CN106377547A - Extraction method and purpose of renewable particles of umbilical cord blood - Google Patents
Extraction method and purpose of renewable particles of umbilical cord blood Download PDFInfo
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- CN106377547A CN106377547A CN201610871433.3A CN201610871433A CN106377547A CN 106377547 A CN106377547 A CN 106377547A CN 201610871433 A CN201610871433 A CN 201610871433A CN 106377547 A CN106377547 A CN 106377547A
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
Abstract
The invention provides an extraction method of renewable particles of umbilical cord blood. The extraction method comprises the following steps of taking the umbilical cord blood, carrying out low-speed centrifugation, adding a buffer solution in lower layer precipitates A to carry out washing, carrying out low-speed centrifugation, adding erythrocyte lysate in lower layer precipitates B to carry out cracking, carrying out middle-speed centrifugation, adding the buffer solution in lower layer precipitates C, violently shaking, and carrying out low-speed centrifugation; respectively taking an upper layer solution A, an upper layer solution B, and an upper layer solution D, and carrying out high-speed centrifugation; collecting all the precipitates, and cultivating in an MEM (minimum essential medium); and collecting the medium after being cultivated for 10-30 days, and carrying out 3000-7000g centrifugation so as to enrich the renewable particles of the umbilical cord blood. The renewable particles of the umbilical cord blood can be used for treating ischemic myocardial injury as one of major diseases.
Description
Technical field
The present invention relates to biomedical sector, especially histocytology, regenerative medicine field.A kind of specifically related to umbilical cord
Blood regenerated particle, its extracting method and application thereof.
Background technology
In the past few decades, the related medical research of the various disease for the treatment of has a great development.Scientists are entered
Go various researchs and discussion, chemically medicine is to gene therapy, so that the stem-cell therapy generally acknowledged at present.The reason of stem cell
Just propose by early in decades ago.In the more than ten years in past, whole world various countries stem cell field scientific research have very heavy
The discovery wanted.First, the scientific research personnel of all stem cell field recognizes that stem cell has very strong power of regeneration.That is exactly dry thin
Born of the same parents have the ability to be divided into further cell or the organ of each tissue of human body.Such as stem cell can be divided into cardiac muscle cell to because of the heart
Muscular tissue ischemic and the cardiac muscle cell of necrosis that causes are regenerated.In addition stem cell can repair the liver causing due to cirrhosis
Meronecrosis, can reproduce liver cell.Have is exactly that stem cell can reproduce marrow, blood vessel again.Sum it up, the function of stem cell
Have a lot, they almost can rebuild all of clone.
Although stem cell has great clinical treatment potentiality, the quantity of adult stem cell seldom, fresh bone marrow or blood
The stem cell that liquid extracts is used for treatment and receives the restriction of quantity, and the technology of amplification in vitro adult stem cell at present not ten
It is divided into ripe.Up to the present, the detached adult stem cell of marrow can't carry out successful amplification in vitro, almost all of document
The adult stem cell of report is all divided into derived mesenchymal-like cells in cultivation period, rather than is divided into and represents the thin of three germinal layers
Born of the same parents.In vitro in amplification procedure, these lose the mark of stem cell surface through the cell cultivated, that is, lose dry thin
Self amplification of born of the same parents and the ability of many differentiation.
By contrast, present invention discover that Cord blood regenerated particle be naturally occurring the activity in blood of human body and marrow
Composition, in addition Cord blood regenerated particle of the present invention there is not yet risk that the external substances such as any rotaring redyeing gene bring etc..
Content of the invention
The purpose of the present invention is aimed to provide a kind of Cord blood regenerated particle, its extracting method and is obtained by the method
The purposes of Cord blood regenerated particle.The Cord blood regenerated particle that the present invention obtains can be repaired the heart causing because of ischemic and be damaged
Wound, regeneration Cardiac Stem Cells and cardiac muscular tissue, provide a new method for treatment ischemia myocardial damage and road.The present invention
Regenerated particle be specifically for use in one of major disease the treatment of ischemia myocardial damage among.The regenerated particle of the present invention is also
It is specifically for use in and promote among the treatment of cerebrovascular regeneration.
