CN115154399A - Platelet active factor, active component for promoting hair follicle growth, preparation method and application thereof - Google Patents
Platelet active factor, active component for promoting hair follicle growth, preparation method and application thereof Download PDFInfo
- Publication number
- CN115154399A CN115154399A CN202210781361.9A CN202210781361A CN115154399A CN 115154399 A CN115154399 A CN 115154399A CN 202210781361 A CN202210781361 A CN 202210781361A CN 115154399 A CN115154399 A CN 115154399A
- Authority
- CN
- China
- Prior art keywords
- platelet
- centrifugation
- hair follicle
- active
- follicle growth
- 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.)
- Pending
Links
- 230000001737 promoting effect Effects 0.000 title claims abstract description 54
- 230000009583 hair follicle growth Effects 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 210000001772 blood platelet Anatomy 0.000 claims abstract description 78
- 108090000190 Thrombin Proteins 0.000 claims abstract description 33
- 229960004072 thrombin Drugs 0.000 claims abstract description 33
- 210000004027 cell Anatomy 0.000 claims abstract description 27
- 230000000694 effects Effects 0.000 claims abstract description 25
- 238000010257 thawing Methods 0.000 claims abstract description 25
- 210000002381 plasma Anatomy 0.000 claims abstract description 24
- 239000004480 active ingredient Substances 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 238000000926 separation method Methods 0.000 claims abstract description 18
- 210000004700 fetal blood Anatomy 0.000 claims abstract description 17
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- 238000005119 centrifugation Methods 0.000 claims description 50
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 claims description 19
- 108010003541 Platelet Activating Factor Proteins 0.000 claims description 19
- 230000008014 freezing Effects 0.000 claims description 17
- 238000007710 freezing Methods 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 229960004494 calcium gluconate Drugs 0.000 claims description 4
- 239000004227 calcium gluconate Substances 0.000 claims description 4
- 235000013927 calcium gluconate Nutrition 0.000 claims description 4
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000013543 active substance Substances 0.000 abstract description 12
- 201000004384 Alopecia Diseases 0.000 abstract description 10
- 231100000360 alopecia Toxicity 0.000 abstract description 9
- 239000000243 solution Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 7
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 6
- 102000013275 Somatomedins Human genes 0.000 description 6
- 238000001994 activation Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 4
- 102000009618 Transforming Growth Factors Human genes 0.000 description 4
- 108010009583 Transforming Growth Factors Proteins 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 210000004209 hair Anatomy 0.000 description 4
- 210000003780 hair follicle Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 3
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 239000000262 estrogen Substances 0.000 description 3
- 229940011871 estrogen Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 2
- 206010003694 Atrophy Diseases 0.000 description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 2
- 239000003098 androgen Substances 0.000 description 2
- 229960003473 androstanolone Drugs 0.000 description 2
- 230000037444 atrophy Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 108010017843 platelet-derived growth factor A Proteins 0.000 description 2
- 230000003658 preventing hair loss Effects 0.000 description 2
- 229960003604 testosterone Drugs 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102100033875 3-oxo-5-alpha-steroid 4-dehydrogenase 2 Human genes 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 108010066551 Cholestenone 5 alpha-Reductase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101000640851 Homo sapiens 3-oxo-5-alpha-steroid 4-dehydrogenase 2 Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 102000001307 androgen receptors Human genes 0.000 description 1
- 108010080146 androgen receptors Proteins 0.000 description 1
- 206010068168 androgenetic alopecia Diseases 0.000 description 1
- 201000002996 androgenic alopecia Diseases 0.000 description 1
- 229940030486 androgens Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000031774 hair cycle Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000004904 shortening Methods 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
- 239000007790 solid phase Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
- A61K8/983—Blood, e.g. plasma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Dermatology (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a platelet active factor, an active component for promoting hair follicle growth, a preparation method and an application thereof, relating to the technical field of biology. The preparation method of the platelet activity factor provided by the invention comprises the following steps of removing most of red blood cells in umbilical cord blood, and obtaining cells and plasma after solid-liquid separation, so as to improve the content of platelets in the cells; and then, performing thrombin treatment and repeated freeze thawing on the obtained cells to fully release platelet active substances, and collecting supernatant after solid-liquid separation to obtain the platelet active factors. The preparation method is simple and convenient, and has high content of platelet active factor. The preparation method of the active component for promoting hair follicle growth provided by the invention mixes the platelet active factor and the plasma to prepare the active component for promoting hair follicle growth. The preparation method is simple and convenient, does not need special equipment, and the prepared active ingredients for promoting hair follicle growth are safe and harmless, and have good effects of preventing alopecia and promoting hair follicle growth.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a platelet active factor, an active component for promoting hair follicle growth, a preparation method and an application thereof.
