CN109097336A - A kind of stem cell excretion body, preparation method and application - Google Patents
A kind of stem cell excretion body, preparation method and application Download PDFInfo
- Publication number
- CN109097336A CN109097336A CN201811049325.3A CN201811049325A CN109097336A CN 109097336 A CN109097336 A CN 109097336A CN 201811049325 A CN201811049325 A CN 201811049325A CN 109097336 A CN109097336 A CN 109097336A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- excretion
- target gene
- excretion body
- skin
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
-
- 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
- A61K8/982—Reproductive organs; Embryos, Eggs
-
- 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
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Reproductive Health (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Dermatology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of stem cell excretion body, preparation method and application, the genetic test about skin is carried out to user's skin problem first, according to the result of genetic test, search out the target gene for improving its skin problem, the target gene is expanded by external synthesizing mean, then target gene is gone into carrier and is prepared into target gene carrier, the target gene carrier is transfected into mescenchymal stem cell and obtains target gene mescenchymal stem cell, finally the target gene mescenchymal stem cell is cultivated, collect its cell culture fluid, extract the excretion body in the cell culture fluid, the excretion body extracted is sterilized, it dispenses and saves after quantitative；Compared with prior art, the beneficial effects of the present invention are starting with from gene root, problem gene is repaired to be effectively improved skin problem, is alleviated " internal aging ".
Description
Technical field
The present invention relates to stem cell fields, and in particular to a kind of stem cell excretion body, preparation method and application.
Background technique
MSC is the stem cell of current most study, current many clinical tests to confirm that MSC can treat a variety of diseases,
And there are better effects, while MSC has stronger safety, and in different acid or alkali environments, anoxic, high temperature, multigelation
It is still able to maintain higher stability under equal mal-conditions, functionality beauty product made of it can keep cell to greatest extent
The activity and function of regeneration factor, the research of stem cell excretion body skin-protection product now is existing very much, is mostly to utilize its excretion
The activity of body improves skin condition.
However, skin problem is varied, user demand is also varied, anti-ageing, whitening, antiallergic etc., but causes
The reason of skin problem be then by many factors, such as it is sun exposure, air pollution, smoking, excessive drinking, malnutrition, unhealthy
The environmental resistance factors such as diet, chronic disease, it is another then be to have we itself gene regulation, either aging, black
Element deposition or anaphylaxis skin, have much relations with the expression activity of related gene.
Skin care item are set about from protection now, improve or repair the damage of external environment bring, can not be from root solution
Certainly gene problem bring skin problem.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of stem cell excretion body, preparation method and application.
Specific technical solution is as follows:
A kind of stem cell excretion body, the difference is that, the stem cell excretion body is by containing the target base for improving skin
Because carrier mescenchymal stem cell secrete, the target gene carrier include KIF3A, OVOL1, SOD1, COL5A1, MC1R, TYR,
One of GHRHR, EDAR, FLG, MMP9, XRCC1, RAD51, HBE1 or KL or several genes.
It is a kind of to prepare above-mentioned stem cell excretion body method, the difference is that, user's skin problem is closed first
In the genetic test of skin, according to genetic test as a result, the target gene for improving its skin problem is searched out, by closing in vitro
The target gene is expanded at means, target gene is then gone into carrier and is prepared into target gene carrier, by the target
Genophore transfects into mescenchymal stem cell and obtains target gene mescenchymal stem cell, finally by the target gene mescenchymal stem cell
It is cultivated, collects its cell culture fluid, extract the excretion body in the cell culture fluid, the excretion body extracted is gone out
Bacterium dispenses preservation after quantifying；
The gene of the test is one of following or several genes combination:
The related gene formed with whelk combines: EDAR, CYP21A2, KIF3A, OVOL1, ATP2A2；
The related gene of antitan agent ability combines: MC1R, OCA2, SOD1, CYP21A2, KIF3A, OVOL1；
The related gene of skin antiallergic combines: COL5A1, MC1R, KIF3A, OVOL1, SOD1；
The relevant assortment of genes: MC1R, OCA2, BNC2, TYR is formed to freckle melanin；
The relevant assortment of genes of wrinkling resistance: SOD1, GHRHR, EDAR, FLG, MC1R, MMP9；
The assortment of genes relevant to skin radiation-preventing ability and anti-aging ability: XRCC1, RAD51, HBE1, MMP9,
SOD1、CHRHR、KL。
In above-mentioned technical proposal, the target gene include KIF3A, OVOL1, SOD1, COL5A1, MC1R, TYR,
One of GHRHR, EDAR, FLG, MMP9, XRCC1, RAD51, HBE1 or KL or a variety of.