The present invention provides a kind of extracting method of Cord blood regenerated particle, comprises the steps：Cord blood is taken to carry out 50-
300g is centrifuged, and is divided into upper solution A and lower sediment A after centrifugation；Then take off a layer precipitate A, add buffer solution slightly to wash, enter
Row 50-300g is centrifuged, and is divided into upper solution B and lower sediment B after centrifugation；Then take off a layer precipitate B, add erythrocyte cracked liquid
Shake, carries out 1000-3000g centrifugation after cracking, is divided into upper solution C and lower sediment C after centrifugation；Then take lower sediment
C, adds buffer solution acutely to rock, carries out 50-300g centrifugation afterwards, be divided into upper solution D and lower sediment D after centrifugation；Then
Take upper solution A, upper solution B, upper solution D respectively, carry out 3000-7000g centrifugation, collect all precipitations and be placed in MEM's
Cultivated in culture medium；Finally collect the culture culture medium of 10-30 days, carry out 3000-7000g centrifugation, to be enriched with Cord blood
Regenerated particle.Wherein said Cord blood is preferably fresh.
In the inventive solutions, take off a layer precipitate A, by volume for 1:2-5 adds buffer solution.
In the inventive solutions, take off a layer precipitate B, by volume for 1:5-10 adds erythrocyte cracked liquid to be placed in
Horizontal shaker, shakes 20 minutes, carries out 1000-3000g centrifugation afterwards, and centrifugation time is 5-20 minute, is divided into two after centrifugation
Point, upper solution C and lower sediment C, upper solution C discards.
In the inventive solutions, take off a layer precipitate C, by volume for 1:5-10 adds buffer solution acutely to rock,
Jitter time is 1-2 minute, carries out 50-300g centrifugation afterwards, and centrifugation time is 5-20 minute, is divided into two parts after centrifugation, on
Layer solution D and lower sediment D, lower sediment D discards.
In the inventive solutions, take upper solution A, upper solution B, upper solution D respectively, carry out 3000-
7000g is centrifuged, and centrifugation time is 5-20 minute, after centrifugation, collects precipitation.
In the inventive solutions, the precipitation collected is placed in the culture medium of MEM (containing 20% mixing Cord blood
Serum) to be cultivated, condition of culture is 37 DEG C, in 5%CO2 incubator, changes culture medium once within every 2-3 days；Culture 10-30 days
Culture medium collect, carry out 3000-7000g centrifugation, to be enriched with Cord blood regenerated particle.
Present invention also offers grain is regenerated by the Cord blood that the extracting method of the Cord blood regenerated particle of the present invention obtains
Son, described extracting method comprises the steps：Take Cord blood to carry out 50-300g centrifugation, after centrifugation, be divided into upper solution A and lower floor
Precipitate A；Then take off a layer precipitate A, add buffer solution slightly to wash, carry out 50-300g centrifugation, after centrifugation, be divided into upper solution B
With lower sediment B；Then take off a layer precipitate B, add erythrocyte cracked liquid shake, carry out 1000-3000g centrifugation afterwards, centrifugation
After be divided into upper solution C and lower sediment C；Then take off a layer precipitate C, add buffer solution acutely to rock, carry out 50-300g afterwards
Centrifugation, is divided into upper solution D and lower sediment D after centrifugation；Then upper solution A, upper solution B, upper solution D are taken respectively,
Carry out 3000-7000g centrifugation, all precipitations of collection are placed in the culture medium of MEM is cultivated；Finally collect culture 10-30 days
Culture medium, carries out 3000-7000g centrifugation, to be enriched with Cord blood regenerated particle.Wherein said Cord blood is preferably fresh.
In the inventive solutions, take off a layer precipitate A, by volume for 1:2-5 adds buffer solution.