Background
Platelets, upon activation, release bioactive substances therein, such as: platelet Derived Growth Factor (PDGF), transforming Growth Factor (TGF), insulin-like growth factor (IGF) and the like, which have synergistic effect, can promote the proliferation and differentiation of cells, can effectively improve local microenvironment to facilitate tissue repair, and are research hotspots in the fields of intractable ulcer, burn, oral medicine, plastic surgery and the like. The ability of these bioactive factors to promote cell and tissue repair depends on the concentration of growth factors released by high concentrations of platelets. In addition, high speed centrifugation easily leads to partial activation of platelets, and active factors are released in advance. Therefore, how to improve the enrichment degree of the blood platelets and increase the concentration of the bioactive factors has important significance.
In the case of androgenetic alopecia (seborrheic alopecia), testosterone in androgens is converted to dihydrotestosterone by the action of 5-alpha reductase, resulting in atrophy of hair follicles and thus in the appearance of alopecia.
(cells of susceptible hair follicles contain specific type II 5 alpha reductase, which converts testosterone androgen circulating in the blood to this region into dihydrotestosterone, which causes a series of reactions by binding to androgen receptors in the cells, thereby causing progressive miniaturization of hair follicles, gradual thinning of hair in the anagen phase, shortening of the hair growth cycle, and as a result, the hair, which is originally thick and dark, gradually turns into pale vellus hair, and finally, vellus hair is also shed due to atrophy and disappearance of hair follicles.
However, there is still a lack of a product for promoting hair follicle growth of human origin which is effective in preventing hair loss.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
A first object of the present invention is to provide a method for producing a platelet-activating factor, which solves at least one of the above problems.
The second object of the present invention is to provide a platelet-activating factor.
A third object of the present invention is to provide a method for preparing an active ingredient for promoting hair follicle growth, so as to solve at least one of the above problems.
It is a fourth object of the present invention to provide an active ingredient for promoting hair follicle growth.
The fifth purpose of the invention is to provide the application of the platelet activity factor or the hair follicle growth promoting active ingredient in preparing a product for promoting hair follicle growth.
In a first aspect, the present invention provides a method for preparing platelet-activating factor, comprising the steps of:
a. removing red blood cells in the umbilical cord blood, and then carrying out solid-liquid separation to respectively obtain cells and blood plasma;
b. and (b) performing thrombin treatment on the cells obtained in the step (a), then performing freeze thawing repeatedly, performing solid-liquid separation, and then reserving the supernatant to prepare the platelet activity factor.
As a further technical scheme, the method for removing the red blood cells in the umbilical cord blood comprises centrifugation;
preferably, the centrifugal force of the centrifugation is 100 to 600g, preferably 300g;
preferably, the centrifugation time is 8-12 min, preferably 10min;
preferably, the temperature of the centrifugation is 0 to 25 ℃.
As a further technical scheme, in the step a, the solid-liquid separation comprises centrifugation or filtration;
preferably, the centrifugal force of the centrifugation is 100 to 600g, preferably 300g;
preferably, the centrifugation time is 8-12 min, preferably 10min;
preferably, the temperature of the centrifugation is 0 to 25 ℃.
As a further technical scheme, the thrombin treatment is to mix the cells obtained in the step a with a thrombin solution;
preferably, the concentration of the thrombin solution is 800-1200U/mL, preferably 1000U/mL;
preferably, the volume ratio of the cells to thrombin solution is 8;
preferably, the thrombin solution is a calcium gluconate solution containing thrombin;
preferably, the thrombin treatment time is 0.5 to 2 hours, preferably 1 hour.