In above-mentioned technical proposal, the stem cell excretion preparation the following steps are included:
(1) mescenchymal stem cell is subjected to secondary culture, is screened out from it between cell state is good, proliferative capacity is strong and fills
Matter stem cell；
(2) target gene is expanded, and restriction enzyme site is added, be packed into carrier, target gene carrier is obtained, by institute
It states target gene carrier and goes to step (1) described mescenchymal stem cell, obtain target gene mescenchymal stem cell；
(3) mescenchymal stem cell chosen in step (2) is cultivated using ɑ-MEM sky culture medium, when cultivating one section
Between after, remove impurity, isolate culture supernatant；
(4) after step (3) culture supernatant being mixed with Total exosome Isolation excretion body extracting solution
It is incubated for, the first incubation fluid is obtained after incubation, centrifugal concentrating is carried out to the first incubation fluid, obtains the concentrate of the body containing excretion；
(5) Total exosome is added after sterile PBS dissolution being added in the concentrate of the body containing excretion described in step (4)
Isolation excretion body extracting solution is incubated for, and the second incubation fluid is obtained after incubation, the second incubation fluid is carried out bottom is collected by centrifugation
Precipitating obtains the excretion body of mescenchymal stem cell secretion.
In above-mentioned technical proposal, the mescenchymal stem cell be umbilical cord mesenchymal stem cells, mesenchymal stem cell or
One of peripheral blood mescenchymal stem cell.
In above-mentioned technical proposal, the carrier is plasmid, and the target gene carrier transfection method is lipo2000 infection protocol
Or electric robin.
In above-mentioned technical proposal, the stem cell excretion preparation step (3) is completed by following steps:
Step 3A: the blank stem cell ɑ-MEM culture medium without excretion body serum is added to the target of step (2) described screening
In gene mescenchymal stem cell, it is conditioned medium that cell culture supernatant is collected after 24 hours to 48 hours；
3B: by conditioned medium described in step 3A at 4 DEG C, being centrifuged 30min under the conditions of 2000g, discards precipitated impurities, receives
Collect supernatant liquid, i.e. culture supernatant.
In above-mentioned technical proposal, the stem cell excretion preparation step (4) is completed by following steps:
Step 4A: by culture supernatant described in step 3B and Total exosome Isolation excretion body extracting solution
It is after 2:1 is mixed, to be incubated overnight under the conditions of 4 DEG C, the first Incubating Solution is obtained after incubation with volume ratio；
Step 4B: by the first Incubating Solution obtained by step 4A at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained naked eyes can not
The bottom precipitation seen, the as concentrate of the body containing excretion.
In above-mentioned technical proposal, the stem cell excretion preparation step (5) is completed by following steps:
5A: the concentrate of the body containing excretion described in step 4B is dissolved with phosphate buffered saline solution (PBS), is obtained outer
Secrete body salting liquid；
5B: excretion body extracting solution is added in the excretion body salting liquid described in step 5A, 1 hour is incubated under the conditions of 4 DEG C extremely
12 hours, the volume ratio of the excretion body salting liquid and Total exosome Isolation excretion body extracting solution was 2:1, was incubated
The second Incubating Solution is obtained after educating；
5C: by the second Incubating Solution described in step 5B at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained lower layer naked eyes are not
Visible precipitating is the excretion body of mescenchymal stem cell secretion.
Application of the above-mentioned stem cell excretion body in skin care.
Compared with prior art, the beneficial effects of the present invention are starting with from gene root, problem gene is repaired to have
Effect improves skin problem, alleviates " internal aging ", accurate skin care is carried out according to the skin problem of different user, to reach more preferable
Skin effect.
Specific embodiment
The present invention will be further described combined with specific embodiments below.
Embodiment one
User A: female, 25 years old, sensibility skin occurred the problem of red capillary, pruitus throughout the year, surveys first with gene
Following gene is tested in examination:
The related gene of skin antiallergic: COL5A1, MC1R, KIF3A, OVOL1, SOD1；
Test result is shown: user's A COL5A1 activity of gene expression is relatively low to cause skin thickness partially thin, while KIF3A,
OVOL1 activity of gene expression is higher to cause cutaneous anaphylaxis serious compared to ordinary person.
Target gene screening: COL5A1, KIF3A, OVOL1 and SOD1, it is intended to increase skin thickness, increase skin light antiallergic
Property and melanin degradation capability, and then improve skin repellence.
