WO2023103744A1

WO2023103744A1 - Method for extracting growth factors from platelets

Info

Publication number: WO2023103744A1
Application number: PCT/CN2022/132786
Authority: WO
Inventors: 蔡慧思; 蔡慧琪; 董婷霞; 詹华强
Original assignee: 无限发展有限公司
Priority date: 2021-12-07
Filing date: 2022-11-18
Publication date: 2023-06-15

Abstract

A method for extracting growth factors from platelets, comprising: centrifuging a blood sample of a preset volume to obtain platelet-rich plasma; removing iron ions in the platelet-rich plasma by using a magnetic base method; performing ultrasonic lysis on the platelet-rich plasma to obtain growth group platelet-rich plasma; adding non-protein inhibitors into the growth group platelet-rich plasma; using ultraviolet light to disinfect and sterilize the growth group platelet-rich plasma for a preset period of time; aliquoting and freezing the growth group platelet-rich plasma. The growth group platelet-rich plasma comprises platelet factor 4, β-thromboglobulin, epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor. The growth group platelet-rich plasma having a large amount of growth factors can be obtained by means of simple steps, and can be used for preparing skin regeneration products, skin moisturizing products, skin anti-aging products and products for promoting skin wound healing.

Description

Extraction method of platelet growth factor

technical field

The invention relates to the field of biotechnology, in particular to a method for extracting growth factors in platelets.

Background technique

Platelets in the blood store a large number of active growth factors, which play an extremely important role in promoting wound healing, cell proliferation and differentiation, and tissue formation. After the platelets rupture, they can release a large number of growth factors and cytokines. At present, platelet rich plasma (Platelet Rich Plasma, PRP), that is, PRP is more and more widely used in clinics, including orthopedics, ophthalmology, and plastic surgery.

However, PRP extracted from patients must be fresh and cannot be stored for a long time. Among them, the average lifespan of platelets in PRP is usually only 5 to 9 days after extraction, but a normal PRP treatment cycle takes 3 to 6 months. Usually, in order to extract PRP, the patient must undergo venipuncture and wait for more than 30 minutes each time receiving PRP treatment.

Contents of the invention

A method for extracting growth factors in platelets disclosed in the embodiments of the present invention can obtain growth group platelet-rich plasma containing a large amount of growth factors.

On the one hand, an embodiment of the present invention provides a method for extracting growth factors in platelets, comprising: centrifuging a blood sample with a preset volume to obtain platelet-rich plasma; using a magnetic seat method to remove iron in the platelet-rich plasma ions; the platelet-rich plasma is ultrasonically cracked to obtain the growth group platelet-rich plasma; non-protein inhibitors are added to the growth group platelet-rich plasma; Disinfection and sterilization; and aliquoting and freezing the growth group platelet-rich plasma; wherein, the growth group platelet-rich plasma includes platelet factor 4, β-thromboglobulin, epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor .

On the other hand, an embodiment of the present invention provides a method for extracting growth factors in platelets, comprising: separating the components of the blood sample to obtain platelet-rich plasma; removing impurities in the platelet-rich plasma; The platelet-rich plasma is subjected to ultrasonic cracking, so that the platelets in the platelet-rich plasma burst and release growth factors to obtain growth group platelet-rich plasma.

In one embodiment of the present invention, it includes: said growth group platelet-rich plasma includes platelet factor 4, β-thromboglobulin, epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor.

In one embodiment of the present invention, the extraction method further includes: adding non-protein inhibitors to the platelet-rich plasma of the growth population.

In one embodiment of the present invention, the non-protein inhibitor is ethylenediaminetetraacetic acid or ethylene glycol bis(2-aminoethyl ether)tetraacetic acid.

In one embodiment of the present invention, the extraction method further includes: performing disinfection and sterilization on the growth group platelet-rich plasma.

In an embodiment of the present invention, the sterilizing treatment of the growth group platelet-rich plasma specifically includes: using ultraviolet rays to sterilize the growth group platelet-rich plasma for 25-35 minutes.

In one embodiment of the present invention, the extraction method further includes: measuring the protein content in the platelet-rich plasma of the growth group by Bradford method.

