CN111467374A - Novel leukocyte extract mixed factor and preparation method thereof - Google Patents
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Abstract
The invention discloses a novel leukocyte extract mixed factor and a preparation method thereof, wherein the novel leukocyte extract mixed factor comprises 10-30% by volume of leukocyte extracts, 20-40% by volume of platelet lysates, 10-20% by volume of stem cell extracts a, 10-20% by volume of stem cell extracts b and 20-40% by volume of stem cell extracts c. The invention takes the white blood cells as the main components, obtains the white blood cell extract after separation and extraction, and obtains the novel white blood cell extract mixed factor after mixing with the platelet lysate and the stem cell extract according to a certain proportion, and the novel white blood cell extract mixed factor contains a large amount of natural cell factors and has obvious effect on repairing skin injury.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a novel leukocyte extract mixed factor and a preparation method thereof.
Background
The leucocytes mainly comprise T lymphocytes, B lymphocytes, NK cells and the like. The leucocyte plays a role in paracrine and endocrine regulation on adjacent cells and distant cells by releasing cytokines, and plays a good role in the growth of tissue cells, the balance of immunity and anti-inflammation. The Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF) and Nerve Growth Factor (NGF) released by the polypeptide can directionally promote the growth of various cells, promote the formation of intercellular matrix and promote the repair of damaged tissues. However, the traditional leukocyte extracts still have more problems, the repair needs to activate endogenous stem cells in the process of skin formation and repair, and the exosomal synergistic effect is needed for the presentation of external factors. In recent years, research shows that a large amount of cytokines including fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF) and the like can be released in the mesenchymal stem cell culture process, so that the mesenchymal stem cell culture method is beneficial to angiogenesis of skin microenvironment and accelerates the skin repair process. The culture supernatant of the stem cells can secrete a large number of exosomes, has a good synergistic effect on DNA, RNA and factor delivery processes in the skin repair stage, and meanwhile, intracellular factors and gene fragments of the stem cells can be rapidly presented to the damaged parts of the skin under the synergistic effect of the exosomes.
Disclosure of Invention
The invention provides a novel leukocyte extract mixed factor and a preparation method thereof for better repairing skin injury.
The invention is realized by the following technical scheme:
a novel leukocyte extract mixed factor comprises 10-30% by volume of leukocyte extract, 20-40% by volume of platelet lysate, 10-20% by volume of stem cell extract a, 10-20% by volume of stem cell extract b and 20-40% by volume of stem cell extract c.
A preparation method of a novel leukocyte extract mixed factor comprises the following steps:
a. the leukocyte extract is prepared by treating leukocytes, adding sterile ultrapure water to adjust density to 2 × 106~3×106Performing freeze dissolution by liquid nitrogen, and filtering by 0.45um filter to obtain leukocyte extract;
b. preparation of platelet lysate: freezing platelet plasma at-80 deg.C, dissolving in water bath at normal temperature, and filtering with 0.45um filter to obtain platelet lysate;
c. preparation of stem cell extract a: culturing the mesenchymal stem cells for 48-96 hours at 37 ℃, under the conditions of 5% of volume concentration of carbon dioxide and 2-8% of volume concentration of oxygen, and collecting supernatant to obtain a stem cell extract a;
d. preparation of stem cell extract b: culturing the mesenchymal stem cells for 48-96 hours under the condition that the oxygen volume concentration is 2-8%, adding a compound electrolyte injection after the normal atmospheric oxygen partial pressure is recovered to 17-20% of the oxygen concentration, culturing for 24-36 hours, and collecting a supernatant to obtain a stem cell extract b;
e. preparation of stem cell extract c: after the mesenchymal stem cells are subjected to hypoxic culture and normal oxygen partial pressure is restored, quickly freezing the cell suspension at-60 to-90 ℃ until the cell suspension is completely frozen, slowly thawing at 33 to 40 ℃ to break the cells and release intracellular factors, centrifuging, and filtering through a 0.45um filter to obtain a stem cell extract c;
f. uniformly mixing the leukocyte extract, the platelet lysate, the stem cell extract a, the stem cell extract b and the stem cell extract c in proportion, and filtering by using a 0.22um filter to obtain the novel leukocyte extract mixed factor.
