0.0001% of ~ 0.01.01% of nucleated cells in adult bone marrow tissue, and adipose tissue, as the main cell type, was easily removed by buoyancy after collagenase digestion, and the clone formation rate of the obtained cells was 100 ~ 500 times that of mesenchymal stem cells, and at the same time, the desired stem cell concentration was obtained at one time without in vitro expansion due to the fact that the liposuction amount was multiplied as required, thereby greatly reducing the risk of passage variation and various contaminations.

[0014] From the clinical point of view, a large amount of ADSCs can be easily obtained in a short time, and the method has important significance for the rapid treatment of clinical diseases. Under local anesthesia, adult bone marrow is usually obtained in amounts not exceeding 40ml, yielding about 1.2x109 nucleated cells containing about 2.4x104 MSCs. Adipose tissue, on the other hand, can obtain about 2x108 nucleated cell phases per 100m, where the stem cell content is 40 times that in 40ml bone marrow. Therefore, the ADSCs have the advantage of higher clinical application potential than the MSCs under the same conditions.

[0015] Application status of ADSCs in the field of plastic and beauty treatment

[0016] Application of skin scarless and reparative

[0017] The application of the skin scarless in ADSCs is not yet available and is always the research focus in the field of plastic repair and reconstruction. Complete regeneration of damaged tissue cells and complete replacement of damaged tissue cells are the core content of regenerative medicine research in the medical field. ADSCs not only quickly replace common seed cells in tissue engineering, but also show an important role in entering scar-free research.

[0018]2, ADSCs and rejuvenation

[0019] Thirdly, local and systemic application of stem cells, many cases show surprising effects. The rat subacute aging and common aging model is adopted, and after the ADSCs are transplanted in a variant, the influence of the ADSCs on the free radicals and the immune function in the rat body of the aging model is observed. Compared with a blank control group, the results show that the serum SOD, NO and IL-2 levels and spleen indexes of rats in the model group are obviously reduced, and the serum MDA level is obviously increased. After the ADSCs are transplanted, the serum SOD, NO and IL-2 levels and spleen indexes of the rats in the treatment group are improved compared with those of the rats in the model group, and the MDA content is obviously reduced. The allografting ADSCs can effectively enhance the free radical scavenging and oxidation resistance of a rat body and improve the immune function of the body, thereby delaying the aging of the rat induced by D-gal.

[0020] Therefore, the selection of the adipose-derived stem cells as the source of the multifunctional stem cells for medical cosmetology has obvious advantages, and the invention also has the advantages of the application of the adipose-derived stem cells.

Summary of The Invention

Technical problem

Solution to the problem

Technical solution

[0021] The invention aims to provide a cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells

Can improve the stability of cell factor, can be applied to medical cosmetology, and realizes the regeneration and repair of organism

[0022] In order to achieve the above objects, one embodiment of the present invention provides a cosmetic preparation for delaying aging of the body based on differentiation and regeneration of pluripotent stem cells, the cosmetic preparation comprising per ml:

[0023] pluripotent stem cells: 5x 10510 x105 autologous adipose-derived stem cells; [0024] fibronectin FN5 μ g-10 μ g;

[0025] an electrolyte solution;

[0026] cytokines:

[0027] vascular endothelial growth factor VEGF5 μ g-10 μ g;

[0028] insulin-like growth factor IGF5 μ g-10 μ g;

[0029] hepatocyte growth factor HGF5 microgram ~ 10 microgram;

[0030] -50 μ g of epidermal growth factor EGF20 μ g;

[0031] acid fibroblast growth factor aFGF5 microgram ~ 10 microgram;

[0032] auxiliary reagents:

[0033] 20 mg-50 mg hyaluronic acid;

[0034] arachidonic acid 20 mg-50 mg.

[0035] In one of the optimization schemes of the invention, the electrolyte solution is normal saline.