Follow these steps the stem cell excretion body that preparation is directed to user A:
Mescenchymal stem cell is separately cultured
1) fresh umbilical cord is fetched with aseptic bottle (+one gentamicin of 200ml physiological saline)；
2) jelly of Wharton around separating blood vessel (Wharton ' sjelly), shreds；
3) it is put into 2mg/ml Type I collagen enzyme (about 15ml)；
4) dual anti-and 200 μ l the amphotericin B of 200 μ l is added, is uniformly mixed and is placed in incubator (37 DEG C) overnight；
5) about 20ml/ ware), every ware adds 1ml additive, and dual anti-and 200 μ l the amphotericin B of 200 μ l is set in incubator
Culture；
6) liquid is changed according to cultivation conditions timely and appropriate discovery, the secondary culture after cell covers with.
Mescenchymal stem cell passage
1) (about 1 × 10 after cell covers with7) secondary culture is carried out to 3~5 generations.
2) for partial medium to 50ml centrifuge tube (3ml/ ware), residue goes to waste liquid cylinder in absorption ware；
3) every ware adds 8ml physiological saline and the pancreatin (pancreatin final concentration 0.05%) of 2ml 0.25%；
4) digestion amount of time (general 1min or so) is mixed, it is seen that cell floats up or disappears in microscopic observation confirmation cell
Culture medium is added after changing well and terminates digestion, every ware adds 3ml；
5) cell is washed (general 5~6 times) with pipette piping and druming ware bottom, is then collected into 50ml centrifuge tube,
1800rpm is centrifuged 5min；
6) supernatant is abandoned, culture medium (containing additive) is added and is suspended, by being transferred in ware after the dilution of required cell density, every ware is about
18ml (16~20ml)；
7) it is put into incubator and cultivates.
Select 3~5 passage umbilical cord stem cells activity preferably, the excretion body of secretion is best to the repair function of skin.
Target gene vector construction
(1) target gene COL5A1, KIF3A, OVOL1 and SOD1 gene are synthesized；
(2) target fragment is expanded；
(3) DNA electrophoresis recycles PCR product；
(4) said gene segment is packed into pUC plasmid vector.
Conversion
1) competence is taken, is melted on ice half an hour, every pipe adds 10 μ L connection liquid, gently rotates to mix, in ice
It is middle to place 30 minutes；
2) water-bath is preheating to 42 DEG C, heat shock 90 seconds；
3) quickly pipe is transferred in ice bath, it is 5 minutes cooling；
4) every pipe adds 600 μ L non-resistant LB culture mediums, and then centrifuge tube is transferred on 37 DEG C of shaking tables, incubates 45 minutes again
Soviet Union；
5) 6000g is centrifuged 2min, and the competent cell converted is resuspended with 100 μ L LB, is then transferred into resistant LB
On agar medium；
6) with smoothen until liquid be absorbed；
7) it is inverted plate, is cultivated in 37 DEG C, 16 hours；
8) positive colony PCR is identified；
9) by the lipo2000 infection protocol of safety or electric robin by the plasmid transfection stem cell with target gene, so that
Stem cell becomes positive colony cell, and then the product of expression alien gene.
The extraction of umbilical cord mesenchymal stem cells excretion body and and purifying
1) 3-5 that selection cell state is good, proliferative capacity is strong is for human umbilical cord mesenchymal stem cells；
2) serum free medium Hylone MEM sky culture medium culture 24-48H is added, cell supernatant 5mL is collected；
3) 2000g is centrifuged 30min and removes cell fragment；
4) stem cell supernatant after centrifugation is moved in ultra-filtration centrifuge tube, the invitogen company of 1/2 volume is added
Total exosome Isolation excretion body extracting solution is incubated overnight under the conditions of 4 DEG C, obtains the first incubation fluid；
5) second day 10000g, 4 DEG C of centrifugation 60min, obtains the concentrate of the body containing excretion；
6) the invitogen company's T otal exosome of 1/2 volume is added after dissolving concentrate with sterile PBS
Isolation excretion body extracting solution, 4 DEG C of refrigerators are incubated for 1H, obtain the second incubation fluid；
7) 10000g, 4 DEG C of centrifugation 60min again collect bottom precipitation, 0.22 μ l membrane filtration degerming, BCA protein quantification
It is divided in sterile cillin bottle and freezes for -80 DEG C；
Comparative example 1
Its stem cell excretion body is compared with one stem cell excretion body of embodiment, and except blank pUC plasmid has been accessed, remaining is prepared
Method is all the same.