In the inventive solutions, take off a layer precipitate B, by volume for 1:5-10 adds erythrocyte cracked liquid to be placed in
Horizontal shaker, shakes 20 minutes, carries out 1000-3000g centrifugation afterwards, and centrifugation time is 5-20 minute, is divided into two after centrifugation
Point, upper solution C and lower sediment C, upper solution C discards.
In the inventive solutions, take off a layer precipitate C, by volume for 1:5-10 adds buffer solution acutely to rock,
Jitter time is 1-2 minute, carries out 50-300g centrifugation afterwards, and centrifugation time is 5-20 minute, is divided into two parts after centrifugation, on
Layer solution D and lower sediment D, lower sediment D discards.
In the inventive solutions, take upper solution A, upper solution B, upper solution D respectively, carry out 3000-
7000g is centrifuged, and centrifugation time is 5-20 minute, after centrifugation, collects precipitation.
In the inventive solutions, the precipitation collected is placed in the culture medium of MEM (containing 20% mixing Cord blood
Serum) to be cultivated, condition of culture is 37 DEG C, in 5%CO2 incubator, changes culture medium once within every 2-3 days；Culture 10-30 days
Culture medium collect, carry out 3000-7000g centrifugation, to be enriched with Cord blood regenerated particle.
On the one hand, the present invention provides a kind of extracting method of Cord blood regenerated particle, comprises the steps：
1) Cord blood is taken to carry out 50-300g centrifugation, centrifugation time is 5-20 minute, is divided into two parts, upper strata is molten after centrifugation
Liquid A and lower sediment A.
2) take lower sediment A in step 1, by volume for 1:2-5 add buffer solution slightly wash, carry out 50-300g from
The heart, centrifugation time is 5-20 minute, is divided into two parts, upper solution B and lower sediment B after centrifugation.
3) take lower sediment B in step 2, by volume for 1:5-10 adds erythrocyte cracked liquid to be placed in horizontal shaker, shakes
Dynamic 20 minutes, carry out 1000-3000g centrifugation afterwards, centrifugation time is 5-20 minute, is divided into two parts, upper solution C after centrifugation
With lower sediment C, upper solution C discards.
4) take lower sediment C in step 3, by volume for 1:5-10 adds buffer solution acutely to rock, and jitter time is 1-2
Minute, carry out 50-300g centrifugation afterwards, centrifugation time is 5-20 minute, is divided into two parts, upper solution D and lower floor after centrifugation
Precipitation D, lower sediment D discards.
5) take step 1 solution A, step 2 solution B, step 3 solution D at the middle and upper levels at the middle and upper levels at the middle and upper levels respectively, carry out 3000-
7000g is centrifuged, and centrifugation time is 5-20 minute, after centrifugation, collects all precipitations.
6) culture medium (the mixing serum of umbilical cord blood containing 20%) that the precipitation that step 5 is collected is placed in MEM is cultivated,
Condition of culture is 37 DEG C, in 5%CO2 incubator, changes culture medium once within every 2-3 days.
7) culture medium cultivating step 6 10-30 days is collected, and carries out 3000-7000g centrifugation, to be enriched with Cord blood regeneration
Particle.
Described in the extracting method of above-mentioned Cord blood regenerated particle, the fresh Cord blood of collection is former is saved in blood preseration bag
In, containing anti-coagulants.
In step 1, Cord blood used meets Decree of the State Council of the People's Republic of China the 208th《Blood product management rules》,
The production technology of Cord blood and test basis screening meet state food pharmaceuticals administration general bureau and sent out on 01 25th, 2007
Cloth《How examination blood donor？》, screening process carries out by the delivery room office worker of chain hospital：First, by inquiring medical history and body
The unsuitable blood donor of inspection exclusion, particularly exclusion people at highest risk, all kinds of hepatitis and AIDS, syphilitic.Secondly, to blood donor liver
Dirty function [including ALT (ALT) and bilirubin], hepatitis B surface antibody (HBsAg), HIV-1/HIV-2
Antibody, HCV antibody and syphilis are detected.Again, blood donor is signed after screening《Blood donor's Informed Consent Form》.
