CN114469876B - Preparation method of cell factor freeze-dried powder for skin tissue injury - Google Patents
Preparation method of cell factor freeze-dried powder for skin tissue injury Download PDFInfo
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- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
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- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
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- CDQSJQSWAWPGKG-UHFFFAOYSA-N butane-1,1-diol Chemical compound CCCC(O)O CDQSJQSWAWPGKG-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Dermatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of cell factor freeze-dried powder, and particularly relates to a preparation method of cell factor freeze-dried powder for skin tissue injury. Obtaining macrophages through the culture of mononuclear-macrophages, culturing THP-1 monocytes for establishing a macrophage line, adding phorbol ester into a culture medium, and differentiating the THP-1 monocytes into the macrophages after the culture; adding lipopolysaccharide, interferon-gamma and IL-4, polarizing into M2 type macrophage, inducing inflammation inhibiting macrophage with VB12 and VC to obtain mesenchymal stem cell, adding adjuvant, and lyophilizing. The cytokine obtained by the method has high specific gravity and good activity.
Description
Technical Field
The invention belongs to the field of cell factor freeze-dried powder, and particularly relates to a preparation method of cell factor freeze-dried powder for skin tissue injury.
Background
Damage to skin tissue is often caused by mechanical, physical, chemical, biological, and other factors, and the process by which the body repairs the formed damage is called tissue repair. The traditional beauty industry aims at skin injury mainly through modes of dermatography, laser, refrigeration, surgical excision, grinding, dexamethasone injection and the like. Skin grafting is the removal of a portion of skin from its healthy skin to cover the area from which the scar was excised. The skin in the donor area needs to be supplied with new blood vessels in the recipient area to survive. In general, the probability of success of autologous skin grafting is high; the laser is a well known optical weapon for modern people, has a certain effect on dehairing, but has no effect on scars, and many patients with scar hyperplasia or keloids are strikingly finished by adopting the laser, so that the scars are flattened at the time, but along with red swelling and bleeding, anti-inflammatory drugs are required to be taken, the patient is carelessly easy to infect, secondary severe hyperplasia is most likely to occur, and the scars are long, rapid and high. The intensity and depth of freezing operation are difficult to control, meanwhile, the resistance of tissues to low temperature is different, the treatment is incomplete, the recurrence rate is high, and the defects of the tissues and pigment loss are easy to cause. The method is generally used for treating pits and inflammatory hyperplasia scars left by acnes, and can be called as light grinding, unobvious grinding, heavy grinding, skin tissue defect and extremely easy pigmentation. The dexamethasone injection is easy to rebound, the immunity of a long-term user is reduced, infertility and keloids are easy to occur, and the method is adopted.
With the development of molecular biology, the repair of tissue injury has been known in a deep manner, and the process of tissue repair is regarded as the process of proliferation, differentiation, migration and disappearance of various repair cells. At the same time, tissue repair is also the result of a series of network interactions formed by different types of repair cells, structural proteins, growth factors, and the like. Repair of structures and functions by the same cell around the injury is called regeneration, and if the organization and functions of the tissue before the injury are completely recovered, the repair is performed by the connective tissue of the county, and the repair is called county commission repair or scar repair.
Many pieces of evidence suggest that cytokines play an important role in wound healing, and that these cells play an important role in wound inflammatory response, epithelial regeneration, granulation tissue formation, angiogenesis and extracellular matrix production by different mechanisms. Cytokines include various growth factors including Epidermal Growth Factor (EGF), fibroblast Growth Factor (FGF), keratinocyte Growth Factor (KGF), transforming growth factor (TGF-. Beta.), vascular endothelial growth factor, and the like (VEGF). The existing cytokines are used for preparing skin tissue injury, and have the defects of complex extraction method and low activity.
Disclosure of Invention
Aiming at the problems of complex extraction method and low activity of cytokines for treating skin tissue injury at the present stage, the invention provides a preparation method of the cytokine freeze-dried powder for treating skin tissue injury, which is prepared by culturing mononuclear-macrophages to induce mesenchymal stem cells, adding auxiliary materials and freeze-drying. The cytokine obtained by the method has good activity.
The technical scheme of the invention is as follows:
a preparation method of a cell factor freeze-dried powder for skin tissue injury comprises the following steps:
(1) Culturing of mononuclear-macrophages: taking fresh umbilical cord blood, obtaining THP-1 mononuclear cells by a density gradient centrifugation method, culturing the THP-1 mononuclear cells for establishing a macrophage system, adding phorbol ester into a culture medium, and differentiating the THP-1 mononuclear cells into macrophages after culturing;
(2) Induction culture of mesenchymal stem cells: adding VB12 and VC into the cell culture medium for culturing the macrophages in the step (1), continuously culturing, and collecting cell supernatant after the culturing is finished;
(3) Preparation of cytokine freeze-dried powder: adding mannitol, trehalose, polysorbate 80 and nicotinamide into the supernatant obtained in the step (2), and freeze-drying to obtain the product.