Further, the specific preparation method of the leukocyte extract in the step a is as follows:
① collecting umbilical cord blood of newborn, hepatitis B, hepatitis C, syphilis serum, and HIV, centrifuging, removing upper layer blood plasma layer containing platelet, and collecting lower layer containing erythrocyte and leukocyte;
② adding the compound electrolyte injection with the same volume, centrifuging for 15-20 min at 1500-2000 rpm/min by using a centrifuge, and collecting the middle leucocyte layer;
③ adding erythrocyte lysate, cracking erythrocytes for 3-5 min, adding compound electrolyte injection, centrifuging, washing, and collecting bottom layer leukocytes;
④ discarding supernatant, adding sterile ultrapure water, adjusting leukocyte concentration to 2 × 106~3×106Transferring the cells/ml into a plasma bag, placing the plasma bag at the temperature of 30 ℃ for 30-60 min, then quickly transferring the cells/ml into liquid nitrogen, and after the cells/ml are completely frozen, placing the cells/ml in a constant temperature box at the temperature of 25 ℃ until the cells/ml are completely melted;
⑤ centrifuging the leukocyte solution at 3000-4000 rpm, collecting the supernatant, filtering with 0.45um sterile filter membrane, and collecting the solution, i.e. leukocyte extract.
Further, the specific preparation method of the platelet lysate in step b is as follows:
① collecting blood plasma layer containing platelet in umbilical cord blood of newborn, placing into a refrigerator of-80 deg.C for quick freezing, placing into water bath of 25-33 deg.C for quick thawing to make platelet in blood plasma crack and release factor, and repeatedly freezing and thawing for 2-4 times;
② 2500-3500 rpm/min for 15-20 min, collecting supernatant, filtering with 0.45um sterile filter membrane, and collecting solution, i.e. platelet lysate.
Further, the specific preparation method of the stem cell extract a and the stem cell extract b in the steps c and d is as follows:
① reviving the human mesenchymal stem cells of P3-P5 generation in the cell bank, inoculating 45000-80000 cells/ml, adding a serum-free culture solution, and culturing under the condition of a hypoxia loop, wherein the concentration of carbon dioxide is 5% by volume and the concentration of oxygen is 2-8% by volume at 37 ℃;
②, culturing for 48-96 hours, and collecting cell supernatant, namely the stem cell extract a, when the cell fusion degree reaches 85-95%;
③ washing cells with normal saline for 3 times, adding 80000-150000 cells/ml compound electrolyte injection, culturing at 37 deg.C and 5% carbon dioxide volume under normal oxygen partial pressure;
④ for 24-36 hours, and collecting cell supernatant, namely stem cell extract b.
Further, the specific preparation method of the stem cell extract c in the step e is as follows:
① adding prepared 1-4 ℃ physiological saline into a cell culture bottle, adding the physiological saline into the cell culture bottle at 200000-300000 cells/ml, gently blowing and beating the cells by using a pipette, and collecting the cells into a centrifuge tube;
rapidly freezing at ② -60 to-90 ℃ until the cells are completely frozen, keeping for 3-5 min, and slowly thawing in a thermostat at 33-40 ℃ to break the cells and release intracellular factors;
③ 3500-4500 rpm/min, centrifuging at 2-8 deg.C for 25-35 min, collecting supernatant, and filtering with 0.45um sterile filter to obtain stem cell extract c.
Further, in the step f, 10-30% by volume of the leukocyte extract, 20-40% by volume of the platelet lysate, 10-20% by volume of the stem cell extract a, 10-20% by volume of the stem cell extract b and 20-40% by volume of the stem cell extract c are mixed, and the mixture is filtered by using a 0.22um filter to obtain the novel leukocyte extract mixed factor.
The invention has the beneficial effects that:
in the skin repair process of the leukocyte extract mixed factor, the exosome (stem cell extract a) can promote the cell factor (stem cell extract b), the platelet lysate and the leukocyte extract to be transported to the damaged cortex, so that sufficient DNA, RNA, protein factors and the like are provided for the cell growth process, and the skin repair process is accelerated. The leukocyte extract mixed factor has good effects of repairing tissue injury and improving skin, and has wide clinical application prospect.
Drawings
FIG. 1 is a diagram of human adipose-derived mesenchymal stem cells cultured by the method of example 1 of the present invention;
FIG. 2 is a graph showing a comparison between the amounts of cytokines secreted by the leukocyte extracts and the novel leukocyte extract mixed factor in example 1 of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions of the present invention are further described below by way of specific embodiments with reference to the drawings of the specification.
