CN109939224A - A kind of biological agent and preparation method thereof for repairing skin injury - Google Patents
A kind of biological agent and preparation method thereof for repairing skin injury Download PDFInfo
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- 208000028990 Skin injury Diseases 0.000 title claims abstract description 35
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- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims abstract description 15
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims abstract description 15
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- 108010067306 Fibronectins Proteins 0.000 claims abstract description 15
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of biological agents and preparation method thereof for repairing skin injury, belong to field of biotechnology, the biological agent of this reparation skin injury, including following components by weight: fat stem cell active matter 8-12g, amnion 4-6g, epithelical cell growth factor 0.05-0.1g, fibroblast growth factor 0.05-0.1g, fibroblast 4-8g, fibronectin 0.005-0.01g, deionized water 40-60ml.The beneficial effect of the biological agent of present invention reparation skin injury is can be to injured skin repair, and accelerates the healing efficiency of skin.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of repair skin injury biological agent and its preparation side
Method.
Background technique
Skin is the organ that human body is directly contacted with the external world, is the protective barrier of human body.Since skin directly connects with the external world
Touching, so skin is also to be most susceptible to damage, general small damage, skin can spontaneous slow reparation, but time of reparation
Very slow length 2 is just relatively difficult by the reparation of skin itself when widespread skin damages, and needs using external auxiliary medical treatment
To repair.
Common skin repair mode is that skin injury is repaired by Biotype artificial dermatoplasty at present, but bion
Artificial skin has different immunogenicities from the skin of former patient, so that the therapeutic modality of the skin of transplanting has very big office
It is sex-limited.
In addition current skin repair mode is slowly improved also by Chinese medicine, so that skin is voluntarily slowly repaired, but is controlled
The treatment course for the treatment of period is long, and patient needs to endure the pain of long period.
Recently as the further investigation to stem cell, fat stem cell is isolated a kind of tool from adipose tissue
There is the stem cell of multi-lineage potential, and fat stem cell is extracted and is easy, it is from a wealth of sources, it is appropriate for promoting the use of, at present rouge
Fat stem cell is mainly used in beauty industry, is mainly used for chest enlarge, repairs skin blemishes to wrinkle of skin removal and small range,
Therefore biological agent can be made in fat stem cell, repairing skin injury for patient has very big meaning.
Summary of the invention
In order to solve the above-mentioned technical problem the present invention provides a kind of biological agent and preparation method thereof for repairing skin injury,
Can be to injured skin repair, and accelerate the healing efficiency of skin.
The technical scheme to solve the above technical problems is that a kind of biological agent for repairing skin injury, including
Following components by weight: fat stem cell active matter 8-12g, amnion 4-6g, epithelical cell growth factor 0.05-
0.1g, fibroblast growth factor 0.05-0.1g, fibroblast 4-8g, fibronectin 0.005-0.01g, deionized water
40-60ml。
Biological agent beneficial effect of the invention is: can effectively promote skin by biological agent prepared by this formula
The reparation on surface, so that skin is dynamic, while accelerating to promote skin fibroblasts proliferation, so that skin repair speed
Fastly, wound healing.
The present invention also provides a kind of preparation methods of biological agent for repairing skin injury, which is characterized in that including following
Step:
S1, acquisition adipose tissue, from the extraction fat stem cell in adipose tissue；
S2, culture medium culture is used to the fat stem cell that step S1 is extracted, by the fat stem cell after culture through ultrasound
Broken, centrifuging and taking supernatant is concentrated and fat stem cell active matter is made；
S3, amnion, epithelical cell growth factor, fibroblast growth factor, fibre are taken by the parts by weight in claim 1
Fat stem cell active matter obtained in dimension mother cell, fibronectin and deionized water and step S2, weighed substance is mixed
Biological agent is made after conjunction.
Preparation method beneficial effect of the invention is: by first extracting to fat stem cell, by turning out mostly generation
It after fat stem cell, is made after active matter is mixed with other repair materials and biological agent is made, manufactured biological agent can have
Effect rapidly promotes skin repair, and repairing effect is repaired significantly better than General Medicine and skin itself.
Based on the above technical solution, the present invention can also be improved as follows.
It further, further include by adipose tissue cutting and grinding, then through physiological saline oscillation cleaning 1- in the step S1
5min stands 10-20 minutes, removes supernatant, residue is put into collagenase solution and is digested, and keeps 37 DEG C of constant temperature and continues
Oscillation stirring 30-50min, filters to take liquid, then plus physiological saline cleaning, centrifugation obtain fat stem cell after removing supernatant.
