A biological preparation and its preparation method and application

Abstract

The present application discloses a biological preparation and its preparation method and application, belonging to the field of biomedical technology. The biological preparation is the culture supernatant after culturing fibroblasts using the special culture medium of the present application (containing basal culture medium, human platelet lysate, vitamin C, vitamin E, cysteine ​​and glutathione). While promoting skin wound healing, the biological preparation can inhibit the expression of inflammatory factors around the wound, reduce inflammatory reactions, relieve the discomfort caused by the wound healing process, and improve the comfort during the wound healing period. At the same time, the culture medium provided by the present application, when used for fibroblast culture, can enhance the amplification ability of fibroblasts and enhance the expression of functional genes, and secrete a variety of growth factors and cytokines that promote wound healing. The amplification multiples of fibroblasts obtained by culture with this culture medium will be significantly improved, and the expression of functional genes will also be significantly increased.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a biological preparation and a preparation method and application thereof. Background Art

[0002] As the first line of defense of the human body, the skin has the functions of preventing water loss, protecting the body from UV damage, and preventing microbial invasion, thereby protecting the internal organs of the body. Once the skin is damaged, especially wounds, such as skin damage, microorganisms will take advantage of the opportunity to enter and cause bacterial infections and other problems. Therefore, rapid healing of skin damage plays an important role in maintaining the body's homeostasis.

[0003] Skin wound healing is a dynamic regulatory process, and many cells, cytokines, and extracellular matrices participate in the healing process in an orderly manner in time and space. The healing process can be roughly divided into four consecutive stages, namely: blood coagulation stage, inflammation stage, proliferation stage, and tissue remodeling stage. Different cells participate in each stage, such as epithelial cells, immune cells, endothelial cells, and fibroblasts. These cells can synergistically regulate the wound healing process by changing their own gene expression and cell phenotype. Among them, fibroblasts are particularly worthy of attention.

[0004] In a broad sense, fibroblasts reside in the dermis and have the functions of synthesizing and secreting collagen and extracellular matrix. In the healing of skin wounds, fibroblasts can release extracellular matrix (ECM) containing a large amount of collagen. Platelet-derived growth factor (PDGF), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and other inflammatory factors released by macrophages can recruit fibroblasts to gather at the site of inflammation. Fibroblast protease degrades fibrin clots and secretes a large amount of synthesized collagen to provide signal molecules and epidermal support for newly generated blood vessels and granulation tissue. About a week later, fibroblasts undergo structural and functional changes under the stimulation of transforming growth factor-β (TGF-β) and differentiate into myofibroblasts. The adhesive proteins, glycosaminoglycans, secreted by myofibroblasts, and α-smooth muscle actin (α-SMA) expressed by them can cause epithelial cells to produce contractile pressure to connect the wound edges together, thereby accelerating wound healing. After the wound heals, myofibroblasts undergo apoptosis and are cleared from the wound.

[0005] In the related art, for example, a Chinese invention patent with publication number CN108865990A records a biological preparation for skin damage repair and a preparation method thereof, wherein the preparation method is as follows: resuscitating P3 adipose-derived mesenchymal stem cells, adding DMEM high-glucose complete medium containing 10-12% FBS, culturing until the cell confluence reaches 80-90%, and collecting the supernatant; washing the P3 adipose-derived mesenchymal stem cells, adding DMEM / F12 basal medium containing 50-55 μg / ml β-glucan, culturing for 24-48 hours, and collecting the supernatant; taking the cell culture after collecting the supernatant, adding DMEM / F12 basal medium containing 50-55 μg / ml β-glucan, continuing to culture, collecting the culture fluid, and repeating 1-2 times; combining the supernatants to obtain a biological preparation for skin damage repair; the biological preparation prepared by this method can significantly promote the proliferation of skin fibroblasts and collagen secretion, and has a good effect of promoting skin damage repair. However, it is difficult to extract and separate adipose-derived mesenchymal stem cells, and the study only examined the ability of this biological preparation to promote the proliferation and collagen secretion of human skin fibroblasts when the cells were cultured in vitro.