In one embodiment of the present invention, the extraction method further includes: subpackaging and freezing the growth group platelet-rich plasma.

In one embodiment of the present invention, the growth group platelet-rich plasma can be used to prepare skin regeneration products, skin moisturizing products, skin anti-aging products, and products for promoting skin wound healing.

The above one or more technical solutions have the following advantages or beneficial effects: obtaining platelet-rich plasma by separating components of the blood sample; removing impurities in the platelet-rich plasma; performing ultrasonic cracking on the platelet-rich plasma, The simple steps of causing platelets in the platelet-rich plasma to burst and release growth factors can obtain growth group platelet-rich plasma with a large amount of growth factors, thereby avoiding multiple blood draws for patients during the PRP treatment cycle, and saving doctors and patients at the same time time and effort, and can reduce the production cost of PRP; in addition, by adding non-protein inhibitors to the platelet-rich plasma and disinfecting and sterilizing the growth group platelet-rich plasma for a preset time, the growth of bacteria can be inhibited, Further extend its shelf life for repeated use by patients; moreover, the obtained growth group platelet-rich plasma is widely used, which can significantly reduce skin pores, increase hemoglobin, skin firmness and elasticity, etc., and can be used to prepare Skin regeneration products, skin moisturizing products, skin anti-aging products, products for promoting skin wound healing.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For Those of ordinary skill in the art can also obtain other drawings based on these drawings without making creative efforts.

Fig. 1 is a schematic diagram of a method for extracting growth factors in platelets provided by an embodiment of the present invention.

Fig. 2 is a schematic diagram of a method for extracting growth factors in platelets provided by another embodiment of the present invention.

Figure 3 is a schematic diagram of platelet changes measured by a scanning electron microscope in a method for extracting growth factors in platelets provided by an embodiment of the present invention, and the results show that the platelets in the platelet-rich plasma are intact, and the platelets in the growth group platelet-rich plasma are completely broken .

Fig. 4 is a bar graph showing the release rate of platelet-released growth factors after platelet-rich plasma is ultrasonically treated for different times in a method for extracting growth factors from platelets according to an embodiment of the present invention.

Fig. 5 is a bar graph showing the release rate of each growth factor after adding EDTA and EGTA inhibitors to platelet-rich plasma and performing freeze-thaw cycles in a method for extracting growth factors in platelets provided by an embodiment of the present invention.

Fig. 6 is a schematic diagram showing the comparison of the contents of cytokines and growth factors in homologous whole blood WB, platelet-rich plasma and growth group platelet-rich plasma measured by ELISA.

Fig. 7 is a schematic diagram showing the comparison of the contents of growth factors EGF, VEGF and PDEGF in platelet-rich plasma and growth group platelet-rich plasma products stored at different temperatures for 1-6 months.

Fig. 8 is an experimental diagram and a schematic diagram of the analysis of the cell layer coverage of the wound healing experiment performed on HaCaT cells by platelet-rich plasma and growth group platelet-rich plasma products.

Figure 9 is a schematic diagram showing the comparison of transcription levels of COX2, COL1A1, MM2, TNF-α, COL2A1, COL4A1, COL5A1, COL5A2 and MMP9 genes related to HaCaT cell wound healing by platelet-rich plasma and growth group platelet-rich plasma.

Figure 10 is a schematic diagram of the comparison of the changes in the pores of the cheeks before and after 6 months of application of platelet-rich plasma or growth group platelet-rich plasma to the skin of volunteers.

Figure 11 shows the facial skin moisture, water retention capacity, whitening effect, hemoglobin level, oil level, skin gloss, firmness and elasticity of volunteers who received platelet-rich plasma or growth group platelet-rich plasma for 6 months. Schematic diagram of the comprehensive evaluation results of the skin before and without use.

12 is a schematic diagram of experiments comparing the T0, T4, and T8 cell layer coverages of HaCaT cells in growth group platelet-rich plasma and platelet-rich plasma of eel blood.

Fig. 13 is a schematic diagram of experiments comparing T0, T10, and T24 cell layer coverages of HaCaT cells in the growth group platelet-rich plasma and platelet-rich plasma of deer blood in the wound healing experiment.