Beneficial effect using above-mentioned further scheme is: it is more preferable to fat stem cell extraction effect, it can sufficiently will be fatty
Fat stem cell in tissue extracts.
Further, the filtering is sterile filtered using the strainer of 80-120 mesh, takes filtered fluid, and it is clear that physiological saline is added
It washes, 5-10min is centrifuged using 1000-1500r/min revolving speed, removal supernatant obtains fat stem cell.
Beneficial effect using above-mentioned further scheme is: can remove and be mixed into tiny adipose tissue in liquid, so that rouge
Fat stem cell purity is high.
Further, in the step S2, further includes: fat stem cell is put into the training of the DMEM containing 8%-10% serum
It supports in base, keeps 5%CO2With cultivate 24-48h under 37 DEG C of environment, collect supernatant；Supernatant is collected into place into containing 50-
In the culture medium of the DMEM/F12 of 55ug/ml beta glucan, 5%CO is kept2With continue under 37 DEG C of environment cultivate 24-48h, collect
Supernatant.
Beneficial effect using above-mentioned further scheme is: it is dry to be conducive to fat for two generations of culture and three generations's fat stem cell
Cell promotes skin repair.
Further, the fat stem cell of culture is dissolved in 50ml physiological saline, through sonicated cells 10-15s, is repeated
It is 3-4 times broken, fat stem cell active matter is made through centrifuging and taking supernatant, then after being concentrated.
Beneficial effect using above-mentioned further scheme is: extracting live part, the objects such as useless cell wall are removed.
Further, the revolving speed of the centrifugation is 800-1000r/min, is centrifuged 4-5min, and repeated centrifugation is twice.
Beneficial effect using above-mentioned further scheme is: separating effect is more preferable.
In addition existed the present invention also provides a kind of using the biological agent for repairing skin injury made from above-mentioned preparation method
Application in skin injury reparation.
In addition the present invention also provides a kind of biological agents using above-mentioned reparation skin injury in skin injury reparation
Application.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit
Determine the scope of the present invention.
Embodiment 1
The present embodiment provides a kind of biological agents for repairing skin injury, including following components by weight: fat
Stem Cell Activity object 8g, amnion 4g, epithelical cell growth factor 0.05g, fibroblast growth factor 0.05g, fiber mother is carefully
Born of the same parents 4g, fibronectin 0.005g, deionized water 40ml.
The present embodiment also provides a kind of preparation method of biological agent for repairing skin injury, specifically includes the following steps:
S1, acquisition adipose tissue are ground into the bulk of 1mm by adipose tissue by scissors or cutting machine cutting and grinding, then
100ml physiological saline is added, the oscillation shaking table oscillation cleaning 1min for being 1200r/min through revolving speed, removal residual bloodstain, through over vibration
10 minutes are stood after swinging, after adipose tissue precipitating, removes supernatant, repeated washing is put into 100ml's twice, by remaining sediment
It is digested in collagenase solution, keeps 37 DEG C of constant temperature and 30min is stirred in persistent oscillation, fat stem cell digests in clostridiopetidase A, glue
Protoenzyme is that collagenase type I and II Collagenase Type press 1: 1 mixture, is sterile filtered using the strainer of 80 mesh, and mixed fat is broken
Tissue filter takes filtered fluid, and physiological saline cleaning is added, and is centrifuged 5min using 1000r/min revolving speed, removal supernatant obtains
To fat stem cell；
S2, fat stem cell is put into the DMEM culture medium containing 8% serum to what step S1 was extracted, keeps 5%
CO2With cultivated under 37 DEG C of environment for 24 hours, collect supernatant；The supernatant being collected into is taken to place into containing 50ug/m | beta glucan
In the culture medium of DMEM/F12, keeps continuing culture for 24 hours under 5%CO2 and 37 DEG C of environment, collect supernatant；By the fat of culture
Stem cell is dissolved in 50ml physiological saline, through sonicated cells 10s, repeats 3 times broken, the revolving speed centrifugation through 800r/min
4min takes supernatant, and repeated centrifugation is twice, then fat stem cell active matter is made after being concentrated by ultrafiltration membrane；
S3, take amnion, epithelical cell growth factor, fibroblast growth factor, fiber female by parts by weight among the above
Fat stem cell active matter obtained in cell, fibronectin and deionized water and step S2, after weighed material mixing
Biological agent is made.
Embodiment 2
The present embodiment provides a kind of biological agents for repairing skin injury, including following components by weight: fat
Stem Cell Activity object 10g, amnion 5g, epithelical cell growth factor 0.06g, fibroblast growth factor 0.06g, fiber mother is carefully
Born of the same parents 6g, fibronectin 0.008g, deionized water 50ml.