As a further technical scheme, the repeated freezing and thawing is freezing and thawing circularly;
preferably, the freezing temperature is-196 ℃ to-20 ℃, and the freezing time is 5-10 min;
preferably, the thawing temperature is 35-40 ℃, and the thawing time is 5-10 min;
preferably, the number of repeated freeze-thawing is 4 to 8, preferably 5.
As a further technical scheme, in the step b, the solid-liquid separation comprises centrifugation or filtration;
preferably, the centrifugal force of the centrifugation is 2500-3500 g, preferably 3000g;
preferably, the centrifugation time is 16-24 min, preferably 20min;
preferably, the temperature of the centrifugation is 0 to 25 ℃.
In a second aspect, the invention provides a platelet active factor prepared by the above preparation method.
In a third aspect, the present invention provides a method for preparing an active ingredient for promoting hair follicle growth, comprising: mixing the platelet activity factor with the cord blood plasma to prepare active components for promoting hair follicle growth;
preferably, the volume ratio of the platelet active factor to the cord blood plasma mixture is 3.
In a fourth aspect, the invention provides an active ingredient for promoting hair follicle growth, which is prepared by the preparation method.
In a fifth aspect, the invention provides an application of the platelet activity factor or the hair follicle growth promoting active ingredient in preparing a product for promoting hair follicle growth.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the platelet active factor provided by the invention comprises the following steps of removing most of red blood cells in umbilical cord blood, obtaining cells and plasma after solid-liquid separation, improving the content of platelets in the cells, and obtaining plasma rich in estrogen; and then performing thrombin treatment and repeated freeze thawing on the obtained cells to fully release platelet active substances, performing solid-liquid separation, collecting supernatant rich in the platelet active substances, and preparing the platelet active factors. The preparation method is simple and convenient, does not need special equipment, and the prepared platelet active factor has high content of effective components and good cell proliferation promoting effect.
The preparation method of the active component for promoting hair follicle growth provided by the invention mixes the platelet active factor and the plasma to prepare the active component for promoting hair follicle growth. The preparation method is simple and convenient, no special equipment is needed, the prepared active ingredients for promoting hair follicle growth are safe and harmless, and the active ingredients have good effects of preventing hair loss and promoting hair follicle growth, and can be used for preparing products for promoting hair follicle growth.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the effect of different concentrations of platelet-activating factor on the proliferation of mesenchymal stem cells, as provided in Experimental example 2;
FIG. 2 is a photograph before use in case 1;
FIG. 3 is a photograph after use in case 1;
fig. 4 is a photograph before use in case 2;
fig. 5 is a photograph after use in case 2.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the present invention provides a method for preparing a platelet-activating factor, comprising the steps of:
a. removing red blood cells in the umbilical cord blood, and then carrying out solid-liquid separation to respectively obtain cells and blood plasma;
b. and (b) performing thrombin treatment on the cells obtained in the step (a), then performing freeze thawing repeatedly, performing solid-liquid separation, and then reserving the supernatant to prepare the platelet activity factor.
The preparation method of the platelet active factor provided by the invention comprises the following steps of removing most of red blood cells in umbilical cord blood, obtaining cells and plasma after solid-liquid separation, improving the content of platelets in the cells, and obtaining plasma rich in estrogen; and then performing thrombin treatment and repeated freeze thawing on the obtained cells to fully release platelet active substances, performing solid-liquid separation, collecting supernatant rich in the platelet active substances, and preparing the platelet active factors. The preparation method is simple and convenient, does not need special equipment, and the prepared platelet active factor has high content of effective components and good cell proliferation promoting effect.
In some preferred embodiments, the method for removing red blood cells from cord blood comprises centrifugation.
Preferably, the centrifugal force of the centrifugation may be, for example, but not limited to, 100g, 200g, 300g, 400g, 500g or 600g, preferably 300g. The inventor researches and discovers that when the centrifugal force of the centrifugal machine is in the range of 100-600 g, the release of platelet active factors (platelet-derived growth factors (PDGF), transforming Growth Factors (TGF), insulin-like growth factors (IGF) and the like) can be avoided, and the content of the platelet active factors in a final product can be improved.
