Facial beautifying composition as well as component preparation method and application thereof

Abstract

The invention provides a facial beautifying composition as well as a preparation method and application of components thereof, and relates to the technical field of medical treatment. The invention provides a composition for facial beautification. The composition comprises platelet rich plasma (PRP) and fibroblast stem cells, the PRP is prepared by detecting specific growth factors, the fibroblast stem cells are prepared by adopting a specific culture medium, and a synergistic method of injecting the PRP and the fibroblast stem cells into the skin of a subject in sequence is adopted, so that the collagen generation amount of facial skin and the skin elasticity can be remarkably improved, the melanin content of the skin is reduced, and the skin quality is improved. The composition has practicability and safety and can be used for preparing medicines for improving skin conditions.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, relates to platelet-rich plasma and fibroblast stem cells, and specifically relates to a facial beauty composition and a preparation method and application of its components. Background Art

[0002] Platelet-rich plasma (PRP) is a high-concentration platelet plasma obtained by centrifuging autologous whole blood. It is rich in a variety of growth factors, such as PDGF (platelet-derived growth factor), TGF-β (transforming growth factor), IGF (insulin-like growth factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), VEGF (vascular endothelial growth factor), etc. These growth factors play an extremely important role in promoting cell proliferation and differentiation, increasing collagen synthesis ability, promoting matrix synthesis and sedimentation and tissue formation, and there is a good synergistic effect between growth factors.

[0003] The production of platelet-rich plasma is to extract high-concentration platelet plasma from whole blood drawn from autologous veins by centrifugation. There are many production methods at present. According to the preparation procedures, it can be divided into one-time centrifugation, two-time centrifugation and three-time centrifugation. A large number of experiments have proved that the extraction rate of PRP by one-time centrifugation and two-time centrifugation is the highest and the clinical application is the widest. Its basic principle is that according to the different sedimentation coefficients of the components in the blood, the blood is divided into three layers after the first centrifugation. The bottom layer is the red blood cells with the largest sedimentation coefficient, the top layer is the supernatant layer, and there is a layer at the junction that is difficult to distinguish with the naked eye, that is, the platelet-rich layer. The concentration of platelets and growth factors in PRP produced by different centrifugal forces, centrifugation times and centrifugation times is different (Yuan Ting, Zhang Changqing. Production and principle of platelet-rich plasma in bone tissue and soft tissue repair [J]. Chinese Clinical Rehabilitation, 2004). Platelet-rich plasma has received widespread attention in the fields of skin regeneration, tissue repair and beauty. However, studies on existing technologies have shown that the use of PRP alone has limited effects in terms of skin repair, collagen synthesis, etc., especially in promoting skin elasticity and improving facial wrinkles.

[0004] Fibroblast stem cells (FSCs) are a type of mesenchymal stem cells with self-renewal and multidirectional differentiation potentials. They are widely distributed in connective tissues (such as skin, bones, cartilage, blood vessels, etc.) and can differentiate into osteocytes, chondrocytes, adipocytes, myofibroblasts, etc. Relevant experiments have found that stem cells implanted in the body after autologous culture will secrete some cytokines, which can promote fibroblast proliferation and migration, promote collagen fiber synthesis, reduce the content of matrix metalloproteinases, and thus increase the dermal collagen content. Most of the existing studies focus on the effects of stem cells alone, and lack systematic research on the combined use of fibroblast stem cells and PRP for skin regeneration.

[0005] Prior art CN106727700B discloses a method for preparing platelet-rich plasma PRP and the use of the platelet-rich plasma. The invention extracts platelet-rich plasma from blood, including the following steps: (a) placing the collected whole blood in a container containing an anticoagulant to fully mix the blood and the anticoagulant; (b) placing the blood mixed with the anticoagulant in a centrifuge tube and performing a first centrifugation to separate the blood into three layers; (c) extracting the top layer and most of the middle layer and transferring them to a new centrifuge tube, mixing them evenly, and performing a second centrifugation; (d) discarding the plasma on the upper layer of the centrifuge tube, and using the remaining plasma to resuspend the precipitated platelets to obtain platelet-rich plasma. The prepared platelet-rich plasma can be used to prepare health products, such as orthopedics, dentistry, maxillofacial surgery, sports medicine, and cosmetic medicine. The disadvantage of this method is that in the preparation process of PRP, only the platelet concentration is usually considered, and the concentration of growth factors is not optimized, and the effect of improving skin conditions is not reflected, and the effectiveness and safety cannot be determined.