Detailed ways

In order to facilitate the understanding of the present invention, the following will describe the present invention more fully. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The "growth group" mentioned in the embodiment of the invention refers to various growth factors contained in platelets in the blood, which can be collected by ultrasonically destroying the platelet membrane and then fully released, mainly including platelet-derived growth factor (PDGF), Vascular endothelial cytokine (VEGF) and epidermal growth factor (EGF), etc.

In the embodiment of the present invention, the blood (or blood sample) used is from human or other animals. The prepared growth population platelet-rich plasma can be used for the individual from whom the blood was drawn.

It should also be noted that the division of multiple embodiments in the present invention is only for the convenience of description, and should not constitute a special limitation, and features in various embodiments can be combined and referred to each other if there is no contradiction.

[Example 1]

Referring to Fig. 1, Embodiment 1 of the present invention proposes a method for extracting platelet growth factor in blood. The method for extracting growth factors in platelets includes, for example, the following steps S11 to S21.

S11: Centrifuge the blood sample with a preset volume to obtain platelet rich plasma (Platelet Rich Plasma, PRP);

S13: Using a magnetic seat method to remove iron ions in the platelet-rich plasma;

S15: ultrasonically cracking the platelet-rich plasma to obtain growth group platelet-rich plasma (Platelet Rich Plasma Plus, PRP Plus);

S17: Adding non-protein inhibitors to the platelet-rich plasma of the growth population;

S19: Disinfect and sterilize the platelet-rich plasma of the growth group for a preset time by using ultraviolet rays;

S21: Aliquoting and freezing the platelet-rich plasma of the growth population.

Wherein, the growth group platelet-rich plasma includes platelet factor 4 (PF4), β-thromboglobulin (β-TG), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) .

Specifically, the preset volume of blood sample mentioned in step S11 is, for example, a blood sample of 30ml-60ml. The referenced blood can be of human or other animal origin. The centrifugation mentioned can be understood as the separation of components with different specific gravity in blood by means of centrifugal force. The ultrasonic lysis mentioned in S15 can be understood as the complete rupture of platelets to release the internal growth factors and cytokines, as shown in Figure 3 . The preset time mentioned in step S19 is, for example, 30 minutes.

To sum up, in the method for extracting platelet growth factor provided in Example 1 of the present invention, a blood sample with a preset volume is centrifuged to obtain platelet-rich plasma, and the platelet-rich plasma is removed by using a magnetic seat method. Iron ions of the platelet-rich plasma were ultrasonically cracked to obtain growth group platelet-rich plasma, non-protein inhibitors were added to the growth group platelet-rich plasma, and ultraviolet rays were used to preset the growth group platelet-rich plasma Timely disinfection and sterilization, as well as aliquoting and freezing the growth group platelet-rich plasma, can obtain growth group platelet-rich plasma with a large amount of growth factors and a longer shelf life.

In this embodiment, the method for extracting the growth factor in platelets further includes, for example: measuring the protein content in the platelet-rich plasma of the growth group by using the Bradford method.

[Example 2]

Referring to Fig. 2, Embodiment 1 of the present invention proposes a method for extracting growth factors in platelets. The method for extracting growth factors in platelets includes, for example, the following steps S31 to S35.

S31: Separate the components of the blood sample to obtain platelet rich plasma (Platelet Rich Plasma, PRP);

S33: removing impurities in the platelet-rich plasma;

S35: Ultrasonic lysing the platelet-rich plasma to burst platelets in the platelet-rich plasma and release growth factors to obtain growth group platelet rich plasma (Platelet Rich Plasma Plus, PRP Plus).

Wherein, the blood sample mentioned in step S31 may be from human beings or from other animals. The separation mentioned is, for example, to separate blood components by centrifugation, but it is not limited here, as long as the same or similar functions can be achieved. The impurities mentioned in step S33 are, for example, iron ions, which are removed by, for example, a magnetic seat method. The ultrasonic lysis mentioned in step S35 can be understood as complete rupture of platelets to release internal growth factors and cytokines, as shown in FIG. 3 .

Further, the growth group platelet-rich plasma mentioned in step S35 includes, for example, platelet factor 4 (PF4), β-thromboglobulin (β-TG), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and platelet Derived growth factor (PDGF).

