CN115125193B - Animal umbilical cord extract and extraction method and application thereof - Google Patents
Animal umbilical cord extract and extraction method and application thereof Download PDFInfo
- Publication number
- CN115125193B CN115125193B CN202110779475.5A CN202110779475A CN115125193B CN 115125193 B CN115125193 B CN 115125193B CN 202110779475 A CN202110779475 A CN 202110779475A CN 115125193 B CN115125193 B CN 115125193B
- Authority
- CN
- China
- Prior art keywords
- umbilical cord
- extraction method
- animal
- exosomes
- tissues
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 65
- 238000000605 extraction Methods 0.000 title claims abstract description 34
- 241001465754 Metazoa Species 0.000 title claims abstract description 25
- 239000000284 extract Substances 0.000 title abstract description 16
- 210000001808 exosome Anatomy 0.000 claims abstract description 45
- 210000001519 tissue Anatomy 0.000 claims abstract description 37
- 238000010257 thawing Methods 0.000 claims abstract description 17
- 238000001914 filtration Methods 0.000 claims abstract description 15
- 239000000047 product Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 13
- 238000007710 freezing Methods 0.000 claims abstract description 11
- 238000005119 centrifugation Methods 0.000 claims abstract description 10
- 230000008014 freezing Effects 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- 238000000227 grinding Methods 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 241000283690 Bos taurus Species 0.000 claims description 22
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 210000001626 skin fibroblast Anatomy 0.000 claims description 9
- 230000035755 proliferation Effects 0.000 claims description 8
- 230000001737 promoting effect Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 8
- 238000002474 experimental method Methods 0.000 abstract description 8
- 102000004190 Enzymes Human genes 0.000 abstract description 4
- 108090000790 Enzymes Proteins 0.000 abstract description 4
- 230000002411 adverse Effects 0.000 abstract description 2
- 239000012467 final product Substances 0.000 abstract description 2
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 238000000053 physical method Methods 0.000 abstract description 2
- 230000000087 stabilizing effect Effects 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 238000000034 method Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 4
- 239000004570 mortar (masonry) Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000003921 particle size analysis Methods 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000005299 abrasion Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 230000010435 extracellular transport Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Rheumatology (AREA)
- Pregnancy & Childbirth (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to the technical field of animal umbilical cord extraction, and discloses an animal umbilical cord extract, an extraction method and application thereof. Quick-freezing animal umbilical cord tissues, grinding the umbilical cord tissues into uniform powdery tissues, adding buffer solution, and repeatedly freezing and thawing for 3 times at-80 ℃ and room temperature; and carrying out ultrasonic treatment and centrifugation on the frozen and thawed product, continuing ultrasonic treatment on supernatant, and filtering to obtain the exosomes. The extraction method of the invention completely adopts a physical extraction method and maintains the low-temperature environment of the whole extraction process, thereby avoiding adverse factors which influence the quality and activity of the final product, such as enzymes, organic solvents or high temperature and the like which are introduced by other preparation processes at present; meanwhile, the extraction process of the invention combines the structural characteristics of the umbilical cord, gradually removes other components through physical methods such as freeze thawing, ultrasonic treatment, filtration and the like, finally obtains a sterile product rich in exosomes, and fully verifies through identification experiments and functional verification experiments that the invention has established a preparation process for stabilizing exosomes of the umbilical cord.
Description
Technical Field
The invention relates to the technical field of animal umbilical cord extraction, in particular to an animal umbilical cord extract and an extraction method and application thereof.
