CN110028571A - The preparation method and applications of blood broken wall growth factor - Google Patents
The preparation method and applications of blood broken wall growth factor Download PDFInfo
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- CN110028571A CN110028571A CN201810029948.8A CN201810029948A CN110028571A CN 110028571 A CN110028571 A CN 110028571A CN 201810029948 A CN201810029948 A CN 201810029948A CN 110028571 A CN110028571 A CN 110028571A
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- 239000003102 growth factor Substances 0.000 title claims abstract description 121
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- 239000003963 antioxidant agent Substances 0.000 claims abstract description 8
- 235000006708 antioxidants Nutrition 0.000 claims abstract description 8
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- 238000000034 method Methods 0.000 claims description 27
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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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/49—Platelet-derived growth factor [PDGF]
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
This application provides the preparation method and applications of blood broken wall growth factor.The non-human blood's broken wall growth factor obtained by preparation method provided by the present application has high yield and marketization value, be mainly used in skin again, skin-whitening or skin it is anti-oxidant/anti-aging etc..
Description
Technical field
The application belongs to field of biotechnology, is related to the preparation method and applications of blood broken wall growth factor.
Background technique
Growth factor is naturally present in intracorporal protein, irritating and enhancing cell growth and tissue repair energy
Power.Platelet reserve in blood a large amount of active growth factors.
High blood concentrations thrombocyte plasma, i.e. PRP are largely used currently used for wound healing and athletic injury.However,
The PRP extracted from patient must be unable to long-time storage using fresh.The average life span of blood platelet is usually only mentioning
5 to 9 days after taking, however normal PRP treatment procedure needs carry out 3 to 6 months by a definite date.In general, being controlled before receiving PRP every time
When treatment, patient must be subjected to venipuncture waiting 30 minutes above for extraction PRP.Therefore, growth factor in PRP is guaranteed the quality
Phase, which extends to allow in therapeutic process, to be avoided repeatedly drawing blood, and saves time and the labour of doctor and patient, and reduce PRP and be produced into
This.
Summary of the invention
On the one hand, this application provides the preparation methods of blood broken wall growth factor comprising following steps: separation blood
Liquid, to obtain high concentration thrombocyte plasma；And cracking high concentration thrombocyte plasma, to obtain growth factor extracting solution.
In one embodiment, the preparation method of blood broken wall growth factor provided by the present application is the following steps are included: divide
From blood, to obtain high concentration thrombocyte plasma；High concentration thrombocyte plasma is cracked, to obtain growth factor extracting solution；And
Growth factor extracting solution is filtered, to remove pollutant.
In another embodiment, blood broken wall growth factor provided by the present application preparation method the following steps are included:
Blood is separated, to obtain high concentration thrombocyte plasma；High concentration thrombocyte plasma is cracked, to obtain growth factor extracting solution；It crosses
Growth factor extracting solution is filtered, to remove pollutant；And the filtered growth factor extracting solution of freeze-drying.
In another embodiment, preparation method provided herein further includes in high concentration blood platelet blood obtained
Buffer is added in slurry so that the step of its pH stable.In a particular embodiment, the buffer is HEPES, phosphoric acid delays
Rush salting liquid (PBS) or physiological saline etc..
In one embodiment, the step of blood is separated in the preparation method is realized by being centrifuged.It is being embodied
In scheme, the centrifugal condition is as follows: 800-2300 gram/minute.In another embodiment, the centrifugation carries out 3 times.
In one embodiment, in the preparation method crack high concentration thrombocyte plasma the step of by freezing/weight it is molten,
Ultrasound or grinding are to realize.In a particular embodiment, the molten condition of freezing/weight is as follows: -50 DEG C to -60 DEG C freezing 20-30
Minute, 35 DEG C to 40 DEG C weights are 5-15 minutes molten.In another embodiment, described freeze/to dissolve into row again at least 2 times.?
In another specific embodiment, the ultrasound condition is as follows: 185W, 40kHz ultrasound 10-20 minutes.