Referring to Figure 4, the time of ultrasonic lysis will affect the release rate of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF). As can be seen from Figure 4, different ultrasound times are listed in the figure: 15 minutes, 30 minutes, 69 minutes, and 120 minutes. highest release rate. Therefore, preferably, the ultrasonic lysis time is 30 minutes.

Referring to Fig. 5 and Fig. 6, in this embodiment, the method for extracting growth factors in platelets further includes, for example: adding non-protein inhibitors to the platelet-rich plasma of the growth group.

Among them, non-protein inhibitors are, for example, ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA), which are inhibitors that do not cause skin allergies, and specific inhibitors The species here are not limited to EGTA and EDTA, as long as the same effect can be achieved, the specific concentration is not particularly limited, and can be any effective concentration that can inhibit microorganisms. In this example, the effect of adding non-protein inhibitors here is not only to prevent skin irritation, but also to inhibit microbial growth to further extend its shelf life. Comparing Figures 5 and 6, it can be seen that the addition of inhibitors EDTA and EGTA can promote the release of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) in platelets. In Figure 5, the "freeze-thaw" of the freeze-thaw cycle refers to freezing and then thawing, that is, freezing once and thawing once, which is different from the "repeated freezing and thawing" of the traditional technology (ie, freezing and thawing repeatedly).

In this embodiment, the method for extracting growth factors in platelets further includes, for example: disinfecting and sterilizing the growth group platelet-rich plasma.

Wherein, the disinfection and sterilization treatment of the growth group platelet-rich plasma is specifically: using ultraviolet rays to disinfect and sterilize the growth group platelet-rich plasma for 25-35 minutes, preferably 30 minutes, so as to achieve sterilization, and then prolong its life. The role of shelf life. It is worth noting that after the platelet-rich plasma of the growth group is sterilized, it needs to be immediately divided into sterilized microcentrifuge tubes, freeze-dried, and stored in a -80°C low-temperature refrigerator for later use.

Wherein, the protein content of the test solution is measured by the Bradford method, so that the samples distributed in each sterilized microcentrifuge tube have about the same amount of protein, such as 1 mg of protein. Note here that before using the growth group platelet-rich plasma sample, it is necessary to add sterile saline (0.9% NaCl) to dissolve the protein.

In this embodiment, the method for extracting growth factors in platelets further includes, for example: subpackaging and freezing the growth group platelet-rich plasma, so as to prolong its shelf life and use it multiple times.

Furthermore, the growth group platelet-rich plasma obtained based on the above method can be used to prepare skin regeneration products, skin moisturizing products, skin anti-aging products, and products for promoting skin wound healing.

To sum up, in the method for extracting platelet growth factor provided by Example 2 of the present invention, each component of the blood sample is separated to obtain platelet-rich plasma, and impurities in the platelet-rich plasma are removed; The simple steps of performing ultrasonic cracking of the platelet-rich plasma to make the platelets in the platelet-rich plasma burst and release growth factors can obtain growth group platelet-rich plasma with a large amount of growth factors, thereby avoiding multiple times of PRP treatment cycles for patients. Blood drawing saves the time and energy of doctors and patients, and can reduce the production cost of PRP; in addition, by adding non-protein inhibitors to the growth group platelet-rich plasma, the steps of disinfection and sterilization of the growth group platelet-rich plasma , can inhibit the growth of bacteria, and greatly prolong the shelf life of the growth group platelet-rich plasma; in addition, the growth group platelet-rich plasma can be subpackaged and frozen for users to take multiple times; moreover, based on the extraction method of the growth factor in the platelets, the The growth group platelet-rich plasma is widely used, and it can be applied to the preparation of skin regeneration products, skin moisturizing products, skin anti-aging products, and skin wound healing products.

[Embodiment 3]

Referring to Figure 6, by using the ELISA (enzyme linked immunosorbent assay, enzyme-linked immunosorbent assay) kit to detect the cells in PRP, PRP Plus prepared in the above-mentioned Example 1 or Example 2, and homologous whole blood WB without any treatment factors and growth factors.

Among them, specific examples of cytokines and growth factors include: platelet factor-4 (PF 4), β-thromboglobulin (β-TG), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) content, its specific detection steps are as follows:

(1) WB of PRP, PRP Plus prepared by the above method and homologous whole blood without any treatment.