[0006] Moetaz et al. (Microneedling combined with platelet-rich plasma or trichloroacetic acid peeling for management of acne scarring: A split-face clinical and histologic comparison. [J]. John Wiley & Sons, Ltd, 2018 (1).) conducted microneedling alone and microneedling combined with platelet-rich plasma on volunteers with post-acne atrophic scars. They took photos and puncture biopsies before and 3 months after treatment, and performed clinical, histologic, and histologic evaluations. The results showed that microneedling combined with PRP significantly improved the effect of microneedling alone. The disadvantage of this method is that microneedling mechanically stimulates the dermis to activate skin repair, which is a minimally invasive operation. It is difficult to avoid damaging part of the subject's skin, and the overall improvement effect on skin condition is average, and long-term effectiveness and safety are difficult to guarantee.

[0007] Although studies have shown that fibroblast stem cells can improve skin elasticity and repair function, most of these studies focus on the use of stem cells alone, lacking experimental data and effect analysis of the combined use of stem cells and PRP. Therefore, there is a large gap in the existing technology in the technical path and effect optimization of the combined use of PRP and fibroblast stem cells, and no specific combination plan and its effectiveness and safety verification have been proposed. Summary of the invention

[0008] The present invention aims at the problems existing in the prior art and provides a facial beauty composition and a preparation method and application of its components. The present invention provides a facial beauty composition, the composition comprising PRP and fibroblast stem cells; the PRP is prepared by detecting a specific growth factor, the fibroblast stem cells are prepared by using a specific culture medium, and a synergistic method of sequentially injecting the PRP and the fibroblast stem cells into the skin of a subject can significantly increase the amount of collagen produced in the facial skin and the elasticity of the skin, and significantly reduce the melanin content in the skin, and can be used to prepare a drug for improving skin conditions.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] In one aspect, the present invention provides a composition for facial beauty, comprising platelet-rich plasma and fibroblast stem cells.

[0011] The method for preparing platelet-rich plasma comprises the following steps:

[0012] S1. Obtain whole blood for use;

[0013] S2, centrifuging whole blood to separate platelet-rich plasma; the growth factors in the platelet-rich plasma include VEGF, bFGF and PDGF, and the concentration requirements of the growth factors are VEGF ≥ 200 ng / mL, bFGF ≥ 100 ng / mL and PDGF ≥ 60 ng / mL;

[0014] The method for preparing fibroblast stem cells comprises the following steps:

[0015] a. Isolation of fibroblasts;

[0016] b. Fibroblast stem cells are obtained by culture in a culture medium, wherein more than 95% of the fibroblast stem cells express CD90, CD105 and CD73, and more than 95% of the cells do not express CD45, CD34 and CD14.

[0017] Preferably, the culture medium contains growth factors including VEGF, bFGF, TGF-β and PDGF.

[0018] Preferably, the volume ratio of the platelet-rich plasma to the fibroblast stem cells is 5:1-10:1.

[0019] Preferably, in step S1, 5-10 mL of whole blood is collected from the subject's vein using a sterile blood collection tube, and an anticoagulant is added and mixed.

[0020] Preferably, the anticoagulant comprises ethylenediaminetetraacetic acid, ethylenediaminetetraacetate, sodium citrate or citrate dextrose solution.

[0021] Preferably, the centrifugation in step S2 comprises the following steps:

[0022] Use a centrifuge to separate the blood. First, centrifuge at 1000-2000g for 5-15 minutes. After centrifugation, it will be divided into three layers. Take the top two layers and centrifuge at 2000-4000g for 10-20 minutes to separate the platelet-rich plasma.

[0023] Preferably, the method for detecting the growth factor concentration in step S2 is ELISA.

[0024] Traditional PRP preparation methods often lack precise control of growth factor concentration, resulting in unstable effects. The present invention ensures that the concentration of key growth factors in PRP reaches the optimal level by optimizing centrifugation conditions and strict quality control processes (such as using ELISA testing), thereby improving the effect.

[0025] Preferably, the tissue sample in step a includes skin tissue or fat tissue.

[0026] Preferably, in step a, trypsin digestion is used to separate fibroblasts from the tissue sample.