In a particular embodiment, the preparation method of broken wall growth factor provided herein comprising following steps:
Blood obtains high concentration thrombocyte plasma with 800-2300 gram/minute centrifugation 3 times；And high concentration thrombocyte plasma is by extremely
Few 2 freezing/weights are molten, or ultrasound cracking at least 10 minutes, obtain growth factor extracting solution.
In another embodiment, the preparation method of broken wall growth factor provided herein comprising following
Step: blood obtains high concentration thrombocyte plasma with 800-2300 gram/minute centrifugation 3 times；High concentration thrombocyte plasma is by extremely
Few 2 freezing/weights are molten, or ultrasound cracking at least 10 minutes, obtain growth factor extracting solution；And growth factor extracting solution warp
After filtering, freeze-drying preservation is carried out.
In the preparation method of above-mentioned broken wall growth factor, the 1st progress of centrifugation 5-10 minutes, the 2nd progress
5-10 minutes, the 3rd progress 10-20 minutes,
In the preparation method of above-mentioned broken wall growth factor, the molten condition of freezing/weight is as follows: -50 DEG C to -60 DEG C cold
Freeze 20-30 minutes, 35 DEG C to 40 DEG C weights are 5-15 minutes molten, and the molten preferred progress of the freezing/weight 2 to 5 times.
In the preparation method of above-mentioned broken wall growth factor, the ultrasound condition is as follows: 185W, 40kHz ultrasound 10-20
Minute.
In another embodiment, in preparation method provided herein, the blood is originated from following animal: feeding
Newborn class, birds, reptiles, amphibian animal and fish.In a particular embodiment, the blood is originated from fish.
In addition, broken wall growth factor prepared by method provided herein mainly includes VEGF, EGF and PDGF.Institute
The half-life period for stating broken wall growth factor is 12 months or more.
On the other hand, it this application provides the broken wall growth factor by above-mentioned method preparation, is used for skin trauma and is cured
Conjunction, skin regeneration, skin-whitening or skin it is anti-oxidant/anti-aging.
Another aspect is used to prepare in following articles this application provides the broken wall growth factor by above-mentioned method preparation
Purposes: skin regeneration articles；Skin-whitening articles；Skin is anti-oxidant/anti-aging articles；Or assist skin wound healing
Articles.
Detailed description of the invention
Fig. 1 is the optimum results that centrifugal condition in blood step is separated in embodiment 1.
Fig. 2 is the optimization that molten (Frozen-thawed cycled) condition of freezing/weight in high concentration thrombocyte plasma step is cracked in embodiment 1
As a result.
Fig. 3 is the optimum results that ultrasound condition in high concentration thrombocyte plasma step is cracked in embodiment 1.
Fig. 4 is the effect of the mankind and non-human broken wall growth factor to hyperplasia in embodiment 2.
Fig. 5 is the TScratch analysis chart of the cellular layer coverage rate of HaCaT cell in embodiment 3.
Fig. 6 is the test result that 3 Mesichthyes broken wall growth factor of embodiment acts on wound healing.
Fig. 7 be in embodiment 4 mankind and non-human broken wall growth factor to the inhibiting effect of tyrosinase activity.
Fig. 8 is inhibiting effect of the 4 Mesichthyes broken wall growth factor of embodiment to tyrosinase activity.
Fig. 9 is the oxidation resistance test result of mankind's broken wall growth factor in embodiment 5.
Figure 10 is the oxidation resistance test result of 5 Mesichthyes broken wall growth factor of embodiment.
Specific embodiment
Defined below and method is provided preferably to define the application and instruct this field general in the application practice
Logical technical staff.Unless otherwise mentioned, term understands according to the common usage of person of ordinary skill in the relevant.It is cited herein
All patent documents, academic paper, professional standard and other public publications etc., full content therein is integrally incorporated herein
As reference.
In this application, " blood broken wall growth factor " or " broken wall growth factor " mean to be contained in blood in blood platelet
Various growth factors, can by ultrasound or freezing/weight molten (Frozen-thawed cycled) puncture platelet membrane and collect acquisitions, mainly packet
Include platelet derived growth factor (PDGF), the vascular endothelial cell factor (VEGF) and epidermal growth factor (EGF) etc..