The qualified Cord blood cold chain transportation receipt position of collection in step 1, temperature is maintained at 2 to 8 DEG C.
In step 2 and step 4, buffer solution used can be any one known in the art, preferably PBS, is
Life Invitrogen, lot number is 1747277, PBS, PH7.4,10X, 500ML.
In step 3, erythrocyte cracked liquid can be any one known in the art, preferably 155mM NH4CL
(Sigma, CAS 12125-02-9), 10mM KHCO3(Sigma, CAS 298-14-6) and 0.1mM EDTA (Life
Invitrogen, 0.5M, No. LOT 1737880, PH8.0).
MEM culture medium used in step 6 is MEM ALPHA, is Life Invitrogen, and lot number is 1713957, MEM
ALPHA, 500ML.
Mixing serum of umbilical cord blood used in step 6 obtains in accordance with the following methods：By umbilical cord blood and 100mg/
Ml maltonic acid calcium (Sigma-Aldrich, CAS 299-28-5) is 10：1 ratio mixes, and is placed in 56 DEG C and inactivates 30 points
Clock, mixes afterwards, 4000rpm/ minute, is centrifuged 5 minutes, abandons precipitation, -80 DEG C save backup by supernatant, and the method is aseptic behaviour
Make.
Step 1 should be sterile working to all operationss of step 7.
On the other hand, the invention provides the Cord blood regenerated particle being obtained by following extracting methods, methods described bag
Include following steps：
1) Cord blood is taken to carry out 50-300g centrifugation, centrifugation time is 5-20 minute, is divided into two parts, upper strata is molten after centrifugation
Liquid A and lower sediment A.
2) take lower sediment A in step 1, by volume for 1:2-5 add buffer solution slightly wash, carry out 50-300g from
The heart, centrifugation time is 5-20 minute, is divided into two parts, upper solution B and lower sediment B after centrifugation.
3) take lower sediment B in step 2, by volume for 1:5-10 adds erythrocyte cracked liquid to be placed in horizontal shaker, shakes
Dynamic 20 minutes, carry out 1000-3000g centrifugation afterwards, centrifugation time is 5-20 minute, is divided into two parts, upper solution C after centrifugation
With lower sediment C, upper solution C discards.
4) take lower sediment C in step 3, by volume for 1:5-10 adds PBS acutely to rock, and jitter time divides for 1-2
Clock, carries out 50-300g centrifugation afterwards, and centrifugation time is 5-20 minute, is divided into two parts, upper solution D and lower floor to sink after centrifugation
Shallow lake D, lower sediment D discards.
5) take step 1 solution A, step 2 solution B, step 3 solution D at the middle and upper levels at the middle and upper levels at the middle and upper levels respectively, carry out 3000-
7000g is centrifuged, and centrifugation time is 5-20 minute, after centrifugation, collects all precipitations.
6) culture medium (the mixing serum of umbilical cord blood containing 20%) that the precipitation that step 5 is collected is placed in MEM is cultivated,
Condition of culture is 37 DEG C, in 5%CO2 incubator, changes culture medium once within every 2-3 days.
7) culture medium cultivating step 6 10-30 days is collected, and carries out 3000-7000g centrifugation, to be enriched with Cord blood regeneration
Particle.
Described in the extracting method of above-mentioned Cord blood regenerated particle, the fresh Cord blood of collection is former is saved in blood preseration bag
In, containing anti-coagulants.
In step 1, Cord blood used meets Decree of the State Council of the People's Republic of China the 208th《Blood product management rules》,
The production technology of Cord blood and test basis screening meet state food pharmaceuticals administration general bureau and sent out on 01 25th, 2007
Cloth《How examination blood donor？》, screening process carries out by the delivery room office worker of chain hospital：First, by inquiring medical history and body
The unsuitable blood donor of inspection exclusion, particularly exclusion people at highest risk, all kinds of hepatitis and AIDS, syphilitic.Secondly, to blood donor liver
Dirty function [including ALT (ALT) and bilirubin], hepatitis B surface antibody (HBsAg), HIV-1/HIV-2
Antibody, HCV antibody and syphilis are detected.Again, blood donor is signed after screening《Blood donor's Informed Consent Form》.