Background
In recent years, studies on animal tissues such as placenta and umbilical cord have been increasing, and physiological activities of many animal placenta and umbilical cord such as cow, sheep, deer, donkey, etc. have been found. The animal umbilical cord extract is a compound active ingredient derived from an animal umbilical cord, contains rich amino acids, proteins, lecithin, polysaccharide, vitamins, mineral elements and the like, has the effects of enhancing memory, resisting stress, regulating endocrine, enhancing immunity, delaying aging and the like, and has various biological effects of promoting peripheral blood circulation, promoting cell and tissue respiration, removing free radicals, preserving moisture, activating immunity, beautifying skin and the like according to the current relevant research reports. Currently, there are various patents for extracting effective active substances using umbilical cord. The methods for obtaining placenta extract in these patents mainly comprise the following 3 methods: (1) direct extraction method: homogenizing umbilical cord tissue, extracting with extractive solution, or repeatedly freeze thawing, centrifuging extractive solution, and lyophilizing. (2) supercritical CO2 extraction method: freeze-drying umbilical cord tissue, pulverizing, supercritical extracting, homogenizing, centrifuging, and freeze-drying the centrifugate. (3) enzyme-assisted extraction method: after the umbilical cord is subjected to moderate enzymolysis by 1-2 proteases, centrifuging and freeze-drying the centrifugate. The preparation method and the products of the umbilical cord extracts have the following disadvantages: (1) The obtained umbilical cord extract mainly contains macromolecular proteins, and has low human absorption efficiency; (2) The lack of an extracting solution refining process has complex components and low quality; (3) The extraction method is simple and extensive, the extraction efficiency is low, and the raw material utilization rate is low. Therefore, how to effectively obtain the bioactive components in the umbilical cord of the animal and fully exert the effects of the bioactive components has important scientific and industrial significance.
Exosomes (exosomes) are a membranous vesicle released from the intracellular vesicle structure outside the cell, about 30-150nm in diameter, which are secreted by the cell under normal physiological conditions, as a result of fusion between the plasma membrane and the multivesicles, and are secreted into the extracellular space by exocytosis. Exosome-like vesicles are divided into inner and outer portions by lipid bilayers, which, due to their inclusion of membrane lipids, membrane proteins, genetic material and cytoplasmic components, indirectly reflect the nature and status of the cell. Meanwhile, exosome-like vesicles act as extracellular transport proteins that mediate cell-cell communication by binding to other cells and tissues and transferring membrane components, mRNA, miRNA, proteins (growth hormone, cytokines, etc.), etc. to recipient cells.
The animal exosome is widely in living animal body, selectively wraps lipid, protein, mRNA, miRNA and other bioactive substances, can be absorbed transdermally and absorbed by target cells, can clearly lighten the damage degree of tissues, promote the repair of damaged tissue morphology and function, and can have the potential biological functions of regulating organism immunity, regulating cell growth and differentiation and the like. Therefore, the establishment of the preparation process of the animal umbilical cord exosomes, in particular to the preparation process of the bovine umbilical cord exosomes, can lay a foundation for the safe, stable and effective research and development and industrialization of new raw materials of cosmetics based on the animal umbilical cord exosomes.
Disclosure of Invention
Therefore, the invention aims to provide an animal umbilical cord extract, and an extraction method and application thereof, so that the exosome obtained by the extraction process has higher protein concentration and has obvious effect of promoting human skin fibroblast proliferation, and can be applied to the field of cosmetics.
In order to achieve the above object, the present invention provides the following technical solutions:
An extraction method of an animal umbilical cord exosome comprises the following steps:
step 1, quick freezing animal umbilical cord tissues with liquid nitrogen, and then grinding the umbilical cord tissues into uniform powdery tissues;
step 2, adding the buffer solution into the powdery tissue, and repeatedly freezing and thawing for 3 times at the temperature of-80 ℃ and the room temperature;
Step 3, ultrasonically treating the frozen and thawed product;
And step 4, centrifuging after ultrasonic treatment, continuing ultrasonic treatment of supernatant, and filtering to obtain the exosomes.
The existing preparation process mainly comprises the preparation of the umbilical cord extract, and has the advantages of simple and extensive preparation process, low extraction efficiency, low raw material utilization rate, poor process stability, lack of an extracting solution refining process, complex product components and low quality. In order to solve the problem, the invention extracts exosomes with obvious promotion of human skin fibroblast proliferation and high protein concentration from animal umbilical cord tissues through a series of physical means.
In a specific embodiment of the invention, the extraction method of the invention is subjected to a correlation test by means of bovine umbilical cord tissue; preferably, the time of freeze thawing is a freeze time/thaw time: 2h/2h. Preferably, the buffer is a PBS buffer.