Skin test experiment:
Stem cell excretion body and market water laser accunputure improve human facial skin；
Experimental procedure: first (CBS-807 skin analysis system) is used to choose another two and user A skin skin before experiment
The young woman that matter is not much different does check experiment, wherein a people with facial cleanser by Irrigation it is clean after, pumped with Needleless injection
Customization stem cell excretion body is dissolved into liquid pump on skin of face, T-shaped full face smearing is scratched.Another two people then a use pump into
Common brand water laser accunputure in the market, one is then pumped using 1 excretion body of comparative example；It is examined with behind latter 2 weeks, 4 weeks, 2 months with skin
Survey moisture, elasticity, skin thickness, red capillary and the elastic situation of instrument (CBS-807 skin analysis system) detection skin.As a result it shows
Show that stem cell excretion body of the present invention compared to 1 excretion body of comparative example and water laser accunputure, has bright for user A anaphylaxis skin situation
Aobvious improvement.
Embodiment two
The problems such as user B: female, 38 years old, skin relaxed, and generated wrinkle, drying and color spot, first with genetic test
Following gene is tested.
Gene relevant to skin radiation-preventing ability and anti-aging ability: XRCC1, RAD51, HBE1, MMP9, SOD1,
CHRHR、KL。
The assortment of genes relevant to wrinkling resistance: SOD1, GHRHR, EDAR, FLG, MC1R, MMP9.
The relevant assortment of genes: MC1R, OCA2, BNC2, TYR is formed to freckle melanin；
Test result is shown: SOD1, CHRHR, MMP9, HBE1 activity of gene expression are relatively low, lead to aging corrugated occur
Condition, TYR activity of gene expression is insufficient, leads to melanin rejection ability deficiency occur.
Target gene screening: it is dry thin as foreign gene immigration mesenchyma that SOD1, CHRHR, MMP9, HBE1 gene are chosen
Born of the same parents, it is intended to which the anti-aging ability for improving skin improves existing aging state.
Follow these steps the stem cell excretion body that preparation is directed to user B
Mescenchymal stem cell is separately cultured
(1) fresh umbilical cord is fetched with aseptic bottle (+one gentamicin of 200ml physiological saline)；
(2) jelly of Wharton around separating blood vessel (Wharton ' sjelly), shreds；
(3) it is put into 2mg/ml Type I collagen enzyme (about 15ml)；
(4) dual anti-and 200 μ l the amphotericin B of 200 μ l is added, is uniformly mixed and is placed in incubator (37 DEG C) mistake
Night；
(5) about 20ml/ ware), every ware adds 1ml additive, and dual anti-and 200 μ l the amphotericin B of 200 μ l sets incubator
Middle culture；
(6) liquid is changed according to cultivation conditions timely and appropriate discovery, the secondary culture after cell covers with.
Mescenchymal stem cell passage
(1) (about 1 × 10 after cell covers with7) secondary culture is carried out to 3~5 generations.
(2) for partial medium to 50ml centrifuge tube (3ml/ ware), residue goes to waste liquid cylinder in absorption ware；
(3) every ware adds 8ml physiological saline and the pancreatin (pancreatin final concentration 0.05%) of 2ml 0.25%；
(4) digestion amount of time (general 1min or so) is mixed, it is seen that cell floats up or confirms cell in microscopic observation
Culture medium is added after digestion is good and terminates digestion, every ware adds 3ml；
(5) cell is washed (general 5~6 times) with pipette piping and druming ware bottom, is then collected into 50ml centrifuge tube,
1800rpm is centrifuged 5min；
(6) supernatant is abandoned, culture medium (containing additive) is added and is suspended, is transferred in ware after being diluted by required cell density, every ware
About 18ml (16~20ml)；
(7) it is put into incubator and cultivates.
Select 3~5 passage umbilical cord stem cells activity preferably, the excretion body of secretion is best to the repair function of skin.
Target gene vector construction
(1) target gene SOD1, CHRHR, MMP9 and HBE1 are synthesized；
(2) target fragment is expanded；
(3) DNA electrophoresis recycles PCR product；
(4) said gene segment is packed into pBR322 plasmid vector.