A method for preparing a novel leukocyte extract mixed factor comprises the following steps:
1. preparing a leukocyte extract:
① collecting umbilical cord blood of newborn, hepatitis B, hepatitis C, syphilis serum, and HIV, centrifuging, removing upper layer blood plasma layer containing platelet, and collecting lower layer containing erythrocyte and leukocyte.
② adding the compound electrolyte injection with the same volume, centrifuging for 15-20 min at 1500-2000 rpm/min by using a centrifuge, and collecting the middle leucocyte layer.
③ adding erythrocyte lysate, cracking erythrocytes for 3-5 min, adding compound electrolyte injection, centrifuging, washing, and collecting leukocytes on the bottom layer.
④ discarding supernatant, adding sterile ultrapure water, adjusting leukocyte concentration to 2 × 106~3×106And (4) transferring the cells/ml into a plasma bag, placing the plasma bag at the temperature of 30 ℃ for 30-60 min, then quickly transferring the cells/ml into liquid nitrogen, and after the cells/ml are completely frozen, placing the cells/ml in a constant temperature box at the temperature of 25 ℃ until the cells/ml are completely melted.
⑤ centrifuging the leukocyte solution at 3000-4000 rpm, collecting the supernatant, filtering with 0.45um sterile filter membrane, and collecting the solution, i.e. leukocyte extract.
2. Platelet lysate preparation
①, collecting the platelet-containing plasma layer obtained in the step 1, freezing the platelet-containing plasma layer in a refrigerator at the temperature of-80 ℃, rapidly thawing the platelet-containing plasma layer in a water bath at the temperature of 25-33 ℃, and repeatedly freezing and thawing for 2-4 times to release factors by platelet lysis.
③ 2500-3500 rpm/min for 15-20 min, collecting supernatant, filtering with 0.45um sterile filter membrane, and collecting solution, i.e. platelet lysate.
3. Cell culture:
① reviving the human mesenchymal stem cells of P3-P5 generation in the cell bank, inoculating 45000-80000 cells/ml, adding serum-free culture solution, and culturing under the condition of hypoxia ring at 37 ℃, wherein the volume concentration of carbon dioxide is 5% and the volume concentration of oxygen is 2-8%.
②, culturing for 48-96 hours, and collecting cell supernatant, namely the stem cell extract a, when the cell fusion degree reaches 85-95%.
③ washing cells with physiological saline for 3 times, adding 80000-150000 cells/ml compound electrolyte injection according to the cell number of 3- ① inoculated cells, culturing under normal oxygen partial pressure, 37 deg.C, and carbon dioxide volume concentration of 5%.
④ for 24-36 hours, and collecting cell supernatant, namely stem cell extract b.
4. Intracellular factor collection
① adding prepared 1-4 ℃ physiological saline into a cell culture bottle, adding the physiological saline into 200000-300000 cells/ml according to the cell number calculation when inoculating cells according to the '3- ①', gently blowing and beating the cells by using a 25ml pipette, and collecting the cells into a centrifuge tube.
Rapidly freezing at ② -60 to-90 ℃ until the cells are completely frozen, keeping for 3-5 min, and slowly freezing in a thermostat at 33-40 ℃ to crack the cells and release intracellular factors.
③ 3500-4500 rpm/min, centrifuging at 2-8 deg.C for 25-35 min, collecting supernatant, and filtering with 0.45um sterile filter to obtain stem cell extract c.
5. Preparation of novel leukocyte extract mixed factor
① leukocyte extract 10-30%, platelet lysate 20-40%, stem cell extract a 10-20%, stem cell extract b 10-20%, and stem cell extract c 20-40%.
② filtering with 0.22um filter to obtain the final product.
EXAMPLE 1 preparation of novel leukocyte extract Mixed factor
1. Under the condition that a hospital signs an informed consent, collecting 50ml of newborn umbilical cord blood, transporting the newborn umbilical cord blood back to a laboratory at 10 ℃ for 2h, detecting hepatitis B, hepatitis C, syphilis serum and HIV, detecting the newborn umbilical cord blood to be negative, centrifuging the newborn umbilical cord blood at 2000rpm/min for 15min, collecting 20ml of platelet-containing plasma on the upper layer, and collecting 20ml of erythrocyte-containing leukocyte-containing layer on the lower layer.