The qualified Cord blood cold chain transportation receipt position of collection in step 1, temperature is maintained at 2 to 8 DEG C.
In step 2 and step 4, buffer solution used can be any one known in the art, preferably PBS, is
Life Invitrogen, lot number is 1747277, PBS, PH7.4,10X, 500ML.
In step 3, erythrocyte cracked liquid can be any one known in the art, preferably 155mM NH4CL
(Sigma, CAS 12125-02-9), 10mM KHCO3 (Sigma, CAS 298-14-6) and 0.1mM EDTA (Life
Invitrogen, 0.5M, No. LOT 1737880, PH8.0).
MEM culture medium used in step 6 is MEM ALPHA, is Life Invitrogen, and lot number is 1713957, MEM
ALPHA, 500ML.
Mixing serum of umbilical cord blood used in step 6 obtains in accordance with the following methods：By umbilical cord blood and 100mg/
Ml maltonic acid calcium (Sigma-Aldrich, CAS 299-28-5) is 10：1 ratio mixes, and is placed in 56 DEG C and inactivates 30 points
Clock, mixes afterwards, 4000rpm/ minute, is centrifuged 5 minutes, abandons precipitation, -80 DEG C save backup by supernatant, and the method is aseptic behaviour
Make.
Step 1 should be sterile working to all operationss of step 7.
Further, the invention provides the biologic product containing Cord blood regenerated particle of the present invention, comprise navel of the present invention
With the composition of blood regenerated particle, and the kit including Cord blood regenerated particle of the present invention.
Present invention also offers the Cord blood regenerated particle biologic product of the present invention is used for treating myocardial damage, brain in preparation
Purposes in the medicine of injury of blood vessel, and it is used for regenerating cardiac muscular tissue, regeneration vessel, regeneration skin and hair follicle, again in preparation
Purposes in raw liver organization cell, the medicine of regeneration renal tissue cell, particularly promotes myocardial damage regeneration to repair in preparation
Purposes in medicine that is multiple or promoting cerebrovascular regeneration.Cord blood regenerated particle of the present invention is especially possible for treating major disease
One of ischemia myocardial damage treatment.
Cord blood regenerated particle of the present invention has wide use in regenerative medicine, drug discovery, cell therapy, cosmetics
On the way.For, in cell therapy, treating myocardial damage, cerebrovascular trauma etc..
Cord blood regenerated particle of the present invention is used in regenerative medicine, regeneration cardiac muscular tissue, regeneration vessel, regeneration skin and hair
Capsule, regeneration liver histocyte, regeneration renal tissue cell.
In embodiments of the present invention, make cell or tissue regeneration method include giving to patient effective amounts this
Bright Cord blood regenerated particle or the composition containing Cord blood regenerated particle of the present invention or medicine, make cell or tissue regenerate.
In a preferred embodiment, before giving to patient, alternatively in vitro culture and amplification Cord blood regenerated particle of the present invention.?
In one embodiment, it is possible to use Cord blood regenerated particle of the present invention is to regenerate cardiac muscle cell (cardiomyocyte) with life
The damaged tissues of heart injury (being for example derived from acute or chronic heart disease) are repaired on reason ground.Can by transplanting, implantation (for example originally
Invention Cord blood regenerated particle itself or as stroma cell composition part implantation), injection (for example direct injection is to disease
Disease or site, such as the ischemic site in the heart of direct injection to the individuality with myocardial damage or the brain of the patient's condition
The ischemic site of injury of blood vessel), infusion, by catheter delivery or any other well known by persons skilled in the art for carrying
The Therapeutic combinations completing Cord blood regenerated particle of the present invention or comprising Cord blood regenerated particle of the present invention for the method for the treatment of
Thing gives to its individuality of needs.