On the one hand the application provides a kind of store method of improved growth factor, can extend guaranteeing the quality for growth factor
Phase makes it across entire treatment stage.Store method provided herein provides stable freeze-drying growth factor concentrate,
Can be with long-time storage more than 6 months, the usual PRP course for the treatment of that continues 3 to 6 months enough.In addition, utilizing guarantor provided by the present application
Method is deposited, is the high bioactivity that can be reserved for growth factor without special storing mode, while without in entire treatment phase
Between repeatedly extract blood and repeatedly processing to obtain fresh growth factor.
Realize that the technical solution of the store method of above-mentioned growth factor is as follows:
(1) blood sample of the blood platelet comprising growth factor to be saved is obtained；
(2) by growth factor release from blood platelet；
(3) growth factor discharged is filtered；
(4) freeze-drying saves filtered growth factor, thus can extend the holding time of growth factor.
On the other hand, this application provides a kind of preparation method of blood broken wall growth factor, this method is at low cost, operation
Simply, and by the method broken wall growth factor extracting solution obtained mainly contained the various growths of VEGF, EGF and PDGF because
Son.
Realize that the preparation method technical solution of above-mentioned blood broken wall growth factor is as follows:
The first step, the collection of blood PRP
Blood sample is collected, including saving the blood platelet for wherein containing growth factor for therapeutical uses in future.In general, blood
Sample mainly passes through venous collection from inhuman animal.The preferably blood sample of 30ml-60ml, and it is stored in lemon
Convenient transportation in sour sodium.
Blood sample is placed in a centrifuge and is centrifuged (3,800 turns -4,000 turns per minute).It is highly preferred that it is described from
The heart repeats three times, and 5 minutes for the first time, second 5 minutes, third time 20 minutes.The purpose of centrifugation be make it is PRP layers thin with blood
Born of the same parents' layer separation, to improve the concentration of growth factor in PRP.
Second step, the stabilization of PRP
The pH value that buffer stablizes PRP is added, needed for being maintained to avoid pH value fluctuation at extremely low temperatures and by pH value
In range, the effect of making growth factor, which not will receive temperature, to be influenced and changes.Buffer, such as 0.6-1.0%4- (2- hydroxyl
Ethyl) -1- piperazine ethanesulfonic acid (HEPES), preferably 0.8%HEPES can be regarded as in terms of PRP volume for freeze drying protectant.
Third step discharges growth factor from blood platelet
In order to obtain growth factor, freezes 30 minutes at a temperature of needing PRP to be placed on -50 DEG C to -60 DEG C, then pass through
Pipe is placed in 37 DEG C of water-bath and is thawed 10 minutes.By freezing and weighing molten PRP, ice crystal will form makes its volume in blood platelet
Expansion and final plaque rupture.In this way, just can be from the growth factor extracting solution obtained in PRP in blood platelet.The molten side of freezing/weight
Case is at least repeated twice, it is therefore an objective to increase the recovery rate of growth factor.Blood platelet is easy to apoptosis at low temperature, may release inhibition
Agent simultaneously destroys growth factor therein.However, by growth factor of this method from intra platelet free calcium, retention period can prolong significantly
It is long.
Platelet lysates, which can be, to be accomplished in several ways, and molten or mechanical lysis is including but not limited to freezed/weigh, packet
Include ultrasonic treatment and hand-ground at low temperature.For example, in 185W, ultrasound 10 minutes or more under 40kHz.
4th step, filtration sterilization
The extracting solution for containing growth factor need to be through filtration treatment (for example, 0.2 μm of cellulose acetate film be as filter).
For the purpose of filtering primarily to reducing pathogen and other pollutants, these pollutants may influence the effective stability of solution
And the service life with growth factor.
5th step, freeze-drying
Selectively, filtered growth factor extracting solution obtained is freeze-dried, to save backup.