Preferably, the ultrasonic treatment is:
Power 500w, time 30min, run/stop time: 8s/8s.
Preferably, the centrifugation uses centrifugal force with a plurality of rotational speed increasing steps. In a specific embodiment of the invention, the centrifugation is:
Centrifugation at 3000rpm for 10min, collecting supernatant, centrifuging at 4000rpm for 15min, and collecting supernatant, centrifuging at 10000rpm for 1h.
Preferably, the filtration adopts multi-stage filtration with decreasing filter screen pore size. In a specific embodiment of the invention, the filtering is:
the filtration was carried out in order according to the pore diameters of 5 μm, 1 μm, 0.45 μm and 0.22. Mu.m.
Compared with a control process, the grinding link of the invention can rapidly obtain a sufficiently small powder structure sample, and can obtain exosomes with higher protein concentration, the concentration is about 2 mg/ml. And through particle size analysis, electron microscope observation and immunoblotting analysis of the prepared umbilical cord extract, the main component of the product obtained by the preparation process of the invention is an exosome, and the particle size is concentrated at about 100 nm. Meanwhile, proliferation experiments of human skin fibroblasts show that the umbilical cord exosome prepared by the invention has obvious effect of promoting proliferation of human skin fibroblasts. Based on the above, the invention provides an exosome prepared by the extraction method and application of the exosome in preparing a product for promoting human skin fibroblast proliferation or preparing cosmetics.
According to the technical scheme, the extraction method disclosed by the invention completely adopts a physical extraction method and maintains the low-temperature environment of the whole extraction process, so that adverse factors of enzymes, organic solvents or high temperature and the like which are introduced by other preparation processes at present and influence the quality and activity of the final product are avoided; meanwhile, the extraction process of the invention combines the structural characteristics of the umbilical cord, gradually removes other components through physical methods such as freeze thawing, ultrasonic treatment, filtration and the like, finally obtains a sterile product rich in exosomes, and fully verifies that the preparation process for stabilizing exosomes outside the umbilical cord is established through a series of identification experiments and functional verification experiments.
Drawings
FIG. 1 shows the effect of bovine umbilical cord exosome extraction freeze-thaw times on protein concentration; * P <0.001, < p <0.0001;
FIG. 2 shows the identification results of the umbilical cord exosomes of cattle; a is a particle size analysis result, B is an electron microscope observation result, and C is western blot analysis;
FIG. 3 shows a bovine umbilical cord exosome cell efficacy experiment; NC represents a negative control group (high-sugar DMEM basal medium group), PC represents a positive control group (high-sugar DMEM basal medium+10% fetal bovine serum), and 25-100 represents bovine umbilical cord band exosome groups added with different concentrations (unit: μg/ml); * P <0.0001 (compared to NC).
Detailed Description
The embodiment of the invention discloses an animal umbilical cord extract, an extraction method and application thereof, and a person skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention. While the exosomes and methods and applications of extracting the exosomes of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the exosomes and methods and applications of extracting the exosomes described herein can be modified or appropriately altered and combined to implement and use the present technology without departing from the spirit and scope of the present invention.
The invention provides an animal umbilical cord extract, and an extraction method and application thereof.
Example 1: comparison of different tissue abrasion operations
The experimental materials are purchased from the umbilical cord of a cow naturally delivered by a cow farm of a well-known milk brand in Guangdong, and after the experimental materials are detected to be qualified by viruses and bacteria, the experimental materials are repeatedly washed by tap water, and then are diced and split-packed, and frozen and stored at the temperature of minus 80 ℃;
weighing a certain mass of bovine umbilical cord according to the output requirement of exosomes, and thawing at room temperature; after the tissue is completely thawed, washing the tissue by flowing water until the tissue is nearly colorless, and then carrying out replacement washing by pure water, and simultaneously precooling a mortar;
the bovine umbilical cord was then split equally into two parts and treated according to the following two grinding means:
(1) Cutting umbilical cord tissue, placing into a mortar, adding sufficient liquid nitrogen, and starting grinding, wherein the liquid nitrogen is continuously added during the period to keep the tissue frozen state;
(2) Paving umbilical cord tissues, placing the umbilical cord tissues in a container filled with liquid nitrogen, quick freezing (5 min), and then further grinding the tissues by using a mortar to prepare uniform powdery tissues;
The results show that: the umbilical cord tissue in the mode (1) is difficult to grind into powder and often agglomerates on a mortar, the umbilical cord tissue in the mode (2) is more beneficial to grinding, and a sufficiently small powder structure sample can be quickly obtained.
