WO2019161540A1

WO2019161540A1 - Pluripotent stem cell differentiation and regeneration-based cosmetic preparation for delaying aging of body

Info

Publication number: WO2019161540A1
Application number: PCT/CN2018/077028
Authority: WO
Inventors: 高扬; 杨芷
Original assignee: 高扬
Priority date: 2018-02-23
Filing date: 2018-02-23
Publication date: 2019-08-29
Also published as: CN110475534A

Abstract

A pluripotent stem cell differentiation and regeneration-based cosmetic preparation for delaying the aging of the body; each ml contains 5×10⁵-10x10⁵ autologous adipose-derived stem cells; 5 µg-10 µg of fibronectin FN5; an electrolyte solution and a plurality of cytokines and auxiliary reagents. Further disclosed is a method for preparing a cosmetic preparation. The cosmetic preparation comprises autologous pluripotent stem cells and active ingredients such as a plurality of cytokines. After being injected or implanted into the body, the cells may be rapidly proliferated and regenerated, and the directed differentiation of the stem cells in corresponding tissues of the body may replace and repair damaged or aging cell tissue of the body, maintain activity and continuously secrete cytokines, and exert great damage repair and therapeutic effects such that the body, for example the skin, looks younger.

Description

A cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells

Technical field

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

Background technique

[0002] Stem cells are seed cells that have not yet differentiated and developed, have unlimited self-renewal and replication ability, and can be differentiated into specific tissues, which is the basis for stem cells for skin care and even tissue regeneration. Stem cells are divided into embryonic stem cells and adult stem cells. Embryonic stem cells exist in the blastocysts and can self-renew and replicate and differentiate into 20 different types of cells. These different types of cells make up the human body; adult stem cells exist in many adults. Within tissues and organs, such stem cells can only differentiate into specific types of cells that are themselves or related to themselves. They are not only used as a self-repairing system for the human body, but also responsible for maintaining the regeneration of some human organs/tissues, such as blood, skin and small intestine.

[0003] Scientific studies have shown that two kinds of adult stem cells are confirmed to exist in human skin, one is epidermal stem cells, which are present in the basal layer of the epidermis, and its main function is to supplement epidermal cells to maintain skin cell balance and repair Damaged tissue; the other is present in dermal dermal dermal stem cells, has a strong self-renewal ability, can induce the formation of hair follicles and migrate into the dermal layer across the hair follicle, they proliferate and differentiate into fibroblasts (fibroblasts can The secretory component of the extracellular matrix), the main function is to make the skin full of elasticity and reduce wrinkles. However, as we age, skin renewal becomes slower, and the activity and quantity of stem cells gradually decrease. Ultraviolet rays produce toxins and reactive oxygen species on the skin, which damage the most sensitive stem cells; at the same time, low temperature, low humidity and environmental Mutations impair the barrier function of the skin. These internal or external environmental factors can affect the function of stem cells.

[0004] Stem cell growth factor is a multifunctional potent cytokine that binds to specific receptors of target cells in the human microenvironment, and exerts biological functions such as stimulating target cell differentiation and proliferation, promoting target cell synthesis and secretion, and chemotaxis and induction of inflammatory cells. , initiate metabolic processes within the cell and transfer of substances and information between cells. Many cytokine receptors have kinase activity, particularly tyrosine kinase activity (eg, PDGF receptor, EGF receptor). Through the combination of cytokines and corresponding receptors, activate receptors, promote the formation of subcutaneous blood vessels, repair the subcutaneous blood microcirculation system, improve the microenvironment of cell growth, induce proliferation and differentiation of various cells, and accelerate the replacement of senescent cells by skin nascent cells. Process, thus achieving the effect of delaying aging.

[0005] Stem cells produce and secrete a large number of biologically active factors, which can effectively regulate cell signaling, activate human stem cells, and thereby physiologically repair or replace body damage, lesions, and aging cells. 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 Interleukin-7 (IL-6 and IL-7), megakaryocyte colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), interferon (IFN) and other factors, these cytokines promote cell proliferation, differentiation, and resistance Apoptosis and other functions; Stem cells can also produce natural immune proteins, such as IgG, IgA, IgM, IgD, IgE, etc., to resist or repair damage to the body cells caused by themselves and external factors.