Conversion
(1) competence is taken, is melted on ice half an hour, every pipe adds 10 μ L connection liquid, gently rotates to mix, in ice
It is middle to place 30 minutes；
(2) water-bath is preheating to 42 DEG C, heat shock 90 seconds；
(3) quickly pipe is transferred in ice bath, it is 5 minutes cooling；
(4) every pipe adds 600 μ L non-resistant LB culture mediums, and then centrifuge tube is transferred on 37 DEG C of shaking tables, incubates 45 minutes
Recovery；
(5) 6000g is centrifuged 2min, and the competent cell converted is resuspended with 100 μ L LB, is then transferred into resistant
On LB agar medium；
(6) with smoothen until liquid be absorbed；
(7) it is inverted plate, is cultivated in 37 DEG C, 16 hours；
(8) positive colony PCR is identified；
(9) made by the lipo2000 infection protocol of safety or electric robin by the plasmid transfection stem cell with target gene
Obtaining stem cell becomes positive colony cell, and then the product of expression alien gene.
The extraction and purifying of umbilical cord mesenchymal stem cells excretion body
(1) 3-5 that selection cell state is good, proliferative capacity is strong is for human umbilical cord mesenchymal stem cells；
(2) serum free medium Hylone MEM sky culture medium culture 24-48H is added, cell supernatant 5mL is collected；
(3) 2000g is centrifuged 30min and removes cell fragment；
(4) stem cell supernatant after centrifugation is moved in ultra-filtration centrifuge tube, the invitogen company of 1/2 volume is added
Total exosome Isolation excretion body extracting solution is incubated overnight under the conditions of 4 DEG C, obtains the first incubation fluid；
(5) second days 10000g, 4 DEG C of centrifugation 60min, obtain the concentrate of the body containing excretion；
(6) the invitogen company's T otal exosome of 1/2 volume is added after dissolving concentrate with sterile PBS
Isolation excretion body extracting solution, 4 DEG C of refrigerators are incubated for 1H, obtain the second incubation fluid；
(7) 10000g, 4 DEG C of centrifugation 60min, collection bottom precipitation, 0.22 μ l membrane filtration degerming, BCA albumen are fixed again
Amount, which is divided in sterile cillin bottle, to be frozen for -80 DEG C.
Comparative example 2
Its stem cell excretion body is compared with two stem cell excretion body of embodiment, except having accessed blank pBR322 plasmid, remaining
Preparation method is all the same.
Skin test experiment:
Stem cell excretion body and market water laser accunputure improve human facial skin；
Experimental procedure: first (CBS-807 skin analysis system) is used to choose another two and user B skin skin before experiment
The young woman that matter is not much different does check experiment, wherein a people with facial cleanser by Irrigation it is clean after, pumped with Needleless injection
Customization stem cell excretion body is dissolved into liquid pump on skin of face, T-shaped full face smearing is scratched.Another two people then a use pump into
Common brand water laser accunputure in the market, one is then pumped using 2 excretion body of comparative example；It is examined with behind latter 2 weeks, 4 weeks, 2 months with skin
Survey moisture, elasticity, wrinkle and the color spot situation of instrument (CBS-807 skin analysis system) detection skin.Stem cell excretion of the present invention
Body is compared to 2 excretion body of comparative example and water laser accunputure, and for user B skin aging, color spot, embodiment is compared to comparative example 2 and water
Laser accunputure improves significantly effect.
The utilization of these examples is merely to illustrate the protection scope that the present invention is not intended to limit the present invention.In addition, reading
After technology contents of the invention, those skilled in the art can make various changes, modification or modification, all this to the present invention
A little equivalent forms also belong within the required protection scope limited of the application.
Claims (10)
1. a kind of stem cell excretion body, which is characterized in that the stem cell excretion body is by containing the target gene carrier for improving skin
Mescenchymal stem cell secretion, the target gene carrier include KIF3A, OVOL1, SOD1, COL5A1, MC1R, TYR, GHRHR,
One of EDAR, FLG, MMP9, XRCC1, RAD51, HBE1 or KL or several genes.