Preferably, the time of the centrifugation can be, but is not limited to, 8min, 9min, 10min, 11min or 12min, preferably 10min;
preferably, the temperature of the centrifugation may be, for example, but not limited to, 0 ℃, 5 ℃,10 ℃, 15 ℃,20 ℃ or 25 ℃.
Through further optimization and adjustment of the centrifugation conditions, erythrocyte impurities are better removed, and the early release of platelet active factors in the centrifugation process is avoided.
In some preferred embodiments, in step a, the solid-liquid separation comprises centrifugation or filtration.
Preferably, the centrifugal force of the centrifugation may be, for example, but not limited to, 100g, 200g, 300g, 400g, 500g or 600g, preferably 300g. The inventor researches and discovers that when the centrifugal force of the centrifugation is in the range of 100-600 g, the release of the platelet active substance can be avoided while the enrichment of the platelets is realized.
Preferably, the time of the centrifugation may be, but is not limited to, 8min, 9min, 10min, 11min or 12min, preferably 10min;
preferably, the temperature of the centrifugation can be, for example, but not limited to, 0 ℃, 5 ℃,10 ℃, 15 ℃,20 ℃ or 25 ℃.
By further optimizing and adjusting the centrifugation conditions, the enrichment of the platelets is realized on the premise of avoiding the release of platelet active substances.
The research of the invention finds that the thrombin is matched with the cells obtained in the step a in a repeated freeze-thawing mode to process, so that the release of effective substances in the platelets can be further improved, and the content of the effective substances in the platelets in the product can be improved.
In some preferred embodiments, the thrombin treatment is to mix the cells obtained in step a with a thrombin solution, and to promote the release of the active substance in the platelets by the thrombin treatment.
Preferably, the concentration of the thrombin solution may be, for example, but not limited to, 800U/mL, 900U/mL, 1000U/mL, 1100U/mL, or 1200U/mL, preferably 1000U/mL;
preferably, the volume ratio of the cells to thrombin solution can be, but is not limited to, 8;
preferably, the thrombin solution is a calcium gluconate solution containing thrombin;
preferably, the thrombin treatment time may be, for example, but not limited to, 0.5h, 1h, 1.5h or 2h, preferably 1h.
In some preferred embodiments, the repeated freezing and thawing is a cycle of freezing and thawing;
preferably, the freezing temperature may be, but not limited to, -196 ℃, -180 ℃, -160 ℃, -140 ℃, -120 ℃, -100 ℃, -80 ℃, -60 ℃, -40 ℃, or-20 ℃, and the freezing time is 5 to 10min.
Preferably, the thawing temperature can be, but is not limited to, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ or 40 ℃, and the thawing time is 5-10 min.
Preferably, the number of repeated freeze-thawing may be, for example, but not limited to, 4, 5, 6, 7 or 8, preferably 5.
By further optimizing and adjusting the thrombin treatment condition and the repeated freeze-thaw treatment condition, the active substances in the blood platelets are fully released, and the content of the active substances in the blood platelets in the product is improved.
In some preferred embodiments, in step b, the solid-liquid separation comprises centrifugation or filtration;
preferably, the centrifugal force of the centrifugation may be, for example, but not limited to 2500, 2700g, 2900g, 3100g, 3300g or 3500g, preferably 3000g;
preferably, the time of the centrifugation may be, for example, but not limited to, 16min, 18min, 20min,22 min or 24min, preferably 20min;
preferably, the temperature of the centrifugation can be, for example, but not limited to, 0 ℃, 5 ℃,10 ℃, 15 ℃,20 ℃ or 25 ℃.
Through further optimization and adjustment of the centrifugation conditions, solid-phase substances in the mixed solution are sufficiently removed.
In a second aspect, the invention provides a platelet active factor prepared by the above preparation method.
The platelet active factor provided by the invention has high content of effective components, and the research of the inventor finds that the platelet active factor has good proliferation promoting effect on mesenchymal stem cells.
In a third aspect, the present invention provides a method for preparing an active ingredient for promoting hair follicle growth, comprising: mixing the platelet active factor with the cord blood plasma to prepare active components for promoting hair follicle growth;
preferably, the volume ratio of the platelet active factor to the cord blood plasma mixture is 3.