The present embodiment also provides a kind of preparation method of biological agent for repairing skin injury, specifically includes the following steps:
S1, acquisition adipose tissue are ground into the bulk of 1mm by adipose tissue by scissors or cutting machine cutting and grinding, then
100ml physiological saline is added, the oscillation shaking table oscillation cleaning 2min for being 1200r/min through revolving speed, removal residual bloodstain, through over vibration
15 minutes are stood after swinging, after adipose tissue precipitating, removes supernatant, repeated washing is put into 100ml's twice, by remaining sediment
It is digested in collagenase solution, keeps 37 DEG C of constant temperature and 40min is stirred in persistent oscillation, fat stem cell digests in clostridiopetidase A, glue
Protoenzyme is that collagenase type I and II Collagenase Type press 1: 1 mixture, is sterile filtered using the strainer of 100 mesh, and mixed fat is broken
Tissue filter takes filtered fluid, and physiological saline cleaning is added, and is centrifuged 6min using 1000r/min revolving speed, removal supernatant obtains
To fat stem cell；
S2, fat stem cell is put into the DMEM culture medium containing 10% serum to what step S1 was extracted, keeps 5%
CO2With cultivate 36h under 37 DEG C of environment, collect supernatant；The supernatant being collected into is taken to place into containing 50ug/ml beta glucan
In the culture medium of DMEM/F12,5%CO is kept2With continue under 37 DEG C of environment cultivate 36h, collect supernatant；By the fat of culture
Stem cell is dissolved in 50ml physiological saline, through sonicated cells 10s, repeats 3 times broken, the revolving speed centrifugation through 900r/min
4min takes supernatant, and repeated centrifugation is twice, then fat stem cell active matter is made after being concentrated by ultrafiltration membrane；
S3, take amnion, epithelical cell growth factor, fibroblast growth factor, fiber female by parts by weight among the above
Fat stem cell active matter obtained in cell, fibronectin and deionized water and step S2, after weighed material mixing
Biological agent is made.
Embodiment 3
The present embodiment provides a kind of biological agents for repairing skin injury, including following components by weight: fat
Stem Cell Activity object 12g, amnion 6g, epithelical cell growth factor 0.1g, fibroblast growth factor 0.1g, fibroblast
8g, fibronectin 0.01g, deionized water 60ml.
The present embodiment also provides a kind of preparation method of biological agent for repairing skin injury, specifically includes the following steps:
S1, acquisition adipose tissue are ground into the bulk of 2mm by adipose tissue by scissors or cutting machine cutting and grinding, then
100ml physiological saline is added, the oscillation shaking table oscillation cleaning 5min for being 1200r/min through revolving speed, removal residual bloodstain, through over vibration
20 minutes are stood after swinging, after adipose tissue precipitating, removes supernatant, repeated washing is put into 100ml's twice, by remaining sediment
It is digested in collagenase solution, keeps 37 DEG C of constant temperature and 50min is stirred in persistent oscillation, fat stem cell digests in clostridiopetidase A, glue
Protoenzyme is that collagenase type I and II Collagenase Type press 1: 1 mixture, is sterile filtered using the strainer of 120 mesh, and mixed fat is broken
Tissue filter takes filtered fluid, and physiological saline cleaning is added, and is centrifuged 10min using 1500r/min revolving speed, removes supernatant
Obtain fat stem cell；
S2, fat stem cell is put into the DMEM culture medium containing 10% serum to what step S1 was extracted, keeps 5%
CO2With cultivate 48h under 37 DEG C of environment, collect supernatant；The supernatant being collected into is taken to place into containing 55ug/ml beta glucan
In the culture medium of DMEM/F12,5%CO is kept2With continue under 37 DEG C of environment cultivate 48h, collect supernatant；By the fat of culture
Stem cell is dissolved in 50ml physiological saline, through sonicated cells 15s, repeats 4 times broken, the revolving speed centrifugation through 1000r/min
5min takes supernatant, and repeated centrifugation is twice, then fat stem cell active matter is made after being concentrated by ultrafiltration membrane；
S3, take amnion, epithelical cell growth factor, fibroblast growth factor, fiber female by parts by weight among the above
Fat stem cell active matter obtained in cell, fibronectin and deionized water and step S2, after weighed material mixing
Biological agent is made.