By the growth of blood broken wall growth factor preparation method disclosed in the present application non-human blood's broken wall obtained because
Son with yield and is easier to market-oriented absolute predominance, and white-skinned face function is more significant, and anti-oxidation efficacy is also relatively more steady
It is fixed.Production cost can be greatly reduced using broken wall growth factor prepared by non-human blood, and drug, health care can be widely used for
Product, food and beauty product have huge market potential.That is, broken wall growth factor is extracted with non-human blood, it can
The yield and marketization value for improving broken wall growth factor, are mainly used for the production of skin regeneration, whitening and anti-aging product etc..
Below with reference to the drawings and specific embodiments, the application is described in further detail.
Embodiment 1
The present embodiment is exemplary to provide the preparation method of broken wall growth factor, and blood therein may originate from the mankind and inhuman
Class species (such as pig, chicken, fish).
Specifically, provided broken wall growth factor preparation method the following steps are included:
A) 30ml-60ml blood sample is rotated into row centrifugation 5 minutes with 3,000-5,000 per minute；Supernatant is taken, then with every
Minute 3,000-5,000 rotates into row centrifugation 5 minutes；Supernatant is taken again, then rotates into 20 points of row centrifugation with per minute 3,000-5,000
Clock, thus to obtain high concentration thrombocyte plasma；
B) Frozen-thawed cycled is utilized, i.e., -50 DEG C to -60 DEG C freeze 30 minutes, 37 DEG C of weights molten 10 minutes；Or 185W,
It is ultrasonic under 40kHz, so that the platelet lysates in high concentration thrombocyte plasma obtained, mention to obtain broken wall growth factor
Liquid is taken, including the various growth factors such as VEGF, EGF and PDGF；
C) after 0.2 μm of cellulose acetate film filtering, filtrate is placed in -80 DEG C, vacuum refrigeration is carried out under 150mTor
It is 16 hours dry, it is placed in 4 DEG C and saves backup.
Condition when the present embodiment is centrifuged blood sample is optimized.Fig. 1 shows third time centrifugation time
Influence to the growth factor content released in high concentration thrombocyte plasma.As seen from Figure 1, with the increasing of third time centrifugation time
Adding, the content of releasing of various growth factors dramatically increases, and when more than 20 minutes, content there is no variation or drop instead
It is low.Therefore, third time centrifugation time was advisable with 10 to 20 minutes, and 20 minutes best.
The blood platelet in multigelation or the method cracking high concentration thrombocyte plasma of ultrasound, the present embodiment pair can be used
Different freeze thawing or ultrasound condition are optimized.
From Figure 2 it can be seen that the content of releasing of various growth factors dramatically increases with the increase of number of freezing and thawing, and freeze thawing time
When number is more than 5 times, content there is no variation or reduce instead.Therefore, number of freezing and thawing is at least 2 times, is advisable with 2 to 5 times,
5 suboptimums.
Fig. 3 shows influence of the ultrasonic time to the growth factor content released.As seen from Figure 3, with ultrasonic time
Increase, the content of releasing of various growth factors dramatically increases.Therefore, ultrasonic time is at least 10 minutes, is with 10 to 20 minutes
Preferably, 10 minutes it is best.
Embodiment 2
The present embodiment carries out HaCaT Skin Cell hyperplasia to prepared broken wall growth factor according to the method for embodiment 1 and makees
With experiment.
Specifically, carried out HaCaT Skin Cell proliferative effect experiment the following steps are included:
A) broken wall growth factor is made referring to the preparation method of embodiment 1.
B) prepared mankind's broken wall growth factor and non-human broken wall growth factor extracting solution addition HaCaT skin is thin
Born of the same parents cultivate together, collect and are studied in different time.
It using MTT experimental test cell survival rate, makes comparisons with blank control, to assess broken wall growth factor to skin
The proliferative effect of cell.
As a result as shown in Figure 4.As it can be seen that mankind's broken wall growth factor has obvious proliferative effect for Skin Cell；However,
In non-human broken wall growth factor, only the broken wall growth factor made of fish blood has obvious proliferative effect for Skin Cell.
Embodiment 3
The present embodiment carries out wound healing assay to broken wall growth factor prepared according to the method for embodiment 1.