Example 2: comparison of different freezing and thawing conditions
A powdered sample was obtained as in (2) in example 1, aliquoted into 3 portions, and treated by the following 3 freeze thawing means by adding the same PBS buffer:
(1) The powdery tissue is not subjected to freeze thawing treatment;
(2) Placing the powdery tissue at-80 ℃ to room temperature for 1 time (12 h) of freeze thawing;
(3) Placing the powdery tissue at-80 ℃ to room temperature, and repeatedly freezing and thawing for 3 times (freezing time/thawing time: 2h/2 h);
the results show that: the protein concentration (BCA method) of the umbilical cord exosomes of the cattle is finally obtained in the freeze thawing mode (3) which is higher than that in modes (1) and (2) (see figure 1), and the invention adopts the mode (3).
Example 3: bovine umbilical cord extract post-treatment and exosome purification
The frozen and thawed product was sonicated (power: 500w, time: 30min, run/stop time: 8s/8 s), further disrupting the cells and releasing umbilical cord exosomes, and the ultrasound product was collected.
And (3) centrifuging: centrifuging the ultrasonic product obtained by operation at different rotational speeds (3000 rpm,10min;4000rpm,15min;10000rpm,1 h), and collecting supernatant of the last centrifugation after each centrifugation to remove insoluble substances in the filtrate;
and (3) ultrasonic treatment: subjecting the supernatant obtained by centrifugation in the last step to ultrasonic treatment (power: 500w, time: 30min, run/stop time: 8s/8 s);
And (3) filtering: the product after ultrasonic treatment is subjected to multistage filtration (the pore diameters of a filter screen are 5 mu m, 1 mu m, 0.45 mu m and 0.22 mu m in sequence) to prepare the bovine umbilical cord exosomes.
Example 4: identification of bovine umbilical cord exosomes
1) Protein concentration test:
Protein concentration detection is carried out on the prepared bovine umbilical cord exosomes by using a BCA method, and the result shows that the protein concentration of the bovine umbilical cord exosomes is 1.99+/-0.10 mg/ml.
2) Exosome identification
Particle size analysis, electron microscope observation and immunoblotting analysis of the obtained bovine umbilical cord extract prove that the main component of the product obtained by the preparation process is exosome. The specific detection results are shown in fig. 2.
Example 5: bovine umbilical cord exosome efficacy test
The experimental procedure was as follows: taking human skin fibroblasts in a logarithmic growth phase with good growth state, inoculating 2500 cells into a 96-well plate, arranging 3 compound wells in 100 mu l each, placing into a cell culture box, culturing at 37 ℃ under the conditions of 5% CO 2 and saturated humidity, absorbing and discarding the culture medium after culturing for 24 hours, and respectively adding a negative control culture medium, a positive control culture medium and a culture medium with corresponding concentration gradients (25, 50 and 100 mu g/ml) of bovine umbilical cord exosomes according to experimental groups. After 48h of incubation, the drug-containing medium was discarded and freshly prepared 10. Mu.l of toxicity test solution CCK8 was added to each well. After the culture is continued for 4 hours in an incubator, an enzyme-labeled instrument is used for measuring an OD value with the wavelength of 450nm, and the average value of the experimental results is taken as a final experimental result for statistical analysis.
The proliferation experiment of human skin fibroblasts shows that the bovine umbilical cord exosome prepared by the invention has remarkable effect of promoting proliferation of human skin fibroblasts (see figure 3).
The foregoing is only for the understanding of the method of the present invention and the core idea thereof, and it should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications also fall within the protection scope of the claims of the invention.