Embodiment 4
The present embodiment provides a kind of biological agents for repairing skin injury, including following components by weight: fat
Stem Cell Activity object 10g, amnion 4g, epithelical cell growth factor 0.05g, fibroblast growth factor 0.05g, fiber mother is carefully
Born of the same parents 6g, fibronectin 0.006g, deionized water 50ml.
The present embodiment also provides a kind of preparation method of biological agent for repairing skin injury, specifically includes the following steps:
S1, acquisition adipose tissue are ground into the bulk of 1mm by adipose tissue by scissors or cutting machine cutting and grinding, then
100ml physiological saline is added, the oscillation shaking table oscillation cleaning 3min for being 1200r/min through revolving speed, removal residual bloodstain, through over vibration
15 minutes are stood after swinging, after adipose tissue precipitating, removes supernatant, repeated washing is put into 100ml's twice, by remaining sediment
It is digested in collagenase solution, keeps 37 DEG C of constant temperature and 40min is stirred in persistent oscillation, fat stem cell digests in clostridiopetidase A, glue
Protoenzyme is that collagenase type I and II Collagenase Type press 1: 1 mixture, is sterile filtered using the strainer of 100 mesh, and mixed fat is broken
Tissue filter takes filtered fluid, and physiological saline cleaning is added, and is centrifuged 8min using 1200r/min revolving speed, removal supernatant obtains
To fat stem cell；
S2, fat stem cell is put into the DMEM culture medium containing 10% serum to what step S1 was extracted, keeps 5%
CO2With cultivate 36h under 37 DEG C of environment, collect supernatant；The supernatant being collected into is taken to place into containing 53ug/ml beta glucan
In the culture medium of DMEM/F12,5%CO is kept2With continue under 37 DEG C of environment cultivate 36h, collect supernatant；By the fat of culture
Stem cell is dissolved in 50ml physiological saline, through sonicated cells 10s, repeats 4 times broken, the revolving speed centrifugation through 1000r/min
5min takes supernatant, and repeated centrifugation is twice, then fat stem cell active matter is made after being concentrated by ultrafiltration membrane；
S3, take amnion, epithelical cell growth factor, fibroblast growth factor, fiber female by parts by weight among the above
Fat stem cell active matter obtained in cell, fibronectin and deionized water and step S2, after weighed material mixing
Biological agent is made.
Embodiment 5
The present embodiment provides a kind of biological agents for repairing skin injury, including following components by weight: fat
Stem Cell Activity object 10g, amnion 6g, epithelical cell growth factor 0.08g, fibroblast growth factor 0.05g, fiber mother is carefully
Born of the same parents 8g, fibronectin 0.006g, deionized water 50ml.
The present embodiment also provides a kind of preparation method of biological agent for repairing skin injury, specifically includes the following steps:
S1, acquisition adipose tissue are ground into the bulk of 1mm by adipose tissue by scissors or cutting machine cutting and grinding, then
100ml physiological saline is added, the oscillation shaking table oscillation cleaning 4min for being 1200r/min through revolving speed, removal residual bloodstain, through over vibration
18 minutes are stood after swinging, after adipose tissue precipitating, removes supernatant, repeated washing is put into 100ml's twice, by remaining sediment
It is digested in collagenase solution, keeps 37 DEG C of constant temperature and 40min is stirred in persistent oscillation, fat stem cell digests in clostridiopetidase A, glue
Protoenzyme is that collagenase type I and II Collagenase Type press 1: 1 mixture, is sterile filtered using the strainer of 120 mesh, and mixed fat is broken
Tissue filter takes filtered fluid, and physiological saline cleaning is added, and is centrifuged 8min using 1200r/min revolving speed, removal supernatant obtains
To fat stem cell；
S2, fat stem cell is put into the DMEM culture medium containing 10% serum to what step S1 was extracted, keeps 5%
CO2With cultivate 36h under 37 DEG C of environment, collect supernatant；The supernatant being collected into is taken to place into containing 55ug/ml beta glucan
In the culture medium of DMEM/F12,5%CO is kept2With continue under 37 DEG C of environment cultivate 36h, collect supernatant；By the fat of culture
Stem cell is dissolved in 50ml physiological saline, through sonicated cells 12s, repeats 4 times broken, the revolving speed centrifugation through 1000r/min
5min takes supernatant, and repeated centrifugation is twice, then fat stem cell active matter 2 is made after being concentrated by ultrafiltration membrane
S3, take amnion, epithelical cell growth factor, fibroblast growth factor, fiber female by parts by weight among the above
Fat stem cell active matter obtained in cell, fibronectin and deionized water and step S2, after weighed material mixing
Biological agent is made.