[0027] Preferably, in step b, the cells are cultured using a culture medium, and each culture cycle is 7-10 days, until the cells reach 10 6 Pieces / square centimeter.

[0028] Preferably, the culture medium comprises DMEM medium containing bFGF.

[0029] Preferably, the culture medium is maintained at 37°C and 5% CO 2 Each culture cycle lasts for 7-10 days until the cells reach 10 6 Pieces / square centimeter.

[0030] Preferably, in step b, the surface markers of the cells are analyzed by flow cytometry to ensure that the cells are of fibroblast stem cell type.

[0031] In another aspect, the present invention provides a method for facial beauty, comprising injecting the above composition into a subject's skin area.

[0032] Preferably, the method comprises separately injecting the platelet-rich plasma and fibroblast stem cells in the above composition into the tested skin area of ​​the subject, and the injection volume ratio of the platelet-rich plasma to the fibroblast stem cells is 5:1-10:1.

[0033] Preferably, the injection method includes subcutaneous injection or dermal injection.

[0034] Preferably, the method further comprises administering a round of injections monthly for a period of 3-6 months.

[0035] Preferably, the volume ratio of platelet-rich plasma to fibroblast stem cells in the composition is 5:1-10:1, and the volume of the platelet-rich plasma is 5-10 mL.

[0036] Specifically, the volume ratio of the platelet-rich plasma to the fibroblast stem cells is 5:1, and the volume of the platelet-rich plasma is 5 mL.

[0037] Preferably, the method comprises first injecting the platelet-rich plasma into the subject's test skin area, and then injecting the fibroblast stem cells.

[0038] Preferably, the method comprises the following steps:

[0039] First, platelet-rich plasma is injected into the tested skin area, and the injection volume of the platelet-rich plasma is 5-10 mL; 5-7 days after the injection, fibroblast stem cells are injected into the tested skin area, and the volume ratio of the injection volume of platelet-rich plasma to the injection volume of fibroblast stem cells is 5:1-10:1.

[0040] Specifically, the injection volume of the platelet-rich plasma is 5 mL, and the volume ratio of the injection volume of the platelet-rich plasma to the injection volume of the fibroblast stem cells is 5:1.

[0041] The present invention combines the optimized PRP with autologous fibroblast stem cells, adopts a scientific injection scheme and treatment time interval, and can achieve better results than single therapy. At the same time, the scheme provided by the present invention is completely based on injection technology, promoting skin regeneration through the combination of PRP and stem cells, and does not involve minimally invasive technology, ensuring that the technical scheme has clear operability and feasibility. All operations are completed through skin injection or other non-invasive means, avoiding damage to the skin caused by traditional minimally invasive surgery.

[0042] In another aspect, the present invention provides use of the above composition in preparing a product for improving skin condition.

[0043] Preferably, the application includes the following applications:

[0044] 1) Application in the preparation of anti-skin aging products;

[0045] 2) Application in the preparation of products for improving skin elasticity;

[0046] 3) Application in the preparation of products for increasing collagen production in skin cells;

[0047] 4) Application in the preparation of products for improving skin wrinkles;

[0048] 5) Application in the preparation of products for improving skin sagging;

[0049] 6) Application in the preparation of products for reducing skin melanin content.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention provides a composition for facial beauty, the composition comprising PRP and fibroblast stem cells; the PRP and fibroblast stem cells are prepared by an optimized method and are synergistically injected into the skin of a subject one after the other, which can significantly increase the amount of collagen produced and skin elasticity in facial skin, significantly reduce the melanin content in the skin, have a synergistic effect, are practical and safe, and can be used to prepare a drug for improving skin conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Comparison of skin of a subject in Group 2 before and after treatment.

[0053] Figure 2 Image of skin testing equipment.

[0054] Figure 3 Computer working interface when detecting skin elasticity recovery rate. DETAILED DESCRIPTION

[0055] PRP is prepared from the patient's own blood, and concentrated platelets and growth factors are extracted through centrifugation. The core of this optimization program is to precisely control the concentration of key growth factors in PRP to promote skin repair and regeneration. Specifically, it includes:

[0056] A specific centrifugal force (centrifugal rate, such as 1500-3000rpm) and centrifugal time (such as 5-15 minutes) are selected to ensure maximum concentration of platelets and growth factors; the centrifugal speed and time can be adjusted according to the different characteristics of the blood. During the quality control process, the concentration of key growth factors such as VEGF, bFGF, PDGF in PRP is determined by ELISA detection to ensure that it can play the best effect during the treatment process. Further regulators of blood components can be selectively added to increase the activity of growth factors in PRP, including but not limited to peptides and small molecule drugs; the PRP is stored under appropriate conditions for subsequent use.