The preparation method of the active component for promoting hair follicle growth provided by the invention mixes the platelet active factor and the plasma to prepare the active component for promoting hair follicle growth. The preparation method is simple and convenient, and does not need special equipment.
In a fourth aspect, the invention provides an active ingredient for promoting hair follicle growth, which is prepared by the preparation method.
The active component for promoting hair follicle growth is safe and harmless, has good effects of preventing alopecia and promoting hair follicle growth, and can be used for preparing products for promoting hair follicle growth.
In a fifth aspect, the invention provides an application of the platelet activity factor or the hair follicle growth promoting active ingredient in preparing a product for promoting hair follicle growth.
The platelet active factor provided by the invention is safe and harmless, has high content of effective components, and has good cell proliferation promoting effect; the active component for promoting hair follicle growth provided by the invention is safe and harmless, and has good effects of preventing alopecia and promoting hair follicle growth, so that the platelet active factor and the active component for promoting hair follicle growth provided by the invention can be used for preparing products for promoting hair follicle growth.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
A preparation method of platelet activity factor comprises the following steps:
1) Taking a public waste cord blood (with maternal blood and no more than 36h, the umbilical blood collection amount is more than 80 mL), sticking a bar code on the maternal blood, and then sending nucleic acid and enzyme immunoassay.
2) The cord blood was centrifuged for the first time using a bonder attached to the overhead triple blood collection bag at 300g,10min,22 ℃, plasma, buffy coat and a small amount of red blood cells were separated in a plasma clamp into another bag of the triple bag, and the majority of red blood cells in the main bag were discarded by heat pooling.
3) The plasma, the buffy coat layer and a small amount of red blood cells centrifuged as above were centrifuged for a second time (at 300g,10mins,22 ℃) and the upper layer of the cord plasma rich in estrogen was separated, and the remaining portion at this time was rich in platelets, a small amount of plasma and a small amount of red blood cells (platelet-rich fraction).
4) The platelet-rich fraction was aspirated by syringe, transferred to a 15mL centrifuge tube, and a 1000U/mL thrombin/calcium gluconate solution 10% in total volume (platelet-rich fraction volume) was added, mixed well and allowed to stand for 1 hour.
5) Standing for 1 hour, placing a 15ml centrifuge tube in liquid nitrogen at-196 ℃ to fully freeze the centrifuge tube for 5 minutes, taking out the centrifuge tube and placing the centrifuge tube in a 37 ℃ water bath box to unfreeze for 5 minutes; repeating the freezing and thawing process for 5 times, centrifuging again at 3000g,20min and 22 deg.C, and sucking supernatant to obtain platelet activity factor.
Example 2
A process for producing platelet-activating factor, which is different from example 1 in that the centrifugal force in the first centrifugation in step 1) is 100g and the centrifugal force in the second centrifugation in step 2) is 100g.
Example 3
A process for producing platelet-activating factor, which is different from example 1 in that the centrifugal force in the first centrifugation in step 1) is 600g and the centrifugal force in the second centrifugation in step 2) is 600g.
Comparative example 1
A method for producing platelet-activating factor, which is different from example 1 in that the centrifugal force of the first centrifugation in step 1) is 1200g, and the centrifugal force of the second centrifugation in step 2) is 1200g.
Comparative example 2
A method for producing platelet-activating factor, which is different from example 1 in that the centrifugal force in the first centrifugation in step 1) is 2400g, and the centrifugal force in the second centrifugation in step 2) is 2400g.
Comparative example 3
A method for preparing platelet-activating factor, which is different from example 1 in that the step 5) is:
after standing for 1 hour, the 15ml centrifuge tube was centrifuged again at 3000g,20min,22 ℃ and the supernatant was aspirated to obtain platelet activity factor.
Comparative example 4
A method for preparing platelet-active factor, which is different from example 1 in that the steps 4) and 5) are as follows:
4) The platelet rich fraction was aspirated by syringe, transferred to a 15mL centrifuge tube, added with 10% of total volume of normal saline, mixed well and then allowed to stand for 1 hour.