[0006] The use of bioactive factors produced by stem cell activation activates body stem cells, and then repairs or replaces body damage, lesions, and aging cells by self-stem cell rationality, and has broad application prospects in disease prevention and health care and beauty fields.

[0007] The skin is the largest organ that constitutes a human body, accounting for about 16% of the body's volume, with an area of 1.8 m2, a thickness of 2 to 4 mm, and a weight of about 3 kg. It consists of the epidermis, dermis, and subcutaneous fat layer. The skin is in direct contact with the external environment, and protects the human body from various harmful factors including harmful microorganisms and external stimuli such as ultraviolet rays. It has an important protective film function and is essential for the maintenance of biochemical functions required for systemic metabolism. An indispensable organ.

[0008] Skin aging is a very complicated process. The main manifestations are shortening of telomeres, decreased DNA methylation levels, and changes in the expression levels of various cytokines, such as changes in the number and function of growth factors and their receptors such as EGF. One of the important reasons is subcutaneous vascular atrophy, which leads to Insufficient blood supply to the skin, causing pathophysiological effects, lack of nutrition in the cells, gradual decrease in reactivity to the outside world, weakening of skin cell proliferation and differentiation, slowing of metabolism, and aging symptoms such as wrinkles and pigmentation on the skin.

[0009] With the expansion of the cosmetic and plastic surgery market, more and more people choose to apply facial freckle, photorejuvenation and other methods for facial beauty. Skin laser cosmetology is a skin treatment by photothermal action. By producing high energy, accurate focusing, high energy is generated locally in human skin tissue, and beauty is achieved by removing and destroying tissue. The purpose of tolerance. However, the number of people who have postoperative complications due to improper treatment selection and improper operation is increasing year by year. Improper handling can result in excessive tissue damage, severe inflammatory reactions, and capillary engorgement. The main side effects include edema, blisters, pigmentation, hypopigmentation, telangiectasia, increased skin sensitivity, and more.

[0010] Therefore, in the current beauty industry, traditional chemical beauty and physical beauty can no longer meet the demand, and natural plant extract ingredients are in full bloom, and stem cell beauty is receiving more and more attention for its remarkable effect and long-lasting effect. Stem cells act on beauty, get rid of the traditional chemical beauty, physical beauty, and bring us a new experience, from the inside of the human body to achieve beautiful, young.

[0011] As an orthopedic surgery with a focus on repair and reconstruction and a medical beauty centered on rejuvenation, regenerative medicine has always been a field of concern and research. Adipose-derived mesenchymal stem cells solve many medical cosmetic problems.

[0012] First of all, the convenience of source is not only the biggest advantage of fat-derived ADSCs, but also the advantage of plastic surgery. On the one hand, adipose tissue is widely distributed in the body and has abundant reserves. On the other hand, liposuction is a routine operation of mature surgery, which has a low risk of surgery and is easy to obtain as a conventional “discarded by-product”. For patients, the liposuction sculpture shape while enjoying the youthful magical effect of stem cells is a win-win life reshaping, pain and fear less than bone marrow extraction, and no risk of blood source pollution and immune rejection, thus forming a clinical application. The advantages.

[0013] According to foreign reports, in the initial stage of liposuction from 1994 to 2000, the mortality rate was 0 in 66,570 cases of liposuction, and the rate of medical accidents was only 0.68%. It can be seen that liposuction is safe and reliable. Another advantage of ADSCs is their high acquisition efficiency. The yield of MSCs in the bone marrow is relatively low, accounting for only 0.0001% to 0.01% in nucleated cells of adult bone marrow tissue. After the adipose tissue is digested by collagenase, the fat cell is the main cell type, and it is easily removed by buoyancy. The obtained cell clone formation rate is 100 to 500 times that of the mesenchymal stem cells. At the same time, since the amount of liposuction can be multiplied as needed, the required concentration of stem cells can be obtained at one time, without in vitro expansion, which greatly reduces the risk of passage variation and various pollution.

[0014] From a clinical point of view, it is easy to obtain a large number of ADSCs in a short period of time, which is of great significance for rapid treatment of clinical diseases. In the case of local anesthesia, the adult bone marrow is usually obtained in an amount of no more than 40 ml, and about 1.2 x 109 nucleated cells, including about 2.4 x 104 MSCs, can be obtained. And fat tissue, every 1 Please get about 2x108 nucleated cell phases at 00m, and the stem cell content is 40 times that of 40ml bone marrow. Thus, under the same conditions, ADSCs have more clinical application potential advantages than MSCs.