[0036] In one of the optimization schemes of the invention, the cosmetic preparation contains per milliliter: [0037] pluripotent stem cells: 6x105 autologous adipose-derived stem cells;

[0038] fibronectin FN10 microgram;

[0039] an electrolyte solution;

[0040] cytokines:

[0041] vascular endothelial growth factor VEGF8 micrograms;

[0042] insulin-like growth factor IGF5 microgram;

[0043] hepatocyte growth factor HGF9 microgram;

[0044] 30 micrograms of epidermal growth factor EGF;

[0045] 6 micrograms of acidic fibroblast growth factor (aFGF);

[0046] auxiliary reagents:

[0047] 50 mg of hyaluronic acid;

[0048] arachidonic acid 30 mg.

[0049] In one of the optimization schemes of the invention, the cosmetic preparation contains per milliliter:

[0050] pluripotent stem cells, namely 5xl05 ~ 10xl05 autologous adipose-derived stem cells;

[0051] fibronectin FN5 μ g-10 μ g; [0052] an electrolyte solution;

[0053] cytokines:

[0054] vascular endothelial growth factor VEGF5 μ g-10 μ g;

[0055] insulin-like growth factor IGF5 μ g-10 μ g;

[0056] hepatocyte growth factor HGF5 microgram ~ 10 microgram;

[0057] -50 μ g of epidermal growth factor EGF20 μ g;

[0058] acid fibroblast growth factor aFGF5 microgram ~ 10 microgram;

[0059] auxiliary reagents:

[0060] 20 mg-50 mg hyaluronic acid;

[0061] arachidonic acid 20 mg-50 mg;

[0062] 20 mg-50 mg spirulina polysaccharide.

[0063] The invention also discloses a method for preparing the cosmetic preparation, which comprises the following steps:

[0064] (1) separating autologous fat, and culturing autologous fat-derived stem cells to obtain autologous fat-derived pluripotent stem cells;

[0065] (2) culturing the mesenchymal stem cells to obtain cell factors;

[0066] (3) adding autologous adipose-derived pluripotent stem cells, cytokines and auxiliary reagents into an electrolyte solution under an aseptic condition; mixing and preserving.

[0067] As a supplement to the above preparation method, the specific process for obtaining autologous adipose-derived pluripotent stem cells in the preferred step (1) of the present invention is:

[0068] a1, extruding autologous adipose tissues, adding PBS solution for cleaning, adding the PBS solution, centrifuging for 5min at the rotating speed of 2000rpm, reserving an upper fat layer and a lower precipitation layer, and absorbing and discarding a middle layer;

[0069] b1, adding digestive juice into the upper fat layer and the lower precipitation layer, placing the mixture in a constant temperature shaking table at 37 ℃, shaking and digesting the mixture for 20min at 190r/m in, and collecting the liquid at the lowest layer to obtain autologous primary pluripotent stem cells;

[0070] c1, transferring the primary pluripotent stem cells into a centrifugal tube containing complete culture medium to terminate digestion, then closing the centrifugal tube, centrifuging lOmin at 1500rpm, sucking the supernatant by a suction tube, removing the supernatant, and supplementing the culture medium to prepare cell suspension for later use;

[0071] DU cultures primary pluripotent stem cells in cell suspension under the conventional conditions, the cell adherence condition is observed, the first liquid change is carried out after 12h ~ 18h, the culture liquid is changed every 3 days, the passage is carried out after the cells grow to be fused, and the autologous adipose-derived pluripotent stem cells which can be used for cosmetic preparations are obtained after the passage culture.

[0072] In addition to the above preparation method, in the present invention, preferably, the method of culturing mesenchymal stem cells to obtain cytokines in step (2) comprises:

[0073] a2, culturing the mesenchymal stem cells under conventional conditions to make them grow adherent, and changing the culture solution after culturing for 12h ~ 18h

Washing with PBS solution;

[0074] b2, adding the washed mesenchymal stem cells into a serum-free culture medium to continue culturing and inducing for 20h ~ 28h

The serum-free culture medium contains 0.2umol/L ~ 0.5.5 umol/L of 1TF anthocyanin and lO.lumol/L O.Su mol/L of wild enzyme, culture solution is collected after the culture is finished, the culture solution is centrifuged and filtered, small molecules with the molecular weight less than 5KD are removed, and concentrated solution containing cell factors is obtained after the concentration.