2. Preparing a leukocyte extract:
① mixing the collected erythrocyte and leukocyte layer 20ml with 20ml compound electrolyte solution, centrifuging at 1850rpm/min for 17min, and collecting the intermediate leukocyte layer.
② adding erythrocyte lysate, lysing erythrocytes for 3min, adding compound electrolyte injection, centrifuging, washing, and collecting leukocyte on bottom layer.
③ discarding supernatant, adding sterile ultrapure water 20ml, adjusting leukocyte concentration to 3 × 106Transferring the cells/ml into a plasma bag, placing the plasma bag at the temperature of 30 ℃ for 30min, then quickly transferring the cells to liquid nitrogen, and placing the cells in a constant temperature box at the temperature of 25 ℃ until the cells are completely thawed after the cells are completely frozen.
④ centrifuging leukocyte solution at 4000rpm for 15min, collecting supernatant, filtering with 0.45um sterile filter membrane, and collecting solution 20ml to obtain leukocyte extract.
2. Platelet lysate preparation
① collecting the blood plasma layer containing platelet in step 1, rapidly thawing in 33 deg.C water bath to release factor for platelet lysis, and repeatedly freezing and thawing for 2 times.
③ 3500rpm/min for 20min, collecting the supernatant, filtering with 0.45um sterile filter membrane, and collecting 20ml of solution, i.e. platelet lysate.
3. Culturing human adipose mesenchymal stem cells:
① recovering human adipose-derived mesenchymal stem cells (P5) from cell bank, inoculating total cells of 2.5 × 106And (3) resuspending the cells by using 50ml of serum-free medium, adjusting the cell density to 50000 cells/ml, aliquoting the cells into 4T 75 culture bottles, and placing the bottles in a carbon dioxide incubator for culture under the conditions of 37 ℃, 5% of carbon dioxide volume concentration and 3% of oxygen volume concentration.
②, culturing for 48h, and when the cell fusion degree is 95%, as shown in figure 1, collecting 50ml of cell supernatant, namely 50ml of stem cell extract a, the main component of the stem cell extract a is mesenchymal stem cell exosome (MSC-Exo), the content of exosome secreted by the mesenchymal stem cells under the hypoxia environment is higher.
③ washing cells with normal saline for 3 times, adding 30ml compound electrolyte injection into 4T 75 culture bottles in equal amount according to cell number during cell recovery, and culturing in carbon dioxide incubator at 37 deg.C, carbon dioxide volume concentration of 5%, and oxygen volume concentration of 20%.
④ after culturing for 24h, collecting 30ml of cell supernatant, namely 30ml of stem cell extract b. the stem cell extract b is mainly cultured under the condition of restoring normal oxygen partial pressure after hypoxia, and simultaneously removing the basic culture medium and serum components, at this time, the mesenchymal stem cells can secrete a large amount of cytokines, such as EGF, FGF, VEFG, SCF and the like.
4. Cell disruption and intracellular factor collection
① after collecting the stem cell extract b, the extract was added to a cell culture flask using 2 ℃ physiological saline, and a total of 10ml of physiological saline (i.e., 2.5ml of physiological saline per T75 culture flask) was added to 4T 75 flasks in equal amounts, as counted by the number of cells at the time of cell recovery, i.e., 250000 cells/ml of physiological saline was added, and the cells were collected by gently pipetting using a 25ml pipette.
② placing the collected cell suspension at-70 deg.C, quickly freezing to completely freeze, keeping for 3min, slowly freezing in a thermostat at 33 deg.C to allow the cells to break and release intracellular factors, centrifuging at 4000 rpm/min at 4 deg.C for 30min, collecting supernatant 10ml, and filtering with 0.45um sterile filter to obtain 10ml of stem cell extract c.
5. Preparation of novel leukocyte extract mixed factor
① taking 10ml of leukocyte extract, 10ml of platelet lysate, 10ml of stem cell extract a, 10ml of stem cell extract b10ml and 10ml of stem cell extract c, and mixing uniformly.
② filtering with 0.22um filter to obtain new leukocyte extract mixed factor.
EXAMPLE 2 determination of cytokine concentration
And (3) using an SCF (Single-channel fluorescent protein), FGF (fibroblast growth factor), VEGF (vascular endothelial growth factor) and EGF (epidermal growth factor) detection kit, adding the leukocyte extract and the novel leukocyte extract mixed factor solution into the coated enzyme label plate respectively, adding an antibody and a substrate respectively, developing, and detecting by using an enzyme label instrument at the wavelength of 450 nm.