Biological agent made from above-described embodiment 1-5 is subjected to experiment test, specific test mode are as follows: 6 monthly ages is taken to exist
The piggy of the 5-8 month, weight irradiate the skin of piggy backbone two sides by linear accelerator in 20-25kg, and depth 1cm absorbs
Dosage is 20cy, irradiated area 20.8cm2.The skin irradiated after irradiation with punch excision, by skin excision same size
It is round or rectangular, oozing of blood, with the antibiotic normal saline flushing surface of a wound, life prepared by embodiment 1-5 are wiped with sterile dry cotton ball
Object preparation is distinguished in mulching to the wound of 5 piggys, and with gauze take-up strap, and the 6th piggy as a comparison case, uses simple oil
Gauze mulching, normal feeding young pigs, and observe after mulching the 3rd day, the 5th day, the 7th day, the 10th day, the 15th day, the 20th day and
22nd day wound situation, and healing area is measured by measuring scale, healing rate is calculated, specific data such as the following table 1:
Table 1
From above data it can be seen that being repaired by skin repair biological agent prepared by this preparation method with simply dealt
It in complex velocity clearly compared to effect, can be quickly to injured skin on wound by the biological agent mulching of this skin repair
Skin reparation is quickly healed.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of biological agent for repairing skin injury, which is characterized in that including following components by weight: fat is dry thin
Cytoactive object 8-12g, amnion 4-6g, epithelical cell growth factor 0.05-0.1g, fibroblast growth factor 0.05-0.1g,
Fibroblast 4-8g, fibronectin 0.005-0.01g, deionized water 40-60ml.
2. a kind of preparation method for the biological agent for repairing skin injury, which comprises the following steps:
S1, acquisition adipose tissue, extract fat stem cell from adipose tissue；
S2, culture medium culture is used to the fat stem cell that step S1 is extracted, by the fat stem cell after culture through ultrasonication,
Centrifuging and taking supernatant is concentrated and fat stem cell active matter is made；
S3, take amnion, epithelical cell growth factor, fibroblast growth factor, fiber female by the parts by weight in claim 1
Fat stem cell active matter obtained in cell, fibronectin and deionized water and step S2, after weighed material mixing
Biological agent is made.
3. the preparation method of the biological agent according to claim 2 for repairing skin injury, which is characterized in that the step
In S1, further includes that adipose tissue cutting and grinding are added into physiological saline oscillation cleaning 1-5min, stand 10-20 minutes, removal
Residue is put into collagenase solution and digests by supernatant, keeps 37 DEG C of constant temperature and 30-50min is stirred in persistent oscillation, filter to take
Liquid, then plus physiological saline cleaning, centrifugation, remove supernatant after obtain fat stem cell.
4. the preparation method of the biological agent according to claim 3 for repairing skin injury, which is characterized in that the filtering
It is sterile filtered using the strainer of 80-120 mesh, takes filtered fluid, physiological saline cleaning is added, is turned using 1000-1500r/min
Speed centrifugation 5-10min, removal supernatant obtain fat stem cell.
5. the preparation method of the biological agent according to claim 2 for repairing skin injury, which is characterized in that the step
In S2, further includes: fat stem cell is put into the DMEM culture medium containing 8%-10% serum, 5%CO is kept2With 37 DEG C
24-48h is cultivated under environment, collection supernatant 2 is collected into supernatant and places into the DMEM/ containing 50-55ug/ml beta glucan
In the culture medium of F12,5%CO is kept2With continue under 37 DEG C of environment cultivate 24-48h, collect supernatant.
6. the preparation method of the biological agent according to claim 2 for repairing skin injury, which is characterized in that by culture
Fat stem cell is dissolved in 50ml physiological saline, through sonicated cells 10-15s, repeats to be crushed 3-4 times, through centrifuging and taking supernatant
Liquid, then fat stem cell active matter is made after being concentrated.
7. the preparation method of the biological agent according to claim 6 for repairing skin injury, which is characterized in that the centrifugation
Revolving speed be 800-1000r/min, be centrifuged 4-5min, and repeated centrifugation is twice.
8. a kind of biological agent of skin injury of repairing as made from the described in any item preparation methods of claim 2-7 is in skin
Application in injury repair.
9. a kind of application of biological agent as described in claim 1 for repairing skin injury in skin injury reparation.
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| CN201910336994.7A CN109939224A (en) | 2019-04-24 | 2019-04-24 | A kind of biological agent and preparation method thereof for repairing skin injury |
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| CN201910336994.7A Pending CN109939224A (en) | 2019-04-24 | 2019-04-24 | A kind of biological agent and preparation method thereof for repairing skin injury |
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