2. a kind of prepare stem cell excretion body method described in claim 1, which is characterized in that carried out first to user's skin problem
About the genetic test of skin, according to genetic test as a result, the target gene for improving its skin problem is searched out, by external
Synthesizing mean expands the target gene, and target gene is then gone to carrier and is prepared into target gene carrier, will be described
Target gene carrier transfects into mescenchymal stem cell and obtains target gene mescenchymal stem cell, finally that the target gene mesenchyma is dry thin
Born of the same parents cultivate, and collect its cell culture fluid, extract the excretion body in the cell culture fluid, and the excretion body extracted is carried out
Packing saves after sterilizing, being quantitative；
The gene of the test is one of following or several genes combination:
The related gene formed with whelk combines: EDAR, CYP21A2, KIF3A, OVOL1, ATP2A2；
The related gene of antitan agent ability combines: MC1R, OCA2, SOD1, CYP21A2, KIF3A, OVOL1；
The related gene of skin antiallergic combines: COL5A1, MC1R, KIF3A, OVOL1, SOD1；
The relevant assortment of genes: MC1R, OCA2, BNC2, TYR is formed to freckle melanin；
The relevant assortment of genes of wrinkling resistance: SOD1, GHRHR, EDAR, FLG, MC1R, MMP9；
The assortment of genes relevant to skin radiation-preventing ability and anti-aging ability: XRCC1, RAD51, HBE1, MMP9, SOD1,
CHRHR、KL。
3. a kind of stem cell excretion preparation according to claim 2, which is characterized in that the target gene includes
In KIF3A, OVOL1, SOD1, COL5A1, MC1R, TYR, GHRHR, EDAR, FLG, MMP9, XRCC1, RAD51, HBE1 or KL
It is one or more.
4. a kind of stem cell excretion preparation according to claim 3, which is characterized in that the stem cell excretion system
Preparation Method the following steps are included:
(1) mescenchymal stem cell is subjected to secondary culture, it is dry is screened out from it the mesenchyma that cell state is good, proliferative capacity is strong
Cell；
(2) target gene is expanded, and restriction enzyme site is added, be packed into carrier, target gene carrier is obtained, by the target
Genophore goes to step (1) described mescenchymal stem cell, obtains target gene mescenchymal stem cell；
(3) mescenchymal stem cell chosen in step (2) is cultivated using ɑ-MEM sky culture medium, after cultivating a period of time,
Impurity is removed, culture supernatant is isolated；
(4) it is carried out after mixing step (3) culture supernatant with Total exosome Isolation excretion body extracting solution
It is incubated for, the first incubation fluid is obtained after incubation, centrifugal concentrating is carried out to the first incubation fluid, obtains the concentrate of the body containing excretion；
(5) Total exosome is added after sterile PBS dissolution being added in the concentrate of the body containing excretion described in step (4)
Isolation excretion body extracting solution is incubated for, and the second incubation fluid is obtained after incubation, the second incubation fluid is carried out bottom is collected by centrifugation
Precipitating obtains the excretion body of mescenchymal stem cell secretion.
5. a kind of stem cell excretion preparation according to claim 4, which is characterized in that the mescenchymal stem cell is
One of umbilical cord mesenchymal stem cells, mesenchymal stem cell or peripheral blood mescenchymal stem cell.
6. a kind of stem cell excretion preparation according to claim 2, which is characterized in that the carrier is plasmid, institute
Stating target gene carrier transfection method is lipo2000 infection protocol or electric robin.
7. a kind of stem cell excretion preparation according to claim 4, which is characterized in that the stem cell excretion system
Preparation Method step (3) is completed by following steps:
Step 3A: the blank stem cell ɑ-MEM culture medium without excretion body serum is added to the target base of step (2) described screening
Because in mescenchymal stem cell, it is conditioned medium that cell culture supernatant is collected after 24 hours to 48 hours；
3B: by conditioned medium described in step 3A at 4 DEG C, being centrifuged 30min under the conditions of 2000g, discards precipitated impurities, in collection
Layer liquid, i.e. culture supernatant.
8. a kind of stem cell excretion preparation according to claim 4, which is characterized in that the stem cell excretion system
Preparation Method step (4) is completed by following steps:
Step 4A: by culture supernatant described in step 3B and Total exosome Isolation excretion body extracting solution with body
Product is than being after 2:1 is mixed, to be incubated overnight under the conditions of 4 DEG C, the first Incubating Solution is obtained after incubation；
Step 4B: by the first Incubating Solution obtained by step 4A at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained naked eyes are sightless
Bottom precipitation, the as concentrate of the body containing excretion.
9. a kind of stem cell excretion preparation according to claim 4, which is characterized in that the stem cell excretion system
Preparation Method step (5) is completed by following steps:
5A: the concentrate of the body containing excretion described in step 4B is dissolved with phosphate buffered saline solution (PBS), obtains excretion body
Salting liquid；
5B: excretion body extracting solution is added in the excretion body salting liquid described in step 5A, is incubated under the conditions of 4 DEG C 1 hour to 12 small
When, the volume ratio of the excretion body salting liquid and Total exosome Isolation excretion body extracting solution is 2:1, after incubation
Obtain the second Incubating Solution；
5C: by the second Incubating Solution described in step 5B at 4 DEG C, 60min is centrifuged under the conditions of 10000g, gained lower layer naked eyes are invisible
Precipitating be mescenchymal stem cell secretion excretion body.