[0075] In addition to the above-mentioned production method, in the present invention, the conventional conditions in the step D1 are preferably cultivation in an incubator at a cultivation temperature of 37 ℃ and a carbon dioxide concentration of 5%.

[0076] In addition to the above-mentioned production method, in the present invention, the conventional conditions in the step A2 are preferably cultivation in an incubator at a cultivation temperature of 37 ℃ and a carbon dioxide concentration of 5%.

[0077] In addition to the above-mentioned production method, in the present invention, preferably, the serum-free medium in step B2 is DME M medium.

[0078] In addition to the above preparation method, the preferred subculture step in step D1 of the present invention is:

[0079] 1) absorbing the old culture medium in the culture bottle in a clean bench, adding PBS for washing for 2-3 times, and then adding l-2m

L digestive juices (0.25% pancreatin and 0.04% EDTA (v/v 1: 1));

[0080] 2) digesting in the incubator (3-5 min), observing morphology change of adherent cells under an inverted microscope, and adding an equal volume of culture medium to terminate digestion when cytoplasm of the adherent adipose-derived stem cells retracts, intercellular spaces continuously increase, and cells are in a nearly spherical shape and a small number of round cells are detached from the wall;

[0081] 3) repeatedly and orderly gently blowing and beating the cells on the bottle wall by using a straw to ensure that the cells are separated from the bottle wall and then form single cell suspension;

[0082] 4) transferring the single cell suspension into a centrifuge tube, centrifuging, (lOOOrpm, 5 min), removing the supernatant, adding a complete culture medium, slightly blowing to loosen the medium, and carrying out subculture according to the ratio of 1: 2.

Advantageous effects of the invention

Advantageous effects

[0083] In summary, the invention has the following advantages: [0084] the beauty preparation comprises effective components such as autologous multifunctional stem cells and various cell factors, can quickly proliferate and regenerate the cells after being injected or implanted into an organism, can replace and repair damaged or aged cell tissues of the organism through the directional differentiation of the stem cells in corresponding tissues of the organism, keeps the activity and continuously secretes the cell factors, and plays a great role in repairing and treating injuries so as to ensure that the organism such as skin is in a youthful state.

[0085] When the preparation is applied to skin repair or beauty, various rich cytokines act on the skin, the traditional constraints of chemical beauty and physical beauty are eliminated, and the problems of skin aging and the like are fundamentally solved by stimulating the self repair function. The method does not add any antiseptic and other substances harmful to organisms. The long-term operation recovery period brought by the operation wrinkle removal is avoided, and the normal life and work are not influenced.

Brief description of the drawings

Drawings

[0086] FIG. 1 shows the result of measuring the EGF content of the epidermal growth factor in one embodiment of the present invention; wherein the abscissa is time, and the ordinate is content percentage;

[0087] FIG. 2 shows the result of detecting the content of HGF in one embodiment of the present invention; wherein the abscissa is time and the ordinate is percentage of content.

Examples of the invention

Modes for carrying out the invention

[0088] The invention provides a cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells, which comprises the following components in each milliliter:

[0089] pluripotent stem cells, namely 5xl05 ~ 10xl05 autologous adipose-derived stem cells;

[0090] fibronectin FN5 microgram ~ 10 microgram, electrolyte solution;

[0091] cytokines:

[0092] vascular endothelial growth factor VEGF5 microgram ~ 10 microgram;

[0093] insulin-like growth factor IGF5 μ g-10 μ g;

[0094] hepatocyte growth factor HGF5 microgram ~ 10 microgram;

[0095] epidermal growth factor EGF20 microgram ~ 50 microgram;

[0096] acid fibroblast growth factor aFGF5 microgram ~ 10 microgram;

[0097] auxiliary reagents: 20 mg-50 mg hyaluronic acid; arachidonic acid 20 mg-50 mg. [0098] Example 1

[0099] The cosmetic preparation contains per ml:

[0100] 6x105 autologous adipose-derived stem cells;

[0101] fibronectin FN10 microgram;

[0102] physiological saline;

[0103] vascular endothelial growth factor VEGF8 micrograms;

[0104] insulin-like growth factor IGF5 microgram;

[0105] hepatocyte growth factor HGF9 microgram;

[0106] 30 micrograms of epidermal growth factor EGF;

[0107] 6 micrograms of acidic fibroblast growth factor (aFGF);

[0108] 50 mg of hyaluronic acid;

[0109] arachidonic acid 30 mg.