[0015] Application status of ADSCs in the field of plastic surgery

[0016] 1, skin without scars and repair applications

[0017] Skin scarless application has been the focus of research in the field of plastic reconstruction and reconstruction in the absence of ADSCs. Complete regeneration of damaged tissue cells and complete replacement of damaged tissue cells is at the core of regenerative medicine research in the medical field. Not only did ADSCs soon replace common seed cells in tissue engineering, but they also showed an important role in entering scar-free research.

[0018] 2. ADSCs and youthfulness

[0019] The third is the local and systemic application of stem cells, and many cases show amazing effects. Rat subacute aging and normal aging models were used. After ADSCs were transplanted, the effects of free radicals and immune function on aging model rats were observed. Results Compared with the blank control group, serum SOD, NO, IL-2 levels and spleen index were significantly decreased in the model group, and serum MDA levels were significantly increased. After transplantation of ADSCs, serum SOD, NO, IL-2 levels and spleen index were increased in the treatment group compared with the model group, while MDA content was significantly decreased. Allogeneic transplantation of ADSCs can effectively enhance the body's ability to scavenge free radicals and anti-oxidation, improve the body's immune function, and thus delay D-gal-induced aging in rats.

[0020] Therefore, the selection of adipose-derived stem cells as a source of versatile stem cells for medical beauty has obvious advantages, and the present invention is also an advantage of applying adipose-derived stem cells.

Summary of invention

technical problem

Problem solution

Technical solution

[0021] The object of the present invention is to provide a cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells.

, can improve the stability of cytokines, can be applied to medical beauty, realize the regeneration and repair of the body

[0022] In order to achieve the above object, an embodiment of the present invention provides a cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells, wherein the cosmetic preparation contains:

[0023] Pluripotent stem cells: autologous adipose-derived stem cells 5×105 10×105; [0024] Fibronectin FN5 micrograms ~ 10 micrograms;

[0025] an electrolyte solution;

Cytokine:

[0027] vascular endothelial growth factor VEGF5 micrograms ~ 10 micrograms;

[0028] insulin-like growth factor IGF5 micrograms ~ 10 micrograms;

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

[0030] epidermal growth factor EGF20 micrograms ~ 50 micrograms;

[0031] acidic fibroblast growth factor aFGF5 micrograms ~ 10 micrograms;

[0032] Auxiliary reagents:

[0033] hyaluronic acid 20 mg ~ 50 mg;

[0034] Arachidonic acid 20 mg ~ 50 mg.

[0035] One of the optimization schemes of the present invention, the electrolyte solution is physiological saline.

[0036] One of the optimization schemes of the present invention, the cosmetic preparation contains: [0037] pluripotent stem cells: autologous adipose-derived stem cells 6×105;

[0038] fibronectin FN10 micrograms;

[0039] an electrolyte solution;

[0040] Cytokines:

[0041] vascular endothelial growth factor VEGF8 micrograms;

[0042] insulin-like growth factor IGF5 micrograms;

[0043] hepatocyte growth factor HGF9 microgram;

[0044] epidermal growth factor EGF30 micrograms;

[0045] acidic fibroblast growth factor aFGF6 micrograms;

[0046] Auxiliary reagents:

[0047] hyaluronic acid 50 mg;

[0048] Arachidonic acid 30 mg.

[0049] One of the optimization schemes of the present invention, the cosmetic preparation contains:

[0050] Pluripotent stem cells: autologous adipose-derived stem cells 5xl05~10xl05;

[0051] Fibronectin FN5 micrograms ~ 10 micrograms; [0052] an electrolyte solution;

Cytokines:

[0054] vascular endothelial growth factor VEGF5 micrograms ~ 10 micrograms;

[0055] insulin-like growth factor IGF5 micrograms ~ 10 micrograms;

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

[0057] Epidermal growth factor EGF20 micrograms ~ 50 micrograms;

[0058] acidic fibroblast growth factor aFGF5 micrograms ~ 10 micrograms;

[0059] Auxiliary reagents:

[0060] hyaluronic acid 20 mg ~ 50 mg;

[0061] arachidonic acid 20 mg ~ 50 mg;

[0062] Spirulina polysaccharide 20 mg ~ 50 mg.