(2) Using Human PF4, β-TG, EGF, VEGF, and PDGF ELISA kits, add PRP, PRP Plus, and homologous whole blood WB without any treatment to the coatings in different ELISA kits, respectively. In a 96-well plate with human PF4, β-TG, EGF, VEGF, and PDGF antibodies, add 50 μL of sample reagent to each well, make 3 replicate wells for each sample, and incubate at room temperature for 30 minutes.

(3) Add the mixture containing capture antibody and detection antibody to a 96-well plate, 50 μL per well, and incubate at room temperature for 2 hours.

(4) Wash 3 times with washing buffer.

(5) Add the substrate (tetramethylbenzidine) to the 96-well plate and incubate for 10 minutes in the dark.

(6) Add the stop solution to the 96-well plate, 100 μL per well.

(7) Measure the absorbance at 450nm, and calculate the content of human PF-4, β-TG, EGF, VEGF, and PDGF.

(8) Compare the content of each factor in WB of PRP, PRP Plus and homologous whole blood without any treatment.

It can be seen from Figure 6 that compared with PRP and homologous whole blood WB without any treatment, PRP Plus contains significantly more cytokines and growth factors. The amount of β-TG, EGF and VEGF released in PRP Plus is almost twice that of whole blood WB or PRP, which is significantly better than PRP and WB. This result shows that the present invention can obtain more cytokines and growth factors, such as growth factors such as PF4, β-TG, EGF, VEGF, PDGF, by preparing PRP Plus. These active growth factors can be quickly detected by ELISA, so they can be used as technical quality control parameters of PRP Plus.

[Example 4]

Referring to Fig. 7, the PRP and PRP Plus prepared by the above-mentioned embodiment one or embodiment two were stored at different temperatures for 6 months by detecting each growth factor: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and platelet-derived Growth factor (PDGF) content to determine the stability of PRP Plus.

Specifically, the levels of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were measured when PRP and PRP Plus were stored at 4°C, -20°C, and -80°C for 1 to 6 months. Activity level, as can be seen from Figure 7, compared with PRP and WB, PRP Plus has significantly better stability of epidermal growth factor (EGF), and can be stored for more than 6 months. Stored at -80°C, the content of each growth factor is relatively stable, which is the best storage condition.

[Embodiment 5]

Referring to Figure 8, by using HaCaT cells (skin keratinocytes), the PRP and PRP Plus obtained in the above-mentioned embodiment one or embodiment two are applied to the wound healing experiment, and the specific steps are as follows:

(1) Adopt the PRP and PRP Plus prepared in the above-mentioned embodiment one.

(2) Plant the HaCaT cells in the wells of a 12-well plate and put them in a cell culture incubator until the cells are completely densely packed.

(3) Use a pipette tip of 200 μL to draw a cross at the center of the well bottom of each 12-well plate to simulate wound formation, and record the coverage of the cell layer under a microscope with a magnification of 50 times, which is designated as T0.

(4) Add blank control, positive control vascular endothelial growth factor (VEGF), PRP, and PRP Plus to the 6-well plate respectively.

(5) After adding the sample and incubating for 8 hours, the coverage rate of the cell layer was photographed again, which was defined as T8.

(6) Use Image J software to analyze and compare cell layer coverage.

See Figure 8 (left) for a schematic diagram of the cell layer coverage of HaCaT cells, which correspond to the blank control, PRP, positive control (VEGF) and PRP Plus from left to right, and T0 and T8 from top to bottom. After the cells were cultured for 8 hours, the morphological observation showed that the wound healing rate of cells treated with PRP Plus was significantly higher than that of PRP, at least doubled. Fig. 8 (right) is an analysis diagram of the cell layer coverage of HaCaT cells, which is the coverage ratio of each group of experiments compared with the blank control after 8 hours. It can be concluded that PRP Plus technology can speed up wound healing.

[Embodiment 6]

Referring to Figure 9, based on the PRP and PRP Plus prepared in Example 1 or Example 2 above, real-time fluorescent quantitative PCR method was used to detect COX2, COL1A1, MM2, TNF-α, COL2A1, COL4A1, COL5A1, COL5A2 and The transcription level of MMP9 gene, the specific steps are as follows:

(1) PRP and PRP Plus prepared in the above-mentioned embodiment 1 are adopted.