Preferably, the medium of step (1) is: RPMI-1640 medium containing 10% fetal bovine serum; phorbol ester was added at a concentration of 10 ng/mL.
Preferably, the macrophages in the step (1) are M0 type macrophages, lipopolysaccharide and interferon-gamma are added into an M0 type macrophage culture system, and the mixture is incubated, so that the mixture is polarized into M1 type macrophages, namely pro-inflammatory type macrophages; IL-4 is added and incubated to polarize the cells into M2 type macrophages, namely anti-inflammatory type macrophages.
Preferably VB12 is added at a concentration of 1.0 μg/mL to 2.0 μg/mL; VC was added at a concentration of 1. Mu.g/mL-10. Mu.g/mL.
Preferably, in the step (3), the concentration mass fraction of mannitol is 2% -15%, the concentration mass fraction of trehalose is 0.1% -5%, the concentration mass fraction of polysorbate 80 is 0.1% -5%, and the concentration mass fraction of nicotinamide is 0.1% -1%.
The beneficial effects of the invention are that
Culturing mononuclear-macrophages to obtain macrophages, inducing VB12 and VC to obtain mesenchymal stem cells, adding auxiliary materials, and freeze-drying. The lyophilized powder with high EGF content obtained by the method can effectively promote the proliferation of epidermal cells and enhance the repair capability of skin.
The specific embodiment is as follows:
the present invention will be described in detail with reference to specific examples, wherein the exemplary embodiments and descriptions of the present invention are provided for the purpose of illustration and are not intended to be limiting. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
a preparation method of a cell factor freeze-dried powder for skin tissue injury comprises the following specific steps:
(1) Culturing of mononuclear-macrophages:
fresh umbilical cord blood is taken, density gradient separation liquid is added according to the concentration of 1:1, THP-1 mononuclear cells are obtained by a density gradient centrifugation method, the THP-1 mononuclear cells are cultured by an RPMI-1640 culture medium containing 10% fetal bovine serum, and the cultured THP-1 mononuclear cells are inoculated into a 6-pore plate for establishing a macrophage system;
phorbol ester was added to the medium at a concentration of 10ng/mL, and THP-1 monocytes differentiated into M0 phase macrophages after 48-h culture. Adding 0.5. 0.5ng/ml lipopolysaccharide and 10. 10ng/ml interferon-gamma into M0 phase macrophage culture system, and incubating 24. 24 h to polarize into M1 type macrophage (pro-inflammatory type macrophage); mu.l IL-4 (10 ng/ml) was added and incubated for 24 h to polarize it into M2 type macrophages (anti-inflammatory type macrophages).
2. Induction culture of mesenchymal stem cells:
the cell culture medium obtained by the macrophage cell culture in the above step was further cultured by adding VB12 at 1.5. Mu.g/mL and LVC at 5. Mu.g/mLVC, followed by 24. 24 h, and after the completion of the culture, the cell supernatant was collected.
3. Preparation of novel cytokine lyophilized powder:
adding 8% mannitol, 1% trehalose, 1% polysorbate 80 and 1% nicotinamide into the cell culture supernatant obtained in the above steps, sub-packaging into penicillin bottles of 7 mL, each bottle of 2 mL, and freeze-drying.
Example 2
A preparation method of a cell factor freeze-dried powder for skin tissue injury comprises the following specific steps:
1. culturing of mononuclear-macrophages:
fresh umbilical cord blood is taken, density gradient separation liquid is added according to the concentration of 1:1, THP-1 mononuclear cells are obtained by a density gradient centrifugation method, the THP-1 mononuclear cells are cultured by an RPMI-1640 culture medium containing 10% fetal bovine serum, and the cultured THP-1 mononuclear cells are inoculated into a 6-pore plate for establishing a macrophage system;
phorbol ester is added into the culture medium according to the concentration of 10ng/mL, and THP-1 mononuclear cells are differentiated into M0 phase macrophages after 48 h culture; 0.5. 0.5ng/ml lipopolysaccharide and 10. 10ng/ml interferon-gamma were added and incubated 24. 24 h to polarize it into M1 type macrophages (pro-inflammatory type macrophages); mu.L of IL-4 (10 ng/ml) was added and incubated for 24 h to polarize it into M2 type macrophages (anti-inflammatory type macrophages).
2. Induction culture of mesenchymal stem cells:
the cell culture medium obtained by the macrophage cell culture in the above step was further cultured by adding VB12 at 1.0. Mu.g/mL and LVC at 10. Mu.g/mLVC, followed by 24. 24 h, and after the completion of the culture, the cell supernatant was collected.
3. Preparation of novel cytokine lyophilized powder:
adding 2% mannitol, 5% trehalose, 0.1% polysorbate 80 and 1% nicotinamide into the cell culture supernatant obtained in the above steps, sub-packaging into penicillin bottles of 7 mL, each bottle of 2 mL, and freeze-drying.