10. a kind of application of the stem cell excretion body described in claim 1 in skin care.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811049325.3A CN109097336A (en) | 2018-09-07 | 2018-09-07 | A kind of stem cell excretion body, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811049325.3A CN109097336A (en) | 2018-09-07 | 2018-09-07 | A kind of stem cell excretion body, preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109097336A true CN109097336A (en) | 2018-12-28 |
Family
ID=64865598
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811049325.3A Pending CN109097336A (en) | 2018-09-07 | 2018-09-07 | A kind of stem cell excretion body, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097336A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110227058A (en) * | 2019-06-10 | 2019-09-13 | 北京恒峰铭成生物科技有限公司 | Pleiotrophic factor composition and its preparation method and application |
| CN111394301A (en) * | 2020-03-30 | 2020-07-10 | 广州瑞铂茵健康管理咨询有限公司 | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity |
| CN111437244A (en) * | 2020-04-15 | 2020-07-24 | 北京本真工坊生物科技有限公司 | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty |
| CN111440821A (en) * | 2020-04-15 | 2020-07-24 | 北京本真工坊生物科技有限公司 | Preparation method and application method of stem cell for efficiently expressing target protein |
| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
| CN113388584A (en) * | 2020-03-10 | 2021-09-14 | 路宝特（南京）环保科技有限公司 | Preparation method and application of engineered exosome for promoting skin tissue damage repair |
| CN113444730A (en) * | 2021-03-17 | 2021-09-28 | 昆明市延安医院 | Screening and constructing method of primary hepatocyte klotho gene transduction stem cells |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886646A (en) * | 2016-05-27 | 2016-08-24 | 福建爱我健康生物科技有限公司 | Primer, probe and kit for evaluating sun protection condition of skin |
| WO2017122095A1 (en) * | 2016-01-15 | 2017-07-20 | Orbsen Therapeutics Limited | Sdc-2 exosome compositions and methods of isolation and use |
| CN107007541A (en) * | 2017-05-23 | 2017-08-04 | 北京希诺赛尔健康科技推广有限公司 | Application of the excretion body in skin-whitening preparation |
| CN107828860A (en) * | 2017-12-12 | 2018-03-23 | 杜立波 | A kind of skin care item method for customizing based on genetic test |
| CN108004328A (en) * | 2017-12-12 | 2018-05-08 | 杜立波 | A kind of Skin whitening care cosmetics method for customizing |
| CN108473973A (en) * | 2016-09-30 | 2018-08-31 | 赛尔莱克斯生命科学公司 | Including the composition of the excretion body of load albumen and preparation and the method for delivering the composition |
-
2018
- 2018-09-07 CN CN201811049325.3A patent/CN109097336A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017122095A1 (en) * | 2016-01-15 | 2017-07-20 | Orbsen Therapeutics Limited | Sdc-2 exosome compositions and methods of isolation and use |
| CN105886646A (en) * | 2016-05-27 | 2016-08-24 | 福建爱我健康生物科技有限公司 | Primer, probe and kit for evaluating sun protection condition of skin |
| CN108473973A (en) * | 2016-09-30 | 2018-08-31 | 赛尔莱克斯生命科学公司 | Including the composition of the excretion body of load albumen and preparation and the method for delivering the composition |
| CN107007541A (en) * | 2017-05-23 | 2017-08-04 | 北京希诺赛尔健康科技推广有限公司 | Application of the excretion body in skin-whitening preparation |
| CN107828860A (en) * | 2017-12-12 | 2018-03-23 | 杜立波 | A kind of skin care item method for customizing based on genetic test |
| CN108004328A (en) * | 2017-12-12 | 2018-05-08 | 杜立波 | A kind of Skin whitening care cosmetics method for customizing |
Non-Patent Citations (5)
| Title |
|---|
| YOON-JIN KIM 等: "Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin", 《BIOCHEM BIOPHY RES COMMUN》 * |
| 张明香等: "《实用肝硬化治疗学 基础与临床治疗矛盾的对策》", 31 March 2016, 辽宁科学技术出版社 * |
| 王沛: "《全国中医药行业高等教育"十三五"规划教材 制药工艺学》", 31 August 2017, 中国中医药出版社 * |
| 陈千一等: "关于痤疮发病机制的基因多态性研究进展", 《湖北民族学院学报(医学版)》 * |
| 陶勇等: "黑色素生成能力基因检测治疗白癜风的临床应用探讨", 《名医》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110227058A (en) * | 2019-06-10 | 2019-09-13 | 北京恒峰铭成生物科技有限公司 | Pleiotrophic factor composition and its preparation method and application |
| CN113388584A (en) * | 2020-03-10 | 2021-09-14 | 路宝特（南京）环保科技有限公司 | Preparation method and application of engineered exosome for promoting skin tissue damage repair |
| CN111394301A (en) * | 2020-03-30 | 2020-07-10 | 广州瑞铂茵健康管理咨询有限公司 | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity |
| CN111394301B (en) * | 2020-03-30 | 2020-11-24 | 广州瑞铂茵健康科技有限公司 | Application of piceatannol in increasing number of secreted exosomes of pluripotent stem cells and bioactivity |
| CN111437244A (en) * | 2020-04-15 | 2020-07-24 | 北京本真工坊生物科技有限公司 | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty |
| CN111440821A (en) * | 2020-04-15 | 2020-07-24 | 北京本真工坊生物科技有限公司 | Preparation method and application method of stem cell for efficiently expressing target protein |
| CN111437244B (en) * | 2020-04-15 | 2022-08-23 | 北京本真工坊生物科技有限公司 | Preparation method and product of high-efficiency stem cell expression exosome for skin beauty |
| CN112007049A (en) * | 2020-09-21 | 2020-12-01 | 济南磐升生物技术有限公司 | Stem cell exosome composition for treating knee osteoarthritis |
| CN113444730A (en) * | 2021-03-17 | 2021-09-28 | 昆明市延安医院 | Screening and constructing method of primary hepatocyte klotho gene transduction stem cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109097336A (en) | A kind of stem cell excretion body, preparation method and application | |
| US20240165015A1 (en) | Lactobacillus rhamnosus, ferment lysate for regulating skin microecology, preparation method, and application | |
| CN110835616B (en) | Active substance of lactobacillus paracasei GKS6, composition containing same and application of active substance in promoting longevity | |
| CN1943425B (en) | Fishy smell removing method for spirulina princeps | |
| CN108624557A (en) | The preparation method and applications of mescenchymal stem cell excretion body | |
| CN105132318A (en) | Lactobacillus plantarum grx16 and application thereof | |
| WO2022222259A1 (en) | Lactobacillus reuteri having improved anti-aging and hair health enhancement effects, and application thereof | |
| CN111172074B (en) | Bifidobacterium lactis Probio-M8 capable of relieving and improving Alzheimer symptoms and application | |
| CN115216422B (en) | Lactobacillus rhamnosus and application thereof | |
| CN114790430B (en) | Lactobacillus rhamnosus E2 for producing hyaluronic acid and application thereof | |
| WO2021143621A1 (en) | Anaerostipes sp b2131 strain and use thereof in inflammatory bowel diseases | |
| CN107794254A (en) | The method for quickly purifying of recombinant high temperature lyases | |
| CN110833565B (en) | Active substance of lactobacillus plantarum GKM3, composition containing same and application of active substance in promoting longevity | |
| CN110623917A (en) | Beautifying and skin repairing sodium hyaluronate gel coated with stem cell complex factor | |
| CN107325997A (en) | Bifidobacterium longum and its application with cephalo resistance and high expression Sir2 albumen | |
| CN114686402A (en) | Lactococcus lactis subsp lactis HFY14 and application thereof | |
| CN114574393B (en) | Lactobacillus delbrueckii SEUNEU-110 and application thereof in skin | |
| CN110835615B (en) | Active substance of bifidobacterium lactis GKK2, composition containing same and application of active substance and composition in promoting longevity | |
| SKJELKVÅLE et al. | Detection of Enterotoxin in Faeces and Anti‐enterotoxin in Serum after Clostridium perfringens Food‐poisoning | |
| CN109486730A (en) | Bacillus H3, by its fermented fish leather for the purposes and collagen polypeptide of collagen polypeptide | |
| CN116162569A (en) | Bifidobacterium animalis subspecies BL03 and application thereof | |
| CN114796310B (en) | Pharmaceutical composition containing ATCC9080 strain fermentation product, and preparation method and application thereof | |
| Fildes | The Classification of Hæmoglobinophilic Bacteria, Based upon their Relation to Blood-Pigment and to the “Vitamine” Factor | |
| CN114457010A (en) | Stem cell derivative and preparation method and application thereof | |
| CN114128891A (en) | Bacterial strain for promoting intestinal development of infants 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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181228 |
|
| RJ01 | Rejection of invention patent application after publication |