[0063] The present invention also discloses a method of preparing the above cosmetic preparation comprising the following steps:

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

(2) culturing mesenchymal stem cells to obtain cytokines;

[0066] (3) adding autologous adipose-derived pluripotent stem cells, cytokines, and auxiliary reagents to the electrolyte solution under aseptic conditions; and mixing after storage.

[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 as follows:

[0068] A1, the autologous adipose tissue is squeezed and then added to the PBS solution for washing, and then added to the PBS solution and centrifuged at 2000 rpm for 5 min, the upper fat layer and the lower layer of the precipitate are retained, and the intermediate layer is discarded;

[0069] B1, adding a digestive juice in the upper fat layer and the lower sediment layer, placed in a 37 ° C constant temperature shaker, 190r / min, shaking for 20min; collecting the lowest layer of liquid, to obtain autologous primary pluripotent stem cells ;

[0070] C1, the primary pluripotent stem cells are transferred into a centrifuge tube containing complete medium to terminate digestion, and then the centrifuge tube is closed, centrifuged at 1500 rpm for 10 minutes; the supernatant is removed by a pipette, and the medium is added to make a cell suspension. use;

[0071] DU cultures primary pluripotent stem cells in cell suspension under normal conditions, observes cell adherence, and performs the first liquid exchange after 12h~18h, and then replaces the culture solution every 3 days, until Cell growth to melting After the passage, the passage was carried out, and after subculture, autologous adipose-derived pluripotent stem cells which can be used for the cosmetic preparation were obtained.

[0072] As a supplement to the above preparation method, the present invention preferably, in the step (2), culturing the cultured mesenchymal stem cells, and obtaining the cytokine comprises:

[0073] A2, mesenchymal stem cells are cultured under normal conditions to grow adherently, and cultured for 12 to 18 hours.

, washed with PBS solution;

[0074] B2, the washed mesenchymal stem cells are added to the serum-free medium to continue the culture and induce culture for 20h~28h

The serum-free medium contains: 1TF amphibious 0.2umol/L~0.5umol/L and wild building lO.lumol/L O.Su mol/L; after the culture is completed, the culture solution is collected, centrifuged, filtered, and the molecular weight is removed. A small molecule of less than 5 kD is concentrated to obtain a concentrate containing cytokines.

[0075] As a supplement to the above preparation method, it is preferred in the present invention that the conventional conditions in the step D1 are culture in an incubator having a culture temperature of 37 ° C and a carbon dioxide concentration of 5%.

[0076] As a supplement to the above preparation method, the present invention preferably, the conventional conditions in the step A2 are culture in an incubator having a culture temperature of 37 ° C and a carbon dioxide concentration of 5%.

[0077] As a supplement to the above preparation method, the present invention preferably, the serum-free medium in the step B2 means DME M medium.

[0078] As a supplement to the above preparation method, the preferred step of subculture in the step D1 of the present invention is:

[0079] 1) Aspirate the old medium in the culture flask in the ultra-clean workbench, rinse with PBS 2-3 times, then add l-2m

L digestive juice (0.25% trypsin and 0.04% EDTA (v/v 1: 1));

[0080] 2) digesting in the incubator (3-5 min), observing the morphological changes of the adherent cells under an inverted microscope, when the cytoplasm of the adherent adipose stem cells is retracted, the intercellular space is continuously increased, and the cells are nearly spherical and have a small amount. When the round cells are detached, an equal volume of medium is added to terminate the digestion;

[0081] 3) repeatedly pipetting the cells on the wall of the bottle with a pipette, so that the cells are separated from the bottle wall and then present as a single cell suspension;

4) The single cell suspension was transferred to a centrifuge tube, centrifuged, (100 rpm, 5 min), the supernatant was discarded, the whole medium was added, and the mixture was lightly blown to loosen, and subcultured at 1:2.

Advantageous effects of the invention

Beneficial effect

[0083] In summary, the present invention has the following advantages: The cosmetic preparation of the invention comprises autologous pluripotent stem cells and various active ingredients such as cytokines, which can be rapidly proliferated and regenerated after being injected or implanted into the body, and can be directed to the body through the directed differentiation of the stem cells in the corresponding tissues of the body. Damaged or senescent cell tissue undergoes replacement and repair, maintains activity and continues to secrete cytokines, exerting great damage repair and therapeutic effects, and making the body, such as the skin, younger.