(2) HaCaT cells were planted in a 100 mm culture dish, and blank control, positive control (VEGF), 10% PRP, 10% PRP Plus were added and cultured for 24 hours.

(3) Total cellular RNA was extracted using RNA zol and reverse transcribed into cDNA (complementary DNA).

(4) use

480 SYBR Green performs real-time fluorescence quantitative polymerase chain reaction on equal amounts of each cDNA sample to detect the relative transcription levels of COX2, COL1A1, MM2, TNF-α, COL2A1, COL4A1, COL5A1, COL5A2 and MMP9 genes, using GAPDH gene level as an internal reference.

It can be seen from Figure 9 that after HaCaT was treated with PRP and PRP Plus, it was similar to the positive control vascular endothelial cytokine VEGF, and the gene expression of inflammatory factors COX2 and COL1A1 was significantly increased. MM2 and TNF-α factor gene expression increased by 10%-20% after PRP Plus or VEGF treatment. But PRP was not shown to turn on and promote the expression of these genes. The gene expressions of COL2A1, COL4A1, COL5A1, COL5A2 and MMP9 were slightly increased after PRP Plus treatment. It can be seen that PRP Plus has a significant effect on wound healing and repair, which is different from that of PRP.

[Embodiment 7]

Referring to Fig. 10, the PRP and PRP Plus prepared in the above-mentioned embodiment 1 or embodiment 2 are applied to the individual facial skin. In this example, 20 healthy volunteers were used to conduct a 6-month experiment on autologous facial skin, using instruments to detect the skin before and after using PRP and PRP Plus products. The age distribution range of the 20 healthy volunteers was: : 20 to 75 years old; 7 males and 13 females. They were randomly divided into two groups, with 10 subjects in each of PRP and PRP Plus. The test period is six months, using PRP or PRP Plus once a month. When evaluating the skin, the room temperature is 22±2°C, the relative humidity is controlled at 50±5%, and the test sites: forehead, left cheek, right cheek, and chin.

The specific steps for carrying out the experiment are as follows:

(1) Select 20 voluntary healthy individuals (age 20-75 years old, including male and female); and evaluate the facial skin of each individual before use.

(2) Prepare autologous PRP and PRP Plus by the above method, and use the Bradford method to determine the protein content before freeze-drying; and record it with a two-dimensional code label.

(3) Before use, dissolve lyophilized PRP or PRP Plus in sterilized normal saline to 1mg/ml.

(4) Apply the dissolved autologous PRP or PRP Plus to the autologous facial skin that has been microneedled. Go through a makeup remover and cleansing treatment before treatment. Treatments were done once a month for 6 months. 6 times in total.

(5) One week after the sixth skin treatment with autologous PRP or PRP Plus, the facial skin of the individual after 6 months of use was evaluated.

Use a skin detector such as VISIA (Canfield Scientific Inc, NJ) to evaluate and analyze the number and diameter of pores on the cheeks of the facial skin.

It can be seen from Figure 10 that after the skin is treated with PRP or PRP Plus, the number of pores can be reduced, and the pores of the subjects treated with PRP Plus are significantly reduced (Figure 10A). The pores of volunteers in the PRP group were reduced by 60%, and the pores of all volunteers in the PRP Plus group were reduced by more than 80% (Fig. 10B). PRP Plus product treatment minimizes pore diameter compared to PRP product treatment.

Secondly, the parameters of the skin surface are tested, eg with the Courage+Khazaka Electronics GmbH (C+K) system (Cologne, Germany). Use the six probes of CK skin tester: moisture test probe, water loss test probe, melanin test probe, oil test probe, gloss test probe, elasticity test probe to test the skin hydration, water retention, whitening effect, Erythema level, sebum level, gloss and elasticity were tested. A series of skin parameters were evaluated on 20 subjects using 6 probes connected to the C+K skin testing system, including skin moisture, water retention capacity (testing water loss rate), whitening effect (reduction of melanin content), erythema Level (level of hemoglobin content), skin oil level, radiance, firmness and elasticity of the skin. After 6 months of treatment with PRP Plus, the skin's ability to retain water, increase hemoglobin levels, reduce skin oil and improve skin gloss is superior to that of PRP. Compared with the skin without PRP or PRP Plus treatment, both PRP and PRP Plus significantly enhanced the firmness and elasticity of the skin.