5) Standing for 1 hour, placing a 15ml centrifuge tube in liquid nitrogen at-196 ℃ to fully freeze the centrifuge tube for 5 minutes, taking out the centrifuge tube and placing the centrifuge tube in a 37 ℃ water bath box to unfreeze for 5 minutes; repeating the freezing and thawing process for 5 times, centrifuging again at 3000g,20min and 22 deg.C, and sucking supernatant to obtain platelet activity factor.
Test example 1
The platelet activity factors prepared in examples 1 to 3 and comparative examples 1 to 4 were examined, and the results are shown in tables 1 and 2.
TABLE 1 Effect of platelet concentration centrifugation acceleration on its subsequent activation
As can be seen from Table 1, under the condition of 300g, the activation of the platelet can be reduced to the maximum extent, and then the platelet active substance is released to the maximum extent in the subsequent activation process.
TABLE 2 Effect of different activation patterns of platelets on their active factor release
As can be seen from Table 2, when comparing example 1 with comparative examples 3 and 4, the content differences of PDGF-AA, IGF and TGF-beta are statistically significant, P is less than 0.05; compared with comparative example 4, the PDGF-AA, IGF and TGF-beta contents have no obvious difference, and P is more than 0.05. It is demonstrated that the treatment of platelet cells with thrombin in combination with repeated freezing and thawing can further improve the release of platelet active substances as compared with the treatment with thrombin alone and repeated freezing and thawing alone.
Test example 2 CCK8 test for promoting proliferation of mesenchymal stem cell by platelet-activating factor
The platelet-activating factor prepared in example 1 was diluted 0 times (i.e., 0mL of physiological saline was added), 1/4 times (i.e., 1/4 volume of physiological saline was added), 1/3 times (i.e., 1/3 volume of physiological saline was added), 1/2 times (i.e., 1/2 volume of physiological saline was added), and 1 time (i.e., the same volume of physiological saline was added) respectively, and then added to the mesenchymal stem cell culture system (Procell DMEM/F12 medium) in an amount of 5% of the total volume of the medium. After 0-96 hours of culture, the proliferation activity of the cells was measured at each time point, and the results are shown in Table 3 and FIG. 1.
TABLE 3 Effect of different concentrations of platelet-activating factor on mesenchymal Stem cell proliferation
Statistical analysis shows that the 1/4-fold dilution of the platelet activity factor has obvious difference with other groups (except 1/3-fold dilution) on the proliferation promotion of the mesenchymal stem cells after 48 hours; the 1/3-time diluted platelet activity factor has obvious difference on the proliferation promoting effect of the mesenchymal stem cells after 36 hours and other groups; the 0-fold dilution, the 1/2-fold dilution and the 1-fold dilution were not significantly different from the control group. Therefore, an appropriate concentration of platelet-activating factor is advantageous for the proliferation of cells.
Example 4
An active ingredient for promoting hair follicle growth is prepared by the following steps:
the platelet-activating factor provided in example 1 was diluted 1/3 fold with the cord plasma collected during the preparation of example 1, filtered through a 0.22um filter, and dispensed into 10 sterile vials, each containing 2mL of platelet-activating factor, and stored at-20 ℃.
Example 5
An active component for promoting hair follicle growth is prepared by the following steps:
the platelet-activating factor provided in example 1 was diluted 1/4 fold with the cord plasma collected during the preparation of example 1, filtered through a 0.22um filter, and dispensed into 10 sterile vials, each containing 2mL of platelet-activating factor, and stored at-20 ℃.
Case 1
The active ingredient for promoting hair follicle growth provided in example 4 was applied to the area of alopecia on a daily basis before use as shown in fig. 2, and after 1 month of use as shown in fig. 3.
Case 2
The active ingredient for promoting hair follicle growth provided in example 5 was applied to the area of alopecia on a daily basis for a male suffering from alopecia, as shown in FIG. 4 before use and in FIG. 5 after 1 month of use.