[0110] Example 2

[0111] The cosmetic preparation contains per ml:

[0112] 6x105 autologous adipose-derived stem cells;

[0113] fibronectin FN10 microgram;

[0114] physiological saline;

[0115] vascular endothelial growth factor VEGF8 micrograms;

[0116] insulin-like growth factor IGF5 microgram;

[0117] hepatocyte growth factor HGF9 microgram;

[0118] 30 micrograms of epidermal growth factor EGF;

[0119] 6 micrograms of acidic fibroblast growth factor (aFGF);

[0120] 50 mg of hyaluronic acid;

[0121] spirulina polysaccharide 30 mg.

[0122] Comparative example 1

[0123] The cosmetic preparation contains per ml:

[0124] 6x105 autologous adipose-derived stem cells;

[0125] fibronectin FN10 microgram; [0126] physiological saline;

[0127] vascular endothelial growth factor VEGF8 micrograms;

[0128] insulin-like growth factor IGF5 microgram;

[0129] hepatocyte growth factor HGF9 microgram;

[0130] 30 micrograms of epidermal growth factor EGF;

[0131] 6 micrograms of acidic fibroblast growth factor (aFGF);

[0132] hyaluronic acid 50 mg.

[0133] Stability test

[0134] The experimental method comprises the following steps:

[0135] adding equal volume of the cosmetic preparations of example 1, example 2 and control 1 into three test tubes, respectively, adding water for injection to 100ml, storing at 37 deg.C, detecting the contents of epidermal growth factor EGF and hepatocyte growth factor HGF once a month with corresponding kit, and the detection results are shown in FIG. 1 and FIG. 2

[0136] As can be seen from FIG. 1, the content of EGF in comparative example 1 is gradually reduced, and the reduction amplitude is obviously different from that of example 1 and example 2, the reduction amplitude of example 1 is larger than that of example 2, and the difference between the two is maintained between 10% ~ 20%.

[0137] As can be seen from FIG. 2, the hepatocyte growth factor HGF content in the control example 1 is gradually reduced, and the reduction amplitude is not significantly different from those of the example 1 and the example 2. Therefore, the stability of the three components is equivalent. From this, it can be seen that both arachidonic acid and spirulina polysaccharides can improve the stability of EGF, and the stability of HGF cannot be improved significantly.

[0138] Clinical test 1

[0139] 200 women aged 40 to 50 years old were selected, all without plastic or other medical surgery on their eyes. The cosmetic preparation of example 1 and example 2 is prepared into injections, 100 injections are respectively injected by a micro-needle injection mode, and the injections are injected for the second time at an interval of 1 week after the first injection; the condition of all cases was observed after 2 months after the injection was completed.

[0140] The experiment is inconvenient to set the contrast due to individual difference, but a photographing contrast mode, namely photographing contrast on the eyes of the female before and after injection, can be used. By comparison, it was found that the elasticity of the eyes of 184 women was improved, wrinkles such as crow's feet were reduced, and the peripheral skin was smooth and fine in all of examples 1 and 2, and 16 cases were not significantly changed. Therefore, the effective rate of the injection of the invention reaches 92 percent, and the injection has very high effective rate.

[0141] The invention also discloses a method for preparing the cosmetic preparation, which comprises the following steps:

[0142] (1) separating autologous fat, and culturing autologous fat-derived stem cells to obtain autologous fat-derived pluripotent stem cells;

[0143] (2) culturing the mesenchymal stem cells to obtain cell factors; when the content of a certain cytokine is detected to be lower than the required proportion of the invention, an externally purchased cytokine finished product can be added or the cytokine can be added after being independently cultured.

[0144] (3) adding autologous adipose-derived pluripotent stem cells, cytokines and auxiliary reagents into an electrolyte solution under an aseptic condition; mixing and preserving.