It can be seen from Figure 11 that compared with PRP, PRP Plus has a stronger effect of shrinking pores, and can improve skin moisturizing ability, reduce sebum, and increase skin rosiness, which can be applied to autologous skin wound healing, skin regeneration, skin whitening, Or skin antioxidant/anti-aging. It can also further prepare articles related to skin regeneration, skin moisturizing, skin anti-aging or assisting skin wound healing according to its efficacy.

[Embodiment Eight]

Referring to FIG. 12 , eel blood was used to prepare PRP and PRP Plus based on the preparation method provided in Example 1 or Example 2 above, so as to perform wound healing experiments on HaCaT cells. Comparing the cell layer coverage of PRP and PRP Plus in eel blood on HaCaT cells in wound healing experiments at T0, T4, and T8, it can be seen from the figure that PRP Plus in eel blood is significantly better than PRP in eel blood in promoting wound healing.

[Embodiment 9]

Referring to FIG. 13 , based on the preparation method provided in the first or second example above, deer blood was used to prepare PRP and PRP Plus, so as to conduct wound healing experiments on HaCaT cells. Comparing the cell layer coverage rate of PRP and PRP Plus of deer blood on HaCaT cells in wound healing experiments at T0, T10, and T24, it can be seen from the figure that PRP Plus of deer blood is significantly better than PRP of deer blood in promoting wound healing.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims

A method for extracting growth factors in platelets, comprising:

Centrifuge a blood sample with a preset volume to obtain platelet-rich plasma;

Using a magnetic seat method to remove iron ions in the platelet-rich plasma;

Ultrasonic lysing the platelet-rich plasma to obtain growth group platelet-rich plasma;

adding a non-proteinaceous inhibitor to said growth population platelet-rich plasma;

Disinfecting and sterilizing the platelet-rich plasma of the growth population for a predetermined time by using ultraviolet light; and

Packing and freezing the growth group platelet-rich plasma;

Wherein, the growth group platelet-rich plasma includes platelet factor 4, β-thromboglobulin, epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor.
A method for extracting growth factors in platelets, comprising:

Separate the components of the blood sample to obtain platelet-rich plasma;

removing impurities from the platelet-rich plasma;

Ultrasonic lysis is performed on the platelet-rich plasma to burst platelets in the platelet-rich plasma and release growth factors to obtain growth group platelet-rich plasma.
The method for extracting growth factors in platelets according to claim 2, wherein said growth group platelet-rich plasma comprises platelet factor 4, β-thromboglobulin, epidermal growth factor, vascular endothelial growth factor and platelet-derived growth factor .
The extraction method of platelet growth factor according to claim 2, is characterized in that, also comprises:

A non-protein inhibitor is added to the growth population platelet rich plasma.
The method for extracting growth factors in platelets according to claim 4, wherein the non-protein inhibitor is ethylenediaminetetraacetic acid or ethylene glycol bis(2-aminoethyl ether)tetraacetic acid.
The extraction method of platelet growth factor according to claim 2, is characterized in that, also comprises:

Disinfect and sterilize the growth group platelet-rich plasma.
The method for extracting growth factors in platelets according to claim 6, wherein said sterilizing and sterilizing said growth group platelet-rich plasma is specifically: using ultraviolet rays to sterilize said growth group platelet-rich plasma Bacteria for 25-35 minutes.
The extraction method of platelet growth factor according to claim 1 or 2, is characterized in that, also comprises:

The protein content in the platelet-rich plasma of the growth population was measured using the Bradford method.
The extraction method of platelet growth factor according to claim 2, is characterized in that, also comprises:

Aliquot and freeze the growth population platelet rich plasma.
The method for extracting growth factors in platelets according to any one of claims 1-9, wherein the growth group platelet-rich plasma is used for preparing skin regeneration products, skin moisturizing products, skin anti-aging products, and promoting skin wounds. Healing supplies.