[0085] When the preparation of the invention is applied to skin repair or beauty, a plurality of cytokines rich in the skin act on the skin, free from the shackles of traditional chemical beauty and physical beauty, and by stimulating the self-repairing function, fundamentally Solve problems such as skin aging. No harmful substances such as preservatives are added to the method. Eliminating the long-term surgical recovery period caused by surgical wrinkles does not affect normal life and work.

Brief description of the drawing

DRAWINGS

1 is a test result of EGF content of epidermal growth factor according to an embodiment of the present invention; wherein the abscissa is time and the ordinate is percentage content;

2 is a test result of the HGF content of hepatocyte growth factor according to an embodiment of the present invention; wherein the abscissa is time and the ordinate is percentage content.

Invention embodiment

Embodiments of the invention

The present invention provides a cosmetic preparation for delaying body aging based on differentiation and regeneration of pluripotent stem cells in one embodiment of the present invention, wherein the cosmetic preparation contains:

[0089] Pluripotent stem cells: autologous adipose-derived stem cells 5xl05~10xl05;

[0090] fibronectin FN5 micrograms ~ 10 micrograms; electrolyte solution;

Cytokines:

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

[0093] insulin-like growth factor IGF5 micrograms ~ 10 micrograms;

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

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

[0096] acidic fibroblast growth factor aFGF5 micrograms ~ 10 micrograms;

[0097] Auxiliary reagent: hyaluronic acid 20 mg ~ 50 mg; arachidonic acid 20 mg ~ 50 mg. Embodiment 1

[0099] The cosmetic preparation contains:

[0100] autologous adipose-derived stem cells 6×105;

Fibronectin FN10 micrograms;

[0102] physiological saline;

Vascular endothelial growth factor VEGF8 micrograms;

Insulin-like growth factor IGF5 microgram;

[0105] hepatocyte growth factor HGF9 microgram;

[0106] epidermal growth factor EGF30 micrograms;

Acidic fibroblast growth factor aFGF6 microgram;

[0108] hyaluronic acid 50 mg;

[0109] Arachidonic acid 30 mg.

Example 2

[0111] The cosmetic preparation contains:

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

[0113] fibronectin FN10 micrograms;

[0114] physiological saline;

Vascular endothelial growth factor VEGF8 micrograms;

[0116] insulin-like growth factor IGF5 micrograms;

[0117] hepatocyte growth factor HGF9 microgram;

[0118] epidermal growth factor EGF30 micrograms;

Acidic fibroblast growth factor aFGF6 microgram;

[0120] hyaluronic acid 50 mg;

Spirulina polysaccharide 30 mg.

Comparative Example 1

[0123] The cosmetic preparation contains:

[0124] autologous adipose-derived stem cells 6×105;

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

Vascular endothelial growth factor VEGF8 micrograms;

[0128] insulin-like growth factor IGF5 micrograms;

[0129] hepatocyte growth factor HGF9 micrograms;

[0130] epidermal growth factor EGF30 micrograms;

Acidic fibroblast growth factor aFGF6 microgram;

[0132] Hyaluronic acid 50 mg.

Stability experiment

[0134] Experimental method:

[0135] Three sets of medium-volume cosmetic preparations of Example 1, Example 2 and Comparative Example 1 were taken into three test tubes, respectively, and water for injection was added to 100 ml; then, it was stored at 37 ° C, and the corresponding one was used every month. The kit detects the levels of epidermal growth factor EGF and hepatocyte growth factor HGF. The results are shown in Figure 1 and Figure 2.

As can be seen from FIG. 1, the content of epidermal growth factor EGF in Comparative Example 1 was gradually decreased, and the extent of the decrease was significantly different from that of Example 1 and Example 2; Example 1 and Example 2 The ratio is larger, and the gap between the two is maintained between 10% and 20%. Thus, the stability of Example 1 and Example 2 was significantly better than that of Comparative Example 1, and the storage stability of Example 2 was also superior to that of Example 1. When the storage time is more than 24 months, the content of the three is low, indicating that the product is basically ineffective.

As can be seen from Fig. 2, the content of hepatocyte growth factor HGF in Comparative Example 1 was gradually decreased, and the extent of the decrease was not significantly different from that of Example 1 and Example 2. This shows that the stability of the three is equivalent. It can be seen that both arachidonic acid and spirulina polysaccharide can improve the stability of EGF and can not significantly improve the stability of HGF.