[0145] Example 3

[0146] Preparing autologous fat-derived pluripotent stem cells:

[0147] a1, extruding autologous adipose tissues, adding PBS solution for cleaning, adding the PBS solution, centrifuging for 5min at the rotating speed of 2000rpm, reserving an upper fat layer and a lower precipitation layer, and absorbing and discarding a middle layer;

[0148] b1, adding digestive juice into the upper fat layer and the lower precipitation layer, placing the mixture in a constant temperature shaking table at 37 ℃, shaking and digesting the mixture for 20min at 190r/m in, and collecting the liquid at the lowest layer to obtain autologous primary pluripotent stem cells;

[0149] c1, transferring the primary pluripotent stem cells into a centrifugal tube containing complete culture medium to terminate digestion, then closing the centrifugal tube, centrifuging lOmin at 1500rpm, sucking the supernatant by a suction tube, removing the supernatant, and supplementing the culture medium to prepare cell suspension for later use;

[0150] DU is cultured in an incubator with the culture temperature of 37 ℃ and the carbon dioxide concentration of 5% under the conventional conditions, the first liquid change is carried out after 12h ~ 18h, the culture liquid is changed every 3 days, the generation is carried out after the cells grow to be fused, and the autologous adipose-derived pluripotent stem cells which can be used for a cosmetic preparation are obtained after subculture.

[0151] Example 4

[0152] The subculture steps are as follows:

[0153]1) removing the old culture medium in the culture bottle in a clean bench, adding PBS for washing for 2-3 times, and then adding l-2mL of digestive juice (0.25% pancreatin and 0.04% EDTA (v/v 1: 1));

[0154] 2) digesting in the incubator (3-5 min), observing morphology change of adherent cells under an inverted microscope, and adding an equal volume of culture medium to terminate digestion when cytoplasm of the adherent adipose-derived stem cells retracts, intercellular spaces continuously increase, and cells are in a nearly spherical shape and a small number of round cells are detached from the wall;

[0155] 3) repeatedly and orderly gently blowing and beating the cells on the bottle wall by using a straw to ensure that the cells are separated from the bottle wall and then form single cell suspension;

[0156] 4) transferring the single cell suspension into a centrifuge tube, centrifuging, (lOOOrpm, 5 min), removing the supernatant, adding a complete culture medium, slightly blowing to loosen the medium, and carrying out subculture according to the ratio of 1: 2.

[0157] Example 5

[0158] Preparation of cytokines:

[0159] a2, culturing the mesenchymal stem cells in an incubator with 37 ℃ and 5% carbon dioxide concentration under the conventional conditions to make the mesenchymal stem cells grow adherent to the wall, changing the culture solution after culturing for 15h, and cleaning with PBS solution;

[0160]b2, adding the washed mesenchymal stem cells into a serum-free DMEM medium to continue culturing and inducing25h, the serum-free culture medium contains 0.3umol/L of iridin and 1f0.4umol/L of wild rice_;Collecting culture solution after culturing, centrifuging, filtering, removing small molecules with molecular weight less than 5KD, and concentrating to obtain concentrated solution containing cell factor.

[0161] Example 6

[0162] Preparation of cytokines:

[0163] a2, culturing the mesenchymal stem cells in an incubator with 37 ℃ and 5% carbon dioxide concentration under the conventional conditions to make the mesenchymal stem cells grow adherent to the wall, changing the culture solution after culturing for 15h, and cleaning with PBS solution;

[0164]b2, adding the washed mesenchymal stem cells into a serum-free DMEM medium to continue culturing and inducing for 25 hours, wherein the serum-free medium contains 0.3um of astaxanthin_Ol/L_;Collecting the culture solution after the culture, centrifuging, filtering, removing small molecules with the molecular weight less than 5KD, and concentrating to obtain the concentrated solution containing the cell factors.

[0165] Example 7

[0166] Preparation of cytokines:

[0167] a2, culturing the mesenchymal stem cells in an incubator with 37 ℃ and 5% carbon dioxide concentration under the conventional conditions to make the mesenchymal stem cells grow adherent to the wall, changing the culture solution after culturing for 15h, and cleaning with PBS solution;

[0168]b2, adding the washed mesenchymal stem cells into a serum-free DMEM medium to continue culturing and inducing for 25 hours, wherein the serum-free medium contains wild 1f0.4umol/L_;Collecting the culture solution after the culture, centrifuging, filtering, removing small molecules with the molecular weight less than 5KD, and concentrating to obtain the concentrated solution containing the cell factors.