The comparison of the secretion of the leukocyte extract and the cytokine secretion of the novel leukocyte extract mixed factor using the method of example 1 with reference to fig. 2 shows that the novel leukocyte extract mixed factor contains a large amount of stem cell growth factor (SCF), Fibroblast Growth Factor (FGF), endothelial cell growth factor (VEGF), Epidermal Growth Factor (EGF), etc. In summary, the novel leukocyte extract cocktail of the present invention has a higher cytokine concentration than a single conventional leukocyte extract.
Example 3 animal experiments
① selecting 6-week-six-year-old SD rats with 20 animals, evenly dividing into 4 groups, a: blank group, b: single leukocyte extract group, c: novel leukocyte extract mixed factor group, d: physiological saline negative control group, and the model of skin injury on the back of rats is wound with diameter of 3cm
② after successful modeling, according to a, blank group is not treated, b, leukocyte extracts of steps 2- ④, c, novel leukocyte extract mixed factors obtained in steps 5- ②, d, normal saline, and the wound is measured and counted on days 1, 4, 7, 10, 13 and 16 after administration
Table 1: statistical table for wound healing of rat skin injury model
In the skin repair process, the exosome (stem cell extract a) can promote the transfer of the cell factor (stem cell extract b), the platelet lysate and the leukocyte extract to the damaged cortex, provide sufficient DNA, RNA, protein factors and the like for the cell growth process and accelerate the skin repair process.
Claims (8)
1. A novel leukocyte extract mixed factor is characterized in that: the composition comprises 10-30% of leukocyte extract, 20-40% of platelet lysate, 10-20% of stem cell extract a, 10-20% of stem cell extract b and 20-40% of stem cell extract c.
2. The novel leukocyte extract cocktail factor of claim 1 wherein: the leukocyte extract, the platelet lysate, the stem cell extract a, the stem cell extract b and the stem cell extract c are mixed according to the volume component ratio of 1:1:1: 1.
3. A preparation method of a novel leukocyte extract mixed factor is characterized by comprising the following steps: the method comprises the following steps:
a. preparation of leukocyte extractThe leucocyte is treated and then added with sterile ultrapure water to adjust the density to 2 × 106~3×106Performing freeze dissolution by liquid nitrogen, and filtering by 0.45um filter to obtain leukocyte extract;
b. preparation of platelet lysate: freezing platelet plasma at-80 deg.C, dissolving in water bath at normal temperature, and filtering with 0.45um filter to obtain platelet lysate;
c. preparation of stem cell extract a: culturing the mesenchymal stem cells for 48-96 hours at 37 ℃, under the conditions of 5% of volume concentration of carbon dioxide and 2-8% of volume concentration of oxygen, and collecting supernatant to obtain a stem cell extract a;
d. preparation of stem cell extract b: culturing the mesenchymal stem cells for 48-96 hours under the condition that the oxygen volume concentration is 2-8%, adding a compound electrolyte injection after the normal atmospheric oxygen partial pressure is recovered to 17-20% of the oxygen concentration, culturing for 24-36 hours, and collecting a supernatant to obtain a stem cell extract b;
e. preparation of stem cell extract c: after the mesenchymal stem cells are subjected to hypoxic culture and normal oxygen partial pressure is restored, quickly freezing the cell suspension at-60 to-90 ℃ until the cell suspension is completely frozen, slowly thawing at 33 to 40 ℃ to break the cells and release intracellular factors, centrifuging, and filtering through a 0.45um filter to obtain a stem cell extract c;
f. uniformly mixing the leukocyte extract, the platelet lysate, the stem cell extract a, the stem cell extract b and the stem cell extract c in proportion, and filtering by using a 0.22um filter to obtain the novel leukocyte extract mixed factor.