[0138] Clinical Trial 1

[0139] A total of 200 women aged 40 to 50 years were selected, and all women's eyes were not undergoing plastic surgery or other medical procedures. The cosmetic preparations of Example 1 and Example 2 were configured as injections, and 100 cases were injected by microneedle injection respectively, and the second injection was performed one week after the first injection; all cases were observed 2 months after the injection was completed. Case.

[0140] Due to individual differences, this experiment is not convenient to set up the comparison, but you can use the way of photograph comparison , that is, photographing the eyes of women before and after injection. By comparison, it was found that a total of 184 females in Example 1 and Example 2 had improved eye elasticity, wrinkles such as crow's feet and the like, and the surrounding skin became smooth and fine, and the other 16 cases were not obvious. From this, it can be seen that the injection solution of the present invention has an efficiency of 92% and has a very high efficiency.

[0141] The present invention also discloses a method of preparing the above cosmetic preparation comprising the following steps:

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

(2) culturing mesenchymal stem cells to obtain cytokines; when detecting a certain cytokine content lower than the ratio required by the present invention, the purchased cytokine may be added or the cytokine may be cultured separately. Join again.

(3) adding autologous adipose-derived pluripotent stem cells, cytokines, and auxiliary reagents to the electrolyte solution under aseptic conditions; and mixing after storage.

Example 3

Preparation of autologous adipose-derived pluripotent stem cells:

[0147] A1, the autologous adipose tissue is squeezed, added to the PBS solution for washing, and then centrifuged at 2000 rpm for 5 min by adding PBS solution, the upper fat layer and the lower layer of the precipitate are retained, and the intermediate layer is discarded;

[0148] B1, adding a digestive juice in the upper fat layer and the lower sediment layer, placed in a 37 ° C constant temperature shaker, 190 r / min, shaking for 20 min; collecting the lowest layer of liquid to obtain autologous primary pluripotent stem cells ;

[0149] C1, the primary pluripotent stem cells are transferred into a centrifuge tube containing complete medium to terminate digestion, and then the centrifuge tube is closed, centrifuged at 1500 rpm for 10 min; the supernatant is aspirated and removed, and the medium is supplemented with a cell suspension. use;

[0150] DU cultures primary pluripotent stem cells in a cell suspension under normal conditions, and observes cell adherence conditions, which are cultured in an incubator at a culture temperature of 37 ° C and a carbon dioxide concentration of 5%; After 12h~18h, the first liquid exchange was performed, and then the culture solution was changed every 3 days. After the cells were grown to the fusion, the cells were passaged, and after subculture, autologous adipose-derived pluripotent stem cells which can be used for the cosmetic preparation were obtained.

Example 4

[0152] The steps of subculture are:

[0153] 1) Aspirate the old medium in the culture bottle in the ultra-clean workbench, rinse 2-3 times with PBS, then add l-2m L digestive juice (0.25% trypsin and 0.04% EDTA (v/v 1: 1));

[0154] 2) digesting in the incubator (3-5 min), observing the morphological changes of the adherent cells under an inverted microscope, when the cytoplasm of the adherent adipose stem cells is retracted, the intercellular space is continuously increased, and the cells are nearly spherical and have a small amount. When the round cells are detached, an equal volume of medium is added to terminate the digestion;

[0155] 3) repeatedly pipetting the cells on the wall of the bottle with a pipette, and then separating the cells from the bottle wall to form a single cell suspension;

4) The single cell suspension was transferred to a centrifuge tube, centrifuged, (100 rpm, 5 min), the supernatant was discarded, the complete medium was added, the mixture was lightly blown, and the cells were subcultured at 1:2.

Example 5

Preparation of cytokines:

[0159] A2, mesenchymal stem cells were cultured in an incubator at 37 ° C and a carbon dioxide concentration of 5% under normal conditions to grow adherently, cultured for 15 hours, then changed to a liquid, and washed with a PBS solution;

[0160] B2, the washed mesenchymal stem cells were added to serum-free DMEM medium and cultured for 25 hours, and the serum-free medium contained 0.3umol/L and Nobel 1f0.4umol/L _; After completion, the culture solution is collected, centrifuged, and filtered to remove small molecules having a molecular weight of less than 5 kD, and concentrated to obtain a concentrate containing cytokines.