[0169] Example 8

[0170] Preparation of cytokines:

[0171] a2, culturing the mesenchymal stem cells in an incubator with 37 ℃ and 5% carbon dioxide concentration under the conventional conditions to make the mesenchymal stem cells grow adherent to the wall, changing the culture solution after culturing for 15h, and cleaning with PBS solution;

[0172] and B2, adding the washed mesenchymal stem cells into a serum-free DMEM medium to continue to culture and induce for 25 hours, collecting a culture solution after the culture is finished, centrifuging, filtering, removing small molecules with the molecular weight of less than 5KD, and concentrating to obtain a concentrated solution containing the cell factors.

[0173] Cytokine content detection

[0174] The concentrated solution of example 5 ~ from example 8 was subjected to cytokine content detection, and mainly detected vascular endothelial growth factor VEGF, insulin-like growth factor IGF, hepatocyte growth factor HGF, epidermal growth factor EG F, and acidic fibroblast growth factor aFGF.

[0175] TABLE 1 cytokine content in example 5 ~ example 8

[0176] As can be seen from Table 1, the vascular endothelial growth factor VEGF content of examples 5 and 6 is much higher than that of examples 7 and 8, which shows that astaxanthin promotes the secretion of vascular endothelial growth factor VEGF; the contents of the epidermal growth factor EGF in the example 5 and the example 7 are much higher than those in the example 6 and the example 8, which shows that the rosaniline has the promotion effect on the secretion of the epidermal growth factor EGF.

[0177] Clinical experiment 2

[0178] The experimental method comprises the following steps: 150 female volunteers with wounds, aged 30 to 40 years, were recruited into 3 groups, the first of which was coated with an effective dilution of the cytokine fluid of example 5, the second of which was coated with scar-removing cream, and the third of which was left untreated. The application amount of each volunteer is lml, 3 times daily, and the application is continued for 1 month.

[0179] The experimental effect is as follows: the skin and scar conditions of the volunteers were observed, wherein the effective rate of the first group reached 89%, the effective rate of the second group reached 56%, and the volunteers of the third group had scars. The clinical experiment takes the fact that the scar area of the volunteer is larger than one fifth of the wound area as ineffective, and the scar area is smaller than one fifth of the wound area as effective. Therefore, the cell factor of the invention can effectively reduce the formation of scars, so that the skin condition is repaired and improved.

Claims

Claims

[ claim 1] A cosmetic preparation for delaying aging in the body based on the differentiation and regeneration of pluripotent stem cells, characterized in that: the cosmetic preparation contains per ml:

pluripotent stem cells: 5x 10510 x105 autologous adipose-derived stem cells;

fibronectin FN5 microgram ~ 10 microgram;

an electrolyte solution;

cytokines:

vascular endothelial growth factor VEGF5 microgram ~ 10 microgram;

insulin-like growth factor IGF5 microgram ~ 10 microgram;

hepatocyte growth factor HGF5 microgram ~ 10 microgram;

epidermal growth factor EGF20 microgram ~ 50 microgram;

acid fibroblast growth factor aFGF5 microgram ~ 10 microgram;

auxiliary reagents:

20 mg-50 mg hyaluronic acid;

arachidonic acid 20 mg-50 mg.

[ claim 2] the cosmetic preparation according to claim 1, characterized in that: the electrolyte solution is physiological saline.

[ claim 3] the cosmetic preparation according to claim 1, characterized in that: the cosmetic preparation contains per ml:

pluripotent stem cells: 6x105 autologous adipose-derived stem cells;

fibronectin FN10 microgram;

an electrolyte solution;

cytokines:

vascular endothelial growth factor VEGF8 micrograms;

insulin-like growth factor IGF5 microgram;

hepatocyte growth factor HGF9 microgram;

30 micrograms of epidermal growth factor EGF;

6 micrograms of acidic fibroblast growth factor (aFGF); auxiliary reagents:

50 mg of hyaluronic acid;

arachidonic acid 30 mg.