[0057] Fibroblast stem cells can be extracted from the patient's own skin tissue, adipose tissue and other sources. According to the patient's needs and treatment site, the most suitable source of stem cells is selected. Autologous fibroblast stem cells are extracted, and the extracted stem cells are amplified by standard cell culture methods to obtain a sufficient number of active cells. During the cell culture process, a culture containing growth factors is used to ensure the proliferation and activity of stem cells. During the stem cell quality control process, the purity and function of stem cells are detected by flow cytometry, immunofluorescence staining and other methods to ensure that the stem cells used meet the treatment standards. Specifically, during the cell expansion process, the surface markers of the cells are analyzed by flow cytometry (FACS) to ensure that the cells are of fibroblast stem cell type. Commonly used markers include CD90, CD105, CD73, etc.

[0058] In the combined application of PRP and fibroblast stem cells, the treatment plan can be adjusted individually according to the specific situation of the patient. For example, according to the specific skin problems of the patient (such as skin sagging, wrinkle depth, etc.), the injection ratio of PRP and stem cells or the treatment cycle can be adjusted. For patients with different skin types or different age groups, subtle adjustments can be made in the growth factor concentration of PRP, the injection amount of stem cells, etc. to achieve the best treatment effect.

[0059] Unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0060] Collection of volunteer subjects:

[0061] 1) This group of experiments selected 80 healthy volunteers with obvious facial wrinkles, all of whom were female, aged 39 to 45 years old, with an average age of 42.1 years old.

[0062] 2) Inclusion criteria: healthy, no bad habits (such as smoking, drinking, drug abuse, etc.), no serious skin diseases, local dermatitis, skin ulcer infection, etc.; ② Venous blood platelet count before the experiment was between (100-300)×10 9 / L is within normal value: ③ No drugs or health products that affect platelet and blood system functions have been used in the 6 months before blood collection.

[0063] 3) Exclusion criteria: ① Those with scar-prone constitution; ② Those with severe diabetes, hypertension, or cardiopulmonary insufficiency; ③ Those with bleeding tendency or coagulation disorder; ④ Women during menstruation, pregnancy, or lactation; ③ Those who have undergone chemical peeling, physical grinding, soft tissue filling, botulinum toxin injection, or other skin rejuvenation procedures within the past year; Those who have used isotretinoin, retinoids, vitamin C, or other drugs that affect collagen synthesis within the past year.

[0064] 4) Experimental statement: This study uses a syringe to inject PRP and fibroblast stem cells prepared from autologous tissue into the dermis of the facial skin to observe the improvement of the facial skin. The entire injection operation process will strictly abide by the principle of aseptic operation. The injection doctors have relevant injection professional qualifications. All procedures are strictly implemented in accordance with the injection room work system and regulations, and all necessary measures are taken to ensure the safety of the subjects during the treatment. Inform the subjects of the precautions before treatment and possible adverse reactions during and after treatment, obtain the understanding of the subjects and agree to participate in the whole process, and sign the informed consent. This experiment has been studied and approved by the Ethics Committee.

[0065] Example 1: Preparation of platelet-rich plasma

[0066] 1) Use a sterile blood collection tube to collect 5-10 mL of whole blood from the subject's vein. Blood should be processed immediately after collection to prevent degradation of blood components.

[0067] 2) Centrifugal separation:

[0068] A centrifuge (Eppendorf 5804R) was used for blood separation. The centrifugation conditions were set as follows:

[0069] Step 1: Centrifuge at 1500 rpm for 5 minutes to separate red blood cells and white blood cells.

[0070] Step 2: Centrifuge at 3000 rpm for 10 minutes to separate platelet-rich fluid (PRP). This stage concentrates the growth factors in platelets and plasma.