Claims (7)
1. The extraction method of the animal umbilical cord exosome is characterized by comprising the following steps:
step 1, quick freezing animal umbilical cord tissues with liquid nitrogen, and then grinding the umbilical cord tissues into uniform powdery tissues;
step 2, adding the buffer solution into the powdery tissue, and repeatedly freezing and thawing for 3 times at the temperature of-80 ℃ and the room temperature;
Step 3, ultrasonically treating the frozen and thawed product;
step 4, centrifuging after ultrasonic treatment, continuing ultrasonic treatment of supernatant, and then filtering to obtain the exosomes;
The animal umbilical cord tissue is bovine umbilical cord tissue;
the time of freeze thawing is freezing time/thawing time: 2h/2h;
The ultrasonic treatment comprises the following steps:
Power 500w, time 30min, run/stop time: 8s/8s.
2. The extraction method according to claim 1, wherein the centrifugation is performed using a centrifugal force with a plurality of rotational speed increases.
3. The extraction method according to claim 1 or 2, characterized in that the centrifugation is:
Centrifugation at 3000rpm for 10min, collecting supernatant, centrifuging at 4000rpm for 15min, and collecting supernatant, centrifuging at 10000rpm for 1h.
4. The extraction method according to claim 1, wherein the filtration is a multi-stage filtration with decreasing screen pore size.
5. The extraction method according to claim 1 or 4, wherein the filtering is:
and filtering according to the filter screen apertures 5 [ mu ] m, 1 [ mu ] m, 0.45 [ mu ] m and 0.22 [ mu ] m in sequence.
6. An exosome obtained by the extraction method of any one of claims 1 to 5.
7. Use of the exosome of claim 6 in the preparation of a cosmetic for promoting proliferation of human skin fibroblasts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110779475.5A CN115125193B (en) | 2021-07-09 | 2021-07-09 | Animal umbilical cord extract and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110779475.5A CN115125193B (en) | 2021-07-09 | 2021-07-09 | Animal umbilical cord extract and extraction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115125193A CN115125193A (en) | 2022-09-30 |
| CN115125193B true CN115125193B (en) | 2024-04-26 |
Family
ID=83375170
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110779475.5A Active CN115125193B (en) | 2021-07-09 | 2021-07-09 | Animal umbilical cord extract and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115125193B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298709A (en) * | 2000-11-03 | 2001-06-13 | 内蒙古草原兴发股份有限公司 | Process for preparing polypeptide-nucleic acid nutritive liquid of sheep placenta |
| CN106038598A (en) * | 2016-05-31 | 2016-10-26 | 张正亮 | Method for preparing human-derived stem cell secretion bioactive factor and lysate |
| CN107012192A (en) * | 2017-04-14 | 2017-08-04 | 杭州观梓健康科技有限公司 | A kind of umbilical cord Collage Activitv peptide combinations and preparation method thereof |
| CN108379294A (en) * | 2018-03-07 | 2018-08-10 | 甘肃龙麒生物科技股份有限公司 | The technique that liquid nitrogen frozen polishing prepares donkey placenta freeze-dried powder |
| CN108823156A (en) * | 2018-07-04 | 2018-11-16 | 陕西神州生物技术有限公司 | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder |
| CN110448572A (en) * | 2019-08-04 | 2019-11-15 | 丰泽康生物医药（深圳）有限公司 | A kind of preparation method of umbilical cord mesenchymal stem cells active matter and the compound of cord blood stem cell active matter |
| CN111973547A (en) * | 2020-09-08 | 2020-11-24 | 深圳市先康达生物科技有限公司 | Stem cell active factor and its freeze-dried powder |
| CN112852718A (en) * | 2021-03-15 | 2021-05-28 | 广州赛莱拉生物基因工程有限公司 | Preparation method of cattle placenta exosome |
| CN113018245A (en) * | 2021-03-15 | 2021-06-25 | 广州赛莱拉生物基因工程有限公司 | Cow placenta cell exosome freeze-dried powder and preparation method thereof |
-
2021
- 2021-07-09 CN CN202110779475.5A patent/CN115125193B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298709A (en) * | 2000-11-03 | 2001-06-13 | 内蒙古草原兴发股份有限公司 | Process for preparing polypeptide-nucleic acid nutritive liquid of sheep placenta |