[0006] Another example is a Chinese invention patent with publication number CN110559468A, which records a medical dressing containing human fibroblast conditioned culture fluid, a preparation method and an application thereof. The medical dressing comprises a liposome freeze-dried powder A containing human fibroblast conditioned culture fluid, and the preparation method of the liposome freeze-dried powder A containing human fibroblast conditioned culture fluid comprises: preparing a liposome suspension containing human fibroblast conditioned culture fluid (dissolving 3-18% soybean lecithin, 1-6% cholesterol and 0.1-3% vitamin E in anhydrous ethanol, removing anhydrous ethanol by reduced pressure rotary evaporation at 0.04-0.06 MPa and 35-40° C. to obtain a uniform film; adding 20-50% human fibroblast conditioned culture concentrate, 5-10% trehalose and 5-10% mannitol to a phosphate buffer solution having a mass percentage of 3-65.9%, mixing and using The mixed solution is rotated to wash the film and shaken for 30 to 60 minutes, and the hydrated mixture is extruded through a liposome extruder for 10 to 20 times to obtain human fibroblast conditioned culture fluid liposomes; preparation of lyophilized powder of liposomes encapsulating human fibroblast conditioned culture fluid (pre-freeze the prepared liposome suspension at -40 to -20°C for 3 to 6 hours, freeze-dry for 24 to 48 hours, and obtain lyophilized powder of liposomes encapsulating active ingredients of human fibroblast conditioned culture fluid), and human fibroblast conditioned culture concentrate is obtained by ultrafiltration and concentration of serum-free human fibroblast conditioned culture fluid (supernatant of human fibroblast conditioned culture during passage (P6 to P15) culture). The preparation method of the above-mentioned medical dressing is complicated, and the symptoms applied in this patent are mainly skin tightness, skin redness, skin itching, and skin pain, and do not involve wound healing.

[0007] In addition, it can be seen from the above wound healing mechanism that inflammation will occur during the wound healing process. Although inflammation is the body's defense mechanism against injury and infection, inflammation can also cause discomfort to the human body, such as TNF, IL-1, etc., which can cause fever in the human body.

[0008] Therefore, if the inflammatory response can be reduced in the process of promoting wound healing, that is, the expression of inflammatory factors can be inhibited, the discomfort caused by the wound healing process can be alleviated and the comfort during the wound healing period can be improved. Summary of the invention

[0009] 1. Purpose of the Invention

[0010] The invention objective of the present application is to provide a biological preparation and a preparation method and application thereof. The biological preparation is the supernatant of the culture fluid after culturing fibroblasts using the culture medium of the present application. While promoting skin wound healing, the biological preparation can inhibit the expression of inflammatory factors around the wound, reduce the inflammatory response, relieve the discomfort caused by the wound healing process, and improve the comfort during the wound healing period.

[0011] 2. Technical solution

[0012] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0013] A method for preparing a biological agent, the method comprising the following steps:

[0014] S1, taking fibroblasts, inoculating them in a culture medium 1 for culture, and performing subculture when the cell density reaches 80-90%; the culture medium 1 includes a basal culture medium, human platelet lysate (hPL), vitamin C (VC), vitamin E (VE), cysteine ​​(Cys) and glutathione (GSH);

[0015] S2, collecting the culture supernatant of each cell culture generation, mixing and filtering to obtain the biological preparation.

[0016] Furthermore, the above-mentioned basal culture medium includes high-glucose DMEM basal culture medium.

[0017] Furthermore, the concentration of the human platelet lysate (hPL) is 5% to 10% (v / v), wherein the human platelet lysate (hPL) can replace fetal bovine serum, thereby avoiding the introduction of exogenous genes.

[0018] Furthermore, the concentration of the vitamin C (VC) is 50-150 μM. Vitamin C can promote the production of collagen in tissues and help repair aging and damaged skin. Furthermore, the concentration of the vitamin C (VC) is 100 μM.

[0019] Furthermore, the concentration of the vitamin E (VE) is 0.5-1.5 μg / mL. Vitamin E has an antioxidant effect and can help resist damage from free radicals. Vitamin E protects cells from oxidative stress by neutralizing free radicals. At the same time, vitamin E has a protective effect on the skin, can reduce damage to the skin caused by ultraviolet rays from sunlight, and help maintain the elasticity and health of the skin. Furthermore, the concentration of the vitamin E (VE) is 1 μg / mL.

[0020] Furthermore, the concentration of cysteine ​​(Cys) is 15-25 μg / mL. Cysteine ​​can remove free radicals in the body and is quickly converted into cystine after entering the body. It is a stable form of sulfur-rich amino acid. Furthermore, the concentration of cysteine ​​(Cys) is 20 μg / mL.