[0071] 3)PRP quality control:

[0072] The concentration of growth factors in PRP was detected using the ELISA method as follows:

[0073] VEGF: target concentration ≥200 ng / mL;

[0074] bFGF: target concentration ≥100 ng / mL;

[0075] PDGF: target concentration ≥60 ng / mL;

[0076] If the growth factor concentration in PRP is not as expected, it can be optimized by adjusting the centrifugation time or rate.

[0077] Finally, the obtained PRP contained VEGF: 202.5 ng / mL; bFGF: 107.2 ng / mL; PDGF: 65.6 ng / mL.

[0078] Example 2: Preparation of fibroblast stem cells

[0079] 1) Pretreatment: Soak the collected skin tissue in a culture dish containing alcohol for 1 minute, rinse with saline with 1% double antibody by mass for 3-5 times, and remove the fat tissue with hemostatic forceps and scissors, and try to remove it as cleanly as possible. Double antibody refers to a solution containing penicillin and streptomycin, where penicillin is 100IU / ml and streptomycin is 100IU / m1. Separate the dermis and epidermis: Cut the skin tissue into strips of 2x3mm and digest it with 0.25% trypsin at 37℃ for 1h. After digestion, remove the epidermis and rinse it with saline.

[0080] 2) Primary culture: After the dermis is infiltrated with the culture medium containing cytokines, the bottom layer of the dermis (the side where the dermis contacts the fat) is spread on the bottom of the T25 culture flask, leaving appropriate spacing between the dermis layers to facilitate the crawling out of tissue cells. Place the laid culture flask in the incubator and culture it for 1 hour. Then add 2 ml of DMEM culture medium containing 100 ng / ml β-FGF to the T25 culture flask and culture it overnight (handle it with care to prevent the adherent tissue blocks from being moved off). The incubator conditions are 37°C and 5% carbon dioxide concentration. Afterwards, replenish the culture medium to 5 ml every day, and handle it with care to prevent the adherent tissue blocks from being moved off. When the tissue block cells crawl out and cover the bottom of the flask, and the cell fusion reaches 50-80%, they can be passaged. Passage is performed after digestion with trypsin, and quality control is performed during the cell expansion process to ensure that they are autologous fibroblast stem cells. Each culture cycle is 7-10 days, until the cells reach 10 6 Cells can be collected at a rate of 1 cell / cm2.

[0081] 3) Quality Control:

[0082] During the cell expansion process, the surface markers of the cells were analyzed by flow cytometry (FACS) to ensure that the cells were fibroblast stem cell types. A small amount of cells were taken and diluted with physiological saline to a density of 1x10 6 5 mL of monoclonal antibodies against CD90, CD105, CD73, CD45, CD34 and CD14 were respectively aspirated into a stem cell suspension of 100 cells / mL, and 500 μL of the cell suspension was added. The cells were incubated at room temperature in the dark for 20 min. A blank isotype control group was set up at the same time: centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS containing 10% FBS, and then resuspended in 500 mL of PBS and sieved for detection by flow cytometry. The quality control standards that more than 95% of the cells expressed CD90, CD105 and CD73, and more than 95% of the cells did not express CD45, CD34 and CD14 were met.

[0083] During the cell expansion process, it is also necessary to observe the cell morphology under a microscope to ensure that the cells do not show atypia or mutation.

[0084] Example 3: Combined application of platelet-rich plasma and fibroblast stem cells

[0085] The PRP prepared in Example 1 and the fibroblast stem cells prepared in Example 2 were injected together, and the same injection method was used for both: after local infiltration anesthesia was performed on the cheek of the subject (2-3 cm below the zygomatic bone), a 30G long needle was selected and connected and fixed with a 1ml syringe, and the needle was inserted into the subcutaneous layer. The change in the position of the needle could be seen through the skin. The injection was performed by withdrawing the needle while injecting. After the injection, a wet compress of saline gauze was applied, and a cold compress of soft ice was applied for more than 2 hours. The injection method was to inject PRP first, and then inject fibroblast stem cells 7 days later; the injection volume was: 5 mL of PRP injection each time, and 1 mL of fibroblast stem cell injection each time; the treatment was performed once a month, and the cycle was 3 months.

[0086] Comparative Example 1: Preparation of Platelet-Rich Plasma

[0087] PRP was prepared according to the method of Example 1, except that the concentration of growth factors in PRP did not meet the standard. Finally, VEGF: 168.5 ng / mL; bFGF: 85.4 ng / mL; PDGF: 54.9 ng / mL were obtained in PRP.