As can be seen from the results of cases 1 and 2, the active ingredient for promoting hair follicle growth provided by the invention has good effects of preventing alopecia and promoting hair follicle growth.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A preparation method of platelet active factor is characterized by comprising the following steps:
a. removing red blood cells in the umbilical cord blood, and then carrying out solid-liquid separation to respectively obtain cells and blood plasma;
b. and (b) treating the cells obtained in the step (a) with thrombin, then repeatedly freezing and thawing, and keeping the supernatant after solid-liquid separation to prepare the platelet active factor.
2. The method for preparing according to claim 1, wherein the method for removing erythrocytes in umbilical cord blood comprises centrifugation;
preferably, the centrifugal force of the centrifugation is 100 to 600g, preferably 300g;
preferably, the centrifugation time is 8-12 min, preferably 10min;
preferably, the temperature of the centrifugation is 0 to 25 ℃.
3. The preparation method according to claim 1, wherein in the step a, the solid-liquid separation comprises centrifugation or filtration;
preferably, the centrifugal force of the centrifugation is 100 to 600g, preferably 300g;
preferably, the centrifugation time is 8-12 min, preferably 10min;
preferably, the temperature of the centrifugation is 0 to 25 ℃.
4. The preparation method according to claim 1, wherein the thrombin treatment is mixing the cells obtained in step a with a thrombin solution;
preferably, the concentration of the thrombin solution is 800-1200U/mL, preferably 1000U/mL;
preferably, the volume ratio of the cells mixed with the thrombin solution is 8;
preferably, the thrombin solution is a calcium gluconate solution containing thrombin;
preferably, the thrombin treatment time is 0.5 to 2 hours, preferably 1 hour.
5. The method of claim 1, wherein the repeated freezing and thawing is a cycle of freezing and thawing;
preferably, the freezing temperature is-196 to-20 ℃, and the freezing time is 5 to 10min;
preferably, the thawing temperature is 35-40 ℃, and the thawing time is 5-10 min;
preferably, the number of repeated freeze-thaw cycles is 4 to 8, preferably 5.
6. The method according to claim 1, wherein in the step b, the solid-liquid separation comprises centrifugation or filtration;
preferably, the centrifugal force of the centrifugation is 2500-3500 g, preferably 3000g;
preferably, the centrifugation time is 16-24 min, preferably 20min;
preferably, the temperature of the centrifugation is 0 to 25 ℃.
7. A platelet-activating factor produced by the production method according to any one of claims 1 to 6.
8. A method for preparing an active ingredient for promoting hair follicle growth, which comprises the following steps: mixing the platelet active factor of claim 7 with the cord blood plasma to prepare an active ingredient for promoting hair follicle growth;
preferably, the volume ratio of the platelet active factor to the cord blood plasma mixture is 3.
9. An active ingredient for promoting hair follicle growth, which is obtained by the production method according to claim 8.
10. Use of the platelet activity factor of claim 7 or the hair follicle growth promoting active ingredient of claim 9 for the preparation of a product for promoting hair follicle growth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210781361.9A CN115154399A (en) | 2022-07-04 | 2022-07-04 | Platelet active factor, active component for promoting hair follicle growth, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210781361.9A CN115154399A (en) | 2022-07-04 | 2022-07-04 | Platelet active factor, active component for promoting hair follicle growth, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115154399A true CN115154399A (en) | 2022-10-11 |
Family
ID=83491972
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210781361.9A Pending CN115154399A (en) | 2022-07-04 | 2022-07-04 | Platelet active factor, active component for promoting hair follicle growth, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115154399A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4957742A (en) * | 1984-11-29 | 1990-09-18 | Regents Of The University Of Minnesota | Method for promoting hair growth |