Specifically, the wound healing assay that is carried out the following steps are included:
A) broken wall growth factor is made referring to the preparation method of embodiment 1.
B) by HaCaT cell kind in 6 orifice plates plate hole until complete Mi Pu.
C) upper cross is drawn in each 6 orifice plates plate hole bottom center with suction pipette head.
D) blank control, positive control (VEGF) and fish broken wall growth factor is added in 6 orifice plates respectively.
E) it shoots cellular layer coverage rate under 50 times of magnifying power microscopes to note down, it is determined as T0.
F) cellular layer coverage rate is re-shoot after 8 hours and 16 hours, it is determined as T8 and T16.
G) cellular layer coverage rate is analyzed and compared using TScratch software.
Fig. 5 is the TScratch analysis chart of the cellular layer coverage rate of HaCaT cell, is corresponding in turn to respectively sky by left-to-right
White control, positive control (VEGF) and negative control (mek inhibitor U0126), be followed successively by from top to bottom T0, T8 and
TScratch analysis chart.
Fig. 6 be each group test after 16 hours (T16) under 50 times of magnifying power microscopes cellular layer coverage rate lab diagram,
Cellular layer coverage rate when wherein the first behavioral experiment starts when (T0), the second behavior each group are tested after 16 hours (T16)
Cellular layer coverage rate is cured by the wound of left-to-right respectively blank control, positive control (VEGF) and fish broken wall growth factor
Close experimental result.
The result shows that the wound healing cells layer coverage rate (40% of fish broken wall growth factor is added after 16 hours
→ 95%) obviously higher than blank control (40% → 50%), effect and positive control VEGF (5ng/mL) are suitable.
Above-mentioned experimental result shows that fish broken wall growth factor can accelerate the healing rate of wound.
Embodiment 4
The present embodiment carries out tyrosinase inhibitory action survey to broken wall growth factor prepared according to the method for embodiment 1
Examination.
Specifically, carried out tyrosinase inhibitory action test the following steps are included:
A) broken wall growth factor is made referring to the preparation method of embodiment 1.
B) extracting solution is respectively used to the test of tyrosinase activity inhibiting effect, while any inhibitor is added with no
Control be blank control, the test result of vitamin C (strong antioxidant action) then as experiment positive control.
The test of tyrosinase activity inhibiting effect is to screen the conventional test methodologies of whitening drug.Since tyrosinase can be with
Dopachrome is converted by tyrosine, thus in the test, dopachrome production quantity is lower, illustrates extracting solution to tyrosinase
Inhibiting effect it is better.Dopachrome has absorptance under 492nm wavelength, can extrapolate tyrosinase according to the amount of dopachrome
Activity, while be also known that sample whether have whitening function.The tyrosinase activity that any inhibitor is not added is
30.61 μM of levodopa/minutes.
As a result as shown in Figure 7 and Figure 8, fish broken wall growth factor can inhibit the active average out to 45-70% of tyrosinase,
And the tyrosinase inhibition rate of mankind's broken wall growth factor is about 14-22%.In addition, the beauty of broken wall growth factor is made with chicken blood
Though white effect is high, however its cell promotes growth result unobvious (see Fig. 4).
From the above results, mankind's broken wall growth factor provided herein and fish broken wall growth factor can pass through
Whitening function is played to the inhibiting effect of tyrosinase.Whitening function of the fish broken wall growth factor than mankind's broken wall growth factor
It imitates more significant.It can be seen that non-human broken wall growth factor can be used for preparing various whitening products.
Embodiment 5
The present embodiment carries out oxidation resistance experiment to broken wall growth factor prepared according to the method for embodiment 1.
Specifically, the experiment of provided oxidation resistance the following steps are included:
A) the broken wall growth factor of different samples is made in the preparation method referring to embodiment 1.
B) mankind's broken wall growth factor of each sample and fish broken wall growth factor are respectively used to the reagent of oxidation resistance
Box (Total Antioxidant Capacity (TAC) Colorimetric Assay Kit, BioVision
Inc.Milpitas, CA) test, while being experiment negative control with no test result that any extracting solution is added.