[0021] Furthermore, the concentration of the glutathione (GSH) is 0.5-1.5 μg / mL. Glutathione can not only eliminate free radicals in the human body, but also improve human immunity, and protect hemoglobin from oxidation by hydrogen peroxide, free radicals, etc., so that it can continue to normally perform its oxygen transport ability. Furthermore, the concentration of the glutathione (GSH) is 1 μg / mL.

[0022] Furthermore, the above-mentioned fibroblasts include skin fibroblasts.

[0023] Furthermore, the skin fibroblasts include skin fibroblasts obtained by digestion and separation of skin tissue. Furthermore, the skin fibroblasts include skin fibroblasts obtained by digestion and separation of foreskin tissue of children under 13 years old. The skin fibroblasts obtained by digestion and separation of tissue and culture can secrete a variety of growth factors and cytokines that promote wound healing, which can help collagen regeneration of skin tissue and accelerate wound healing.

[0024] Furthermore, the above method for obtaining skin fibroblasts by digestion and separation comprises the following steps:

[0025] Obtain skin tissue and rinse it with sterile PBS buffer;

[0026] Povidone-iodine and ethanol disinfection: place the skin tissue in a disinfectant containing 5% povidone-iodine as the active ingredient, soak for 3 to 5 minutes, and rinse with PBS after soaking; add 75% ethanol, soak for 3 to 5 minutes, and rinse with PBS after soaking; use povidone-iodine and ethanol instead of antibiotics to avoid interference caused by the use of antibiotics;

[0027] The washed intact skin tissue was placed in a neutral protease solution at 4°C for digestion; after digestion, the epidermal tissue was removed and rinsed with PBS;

[0028] Add collagenase solution, then cut the dermal tissue into 1 mm × 1 mm size with sterile scissors and digest in a 37°C incubator;

[0029] The digested cells were filtered through a 70 μm filter and centrifuged to obtain skin fibroblasts.

[0030] Furthermore, the number of fibroblasts inoculated in the above-mentioned culture medium 1 is 2.0-4.0×10 4 viable cells / cm 2 .

[0031] Furthermore, the conditions for culture and subculture in the above-mentioned culture medium 1 are: 37° C., 5% CO 2 .

[0032] Furthermore, the cells were cultured in the above culture medium 1, and when the cell density reached 90%, subculture was performed.

[0033] Furthermore, the collecting of the culture supernatant of each cell culture generation includes collecting the culture supernatant of at least the fifth cell culture generation.

[0034] Furthermore, the collecting of the culture supernatant of each cell culture generation includes collecting the culture supernatant of the 1st to 10th cell cultures.

[0035] Furthermore, the above-mentioned collecting the culture supernatant of each generation of cell culture includes collecting the culture supernatant after the 1st to 5th generations, or the 1st to 6th generations, or the 1st to 7th generations, or the 1st to 8th generations, or the 1st to 9th generations, or the 1st to 10th generations of cell culture.

[0036] Furthermore, the collecting of the culture supernatant of each cell culture generation includes collecting the culture supernatant of the first to sixth cell cultures.

[0037] The present application also provides a biological preparation prepared by the above-mentioned method for preparing a biological preparation.

[0038] The present application also provides the use of the above-mentioned method for preparing a biological preparation in the preparation of a drug for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds.

[0039] The present application also provides the use of the above biological preparation in the preparation of a drug for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds.

[0040] Furthermore, the above-mentioned medicine includes a dressing.

[0041] The present application also provides a pharmaceutical composition for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds, wherein the pharmaceutical composition comprises the above-mentioned biological preparation.

[0042] The present application also provides a culture medium for promoting fibroblast proliferation and collagen synthesis, which culture medium includes a basal culture medium, human platelet lysate (hPL), vitamin C (VC), vitamin E (VE), cysteine ​​(Cys) and glutathione (GSH). When culturing fibroblasts, the culture medium can promote the proliferation of fibroblasts and the synthesis of collagen.

[0043] Furthermore, the above-mentioned basal culture medium includes high-glucose DMEM basal culture medium.