Example 3
A preparation method of a cell factor freeze-dried powder for skin tissue injury comprises the following specific steps:
culturing of mononuclear-macrophages:
fresh umbilical cord blood is taken, density gradient separation liquid is added according to the concentration of 1:1, THP-1 mononuclear cells are obtained by a density gradient centrifugation method, the THP-1 mononuclear cells are cultured by an RPMI-1640 culture medium containing 10% fetal bovine serum, and the cultured THP-1 mononuclear cells are inoculated into a 6-pore plate for establishing a macrophage system;
phorbol ester is added into the culture medium according to the concentration of 10ng/mL, and THP-1 mononuclear cells are differentiated into M0 phase macrophages after 48 h culture; 0.5. 0.5ng/ml lipopolysaccharide and 10. 10ng/ml interferon-gamma were added and incubated 24. 24 h to polarize it into M1 type macrophages (pro-inflammatory type macrophages); mu.l IL-4 (10 ng/ml) was added and incubated for 24 h to polarize it into M2 type macrophages (anti-inflammatory type macrophages).
2. Induction culture of mesenchymal stem cells:
the cell culture medium obtained by the macrophage cell culture in the above step was further cultured by adding VB12 at 2. Mu.g/mL and LVC at 1. Mu.g/mLVC, followed by 24-h, and after the completion of the culture, the cell supernatant was collected.
3. Preparation of novel cytokine lyophilized powder:
adding 15% mannitol, 0.1% trehalose, 5% polysorbate 80 and 0.11% nicotinamide into the cell culture supernatant obtained in the above steps, sub-packaging into penicillin bottles of 7 mL, each bottle being 2 mL, and freeze-drying.
Comparative example 1:
culturing mesenchymal stem cells according to conventional culture method, inoculating cells in logarithmic growth phase into new cell culture bottle, culturing for 24 h, collecting supernatant, adding 8% mannitol, 1% trehalose, 1% polysorbate 80 and 1% nicotinamide, packaging into 7 mL penicillin bottles, each bottle being 2 mL, and lyophilizing.
Examples of the effects
The EGF factor content in examples 1-3 and comparative example 1 were examined separately:
preparation of cytokine solvent:
high molecular weight sodium hyaluronate 0.1%
Medium molecular weight sodium hyaluronate 0.1%
Low molecular weight sodium hyaluronate 0.1%
Butanediol 5%
Propylene glycol 1%
Glycerol 3%
0.2% of antibacterial agent
The rest ingredients are ultrapure water, the above components are fully dissolved in the ultrapure water respectively, and are respectively packaged in penicillin bottles of 7 mL, and each bottle is 5 mL.
1. The lyophilized powders obtained in example 1 and comparative example 1 were thoroughly mixed with a solvent solution and dissolved, and the EGF content of the two sets of lyophilized powders was measured by ELISA-EGF kit, respectively, with the following results:
。
Claims (1)
1. a method for preparing a cytokine lyophilized powder for skin tissue injury, comprising the steps of:
(1) Culturing of mononuclear-macrophages: fresh umbilical cord blood is taken, density gradient separation liquid is added according to the density of 1:1, THP-1 mononuclear cells are obtained by a density gradient centrifugation method, the THP-1 mononuclear cells are cultured by an RPMI-1640 culture medium containing 10% fetal bovine serum and used for establishing a macrophage system, phorbol ester is added into the culture medium according to the concentration of 10ng/mL, and after the culture, the THP-1 mononuclear cells are differentiated into M0 type macrophages; adding 0.5ng/ml lipopolysaccharide and 10ng/ml interferon-gamma into an M0 type macrophage culture system, and incubating to polarize the lipopolysaccharide into M1 type macrophages, namely pro-inflammatory type macrophages; adding 2 μl of IL-4 with concentration of 10ng/mL, incubating, and polarizing to M2 type macrophage, namely anti-inflammatory macrophage;
(2) Induction culture of mesenchymal stem cells: adding VB12 with the concentration of 1.5 mug/mL and VC with the concentration of 5 mug/mL into the cell culture medium for culturing the M2 type macrophage in the step (1), continuously culturing, and collecting cell supernatant after the culturing is finished;
(3) Preparation of cytokine freeze-dried powder: adding mannitol, trehalose, polysorbate 80 and nicotinamide which are respectively 8% and 1% of concentration mass fractions into the supernatant obtained in the step (2), and freeze-drying to obtain the product.
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| "M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide;Marie Genin 等;《BMC Cancer》;20150808;第1页摘要，第2页右栏"细胞培养"，第6页右栏倒数第1段，第7页右栏倒数第1段 * |
| The effects of conditioned media generated by polarized macrophages on the cellular behaviours of bone marrow mesenchymal stem cells;Xiao-Tao He 等;《J. Cell. Mol. Med.》;20171106;第22卷(第2期);摘要，第1305页右栏第4段 * |
| 卢春凤 等.维生素.《实用医学研究基本技术与方法》.知识产权出版社,2018, * |
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