[0088] Comparative Example 2: Preparation of fibroblast stem cells

[0089] Fibroblast stem cells were prepared according to the method of Example 2, except that DEME medium without β-FGF was used for culture.

[0090] Effect example 1: Skin injection and testing

[0091] The 80 subjects were randomly divided into 8 groups, each with 10 people, and the average age of each group was basically the same. The injection scheme for each group was as follows:

[0092] Group 1: The method of Example 3 was followed, except that both PRP and fibroblasts were replaced with hyaluronic acid (Restylane, Q-Med AB);

[0093] Group 2: according to the method of Example 3;

[0094] Group 3: The method of Example 3 was followed, except that the amount of PRP injection was changed to 10 ml;

[0095] Group 4: The method of Example 3 was followed, except that the amount of PRP injected was 0;

[0096] Group 5: The method of Example 3 was followed, except that the amount of fibroblast stem cells injected was 0;

[0097] Group 6: The method of Example 3 was followed, except that the PRP prepared in Example 1 was replaced by the PRP prepared in Comparative Example 1;

[0098] Group 7: The method of Example 3 was followed, except that the fibroblast stem cells prepared in Example 2 were replaced by the fibroblast stem cells prepared in Comparative Example 2;

[0099] Group 8: The method of Example 3 was followed, except that the PRP prepared in Example 1 was replaced by the PRP prepared in Comparative Example 1 and the fibroblast stem cells prepared in Example 2 were replaced by the fibroblast stem cells prepared in Comparative Example 2.

[0100] During the 3-month injection cycle and within 1 month after the injection, all 80 subjects experienced no adverse reactions and felt well. Figure 1 Shown are the facial skin conditions of a subject in Group 2 before and after treatment. Figure 1 A shows the overall condition of the subject's facial skin before and after treatment. It can be seen that the skin condition is significantly better after treatment. Figure 1 B shows the changes in the cheek area of ​​the subject before and after treatment. It can be seen that after treatment, the subject's cheek scar became lighter (elasticity increased) and the skin color became whiter (melanin decreased).

[0101] Detection method:

[0102] Before injection and 30 days after the 3-month injection cycle, the Danish Cortex DermaLab skin tester (see Figure 2 ) The cheek area of ​​the volunteers in groups 1-8 was tested, and collagen, elastic recovery rate and melanin index were selected as analysis indicators (the computer operation interface for elastic recovery rate R2 determination is shown in Figure 3 , R2 = Ua / Uf), and measure 4 times in succession and take the average value as the measurement result for one person.

[0103] Data Analysis:

[0104] The index data before and after treatment were statistically analyzed, and SPSS20.0 software was used to perform repeated analysis of variance and t-test on the data. P < 0.05 was considered to be statistically significant, and P < 0.01 represented a significant statistical difference.

[0105] Table 1 Index determination of different groups before and after PRP and fibroblast stem cell combination treatment

[0106]

[0107]

[0108] Note: * indicates that the data before and after treatment have significant differences (P<0.05); ** indicates that the data before and after treatment have strong significant differences (P<0.01).

[0109] The data of each group before and after treatment were compared and the change range was calculated.

[0110] Change range = (average value after treatment - average value before treatment) ÷ average value before treatment × 100%,

[0111] The data of the change range of each group are statistically summarized in Table 2. The analysis results show that:

[0112] Table 2 Statistics of changes in different groups before and after PRP and fibroblast stem cell combination treatment

[0113]

[0114]

[0115] 1) Collagen density

[0116] Before and after treatment with PRP alone (Group 5) and fibroblast stem cell alone (Group 4), the collagen density of the former increased by about 22%, and the latter increased by about 12%; from Group 2, it can be seen that before and after the combined treatment with PRP and fibroblast stem cells, the collagen density increased by an average of about 48%, which was significantly higher than that of the single treatment group and had a synergistic effect; in addition, from Groups 6-8, it can be seen that although the therapeutic effects of other combinations are also higher than those of the single treatment group, they do not have a synergistic effect.