Example 6

Preparation of cytokines:

[0163] A2, mesenchymal stem cells were cultured in an incubator at 37 ° C and a carbon dioxide concentration of 5% under normal conditions to grow adherently, cultured for 15 hours, then changed to a liquid, and washed with a PBS solution;

[0164] B2, the washed mesenchymal stem cells were added to the serum-free DMEM medium to continue the culture for 25 h, and the serum-free medium contained astaxanthin 0.3 um _O l / L _; after the culture was completed, the culture solution was collected. Centrifugation, filtration, removal of small molecules with a molecular weight of less than 5 KD, and concentration to obtain a concentrate containing cytokines.

Example 7

Preparation of cytokines:

[0167] A2, mesenchymal stem cells were cultured in an incubator at 37 ° C and a carbon dioxide concentration of 5% under normal conditions to grow adherently, cultured for 15 hours, then changed to a liquid, and washed with a PBS solution;

[0168] B2, the washed mesenchymal stem cells were added to the serum-free DMEM medium and cultured for 25 hours, and the serum-free medium contained 1f 0.4 umol/L of the wild building _; after the culture was completed, the culture solution was collected and centrifuged. Filtration, removal of small molecules with a molecular weight of less than 5 KD, and concentration to obtain a concentrate containing cytokines.

Example 8

Preparation of cytokines:

[0171] A2, mesenchymal stem cells were cultured in an incubator at 37 ° C and a carbon dioxide concentration of 5% under normal conditions to grow adherently, cultured for 15 hours, then changed to a liquid, and washed with a PBS solution;

B2, the washed mesenchymal stem cells are added to the serum-free DMEM medium, and the culture is further cultured for 25 hours. After the culture is completed, the culture solution is collected, centrifuged, and filtered to remove small molecules having a molecular weight of less than 5 KD, and concentrated to obtain a content. A concentrate of cytokines.

Cytokine content detection

The concentrates of Examples 5 to 8 were tested for cytokine content, mainly detecting vascular endothelial growth factor VEGF, insulin-like growth factor IGF, hepatocyte growth factor HGF, epidermal growth factor EG F, acid fibrillation. Cell growth factor aFGF.

Table 1 : Cytokine content in Examples 5 to 8

As can be seen from Table 1, the vascular endothelial growth factor VEGF content of Example 5 and Example 6 is much higher than that of Example 7 and Example 8, indicating that astaxanthin promotes the secretion of vascular endothelial growth factor VEGF. The epidermal growth factor EGF content of Example 5 and Example 7 was much higher than that of Example 6 and Example 8, indicating that orecanoside promoted the secretion of epidermal growth factor EGF.

Clinical Experiment 2

[0178] Experimental method: 150 female volunteers with wounds were recruited, aged 30 to 40 years old, and randomly divided into 3 groups, wherein the first group was applied with an effective dilution of the cytokine liquid of Example 5, wherein The second group was coated with mites, and the third group was left untreated. The amount of application per volunteer is 1ml, 3 times a day. , continuous use for 1 month.

[0179] Experimental results: The skin and scar condition of the volunteers were observed. The effective rate of the first group was 89%, and the effective rate of the second group was 56%. The third group of volunteers had scar formation. In this clinical trial, the scar area of the volunteer is more than one-fifth of the wound area, and less than one-fifth is effective. Thus, it can be seen that the cytokine of the present invention can effectively reduce the formation of scars, so that the skin condition can be repaired and improved.

Claims

Claim

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

Pluripotent stem cells: autologous adipose-derived stem cells 5x105 10x105;

Fibronectin FN5 micrograms ~10 micrograms;

a;

Cytokines:

Vascular endothelial growth factor VEGF5 micrograms ~10 micrograms;

Insulin-like growth factor IGF5 micrograms ~10 micrograms;

Hepatocyte growth factor HGF5 micrograms ~ 10 micrograms;

Epidermal growth factor EGF20 micrograms ~50 micrograms;

Acidic fibroblast growth factor aFGF5 micrograms ~10 micrograms;

Auxiliary reagents:

Hyaluronic acid 20 mg ~ 50 mg;

Arachidonic acid 20 mg ~ 50 mg.

[Claim 2] The cosmetic preparation according to claim 1, wherein the electrolyte solution is physiological saline.