C) standard curve is established with the anti-copper ion oxidability of Trolox (analog of vitamin E) a kind of, and according to
The standard curve calculates the total antioxidant capacity of extracting solution.In this test, Trolox equivalence is higher, then illustrates that extracting solution is anti-
Oxidability is better.
As a result as shown in Figure 9 and Figure 10.As it can be seen that the antioxidation average value of mankind's broken wall growth factor is about 5-55mM
Trolox is equivalent；And the antioxidation average value of fish broken wall growth factor is about 35-69mM Trolox equivalent.
From the above results, mankind's broken wall growth factor provided herein and fish broken wall growth factor all have
Antioxidation, aging effects caused by capable of preventing because of oxidation.The antioxidation of fish broken wall growth factor compares
Stablize, can be used for preparing various anti-oxidant and anti-aging product.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. the preparation method of blood broken wall growth factor comprising following steps:
Blood is separated, to obtain high concentration thrombocyte plasma；
High concentration thrombocyte plasma is cracked, to obtain growth factor extracting solution；
Growth factor extracting solution is filtered, to remove pollutant；And
Optionally, it is freeze-dried filtered growth factor extracting solution.
2. the method as described in claim 1, wherein further including that buffer is added in high concentration thrombocyte plasma obtained
So that the step of its pH stable；Preferably, the buffer is HEPES buffer solution, phosphate buffer or physiological saline.
3. it is method according to claim 1 or 2, wherein the step of separation blood is realized by being centrifuged；Preferably, institute
It is as follows to state centrifugal condition: 800-2300 gram/minute；It is highly preferred that the centrifugation carries out 3 times.
4. method according to any one of claims 1 to 3, wherein the step of cracking high concentration thrombocyte plasma passes through
Freezing/weight is molten, ultrasonic or grinding is to realize；Preferably, the molten condition of freezing/weight is as follows: -50 DEG C to -60 DEG C freezing 20-30
Minute, 35 DEG C to 40 DEG C weights are 5-15 minutes molten；It is highly preferred that described row freeze/is dissolved into again at least 2 times；Or preferably, described
Ultrasound condition is as follows: 185W, 40kHz ultrasound 10-20 minutes.
5. the preparation method of blood broken wall growth factor comprising following steps:
Blood obtains high concentration thrombocyte plasma with 800-2300 gram/minute centrifugation 3 times；Preferably, the 1st time centrifugation 5-10 points
Clock, the 2nd centrifugation 5-10 minutes, the 3rd centrifugation 10-20 minutes；
High concentration thrombocyte plasma is molten by least 2 freezing/weights, or ultrasound cracking at least 10 minutes, obtains growth factor
Extracting solution；Preferably, the molten condition of freezing/weight is as follows: -50 DEG C to -60 DEG C freezing 20-30 minutes, 35 DEG C to 40 DEG C weights are molten
5-15 minutes；It is highly preferred that described freeze/dissolve into row 2 to 5 times again；Or preferably, the ultrasound condition is as follows: 185W,
40kHz ultrasound 10-20 minutes；And
Optionally, growth factor extracting solution after filtering, carries out freeze-drying preservation.
6. the method as described in any one of claims 1 to 5, wherein the broken wall growth factor mainly include VEGF, EGF and
PDGF。
7. such as method described in any one of claims 1 to 6, wherein the blood is originated from following animal: mammality, birds,
Reptiles, amphibian animal and fish；Preferably, the blood is originated from fish.
8. the method as described in any one of claims 1 to 7, wherein the half-life period of the broken wall growth factor be 12 months or
More than.
9. the method as described in any one of claims 1 to 7 preparation broken wall growth factor, be used for skin wound healing,
Skin regeneration, skin-whitening or skin it is anti-oxidant/anti-aging.
10. the broken wall growth factor of the preparation of the method as described in any one of claims 1 to 7, is being used to prepare following use
Purposes in product:
Skin regeneration articles；
Skin-whitening articles；
Skin is anti-oxidant/anti-aging articles；Or
Assist the articles of skin wound healing.
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