[0044] Furthermore, the concentration of the human platelet lysate (hPL) is 5% to 10% (v / v), wherein the human platelet lysate (hPL) can replace fetal bovine serum, thereby avoiding the introduction of exogenous genes.

[0045] Furthermore, the concentration of the vitamin C (VC) is 50-150 μM. Vitamin C can promote the production of collagen in tissues and help repair aging and damaged skin. Furthermore, the concentration of the vitamin C (VC) is 100 μM.

[0046] Furthermore, the concentration of the vitamin E (VE) is 0.5-1.5 μg / mL. Vitamin E has an antioxidant effect and can help resist damage from free radicals. Vitamin E protects cells from oxidative stress by neutralizing free radicals. At the same time, vitamin E has a protective effect on the skin, can reduce damage to the skin caused by ultraviolet rays from sunlight, and help maintain the elasticity and health of the skin. Furthermore, the concentration of the vitamin E (VE) is 1 μg / mL.

[0047] Furthermore, the concentration of cysteine ​​(Cys) is 15-25 μg / mL. Cysteine ​​can remove free radicals in the body and is quickly converted into cystine after entering the body. It is a stable form of sulfur-rich amino acid. Furthermore, the concentration of cysteine ​​(Cys) is 20 μg / mL.

[0048] Furthermore, the concentration of the glutathione (GSH) is 0.5-1.5 μg / mL. Glutathione can not only eliminate free radicals in the human body, but also improve human immunity, and protect hemoglobin from oxidation by hydrogen peroxide, free radicals, etc., so that it can continue to normally perform its oxygen transport ability. Furthermore, the concentration of the glutathione (GSH) is 1 μg / mL.

[0049] The present application also provides the use of the above-mentioned culture medium for promoting fibroblast proliferation and collagen synthesis in fibroblast culture.

[0050] Furthermore, the above application includes the step of isolating the fibroblasts at a rate of 2.0 to 4.0×10 4viable cells / cm 2 The cells were inoculated into the above-mentioned culture medium for promoting fibroblast proliferation and collagen synthesis, and cultured at 37°C and 5% CO2.

[0051] Furthermore, the above application also includes subculturing after the cell density reaches 80% to 90%.

[0052] Furthermore, the above-mentioned fibroblasts include skin fibroblasts.

[0053] Furthermore, the skin fibroblasts include skin fibroblasts obtained by digestion and separation of skin tissue. Furthermore, the skin fibroblasts include skin fibroblasts obtained by digestion and separation of foreskin tissue of children under 13 years old. The skin fibroblasts obtained by digestion and separation of tissue and culture can secrete a variety of growth factors and cytokines that promote wound healing, which can help collagen regeneration of skin tissue and accelerate wound healing.

[0054] Furthermore, the above method for obtaining skin fibroblasts by digestion and separation comprises the following steps:

[0055] Obtain skin tissue and rinse it with sterile PBS buffer;

[0056] Povidone-iodine and ethanol disinfection: place the skin tissue in a disinfectant containing 5% povidone-iodine as the active ingredient, soak for 3 to 5 minutes, and rinse with PBS after soaking; add 75% ethanol, soak for 3 to 5 minutes, and rinse with PBS after soaking; use povidone-iodine and ethanol instead of antibiotics to avoid interference caused by the use of antibiotics;

[0057] The washed intact skin tissue was placed in a neutral protease solution at 4°C for digestion; after digestion, the epidermal tissue was removed and rinsed with PBS;

[0058] Add collagenase solution, then cut the dermal tissue into 1 mm × 1 mm size with sterile scissors and digest in a 37°C incubator;

[0059] The digested cells were filtered through a 70 μm filter and centrifuged to obtain skin fibroblasts.

[0060] The present application also provides a method for culturing fibroblasts, the method comprising:

[0061] Fibroblasts were cultured at a rate of 2.0 to 4.0 × 10 4 viable cells / cm 2 Inoculate into the above-mentioned culture medium for promoting fibroblast proliferation and collagen synthesis, and culture at 37°C and 5% CO2;

[0062] When the cell density reaches 80% to 90%, subculture is performed.

[0063] Furthermore, the fibroblasts include skin fibroblasts. Furthermore, the skin fibroblasts include skin fibroblasts obtained by digestion and separation of foreskin tissue of children under 13 years old, and the skin fibroblasts obtained by digestion and separation and culture of the tissue can secrete a variety of growth factors and cytokines that promote wound healing, which can help collagen regeneration of skin tissue and accelerate wound healing.

[0064] Furthermore, the above method for obtaining skin fibroblasts by digestion and separation comprises the following steps:

[0065] Obtain skin tissue and rinse it with sterile PBS buffer;

[0066] Povidone-iodine and ethanol disinfection: place the skin tissue in a disinfectant containing 5% povidone-iodine as the active ingredient, soak for 3 to 5 minutes, and rinse with PBS after soaking; add 75% ethanol, soak for 3 to 5 minutes, and rinse with PBS after soaking; use povidone-iodine and ethanol instead of antibiotics to avoid interference caused by the use of antibiotics;

[0067] The washed intact skin tissue was placed in a neutral protease solution at 4°C for digestion; after digestion, the epidermal tissue was removed and rinsed with PBS;

[0068] Add collagenase solution, then cut the dermal tissue into 1 mm × 1 mm size with sterile scissors and digest in a 37°C incubator;

[0069] The digested cells were filtered through a 70 μm filter and centrifuged to obtain skin fibroblasts.

[0070] The present application also provides a fibroblast obtained by culturing the above-mentioned fibroblast culture method, wherein the fibroblast has highly expressed fibroblast-specific protein-1 (FSP-1), type I collagen α1 (Col1A1), vimentin (Vimentin), and α-smooth muscle actin (α-SMA), and has stronger fibrogenic function.

[0071] The present application also provides the use of the above-mentioned fibroblasts in the preparation of drugs for repairing heart damage, repairing skin damage, treating rheumatoid arthritis, repairing oral mucosa and gingival bone, or treating intervertebral disc damage.

[0072] 3. Beneficial effects

[0073] Compared with the prior art, the present application has the following beneficial effects:

[0074] (1) The present application provides a biological preparation and its preparation method and application. The biological preparation is the supernatant of the culture fluid after culturing fibroblasts using the culture medium of the present application. While promoting skin wound healing, the biological preparation can inhibit the expression of inflammatory factors around the wound, reduce inflammatory response, relieve discomfort caused by the wound healing process, and improve the comfort during the wound healing period.

[0075] (2) The present application provides a culture medium for promoting fibroblast proliferation and collagen synthesis, which includes a basal culture medium, human platelet lysate (hPL), vitamin C (VC), vitamin E (VE), cysteine ​​(Cys) and glutathione (GSH). When used for fibroblast culture, the small molecule additive combination can enhance the proliferation ability of fibroblasts and enhance the expression of fibroblast functional genes, and can secrete a variety of growth factors and cytokines that promote wound healing, which can help collagen regeneration in skin tissue and accelerate wound healing; the proliferation multiples of fibroblasts cultured under such conditions will be significantly improved, and the expression of functional genes will also be significantly upregulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is the morphology and density of fibroblasts after one generation of cell culture with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0077] Figure 2 It is the morphology and density of fibroblasts after adding small molecule additives to culture cells for 2 generations. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0078] Figure 3 It is the morphology and density of fibroblasts after the cells were cultured for 3 generations with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0079] Figure 4It is the morphology and density of fibroblasts after the cells were cultured for 4 generations with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0080] Figure 5 It is the morphology and density of fibroblasts after culturing cells for 5 generations with small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0081] Figure 6 It is a graph of cell doubling time PDL and total cell number.

[0082] Figure 7 q-PCR is used to detect the expression of functional genes.

[0083] Figure 8 The figures are the recovery conditions of mouse skin wounds, where A is the blank control group; B is the fibroblast group cultured in ordinary culture medium (without small molecule additives); and C is the fibroblast group cultured with small molecule additives.

[0084] Fig. 9 is the area of ​​the mouse skin wound.

[0085] Fig.10 It is the expression of related inflammatory factors IL-1β, TNF-a, IL-8, and IL-6 in the skin tissue around mouse skin wounds. DETAILED DESCRIPTION

[0086] The present application is further described below in conjunction with specific embodiments.

[0087] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0089] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0090] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0091] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0092] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0093] Example 1

[0094] This example provides the preparation of skin fibroblasts.

[0095] In this example, skin fibroblasts were obtained by digesting and isolating skin tissue.

[0096] In this embodiment, the skin tissue is foreskin tissue from children under 13 years old, and the digestion and separation of skin fibroblasts includes the following steps:

[0097] Obtain the foreskin tissue and rinse it 10 times with sterile PBS buffer;

[0098] Povidone-iodine and ethanol were used for disinfection (the skin tissue was placed in a disinfectant containing 5% povidone-iodine as an active ingredient and soaked in a centrifuge tube for 3 minutes; sterile tweezers were used to take the skin tissue and place it in a new cell culture dish, rinsed with PBS 3 times, and transferred to a centrifuge tube containing 75% ethanol and soaked for 5 minutes (excessive disinfection will cause loss of cell viability). After soaking, it was rinsed with PBS 3 times; povidone-iodine and ethanol were used instead of antibiotics to avoid interference caused by the use of antibiotics;

[0099] Place the washed intact foreskin tissue in a 15 mL centrifuge tube containing 2 U / mL neutral protease (Cat. No. D4693, Sigma) solution and digest at 4°C for 12 hours; take out the 15 mL centrifuge tube for overnight digestion, pour out the digested tissue from the centrifuge tube, remove the epidermal tissue, add 20 mL PBS buffer and rinse 10 times, repeat three times, and pour out the cleaning solution;

[0100] Add 0.1U / mL collagenase NB6 (Nordmark, N0002779) solution, then cut the dermis into 1mm×1mm size with sterile scissors and transfer to a foreskin tissue suspension centrifuge tube. Seal the mouth of the 50mL centrifuge tube with sealing film and place it in a 37℃ incubator with gentle shaking (speed 70r / min) for digestion for 2h;

[0101] The digested cells were filtered through a 70 μm filter and centrifuged at 300 g for 5 min to obtain digested and separated skin fibroblasts.

[0102] Example 2

[0103] This example provides a comparison of skin fibroblasts cultured in culture medium supplemented with different small molecule additives.

[0104] Take the digested and separated skin fibroblasts obtained in Example 1, resuspend them in 1 mL of high-glucose DMEM basal medium, and count them;

[0105] According to 2.0~4.0×10 4 viable cells / cm 2 The cells were inoculated into T25 culture flasks and cultured in a 37°C, 5% CO2 cell culture incubator. The culture medium was a high-glucose DMEM basal medium + 10% human platelet lysate. In addition, the small molecule additives in Table 1 were also included.

[0106] Table 1 Small molecule additives and dosage

[0107] Small molecule additives Use concentration Vitamin C (VC) 100μM Vitamin E (VE) 1 μg / mL Glutathione (GSH) 1 μg / mL Cysteine ​​(Cys) 20 μg / mL

[0108] After adding small molecule additives, the cell morphology and density after 5 subcultures were as follows Figure 1-Figure 5As shown, the morphology of skin fibroblasts did not change significantly after 5 passages, and the cell growth rate increased under the condition of co-culture with multiple small molecule additives.

[0109] During the subculture, the number of cells collected was plotted against the Doubling time (DT), PDL, and total cell number. Figure 6 As shown, after adding various small molecule additives, the cell expansion time was significantly shortened and the total number of cell harvests was significantly increased.

[0110] Sample collection for q-PCR: Use the cell lysis buffer in the Promega total RNA extraction kit (LS1040), add 300 μL to each sample to fully lyse the sample, and store it at -20°C.

[0111] Sample RNA extraction for q-PCR: Strictly follow the instructions of the kit.

[0112] Reverse transcription of RNA samples: Use Promega's RNA reverse transcription kit (LS2052) and strictly follow the kit instructions. Each reaction system is 20 μL, and the amount of RNA added to each reaction is 400 ng. The reverse transcription time is 37°C for 15 minutes and 98°C for 5 minutes.

[0113] q-PCR reaction: The reaction was completed strictly according to the SYBR green qPCR Mix kit (D7265) of Biotech. The components were: Mix 10 μL, cDNA template 200 ng, primer 0.5 μL, and water to 20 μL.

[0114] q-PCR was used to detect the expression of functional genes. Figure 7 As shown, among these molecular combinations, the combination of multiple small molecule additives (VC+VE+GSH+Cys) can significantly increase the expression of multiple genes such as FSP-1, Col1A1, Vimentin, and a-SMA, and can significantly upregulate the function of fibroblast genes. In particular, as shown by the expression of the Vimentin gene, each individual small molecule additive can hardly upregulate the expression of the Vimentin gene, or even downregulate it (VE group), while under the synergistic effect of the four small molecule additives, the expression of the Vimentin gene is significantly upregulated.