| CN106038598A (en) * | 2016-05-31 | 2016-10-26 | 张正亮 | Method for preparing human-derived stem cell secretion bioactive factor and lysate |
| CN107012192A (en) * | 2017-04-14 | 2017-08-04 | 杭州观梓健康科技有限公司 | A kind of umbilical cord Collage Activitv peptide combinations and preparation method thereof |
| CN108379294A (en) * | 2018-03-07 | 2018-08-10 | 甘肃龙麒生物科技股份有限公司 | The technique that liquid nitrogen frozen polishing prepares donkey placenta freeze-dried powder |
| CN108823156A (en) * | 2018-07-04 | 2018-11-16 | 陕西神州生物技术有限公司 | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder |
| CN110448572A (en) * | 2019-08-04 | 2019-11-15 | 丰泽康生物医药（深圳）有限公司 | A kind of preparation method of umbilical cord mesenchymal stem cells active matter and the compound of cord blood stem cell active matter |
| CN111973547A (en) * | 2020-09-08 | 2020-11-24 | 深圳市先康达生物科技有限公司 | Stem cell active factor and its freeze-dried powder |
| CN112852718A (en) * | 2021-03-15 | 2021-05-28 | 广州赛莱拉生物基因工程有限公司 | Preparation method of cattle placenta exosome |
| CN113018245A (en) * | 2021-03-15 | 2021-06-25 | 广州赛莱拉生物基因工程有限公司 | Cow placenta cell exosome freeze-dried powder and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 陈乃宏.药理学实验操作教程（全视频展示）.中国协和医科大学出版社,2021,第136页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115125193A (en) | 2022-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11318168B2 (en) | Human tissue derived compositions and uses thereof | |
| JP2017190336A (en) | Acellular and bioabsorbable tissue regeneration matrix created by incubating acellular blood product | |
| ES2939147T3 (en) | Allogeneic microvascular tissue for soft tissue treatments | |
| CN107126556B (en) | Stem cell extract, preparation method thereof and application thereof in preparation of skin wound repair preparation | |
| CN108486047B (en) | Medical dressing of stem cell extract and preparation method thereof | |
| WO2015127126A1 (en) | Regenerative cell and adipose-derived stem cell processing system and method | |
| US20220287952A1 (en) | Compositions containing exosomes from animal placenta, methods for producing the same and uses thereof | |
| CN111202749A (en) | Preparation method of stem cell active factor composition with muscle cell repair function | |
| CN111690600A (en) | Engineered human umbilical cord mesenchymal stem cell exosome, transdermal preparation and application | |
| CN108619086A (en) | A kind of Cellular gels preparation for treating tissue damage and application thereof and the active gel solution of holding freeze-stored cell used | |
| CN115125193B (en) | Animal umbilical cord extract and extraction method and application thereof | |
| CN112852718B (en) | Preparation method of bovine placenta exosome | |
| CN112426402A (en) | Application based on fibroblast exosomes and preparation method | |
| CN116515762A (en) | Engineered exosome for promoting diabetic wound healing and preparation method and application thereof | |
| US20080102106A1 (en) | Wound Healing Composition Comprising Substances From Diptera Larvae | |
| US20240263141A1 (en) | Tissue Extracts and Related Methods | |
| CN111450039A (en) | Mild and non-irritant moisturizing and anti-aging eye cream and preparation method thereof | |
| CN110028571A (en) | The preparation method and applications of blood broken wall growth factor | |
| CN111675823B (en) | Preparation method and application of silk fibroin freezing sponge capable of slowly releasing exosomes | |
| JP2009513269A (en) | A cell-free, bioabsorbable tissue regeneration matrix produced by incubating cell-free blood products | |
| CN111265550A (en) | Stem cell factor liposome for repairing damaged tissues and preparation method thereof | |
| CN108143751B (en) | Preparation method and application of male silkworm moth exosome | |
| RU2398873C1 (en) | Method for making medical preparations | |
| US20230047934A1 (en) | Human tissue derived compositions and uses thereof | |
| KR20200138682A (en) | Method for micronizing a soft biological tissue and biological tissue graft thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |