CN109971707A - A kind of construction method in cell body source function physiologically active peptide intracellular library - Google Patents
A kind of construction method in cell body source function physiologically active peptide intracellular library Download PDFInfo
- Publication number
- CN109971707A CN109971707A CN201910242284.8A CN201910242284A CN109971707A CN 109971707 A CN109971707 A CN 109971707A CN 201910242284 A CN201910242284 A CN 201910242284A CN 109971707 A CN109971707 A CN 109971707A
- Authority
- CN
- China
- Prior art keywords
- cell
- added
- culture
- culture medium
- active peptide
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- 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
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a kind of construction method in cell body source function physiologically active peptide intracellular library, its include the following steps: first to umbilical cord carry out cell body culture, isolated mescenchymal stem cell is identified later, recovery amplifying cells, collect cleaning cell body be used for the extraction of cell intracellular activity peptide, liquid nitrogen multigelation method smudge cells collect cell intracellular activity peptide, using vacuum freeze drier to cell intracellular activity peptide liquid carry out freeze-drying be concentrated to get active peptide freeze-dried powder.Cell polypeptide library of the present invention has better compatibility with bigger diversity, the source in new natural bioactive peptide library, in human disease treatment and the addition of anti-ageing skin care industry.
Description
Technical field
The present invention relates to biology techniques field more particularly to a kind of cell body source function physiologically active peptide intracellular libraries
Construction method.
Background technique
Peptide is a kind of compound of the molecule structure between amino acid and protein, is that performance is various in life entity
The indispensable substance of physiological function, wherein the peptides of the physiological action beneficial to human life activity are also known as Functional Polypeptides
(functional biopeptide).From material composition structure, peptide is made of amino acid, is arranged by the type of different amino acid
Sequence has significant physiological activity plus some possible second levels even tertiary structure.In the extraction of peptide, at present from
It is dynamic, diversified biologically active peptide is isolated in plant and microorganism.Existing research shows that biologically active peptide is adjusting biology
Internal enzyme, it is antibacterial, adjust body's immunity, antithrombotic, anti-oxidant removings body free radical, improve the material absorbing of body with
The everyways such as transport function play important physiological action.
In recent years, the research because of biologically active peptide in anti-ageing field is more and more abundant, and biologically active peptide is applied in skin care
Also favored in skin care item market.German Bao Weier ﹒ Crewe moral doctor says: " have found a new antiaging agent --- peptide.
Peptide can make one the youth become, health, and great variety has occurred in the Tai Shi cosmetics world." U.S.'s skin aging international research can lead
Xi Nigulasi ﹒ Perry pricks the doctor of medicine, and " peptide, neuropeptide --- this kind of powerful chemicals have activating skin and hair, promote
Into health of heart, many effects such as the incidence probability of many diseases and strengthening immune system are reduced." Ni Gulasi ﹒ Perry bundle doctor
It learns doctor and has published " promise that Perry is pricked " " peptide, neuropeptide, let us is 10 years old young within 28 days " in the U.S..
Life active peptide is screening drug, the natural resources treasure-house of beautifying and antisenility effective component.Biologically active peptide at present
There are mainly two types of modes in source: the first is artificial synthesized by various modes, such as chemical synthesis, enzyme process or extensive at present
The recombinant DNA technology synthesis used.Artificial synthesized current common application, but various methods all have limitation.Chemical synthesis
Method is suitble to the synthesis of the peptide of some short-movie sections, at high cost, has some shadows to environmental and human health impacts in synthetic reaction process
It rings.Recombinant DNA technology only limits the production of some big peptides and protein.The second way is that extract biology intracorporal natural various
Active peptide.Me has been pushed significantly by the research that can be seen that genetic engineering and protein engineering to the basic research of peptide both at home and abroad
The production and application of state's active peptide, it is always that our biologically active peptides are studied that various biologically active peptides are separated from natural component
Mainstream.
Various biologically active peptides, such as beer ferment are mainly separated from various probiotics or saccharomycete in previous technique
Mother, Lactobacillus helveticus are mainly hydrolyzed by protein and are refined in separation come typical process flow is prepared with papain
--- albumen is added in pretreatment --- brewer's yeast is stirred evenly with water --- for polypeptide liquid process route process: brewer's yeast
--- enzyme-deactivating --- centrifugation --- collects supernatant to enzyme enzymatic hydrolysis.
Isolating biologically active polypeptide has been widely used for various aspects from various probiotics and saccharomycete, especially makes
It being added used in food or food additive, but still there is limitation, (1) brewer's yeast contains bulky grain hops, the impurity such as resin,
It needs to pre-process, to environment and people's health, there are some detrimental effects during processing.(2) the peptide master of the method separation
It uses and is added in food or food additive, polypeptide can provide the material element of the human bodies such as amino acid, derive from probiotics and ferment
Mother stock from polypeptide and human body species difference it is too big because the physiological action mechanism in life entity differs greatly, and therefore, it is very difficult to
Play the physiological function of its polypeptide.Therefore now widely used polypeptide source provides base generally as food sense organ peptide application
Plinth nutriment and improvement food sensory properties, such as enhancing flavor peptides, sense of taste peptide, hardness adjust peptide etc..Or it can not be complete
Meets the needs of physiological function peptide in human body diseases and beautifying and antisenility field.
Therefore need to develop more natural bioactive peptides source, so that isolated active peptide more purifies, and
Not only on food or food additives, the demand of human body diseases and beautifying and antisenility field can satisfy.
Summary of the invention
It is an object of the present invention to be directed to the above-mentioned deficiency of the prior art, it is bigger to propose that a kind of cell polypeptide library has
Diversity, the source in new natural bioactive peptide library have preferably in human disease treatment and the addition of anti-ageing skin care industry
The construction method in the cell body source function physiologically active peptide intracellular library of compatibility.
The present invention solves its technical problem, the technical solution adopted is that, propose that a kind of cell body source function intracellular is raw
Manage the construction method in active peptide library comprising following steps:
A, cell body culture: umbilical cord is cut into segment and is put into containing being washed 2~3 times in 2% dual anti-PBS washing lotion, is put into later
It in culture dish and shreds to paste, culture medium is added and stirs evenly, move into culture bottle later and culture medium is added, will cultivate
Bottle is put into CO2Culture medium is added in incubator into culture bottle after 5~9 days, observe that culture bottle locally goes out under the microscope later
When existing cell fusion degree reaches 90% or more, with pancreatin had digestive transfer culture and collect；
B, isolated mescenchymal stem cell is identified: to the postdigestive cell of pancreatin using cell dyeing buffer or
PBS cleaning solution is washed, later 1000~2000rpm centrifuge cell suspension, 5~10min, adds dyeing slow after discarding supernatant
Cell suspension is made in fliud flushing, will be spare in cell suspension addition streaming pipe, carries out block Fc receptors to cell suspension later, and right
The antibody of fluorescent marker is added in cell suspension, mixing is placed on ice, is protected from light 30~40min of incubation, and FACs buffer is added
Or cell is resuspended in PBS cleaning solution, FACs is added after discarding supernatant in 1000~2000 rpm centrifuge cell 5~10min of suspension
Cell is resuspended in buffer or PBS cleaning solution, uses flow cytometer alkalinity detection and analysis later；
C, it recovery amplifying cells: is put into being taken out after cell cryopreservation in 37 DEG C of water-baths, melts it all, be placed with culture medium
Centrifuge tube in be put into the cell suspension of defrosting, 1000~2000rpm is centrifuged 5~10min and removes supernatant later, to centrifugation
Culture medium piping and druming is added in pipe uniformly, centrifuge washing simultaneously removes supernatant, culture medium is added, cell is resuspended, after being transferred to culture bottle
It is put into CO2Culture medium addition PBS cleaning solution is sucked out later and is washed, is sucked out and washes after the completion of washing by incubator 24~48h of culture
It washs liquid and pancreatin is added, until the visible adherent layer of naked eyes is added culture medium and terminates digestion, piping and druming after thering are a large amount of cells to fall
Cellular layer simultaneously draws cell suspension in 1000~2000rpm centrifugation, 5~10min in centrifuge tube, removes supernatant, adds culture
Base weight is outstanding, centrifuge washing, removes supernatant, culture medium is added and is resuspended, is put into CO after being transferred to culture bottle2Incubator culture；
D, it collects the extraction that cleaning cell body is used for cell intracellular activity peptide: observing that the cell fusion degree in culture bottle reaches 85
Culture medium is sucked out after~90%, the washing of PBS cleaning solution is added, cleaning solution is sucked out after the completion of washing and pancreatin is added, until naked eyes can
See after adherent layer there are a large amount of cells to fall and culture medium termination digestion is added, blows and beats cellular layer and simultaneously draw cell suspension in centrifugation
1000~2000rpm is centrifuged 5~10min in pipe, removes supernatant, adds the resuspension of PBS cleaning solution, centrifuge washing is collected carefully
Born of the same parents；
E, liquid nitrogen multigelation method smudge cells collect cell intracellular activity peptide: the small cell of collection being carried out breeding culture, is received
Collection culture is collected by centrifugation after cell body gradient centrifugation washing three times and receives to P10 generation and in the cell of tiny shuttle-type after pancreatin digestion
Collect sediment, sediment adds deionized water, successively carries out liquid nitrogen multigelation method and low-speed centrifugal, supernatant is taken to carry out after centrifugation
Filtrate after ultrafiltration is carried out Solid Phase Extraction by centrifugal ultrafiltration, and eluent is made of the mixing of methanol, water and formic acid, and what is obtained washes
De- liquid blows the clean methanol of volatilization with nitrogen and obtains cell intracellular activity peptide liquid；
F, freeze-drying is carried out to cell intracellular activity peptide liquid using vacuum freeze drier and is concentrated to get active peptide freeze-dried powder.
As a preferred embodiment of the above technical solution, in step a, the umbilical cord after washing is before being put into culture dish, by umbilical cord
Tiao Qu vascular tissue.
As a preferred embodiment of the above technical solution, in step c, the cell frozen need to all melt in water-bath in 1min
Change.
As a preferred embodiment of the above technical solution, in step c and d, when terminating digestion, the culture medium of addition is all larger than used
Pancreatin monoploid product.
As a preferred embodiment of the above technical solution, in step e, three times gradient centrifugation washing concrete operations be for the first time from
The heart is centrifuged 15min at 3000~5000rpm, removes supernatant, then collects sediment, dispels, and PBS cleaning solution is added to 50 millis
It rises, mixing continues second of centrifugation, is centrifuged 10min at 3000~5000rpm, removes supernatant, then collect sediment, blow
It dissipates, PBS cleaning solution is added to 50 milliliters, mixing continues third time and is centrifuged, and is centrifuged 5min at 3000~5000rpm, goes
Supernatant collects sediment, wherein the revolving speed of centrifugation for the first time, for the second time centrifugation, third time centrifugation is consistent.
As a preferred embodiment of the above technical solution, in step e, it is 8* that sediment, which adds deionized water to be configured to cell concentration,
105The cell concentration of a/ml.
As a preferred embodiment of the above technical solution, in step e, the condition of multigelation method are as follows: the rapid freeze thawing of liquid nitrogen, then
37 DEG C melt rapidly, are repeated three times.
As a preferred embodiment of the above technical solution, in step e, the condition of low-speed centrifugal be at 4 DEG C, according to 2000~
3000rpm is centrifuged 30~40min.
As a preferred embodiment of the above technical solution, it in step e, when Solid Phase Extraction, using 1mlC18 solid-phase extraction column, first uses
Methanol activation and distilled water are pre-processed, and after loading, are eluted the protein substance adsorbed with eluent, wherein eluting
The volume ratio of first alcohol and water in liquid is 80:20, and the volume fraction of formic acid is 0.1%.
As a preferred embodiment of the above technical solution, in step f, cell intracellular activity peptide liquid is passed in refrigerator tray and carries out in advance
Freeze, control liquid level thickness is in 5~10mm, and pre-freezing temperature is -30~-15 DEG C, and the pre-freeze time is 2~4h, is then placed in vacuum again
In freeze drier cool chamber, control vacuum degree is less than 20Pa, and the vacuum freeze drying time is that 5~8h obtains active peptide freeze-drying
Powder.
The invention has the following beneficial effects:
1. selecting original of the mescenchymal stem cell cell space of the culture of umbilical cord source separation amplification as activity physiological function peptide intracellular
Expect source, has the advantage that (1) constructs a kind of source in new natural bioactive peptide library, be following disease medicament
The ingredient screening that screening and anti-ageing skin care industry carry out functional activity physiology peptide provides polypeptide resources bank.(2) the present invention program adopts
Separate physiological function peptide intracellular with from cell body, expand significantly only from cell secretion supernatant abstraction function peptide it is wide
Degree can greatly improve drug screening or anti-ageing skin care ingredient screening so that cell polypeptide library has bigger diversity
Range and effective percentage.(3) polypeptide of the present invention program screening derives from human tissue cell's cell space sample, may not only be applied to eat
Product add field, and can apply in human disease treatment and anti-ageing skin care industry.(4) cell body that the present invention program uses
The polypeptide of extraction can be used as physiological function peptide, for example directly act on dermal fibroblasts active cell letter by Transdermal absorption
Number access directly adjusts the secretion of Skin Cell collagen and hyaluronic acid.(5) this technical solution technique passes through ten thousand grades
Clean room culture cell does not add any antibiotic and other chemical reagent in incubation, therefore this process separates
To polypeptide can satisfy high standard medical treatment and anti-decrepit beauty field demand.It not only can be used as function peptide components to be added to
In skin care item, such as facial mask, Essence, surfactant improves skin problem in toner, is also used as the injection such as water laser accunputure
Agent is applied to the micro- integer field of medical cosmetology.
2. obtaining functional activity peptide intracellular using the quick multigelation method of ultralow temperature, there is operation compared with sonioation method
Mildly, the polypeptide active of separation and Extraction will not be influenced because of high temperature caused by operating process.
3. most impurity and fragment after cellular membrane disruption can be effectively removed using centrifugation and ultrafiltration.
It can completely avoid in process of vacuum drying 4. carrying out polypeptide intracellular using vacuum freeze drier and being condensed into powder
Caused by high temperature and make separation after functional activity peptide activity decline.
Detailed description of the invention
Fig. 1 is originally culture umbilical cord mesenchymal stem cells figure from crawling out in tissue under 10 × 20 power microscopes；
Fig. 2 is flow cytomery cell surface marker result figure；
Fig. 3 is cell adherent growth figure after recovering under 10 × 40 power microscopes；
Fig. 4 is cellular morphology figure after cell amplification cultivation 48h under 10 × 40 power microscopes.
Specific embodiment
Following is a specific embodiment of the present invention in conjunction with the accompanying drawings, technical scheme of the present invention will be further described,
However, the present invention is not limited to these examples.
Embodiment 1:
A kind of construction method in cell body source function physiologically active peptide intracellular library comprising following steps:
A, cell body culture: length 9cm umbilical cord is put into batch cultur ware and is cut into segment (by umbilical cord scissors in the present embodiment
It to mono- section of about 2cm) and is put into containing being washed 3 times in 2% dual anti-PBS cleaning solution, blood remaining in umbilical cord is cleaned completely dry
Only, washed umbilical cord is put into new culture dish Zhong Tiaoqu vascular tissue later and shredded to paste, 5ml is added and contains 10% tire ox
The culture medium of serum simultaneously stirs evenly, and moves into culture bottle and is added the culture medium that 2ml contains 10% fetal calf serum later, gently shake
What dynamic culture bottle made even tissue is covered on culture bottle bottom, and culture bottle is put into CO2Incubator adds after 9 days into culture bottle
Enter the culture medium that 2ml contains 10% fetal calf serum, culture bottle cannot be edge-on when liquid feeding, pipette can be protruded into culture bottle and slowly be dripped
Add, avoid tissue mobile, when visible mescenchymal stem cell local cells degrees of fusion reaches 90% or more under the microscope later, uses
Pancreatin had digestive transfer culture is simultaneously collected, as shown in Figure 1, being originally culture umbilical cord mesenchymal stem cells under 10 × 20 power microscopes from tissue
In the observation figure crawled out；
B, isolated mescenchymal stem cell is identified: the postdigestive cell of pancreatin is washed using PBS cleaning solution
It washs, later 1000rpm centrifuge cell suspension 5min, adds dye solution to dispel after discarding supernatant, vortex oscillator concussion 1~
3 seconds, cell suspension is made, cell count is carried out using cell counting board, cell is made 1 × 10 with cell dyeing buffer7
A/mL suspension, will be spare in 100 μ L cell suspensions, 7 fluidic cell pipes of addition, is used respectively 7 cell suspensions later
HLA-DR, CD14, CD34, CD90, CD105, CD73, CD45 carry out block Fc receptors, and to addition fluorescence mark in cell suspension
The antibody of note, mixing are placed on ice, are protected from light and are incubated for 35min, 5 mL PBS cleaning solutions are added, cell, 1000rpm centrifugation is resuspended
Cell suspension 5min is added 0.5 mL PBS cleaning solution resuspension cell after discarding supernatant, is examined later with flow cytometer alkalinity
It surveying and analyzes, such as Fig. 2, testing result are as follows: CD34, CD14, HLA-DR, CD45 are feminine gender, and CD105, CD73, CD90 is the positive,
The type for determining cell is stem cell, has very strong self-renewing and self competence for added value, also has repair ability；
C, it recovery amplifying cells: is put into being taken out after cell cryopreservation in 37 DEG C of water-baths, makes all to melt in its 1min, be placed with
The cell suspension of defrosting is put into the centrifuge tube of culture medium of the 4ml containing 10% fetal calf serum, 1000rpm is centrifuged 5min and falls later
Supernatant is removed, culture medium piping and druming of the 5ml containing 10% fetal calf serum is added into centrifuge tube uniformly, centrifuge washing simultaneously removes supernatant,
Culture medium of the 2ml containing 10% fetal calf serum is added, cell is resuspended, is put into CO after being transferred to culture bottle2Incubator culture 48h, such as Fig. 3
It is shown, cell adherent growth figure after recovery is observed under 10 × 40 power microscopes, culture medium is sucked out later, PBS cleaning solution is added
It is washed, cleaning solution is sucked out after the completion of washing and 3ml pancreatin is added, until the visible adherent layer of naked eyes has a large amount of cells to fall
Fall behind that the culture medium containing 10% fetal calf serum is added and terminates and digest, for the culture medium of addition greater than pancreatin monoploid used product, piping and druming is thin
Born of the same parents layer simultaneously draw cell suspension in centrifuge tube 2000rpm be centrifuged 10min, remove supernatant, add 6ml culture medium be resuspended,
Centrifuge washing removes supernatant, and 7ml culture medium is added and is resuspended, is put into CO after being transferred to culture bottle2Incubator culture, such as Fig. 4 institute
Show cellular morphology figure after observing cell amplification cultivation 48h under 10 × 40 power microscopes；
D, it collects the extraction that cleaning cell body is used for cell intracellular activity peptide: observing that the cell fusion degree in culture bottle reaches 85
Culture medium is sucked out after~90%, the washing of 4mlPBS cleaning solution is added, cleaning solution is sucked out after the completion of washing and 3ml pancreatin is added, until
Culture medium is added after visually visible adherent layer there are a large amount of cells to fall and terminates digestion, the culture medium of addition is greater than pancreatin used
Monoploid product blows and beats cellular layer and draws cell suspension in 2000rpm centrifugation 5min in centrifuge tube, removes supernatant, add
6mlPBS cleaning solution is resuspended, centrifuge washing collects cell；
E, liquid nitrogen multigelation method smudge cells collect cell intracellular activity peptide: the cell of collection being carried out breeding culture, is collected
It cultivates P10 generation and is in the cell of tiny shuttle-type, gradient centrifugation washing three times is collected by centrifugation after cell body after pancreatin digestion and collects
Sediment, the concrete operations of gradient centrifugation washing are that centrifugation is centrifuged 15min at 5000rpm for the first time three times, remove supernatant, then
Sediment is collected, is dispelled, PBS cleaning solution is added to 50 milliliters, mixing continues second of centrifugation, is centrifuged at 5000rpm
10min removes supernatant, then collects sediment, dispels, be added PBS cleaning solution to 50 milliliters, mixing continue third time from
The heart is centrifuged 5min at 5000rpm, removes supernatant, collects sediment, and adding deionized water to be configured to cell concentration is 8*105A/ml
Cell concentration, successively carry out liquid nitrogen multigelation method and low-speed centrifugal, the condition of multigelation method is the rapid freeze thawing of liquid nitrogen, so
To melt rapidly at 37 DEG C afterwards, is repeated three times, the condition of low-speed centrifugal is to be centrifuged 40min according to 3000rpm at 4 DEG C, from
Take supernatant to carry out centrifugal ultrafiltration after the heart, the molecular cut off of filter membrane is 3ku, in ultra-filtration process, at 4 DEG C under 4800rpm from
Filtrate after ultrafiltration is carried out Solid Phase Extraction by heart 30min, and eluent is made of the mixing of methanol, water and formic acid, Solid Phase Extraction
When, it using 1mlC18 solid-phase extraction column, is first pre-processed with the activation of 2ml methanol and 1ml distilled water, after 2ml loading, is used
400ul eluent elutes the protein substance adsorbed, and wherein the volume ratio of the first alcohol and water in eluent is 80:20,
The volume fraction of formic acid is 0.1%, and obtained eluent blows the clean methanol of volatilization with nitrogen and obtains cell intracellular activity peptide liquid；
F, freeze-drying is carried out to cell intracellular activity peptide liquid using vacuum freeze drier and is concentrated to get active peptide freeze-dried powder: cell born of the same parents
Interior active peptide liquid, which is passed in refrigerator tray, carries out pre-freeze, and for control liquid level thickness in 10mm, pre-freezing temperature is -15 DEG C, the pre-freeze time
It for 4h, is then placed in vacuum freeze drier cool chamber again, control vacuum degree is less than 20Pa, and the vacuum freeze drying time is 5h
Obtain active peptide freeze-dried powder.
Embodiment 2:
A kind of construction method in cell body source function physiologically active peptide intracellular library comprising following steps:
A, cell body culture: 10cm umbilical cord is put into batch cultur ware and is cut into segment (in the present embodiment extremely by umbilical cord scissors
Mono- section of about 2cm) and be put into containing being washed 2 times in 2% dual anti-PBS cleaning solution, blood remaining in umbilical cord is cleaned up completely,
Washed umbilical cord is put into new culture dish Zhong Tiaoqu vascular tissue later and is shredded to paste, 10ml is added and contains 10% tire ox blood
Clear culture medium simultaneously stirs evenly, and moves into culture bottle and is added the culture medium that 5ml contains 10% fetal calf serum later, shake gently
What culture bottle made even tissue is covered on culture bottle bottom, and culture bottle is put into CO2Incubator is added after 5 days into culture bottle
5ml contains the culture medium of 10% fetal calf serum, and culture bottle cannot be edge-on when liquid feeding, can protrude into culture bottle pipette and be slowly added dropwise,
It avoids tissue mobile, when observing that culture bottle cell fusion degree locally occurs and reaches 90% or more under the microscope later, uses pancreatin
Had digestive transfer culture is simultaneously collected；
B, isolated mescenchymal stem cell is identified: to the postdigestive cell of pancreatin using cell dyeing buffer into
Row washing, 2000rpm centrifuge cell suspension 10min, adds dye solution to dispel after discarding supernatant later, the shake of vortex oscillator
It swings 1-3 seconds, cell suspension is made, cell count is carried out using cell counting board, cell is made 1 with cell dyeing buffer ×
107A/mL suspension, will be spare in 100 μ L cell suspensions, 7 fluidic cell pipes of addition, is used respectively 7 cell suspensions later
HLA-DR, CD14, CD34, CD90, CD105, CD73, CD45 carry out block Fc receptors, and to addition fluorescence mark in cell suspension
The antibody of note, mixing be placed on ice, be protected from light be incubated for 30min, be added 5 mL FACs buffers be resuspended cell, 2000rpm from
Core cell suspension 10min is added 0.5 mL FACs buffer resuspension cell after discarding supernatant, uses flow cytometer alkali later
Property detection and analysis, testing result are as follows: CD34, CD14, HLA-DR, CD45 be feminine gender, CD105, CD73, CD90 be the positive, really
The type for determining cell is stem cell, has very strong self-renewing and self competence for added value, also has repair ability；
C, it recovery amplifying cells: is put into being taken out after cell cryopreservation in 37 DEG C of water-baths, makes all to melt in its 1min, be placed with
The cell suspension of defrosting is put into the centrifuge tube of culture medium of the 2ml containing 10% fetal calf serum, 2000rpm is centrifuged 10min and falls later
Supernatant is removed, culture medium piping and druming of the 3ml containing 10% fetal calf serum is added into centrifuge tube uniformly, centrifuge washing simultaneously removes supernatant,
Culture medium of the 5ml containing 10% fetal calf serum is added, cell is resuspended, is put into CO after being transferred to culture bottle2Incubator culture 36h, later
Culture medium addition PBS cleaning solution is sucked out to be washed, cleaning solution is sucked out after the completion of washing and 3ml pancreatin is added, until visually can
See after adherent layer there are a large amount of cells to fall that the culture medium containing 10% fetal calf serum, which is added, terminates digestion, the culture medium of addition is big
In pancreatin monoploid used product, blows and beats cellular layer and draw cell suspension in 1000rpm centrifugation 5min in centrifuge tube, remove supernatant
Liquid adds the resuspension of 4ml culture medium, centrifuge washing, removes supernatant, and 5ml culture medium is added and is resuspended, puts after being transferred to culture bottle
Enter CO2Incubator culture；
D, it collects the extraction that cleaning cell body is used for cell intracellular activity peptide: observing that the cell fusion degree in culture bottle reaches 85
Culture medium is sucked out after~90%, the washing of 4mlPBS cleaning solution is added, cleaning solution is sucked out after the completion of washing and 3ml pancreatin is added, until
Culture medium is added after visually visible adherent layer there are a large amount of cells to fall and terminates digestion, the culture medium of addition is greater than pancreatin used
Monoploid product blows and beats cellular layer and draws cell suspension in 1000rpm centrifugation 10min in centrifuge tube, removes supernatant, add
4mlPBS cleaning solution is resuspended, centrifuge washing collects cell；
E, liquid nitrogen multigelation method smudge cells collect cell intracellular activity peptide: the cell of collection being carried out breeding culture, is collected
It cultivates P10 generation and is in the cell of tiny shuttle-type, gradient centrifugation washing three times is collected by centrifugation after cell body after pancreatin digestion and collects
Sediment, the concrete operations of gradient centrifugation washing are that centrifugation is centrifuged 15min at 3,000 rpm for the first time three times, remove supernatant, then
Sediment is collected, is dispelled, PBS cleaning solution is added to 50 milliliters, mixing continues second of centrifugation, is centrifuged at 3,000 rpm
10min removes supernatant, then collects sediment, dispels, be added PBS cleaning solution to 50 milliliters, mixing continue third time from
The heart is centrifuged 5min at 3,000 rpm, removes supernatant, the sediment of collection, it is dense that the sediment of collection adds deionized water to be configured to cell
Degree is 8*105The cell concentration of a/ml successively carries out liquid nitrogen multigelation method and low-speed centrifugal, the condition of multigelation method is
The rapid freeze thawing of liquid nitrogen, then melts rapidly at 37 DEG C, is repeated three times, the condition of low-speed centrifugal be at 4 DEG C, according to
2000rpm is centrifuged 30min, and supernatant is taken to carry out centrifugal ultrafiltration after centrifugation, and the molecular cut off of filter membrane is 3ku, in ultra-filtration process,
It is centrifuged 30min under 4800rpm at 4 DEG C, the filtrate after ultrafiltration is subjected to Solid Phase Extraction, eluent is by methanol, water and formic acid
Mixing composition when Solid Phase Extraction, using 1mlC18 solid-phase extraction column, is first located with the activation of 2ml methanol and 1ml distilled water in advance
Reason after 2ml loading, elutes the protein substance adsorbed with 400ul eluent, wherein the first alcohol and water in eluent
Volume ratio is 80:20, and the volume fraction of formic acid is 0.1%, and obtained eluent blows the clean methanol of volatilization with nitrogen and obtains cell born of the same parents
Interior active peptide liquid；
F, freeze-drying is carried out to cell intracellular activity peptide liquid using vacuum freeze drier and is concentrated to get active peptide freeze-dried powder: cell born of the same parents
Interior active peptide liquid, which is passed in refrigerator tray, carries out pre-freeze, and for control liquid level thickness in 5mm, pre-freezing temperature is -30 DEG C, and the pre-freeze time is
Then 2h is placed in vacuum freeze drier cool chamber again, control vacuum degree is less than 20Pa, and the vacuum freeze drying time obtains for 8h
To active peptide freeze-dried powder.
Embodiment 3:
A kind of construction method in cell body source function physiologically active peptide intracellular library comprising following steps:
A, cell body culture: 8cm umbilical cord is put into batch cultur ware and is cut into segment (by umbilical cord scissors to about in the present embodiment
Mono- section of 2cm) and be put into containing being washed 3 times in 2% dual anti-PBS cleaning solution, blood remaining in umbilical cord is cleaned up completely, it
Washed umbilical cord is put into new culture dish Zhong Tiaoqu vascular tissue afterwards and is shredded to paste, 7ml is added containing 10% fetal calf serum
Culture medium simultaneously stirs evenly, and moves into culture bottle and is added the culture medium that 3ml contains 10% fetal calf serum later, shake gently culture
What bottle made even tissue is covered on culture bottle bottom, and culture bottle is put into CO23ml is added into culture bottle and contains for incubator after 7 days
The culture medium of 10% fetal calf serum, culture bottle cannot be edge-on when liquid feeding, can protrude into culture bottle pipette and be slowly added dropwise, avoid
Tissue movement is digested when observing that culture bottle cell fusion degree locally occurs and reaches 90% or more under the microscope later with pancreatin
It passes on and collects；
B, isolated mescenchymal stem cell is identified: to the postdigestive cell of pancreatin using cell dyeing buffer into
Row washing, 1500rpm centrifuge cell suspension 7min, adds dye solution fliud flushing to dispel after discarding supernatant later, vortex concussion
Device shakes 1-3 second, and cell suspension is made, using cell counting board progress cell count, with cell dyeing buffer by cell system
At 1 × 107A/mL suspension, will be spare in 100 μ L cell suspensions, 7 fluidic cell pipes of addition, later to 7 cell suspensions point
Not Shi Yong HLA-DR, CD14, CD34, CD90, CD105, CD73, CD45 carry out block Fc receptors, and to fluorescence is added in cell
The antibody of label, mixing are placed on ice, are protected from light and are incubated for 40min, 5 mL FACs buffers are added, cell, 1500rpm is resuspended
Centrifuge cell suspension 7min is added 0.5 mL FACs buffer resuspension cell after discarding supernatant, uses flow cytometer later
Alkaline detection and analysis, testing result are as follows: CD34, CD14, HLA-DR, CD45 are feminine gender, and CD105, CD73, CD90 is the positive,
The type for determining cell is stem cell, has very strong self-renewing and self competence for added value, also has repair ability；
C, it recovery amplifying cells: is put into being taken out after cell cryopreservation in 37 DEG C of water-baths, makes all to melt in its 1min, be placed with
The cell suspension of defrosting is put into the centrifuge tube of culture medium of the 3ml containing 10% fetal calf serum, 2000rpm is centrifuged 10min and falls later
Supernatant is removed, culture medium piping and druming of the 4ml containing 10% fetal calf serum is added into centrifuge tube uniformly, centrifuge washing simultaneously removes supernatant,
Culture medium of the 3ml containing 10% fetal calf serum is added, cell is resuspended, is put into CO after being transferred to culture bottle2Incubator culture for 24 hours, later
Culture medium addition PBS cleaning solution is sucked out to be washed, cleaning solution is sucked out after the completion of washing and 3ml pancreatin is added, until visually can
See after adherent layer there are a large amount of cells to fall that the culture medium containing 10% fetal calf serum, which is added, terminates digestion, the culture medium of addition is big
In pancreatin monoploid used product, blows and beats cellular layer and draw cell suspension in 1500rpm centrifugation 7min in centrifuge tube, remove supernatant
Liquid adds the resuspension of 5ml culture medium, centrifuge washing, removes supernatant, and 7ml culture medium is added and is resuspended, puts after being transferred to culture bottle
Enter CO2Incubator culture；
D, it collects the extraction that cleaning cell body is used for cell intracellular activity peptide: observing that the cell fusion degree in culture bottle reaches 85
Culture medium is sucked out after~90%, the washing of 4mlPBS cleaning solution is added, cleaning solution is sucked out after the completion of washing and 3ml pancreatin is added, until
Culture medium is added after visually visible adherent layer there are a large amount of cells to fall and terminates digestion, the culture medium of addition is greater than pancreatin used
Monoploid product blows and beats cellular layer and draws cell suspension in 1500rpm centrifugation 7min in centrifuge tube, removes supernatant, add
4mlPBS cleaning solution is resuspended, centrifuge washing collects cell；
E, liquid nitrogen multigelation method smudge cells collect cell intracellular activity peptide: the cell of collection being carried out breeding culture, is collected
It cultivates P10 generation and is in the cell of tiny shuttle-type, gradient centrifugation washing three times is collected by centrifugation after cell body after pancreatin digestion and collects
Sediment, the concrete operations of gradient centrifugation washing are that centrifugation is centrifuged 15min at 4000rpm for the first time three times, remove supernatant, then
Sediment is collected, is dispelled, PBS cleaning solution is added to 50 milliliters, mixing continues second of centrifugation, is centrifuged at 4000rpm
10min removes supernatant, then collects sediment, dispels, be added PBS cleaning solution to 50 milliliters, mixing continue third time from
The heart is centrifuged 5min at 4000rpm, removes supernatant, the sediment of collection, it is dense that the sediment of collection adds deionized water to be configured to cell
Degree is 8*105The cell concentration of a/ml successively carries out liquid nitrogen multigelation method and low-speed centrifugal, the condition of multigelation method is
The rapid freeze thawing of liquid nitrogen, then melts rapidly at 37 DEG C, is repeated three times, the condition of low-speed centrifugal be at 4 DEG C, according to
2500rpm is centrifuged 35min, and supernatant is taken to carry out centrifugal ultrafiltration after centrifugation, and the molecular cut off of filter membrane is 3ku, in ultra-filtration process,
It is centrifuged 30min under 4800rpm at 4 DEG C, the filtrate after ultrafiltration is subjected to Solid Phase Extraction, eluent is by methanol, water and formic acid
Mixing composition when Solid Phase Extraction, using 1mlC18 solid-phase extraction column, is first located with the activation of 2ml methanol and 1ml distilled water in advance
Reason after 2ml loading, elutes the protein substance adsorbed with 400ul eluent, wherein the first alcohol and water in eluent
Volume ratio is 80:20, and the volume fraction of formic acid is 0.1%, and obtained eluent blows the clean methanol of volatilization with nitrogen and obtains cell born of the same parents
Interior active peptide liquid；
F, freeze-drying is carried out to cell intracellular activity peptide liquid using vacuum freeze drier and is concentrated to get active peptide freeze-dried powder: cell born of the same parents
Interior active peptide liquid, which is passed in refrigerator tray, carries out pre-freeze, and for control liquid level thickness in 7mm, pre-freezing temperature is -22 DEG C, and the pre-freeze time is
Then 3h is placed in vacuum freeze drier cool chamber again, control vacuum degree is less than 20Pa, and the vacuum freeze drying time obtains for 6h
To active peptide freeze-dried powder.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Claims (10)
1. a kind of construction method in cell body source function physiologically active peptide intracellular library, which comprises the steps of:
A, cell body culture: umbilical cord is cut into segment and is put into containing being washed 2~3 times in 2% dual anti-PBS washing lotion, is put into later
It in culture dish and shreds to paste, culture medium is added and stirs evenly, move into culture bottle later and culture medium is added, will cultivate
Bottle is put into CO2Culture medium is added in incubator into culture bottle after 5~9 days, observe that culture bottle locally goes out under the microscope later
When existing cell fusion degree reaches 90% or more, with pancreatin had digestive transfer culture and collect；
B, isolated mescenchymal stem cell is identified: to the postdigestive cell of pancreatin using cell dyeing buffer or
PBS cleaning solution is washed, later 1000~2000rpm centrifuge cell suspension, 5~10min, adds dyeing slow after discarding supernatant
Cell suspension is made in fliud flushing, will be spare in cell suspension addition fluidic cell pipe, carries out block Fc receptors to cell suspension later,
And to the antibody that fluorescent marker is added in cell suspension, mixing is placed on ice, is protected from light 30~40min of incubation, and it is slow that FACs is added
Cell is resuspended in fliud flushing or PBS cleaning solution, and 1000~2000 rpm centrifuge cell 5~10min of suspension are added after discarding supernatant
Cell is resuspended in FACs buffer or PBS cleaning solution, uses flow cytometer alkalinity detection and analysis later；
C, it recovery amplifying cells: is put into being taken out after cell cryopreservation in 37 DEG C of water-baths, melts it all, be placed with culture medium
Centrifuge tube in be put into the cell suspension of defrosting, 1000~2000rpm is centrifuged 5~10min and removes supernatant later, to centrifugation
Culture medium piping and druming is added in pipe uniformly, centrifuge washing simultaneously removes supernatant, culture medium is added, cell is resuspended, after being transferred to culture bottle
It is put into CO2Culture medium addition PBS cleaning solution is sucked out later and is washed, is sucked out and washes after the completion of washing by incubator 24~48h of culture
It washs liquid and pancreatin is added, until the visible adherent layer of naked eyes is added culture medium and terminates digestion, piping and druming after thering are a large amount of cells to fall
Cellular layer simultaneously draws cell suspension in 1000~2000rpm centrifugation, 5~10min in centrifuge tube, removes supernatant, adds culture
Base weight is outstanding, centrifuge washing, removes supernatant, culture medium is added and is resuspended, is put into CO after being transferred to culture bottle2Incubator culture；
D, it collects the extraction that cleaning cell body is used for cell intracellular activity peptide: observing that the cell fusion degree in culture bottle reaches 85
Culture medium is sucked out after~90%, the washing of PBS cleaning solution is added, cleaning solution is sucked out after the completion of washing and pancreatin is added, until naked eyes can
See after adherent layer there are a large amount of cells to fall and culture medium termination digestion is added, blows and beats cellular layer and simultaneously draw cell suspension in centrifugation
1000~2000rpm is centrifuged 5~10min in pipe, removes supernatant, adds the resuspension of PBS cleaning solution, centrifuge washing is collected carefully
Born of the same parents；
E, liquid nitrogen multigelation method smudge cells collect cell intracellular activity peptide: the cell of collection being carried out breeding culture, is collected
It cultivates P10 generation and is in the cell of tiny shuttle-type, gradient centrifugation washing three times is collected by centrifugation after cell body after pancreatin digestion and collects
Sediment, sediment add deionized water, successively carry out liquid nitrogen multigelation method and low-speed centrifugal, taken after centrifugation supernatant carry out from
Filtrate after ultrafiltration is carried out Solid Phase Extraction by heart ultrafiltration, and eluent is made of the mixing of methanol, water and formic acid, obtained elution
Liquid blows the clean methanol of volatilization with nitrogen and obtains cell intracellular activity peptide liquid；
F, freeze-drying is carried out to cell intracellular activity peptide liquid using vacuum freeze drier and is concentrated to get active peptide freeze-dried powder.
2. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step a, the umbilical cord after washing is before being put into culture dish, by umbilical cord Tiao Qu vascular tissue.
3. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step c, the cell frozen need to all melt in water-bath in 1min.
4. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step c and d, when terminating digestion, the culture medium of addition is all larger than pancreatin monoploid product used.
5. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step e, the concrete operations of gradient centrifugation washing are that centrifugation is centrifuged at 3000~5000rpm for the first time three times
15min removes supernatant, then collects sediment, dispels, and PBS cleaning solution is added to 50 milliliters, mixing continue second from
The heart is centrifuged 10min at 3000~5000rpm, removes supernatant, then collects sediment, dispels, and PBS cleaning solution is added to 50 millis
It rising, mixing continues third time and is centrifuged, and is centrifuged 5min at 3000~5000rpm, removes supernatant, sediment is collected, wherein the
Primary centrifugation, second of centrifugation, the revolving speed of third time centrifugation are consistent.
6. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step e, it is 8*10 that sediment, which adds deionized water to be configured to cell concentration,5The cell concentration of a/ml.
7. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step e, the condition of multigelation method are as follows: the then rapid freeze thawing of liquid nitrogen melts rapidly for 37 DEG C, is repeated three
It is secondary.
8. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step e, the condition of low-speed centrifugal is to be centrifuged 30~40min according to 2000~3000rpm/min at 4 DEG C.
9. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step e, when Solid Phase Extraction, using 1mlC18 solid-phase extraction column, first being located in advance with methanol activation and distilled water
It manages, after loading, is eluted the protein substance adsorbed with eluent, wherein the volume ratio of the first alcohol and water in eluent is
80:20, the volume fraction of formic acid are 0.1%.
10. a kind of construction method in cell body source function physiologically active peptide intracellular library according to claim 1, special
Sign is: in step f, cell intracellular activity peptide liquid, which is passed in refrigerator tray, carries out pre-freeze, control liquid level thickness 5~
10mm, pre-freezing temperature are -30~-15 DEG C, and the pre-freeze time is 2~4h, is then placed in vacuum freeze drier cool chamber again, are controlled
Vacuum degree processed is less than 20Pa, and the vacuum freeze drying time is that 5~8h obtains active peptide freeze-dried powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910242284.8A CN109971707A (en) | 2019-03-28 | 2019-03-28 | A kind of construction method in cell body source function physiologically active peptide intracellular library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910242284.8A CN109971707A (en) | 2019-03-28 | 2019-03-28 | A kind of construction method in cell body source function physiologically active peptide intracellular library |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109971707A true CN109971707A (en) | 2019-07-05 |
Family
ID=67081180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910242284.8A Pending CN109971707A (en) | 2019-03-28 | 2019-03-28 | A kind of construction method in cell body source function physiologically active peptide intracellular library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109971707A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586182A (en) * | 2010-12-17 | 2012-07-18 | 张正前 | Preparation and application of human-stem-cell-secreted bioactive factor and lysis solution |
| CN103494865A (en) * | 2013-10-13 | 2014-01-08 | 张正前 | Composition of human stem cell and gynostemma pentaphyllum bioactive substances and preparation method thereof |
| CN106038598A (en) * | 2016-05-31 | 2016-10-26 | 张正亮 | Method for preparing human-derived stem cell secretion bioactive factor and lysate |
| CN108101959A (en) * | 2016-11-24 | 2018-06-01 | 四川科伦药物研究院有限公司 | A kind of method for preparing high-purity polypeptide or its analog |
-
2019
- 2019-03-28 CN CN201910242284.8A patent/CN109971707A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586182A (en) * | 2010-12-17 | 2012-07-18 | 张正前 | Preparation and application of human-stem-cell-secreted bioactive factor and lysis solution |
| CN103494865A (en) * | 2013-10-13 | 2014-01-08 | 张正前 | Composition of human stem cell and gynostemma pentaphyllum bioactive substances and preparation method thereof |
| CN106038598A (en) * | 2016-05-31 | 2016-10-26 | 张正亮 | Method for preparing human-derived stem cell secretion bioactive factor and lysate |
| CN108101959A (en) * | 2016-11-24 | 2018-06-01 | 四川科伦药物研究院有限公司 | A kind of method for preparing high-purity polypeptide or its analog |
Non-Patent Citations (1)
| Title |
|---|
| 边照阳: "《QuEChERS技术及应用》", 31 December 2017, 中国轻工业出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107988153A (en) | The method of mesenchymal stem cells derived from human umbilical blood source separation excretion body and the reagent used | |
| CN103667187B (en) | A kind of isolated culture method of human adipose-derived stem cell and the construction method of stem cell bank | |
| CN101437938A (en) | Cardiac stem cells | |
| CN107034183A (en) | The method for preparing autologous fat stem cell bioactivity peptide freeze-dried powder | |
| KR20100065338A (en) | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof | |
| CN108309921A (en) | A kind of method for preparing freeze-dried powder rich in cell factor | |
| CN108753708A (en) | A kind of preparation method of Stem Cell Activity factor freeze-dried powder | |
| CN107006452A (en) | A kind of human umbilical cord mesenchymal stem cells source excretion body freezes store method and its application | |
| CN108192862A (en) | A kind of preparation method of pilose antler stem cell, pilose antler stem cell and its application | |
| CN109082382A (en) | Cordyceps sinensis Hirsutella sinensis ZJB18002 and its application | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| US5905035A (en) | Fungus useful for chitin production | |
| CN109182414A (en) | A method of producing new fine jade disaccharides | |
| CN107119020A (en) | A kind of hepatic injury targeting mescenchymal stem cell based on miR 9 and preparation method and application | |
| Ulrich | Pectic enzymes of Pseudomonas cepacia and penetration of polygalacturonase into cells | |
| CN110257327A (en) | A kind of isolated culture method of umbilical cord mesenchymal stem cells | |
| CN109971707A (en) | A kind of construction method in cell body source function physiologically active peptide intracellular library | |
| US20170095593A1 (en) | Adipose-derived stem cell product | |
| CN104099269B (en) | Mycoplasma hyopneumoniae virulent strain and application thereof | |
| CN108601799A (en) | High potential human mesenchymal stem cell is enriched with and expanded from older cell mass | |
| CN110237026A (en) | A kind of Sodium Hyaluronate compound fat stem cell conditioned medium lyophilized preparation | |
| Repesh et al. | Interactions of tumor cells with intact capillaries: a model for intravasation | |
| CN111334472A (en) | PBMC (peripheral vascular endothelial cell) in-vitro 3D collagen hydrogel culture medium and preparation method thereof | |
| CN106434546A (en) | Method for preparing mesenchymal stem cell by fully utilizing umbilical cord resources | |
| CN114525211B (en) | Aspergillus versicolor ZLH-1, protease, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190705 |
|
| RJ01 | Rejection of invention patent application after publication |