[ claim 4] the cosmetic preparation according to claim 1, characterized in that: the cosmetic preparation contains per ml:

pluripotent stem cells: 5x 10510 x105 autologous adipose-derived stem cells;

fibronectin FN5 microgram ~ 10 microgram;

an electrolyte solution;

cytokines:

vascular endothelial growth factor VEGF5 microgram ~ 10 microgram;

insulin-like growth factor IGF5 microgram ~ 10 microgram;

hepatocyte growth factor HGF5 microgram ~ 10 microgram;

epidermal growth factor EGF20 microgram ~ 50 microgram;

acid fibroblast growth factor aFGF5 microgram ~ 10 microgram;

auxiliary reagents:

20 mg-50 mg hyaluronic acid;

arachidonic acid 20 mg-50 mg;

spirulina polysaccharide 20 mg ~ 50 mg.

[ claim 5] Process for the preparation of a cosmetic preparation according to claim 1 ~ 4, characterized in that it comprises the following steps

(1) Separating autologous fat, and culturing autologous fat-derived stem cells to obtain autologous fat-derived pluripotent stem cells;

(2) culturing mesenchymal stem cells to obtain cell factors;

(3) adding autologous fat-derived pluripotent stem cells, cell factors and auxiliary reagents into an electrolyte solution under an aseptic condition; mixing and preserving.

[ claim 6] the production method according to claim 5, characterized in that: the specific process for obtaining the body fat-derived pluripotent stem cells in the step (1) is as follows:

a1, extruding autologous adipose tissues, adding PBS solution for cleaning, adding the PBS solution, centrifuging for 5min at the rotating speed of 2000rpm, keeping an upper fat layer and a lower precipitation layer, and absorbing and discarding the middle layer;

b1, adding digestive juice into the upper fat layer and the lower precipitation layer, placing the mixture in a constant temperature shaking table at 37 ℃, digesting the mixture for 20min by shaking, and collecting the liquid at the lowest layer to obtain autologous primary pluripotent stem cells;

c1, transferring the primary pluripotent stem cells into a centrifugal tube containing a complete culture medium to terminate digestion, then closing the centrifugal tube, centrifuging lOmin at 1500rpm, sucking the supernatant by a suction tube, removing the supernatant, and supplementing the culture medium to prepare a cell suspension for later use;

d1, culturing the primary pluripotent stem cells in the cell suspension under the conventional conditions, observing the cell wall sticking condition, carrying out first liquid change after 12h ~ 18h, changing the culture liquid every 3 days, carrying out passage after the cells grow to be fused, and obtaining the autologous adipose-derived pluripotent stem cells which can be used for a beautifying preparation after subculture.

[ claim 7] the production method according to claim 5, characterized in that: the method for culturing and culturing the mesenchymal stem cells to obtain the cell factors in the step (2) comprises the following steps:

a2, culturing the mesenchymal stem cells under the conventional conditions to make the mesenchymal stem cells grow adherent to the wall, changing the solution after culturing for 12h ~ 18h, and cleaning by using a PBS solution;

b2, adding the washed mesenchymal stem cells into a serum-free culture medium to continue culture induction culture for 20h ~ h, wherein the serum-free culture medium contains! 1TF cyanoxin 0.2umol/L ~.5 umol/L and Yefu Dai 1_;Collecting the culture solution after the culture, centrifuging, filtering, removing small molecules with the molecular weight less than 5KD, and concentrating to obtain the concentrated solution containing the cell factors.

[ claim 8] the production method according to claim 6, characterized in that: the conventional conditions in the step D1 refer to the culture in an incubator with the culture temperature of 37 ℃ and the carbon dioxide concentration of 5%.

[ claim 9] the production method according to claim 7, characterized in that: the conventional conditions in the step A2 refer to the culture in an incubator with the culture temperature of 37 ℃ and the carbon dioxide concentration of 5%.

[ claim 10] the production method according to claim 7, characterized in that: the serum-free medium in the step B2 is DMEM medium.