| US9227089B1 (en) * | 2009-01-14 | 2016-01-05 | Pgfx Patent Holdings, Llc | Skin treatment for promoting hair growth |
| CN107412119A (en) * | 2017-04-27 | 2017-12-01 | 博雅干细胞科技有限公司 | The freeze-dried powder prepared from placenta enrichment blood platelet and extraction active factors |
| CN110484498A (en) * | 2019-03-15 | 2019-11-22 | 山东省齐鲁干细胞工程有限公司 | A method of serum substitute is prepared using Cord blood |
| CN111265548A (en) * | 2020-03-20 | 2020-06-12 | 山东省齐鲁细胞治疗工程技术有限公司 | Preparation method of platelet-rich cytokine plasma freeze-dried powder |
-
2022
- 2022-07-04 CN CN202210781361.9A patent/CN115154399A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4957742A (en) * | 1984-11-29 | 1990-09-18 | Regents Of The University Of Minnesota | Method for promoting hair growth |
| US9227089B1 (en) * | 2009-01-14 | 2016-01-05 | Pgfx Patent Holdings, Llc | Skin treatment for promoting hair growth |
| CN107412119A (en) * | 2017-04-27 | 2017-12-01 | 博雅干细胞科技有限公司 | The freeze-dried powder prepared from placenta enrichment blood platelet and extraction active factors |
| CN110484498A (en) * | 2019-03-15 | 2019-11-22 | 山东省齐鲁干细胞工程有限公司 | A method of serum substitute is prepared using Cord blood |
| CN111265548A (en) * | 2020-03-20 | 2020-06-12 | 山东省齐鲁细胞治疗工程技术有限公司 | Preparation method of platelet-rich cytokine plasma freeze-dried powder |
Non-Patent Citations (1)
| Title |
|---|
| 李洪涛等: "液氮冻融促进血小板源性生长因子AA与转化生长因子β1的释放", 《中国组织工程研究与临床康复》, vol. 14, no. 36, 3 September 2010 (2010-09-03), pages 6764 - 6767 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2250528C (en) | Hair treatment composition and method of manufacturing the composition | |
| EP3603683A1 (en) | Composite hemostatic agent containing iron oxide/nano-kaolin and preparation method thereof | |
| Shah et al. | Biological activation of bone grafts using injectable platelet-rich fibrin | |
| CN110051006A (en) | Zeins/Arabic gum composite nanometer particle and preparation method thereof | |
| DE2231676A1 (en) | PROCESS FOR FRACTIONATION OF BLOOD SERUM AND PLASMA PRODUCTS | |
| CN114699446A (en) | Exosome compound liquid for treating alopecia and preparation method thereof | |
| CN107412119A (en) | The freeze-dried powder prepared from placenta enrichment blood platelet and extraction active factors | |
| EP2781224A1 (en) | Implantable preparations comrpsing globin insoluble at physiological pH and serum for regeneration of tissues and treatment of wounds. | |
| CN115154399A (en) | Platelet active factor, active component for promoting hair follicle growth, preparation method and application thereof | |
| ATE234644T1 (en) | METHOD FOR PRODUCING AN AUTOLOGOUS FIBRIN GLUE | |
| CN110974769A (en) | Preparation and application of hair follicle stem cell regeneration peptide nano liposome | |
| CN107050516A (en) | A kind of fatty gel preparation method rich in fat stem cell and extracellular matrix | |
| CN110237012B (en) | Bletilla striata hand cream for preventing and treating chapped skin and preparation method thereof | |
| CN115125191B (en) | Fibroblast transformation medium, application thereof and preparation method of fibroblast | |
| CN110538196A (en) | Platelet-rich plasma and method for extracting platelet-rich plasma | |
| CN106769325A (en) | A kind of blood nanometer coagulant | |
| CN117165520B (en) | Mesenchymal stem cell exosome, gel preparation and application thereof in epidermal wound and freckle removal | |
| KR101313755B1 (en) | Method for Preparing Autologous Thrombin Activated Autologous Plasma for Regeneration of Epithelial Tissues or Subcutaneous Tissues and Composition Containing the Autologous Plasma | |
| Hill et al. | The reduction of haematin and methaemoglobin | |
| Ward et al. | Free erythrocyte protoporphyrin | |
| CN117797171A (en) | Preparation method and application of autologous platelet-rich plasma | |
| CA2617465A1 (en) | Triple spin, double pool and revolumization process for concentrating platelets, and derivative platelet concentrate | |
| TWI670071B (en) | Composition for promoting hair growth or promoting hair cell growth and use thereof | |
| CN111849886B (en) | SVF cells, and preparation method and application thereof | |
| CN115926245B (en) | Chitosan crosslinked graphene oxide composite sponge with high hemostatic efficiency and preparation method and application 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 |