4. The method for preparing a novel leukocyte extract mixed factor according to claim 3, wherein: the specific preparation method of the leukocyte extract in the step a is as follows:
① collecting umbilical cord blood of newborn, hepatitis B, hepatitis C, syphilis serum, and HIV, centrifuging, removing upper layer blood plasma layer containing platelet, and collecting lower layer containing erythrocyte and leukocyte;
② adding the compound electrolyte injection with the same volume, centrifuging for 15-20 min at 1500-2000 rpm/min by using a centrifuge, and collecting the middle leucocyte layer;
③ adding erythrocyte lysate, cracking erythrocytes for 3-5 min, adding compound electrolyte injection, centrifuging, washing, and collecting bottom layer leukocytes;
④ discarding supernatant, adding sterile ultrapure water, adjusting leukocyte concentration to 2 × 106~3×106Transferring the cells/ml into a plasma bag, placing the plasma bag at the temperature of 30 ℃ for 30-60 min, then quickly transferring the cells/ml into liquid nitrogen, and after the cells/ml are completely frozen, placing the cells/ml in a constant temperature box at the temperature of 25 ℃ until the cells/ml are completely melted;
⑤ centrifuging the leukocyte solution at 3000-4000 rpm, collecting the supernatant, filtering with 0.45um sterile filter membrane, and collecting the solution, i.e. leukocyte extract.
5. The method for preparing a novel leukocyte extract mixed factor according to claim 3, wherein: the specific preparation method of the platelet lysate in step b is as follows:
① collecting blood plasma layer containing platelet in umbilical cord blood of newborn, placing into a refrigerator of-80 deg.C for quick freezing, placing into water bath of 25-33 deg.C for quick thawing to make platelet in blood plasma crack and release factor, and repeatedly freezing and thawing for 2-4 times;
② 2500-3500 rpm/min for 15-20 min, collecting supernatant, filtering with 0.45um sterile filter membrane, and collecting solution, i.e. platelet lysate.
6. The method for preparing a novel leukocyte extract mixed factor according to claim 3, wherein: the specific preparation method of the stem cell extract a and the stem cell extract b in the steps c and d is as follows:
① reviving the human mesenchymal stem cells of P3-P5 generation in the cell bank, inoculating 45000-80000 cells/ml, adding a serum-free culture solution, and culturing under the condition of a hypoxia loop, wherein the concentration of carbon dioxide is 5% by volume and the concentration of oxygen is 2-8% by volume at 37 ℃;
②, culturing for 48-96 hours, and collecting cell supernatant, namely the stem cell extract a, when the cell fusion degree reaches 85-95%;
③ washing cells with normal saline for 3 times, adding 80000-150000 cells/ml compound electrolyte injection, culturing at 37 deg.C and 5% carbon dioxide volume under normal oxygen partial pressure;
④ for 24-36 hours, and collecting cell supernatant, namely stem cell extract b.
7. The method for preparing a novel leukocyte extract mixed factor according to claim 3, wherein: the specific preparation method of the stem cell extract c in the step e is as follows:
① adding prepared 1-4 ℃ physiological saline into a cell culture bottle, adding the physiological saline into the cell culture bottle at 200000-300000 cells/ml, gently blowing and beating the cells by using a pipette, and collecting the cells into a centrifuge tube;
rapidly freezing at ② -60 to-90 ℃ until the cells are completely frozen, keeping for 3-5 min, and slowly thawing in a thermostat at 33-40 ℃ to break the cells and release intracellular factors;
③ 3500-4500 rpm/min, centrifuging at 2-8 deg.C for 25-35 min, collecting supernatant, and filtering with 0.45um sterile filter to obtain stem cell extract c.
8. The method for preparing a novel leukocyte extract mixed factor according to claim 3, wherein: in the step f, mixing 10-30% by volume of the leukocyte extract, 20-40% by volume of the platelet lysate, 10-20% by volume of the stem cell extract a, 10-20% by volume of the stem cell extract b and 20-40% by volume of the stem cell extract c, and filtering by using a 0.22um filter after mixing to obtain the novel leukocyte extract mixed factor.
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| CN112843237A (en) * | 2021-01-29 | 2021-05-28 | 宏之俊生物科技(上海)有限公司 | Novel leukocyte extract mixed factor and preparation method thereof |
| CN113750031A (en) * | 2021-10-12 | 2021-12-07 | 深圳市泓浩生物科技有限公司 | Antioxidant composition, preparation method thereof and skin care product |
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| CN113750031A (en) * | 2021-10-12 | 2021-12-07 | 深圳市泓浩生物科技有限公司 | Antioxidant composition, preparation method thereof and skin care product |
| CN113750031B (en) * | 2021-10-12 | 2024-04-05 | 深圳市泓浩生物科技有限公司 | Antioxidant composition and preparation method thereof |
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