[Claim 3] The cosmetic preparation according to claim 1, wherein: the cosmetic preparation contains:

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

Fibronectin FN10 micrograms;

a;

Cytokines:

Vascular endothelial growth factor VEGF8 micrograms;

Insulin-like growth factor IGF5 microgram;

Hepatocyte growth factor HGF9 micrograms;

Epidermal growth factor EGF30 micrograms;

Acidic fibroblast growth factor aFGF6 microgram; Auxiliary reagents:

Hyaluronic acid 50 mg;

Arachidonic acid 30 mg.

[Claim 4] The cosmetic preparation according to claim 1, wherein: the cosmetic preparation contains:

Pluripotent stem cells: autologous adipose-derived stem cells 5x105 10x105;

Fibronectin FN5 micrograms ~10 micrograms;

a;

Cytokines:

Vascular endothelial growth factor VEGF5 micrograms ~10 micrograms;

Insulin-like growth factor IGF5 micrograms ~10 micrograms;

Hepatocyte growth factor HGF5 micrograms ~ 10 micrograms;

Epidermal growth factor EGF20 micrograms ~50 micrograms;

Acidic fibroblast growth factor aFGF5 micrograms ~10 micrograms;

Auxiliary reagents:

Hyaluronic acid 20 mg ~ 50 mg;

Arachidonic acid 20 mg ~ 50 mg;

Spirulina polysaccharide 20 mg ~ 50 mg.

[Claim 5] A method of preparing the cosmetic preparation according to any one of claims 1 to 4, which comprises the following steps

(1) separating autologous fat and culturing autologous adipose-derived stem cells to obtain autologous adipose-derived pluripotent stem cells;

(2) culturing mesenchymal stem cells to obtain cytokines;

(3) Add autologous adipose-derived pluripotent stem cells, cytokines and auxiliary reagents to the electrolyte solution under aseptic conditions;

[Claim 6] The preparation method according to claim 5, wherein the specific process of obtaining autologous adipose-derived pluripotent stem cells in the step (1) is:

A1, the autologous adipose tissue is squeezed, added to the PBS solution, and then added to the PBS solution. Centrifuge at 2000 rpm for 5 min, retain the upper fat layer and the lower precipitate layer, and the middle layer is discarded;

B1, adding the digestive juice in the upper fat layer and the lower sediment layer, placed in a 37 ° C constant temperature shaker, 190 r / min, shaking digestion for 20 min; collecting the lowest layer of liquid to obtain autologous primary pluripotent stem cells;

C1. Transfer the primary pluripotent stem cells into a centrifuge tube containing complete medium to terminate the digestion, then close the centrifuge tube, centrifuge at 1500 rpm for 10 min; remove the supernatant with a pipette and remove it, and supplement the medium to make a cell suspension for use;

D1. The primary pluripotent stem cells in the cell suspension were cultured under normal conditions, and the cell adherence was observed, and the first liquid exchange was performed after 12h~18h, after which the culture medium was changed every 3 days, and the cells were grown. After the fusion, the passage was carried out, and after subculture, autologous adipose-derived pluripotent stem cells which can be used for the cosmetic preparation were obtained.

[Claim 7] The preparation method according to claim 5, wherein the step of culturing the mesenchymal stem cells in the step (2) to obtain the cytokines comprises:

A2. The mesenchymal stem cells are cultured under normal conditions to grow adherently, and cultured for 12h~18h, then changed to liquid and washed with PBS solution;

B2, the washed mesenchymal stem cells are added to the serum-free medium to continue the culture and induced to culture for 20h to 28h, and the serum-free medium contains! 1TF phthalocyanin 0.2umol/L~0.5umol/L and wild f infant 1 0.2umol/L~0.5umol/L _; after the culture is completed, the culture solution is collected, centrifuged, and filtered to remove small molecules with a molecular weight of less than 5KD, and concentrated to obtain a concentrate containing cytokines.

[Claim 8] The preparation method according to claim 6, wherein the conventional condition in the step D1 is culture in an incubator having a culture temperature of 37 ° C and a carbon dioxide concentration of 5%.

[Claim 9] The preparation method according to claim 7, wherein the conventional condition in the step A2 is culture in an incubator having a culture temperature of 37 ° C and a carbon dioxide concentration of 5%.

[Claim 10] The preparation method according to claim 7, wherein the serum-free medium in the step B2 means DMEM medium.