[0117] 2) Elastic recovery rate

[0118] Before and after treatment with PRP alone (Group 5) and fibroblast stem cell alone (Group 4), the elastic recovery rate of the former increased by about 23%, and the latter increased by about 18%; from Group 2, it can be seen that before and after the combined treatment with PRP and fibroblast stem cells, the elastic recovery rate increased by an average of about 52%, which was significantly higher than that of the single treatment group and had a synergistic effect; in addition, from Groups 6-8, it can be seen that although the therapeutic effects of other combinations are also higher than those of the single treatment group, they do not have a synergistic effect.

[0119] 3) Melanin Index

[0120] Before and after treatment, the melanin index of the group treated with PRP alone (Group 5) and the group treated with fibroblast stem cells alone (Group 4) decreased by about 20% and 15% respectively; as can be seen from Group 2, before and after treatment with the combination of PRP and fibroblast stem cells, the melanin index decreased by an average of about 35%, which was significantly higher than that of the single treatment group.

[0121] Based on the above experimental data, the combination product of PRP and fibroblast stem cells provided by the present invention demonstrates its significant technical effect in the field of skin anti-aging. The combination product can effectively improve the skin improvement effect, especially in improving collagen density and skin elasticity, and its synergistic effect is unpredictable.

[0122] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for facial beauty, characterized in that: The composition comprises platelet-rich plasma and fibroblast stem cells, The method for preparing platelet-rich plasma comprises the following steps: S1. Obtain whole blood for use; S2, centrifuging whole blood to separate platelet-rich plasma; the growth factors in the platelet-rich plasma include VEGF, bFGF and PDGF, and the concentration requirements of the growth factors are VEGF ≥ 200 ng / mL, bFGF ≥ 100 ng / mL and PDGF ≥ 60 ng / mL; The method for preparing fibroblast stem cells comprises the following steps: a. Isolation of fibroblasts; b. Fibroblast stem cells are obtained by culturing with a culture medium, wherein more than 95% of the fibroblast stem cells express CD90, CD105 and CD73, and more than 95% of the cells do not express CD45, CD34 and CD14; the culture medium contains growth factors, and the growth factors include VEGF, bFGF, TGF-β and PDGF.

2. The composition according to claim 1, characterized in that The volume ratio of the platelet-rich plasma to the fibroblast stem cells is 5:1-10:

1.

3. The composition according to claim 1, characterized in that The step S2 comprises the following steps: The blood was centrifuged at 1000-2000g for 5-15 minutes, and then separated into three layers. The top two layers were centrifuged at 2000-4000g for 10-20 minutes to separate the platelet-rich plasma. The growth factor concentration was detected by ELISA.

4. The composition according to claim 1, characterized in that Step a comprises separating fibroblasts from a tissue sample by using a trypsin digestion method, wherein the tissue sample comprises skin tissue or adipose tissue.

5. The composition according to claim 1, characterized in that The culture period in step b is 7-10 days, until the cells reach 10 6 / cm2; the surface markers of the cells were analyzed by flow cytometry; the culture medium included DMEM culture medium containing bFGF.

6. A method for facial beauty, characterized in that: The method comprises injecting a composition according to any one of claims 1 to 5 into a subject's skin area.

7. The method according to claim 6, characterized in that The method comprises separately injecting platelet-rich plasma and fibroblast stem cells in the composition into a tested skin area of ​​a subject, and the injection volume ratio of the platelet-rich plasma to the fibroblast stem cells is 5:1-10:

1.

8. The method according to claim 7, characterized in that The method comprises the following steps: First, platelet-rich plasma is injected into the tested skin area, and the injection volume of the platelet-rich plasma is 5-10 mL; 5-7 days after the injection, fibroblast stem cells are injected into the tested skin area, and the volume ratio of the injection volume of platelet-rich plasma to the injection volume of fibroblast stem cells is 5:1-10:

1.

9. The method according to claim 8, characterized in that The injection volume of the platelet-rich plasma is 5 mL, and the volume ratio of the injection volume of the platelet-rich plasma to the injection volume of the fibroblast stem cells is 5:

1.

10. Use of the composition according to any one of claims 1 to 5 in preparing a product for improving skin condition, characterized in that: The applications include the following: 1) Application in the preparation of anti-skin aging products; 2) Application in the preparation of products for improving skin elasticity; 3) Application in the preparation of products for increasing collagen production in skin cells; 4) Application in the preparation of products for improving skin wrinkles; 5) Application in the preparation of products for improving skin sagging; 6) Application in the preparation of products for reducing skin melanin content.

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