[0115] Example 3

[0116] This embodiment provides a biological preparation.

[0117] In Example 2, fibroblasts were cultured in a culture medium containing a variety of small molecule additives (VC+VE+GSH+Cys), and the supernatant of each generation (1st to 6th generation) of cells after 3 days of culture was collected and mixed. The cells were centrifuged at 300g for 5 minutes to obtain the cell culture supernatant, and the cell culture supernatant was passed through a 0.22μm filter to obtain the biological preparation.

[0118] At the same time, fibroblasts were cultured with the control culture medium in Example 2, and the supernatant of each generation (1st to 6th generation) of cells after 3 days of culture was collected, mixed, and centrifuged at 300g for 5 minutes to obtain the cell culture supernatant, which was passed through a 0.22μm filter as a control biological preparation.

[0119] Example 4

[0120] This embodiment provides the use of the biological preparation of Example 3 in promoting skin wound healing, including:

[0121] Construction of mouse skin wound model:

[0122] Mice aged 5 to 7 weeks were anesthetized, and the hair on their backs near the tails was removed. After disinfection with iodine, a 1×1 cm 2 Use a sterilized scalpel to cut the skin tissue at the marked position, disinfect the wound with iodine, and bandage the wound with gauze.

[0123] The biological preparation obtained in Example 3 and the control biological preparation were applied to the wounds of mice, 1 mL / mouse, and the wound healing of the mice was measured after 3 weeks.

[0124] The results are as follows Figure 8 and Fig. 9 As shown, the wounds of mice treated with biological agents ( Figure 8 C) Mouse wounds treated with control biological agents ( Figure 8 B) In comparison, mice treated with biologics had smaller wounds and healed faster.

[0125] Example 5

[0126] This embodiment provides the use of the biological agent of Example 3 in inhibiting the expression of inflammatory factors around a wound, including:

[0127] Referring to Example 4, after the biological agent was applied around the wound for three weeks, the mouse was anesthetized with isoflurane, and the skin tissue around the original wound was cut with high-temperature sterilized surgical scissors and forceps, cut into small pieces, and quickly put into the cell lysis solution to extract RNA. The specific extraction method was carried out according to Example 2.

[0128] After the extraction was completed, the expression of related inflammatory factors IL-1β, TNF-a, IL-8, and IL-6 were detected.

[0129] The results are as follows Fig.10 As shown, q-PCR studies have shown that after application of the biological preparation of the present application, the expression of inflammatory factors around the wound is significantly reduced.

[0130] In summary, the function of fibroblasts cultured in the culture medium with small molecule additives added according to the present application will be significantly improved. At the same time, the supernatant of the culture fluid of fibroblasts cultured under this condition can significantly promote wound healing of skin tissue, and at the same time significantly inhibit the expression of inflammatory factors in the tissue around the wound.

Claims

1. A method for preparing a biological agent, characterized in that: The preparation method comprises the following steps: S1, taking fibroblasts, inoculating them in culture medium 1 for culture, and performing subculture when the cell density reaches 80-90%; the culture medium 1 comprises a basal culture medium, human platelet lysate, vitamin C, vitamin E, cysteine ​​and glutathione; the concentration of the human platelet lysate is 5%-10% (v / v); the fibroblasts are skin fibroblasts; The concentration of vitamin C is 50-150 μM; The concentration of vitamin E is 0.5-1.5 μg / mL; The concentration of cysteine ​​is 15-25 μg / mL; The concentration of glutathione is 0.5-1.5 μg / mL; S2, collecting the culture supernatant of each cell culture generation, mixing and filtering to obtain the biological preparation.

2. The method for preparing a biological agent according to claim 1, characterized in that: The fibroblasts inoculated in the culture medium 1 are 2.0-4.0×10 4 viable cells / cm 2 .

3. A biological agent prepared by the method for preparing a biological agent according to any one of claims 1-2.

4. A method for preparing a biological preparation according to any one of claims 1 to 2, or use of the biological preparation according to claim 3 in the preparation of a drug for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds.

5. A pharmaceutical composition for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds, characterized in that: The pharmaceutical composition comprises the biological agent according to claim 3.

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