In some embodiments, the therapeutic combination comprising Cord blood regenerated particle of the present invention alternatively comprise one kind or
Multiple other cell types, for example, mature cell (such as fibroblast or endothelial cell), or stem cell or CFU-GM.
This kind of therapeutic agent and/or one or more other cell can individually or with two or more this kind of compounds or agent combination
Give to their individuality of needs.
In some embodiments, individuality to be treated is mammal.In a particular embodiment, to be treated
Body is people.In some specific embodiments, individuality is farming animals or domestic animal.In other specific embodiments, treat
The individuality for the treatment of is horse, sheep, ox, pig, dog or cat.
Although the various embodiments of the present invention are described above it should be understood that only with example rather than restriction
Mode proposes.Various changes can be made without departing from the spirit of the present invention or model according to the present invention to disclosed embodiment
Enclose.Therefore, the width of the present invention and scope should not be limited by any implementations described above.
Cord blood regenerated particle of the present invention is a new discovery, solves a difficult problem for adult stem cell.First, Cord blood is again
Raw particle is the internal normal cell existing, and does not have any processing or infects.Second, we have invented Cord blood regenerated particle body
Outer amplification technique, the Cord blood regenerated particle after amplification remains in that its function, does not voluntarily break up.3rd, internal transplanting
Cord blood regenerated particle does not have any side effect.In hundreds of zooperies, we do not find a tumour.Due to this
Bright Cord blood regenerated particle can be extracted from blood, so it is avoided that using embryonic tissue, does not also just have religion and ethics
Problem.In addition Cord blood regenerated particle does not cause immunological rejection.
Unless otherwise defined, all terms (including technology and scientific terminology) used herein all have and art technology
The identical meaning that personnel are generally understood that.Moreover, it is to be understood that term, as limited in universaling dictionary, should be solved
It is interpreted as that there is the consistent implication of the implication in the background of association area with them, and do not idealized or excessively formalize
Meaning explain, so limit unless specifically stated.
Brief description
Fig. 1 is shown that the form of Cord blood regenerated particle, form when Figure 1A just cultivates for Cord blood regenerated particle, figure
1B for Cord blood regenerated particle cultivate 1 week after form.
After Fig. 2 is shown that regenerated particle transplanting, in mouse body, it is located at the many cells chondritic in MI region.Regenerated particle
Transplanting mouse (Fig. 2A, 2C, 2E) and the heart of mouse (2B, 2D, 2F) being compared with salt solution ventricle wall pass through 2.5 ×,
10 × compare with 40 × lens.The thickness of ventricular muscles outer layer is roughly the same (in Fig. 2 C and 2D shown in black line), but
The gross thickness of the cardiac ventricles wall of implantation regenerated particle mouse is thicker.
It is the image that another regenerated particle transplants mouse heart that Fig. 3 shows, Fig. 3 G to 3J respectively 2.5 ×, 10 ×, 40
× and 100 × times lower image, result shows that MI region regenerated particle is transplanted mouse heart and had more Cardiospheres.
Fig. 4 show be regenerated particle transplant mouse heart in Oct4 and CXCR4 expression, it is found that Oct4 and
CXCR4 expresses in nucleus, is not detected by positive staining in control group, and finds Oct4 intensive little by image
The cell expressed in particulate matter and non-fully break up.
Fig. 5 is shown that newly to form the stained photographs of cell.Fig. 5 A is the heart sections of 2 weeks regenerated particle of injection；Fig. 5 B
It is the heart sections of 8 weeks regenerated particle of injection；Fig. 5 C is the expression of Sox2+GFP in the heart of 2 weeks regenerated particle of injection；
Fig. 5 D is the expression of DDX4+GFP in the heart of 2 weeks regenerated particle of injection；Fig. 5 E is the heart of 8 weeks regenerated particle of injection
In, the expression of Sox2+GFP；Fig. 5 F is the expression of DDX4+GFP in the heart of 8 weeks regenerated particle of injection.
Fig. 6 is shown that the brain of 10 weeks is partially sliced, and Fig. 6 A is control group, no Angiogenesiss；Fig. 6 B is experimental group, has
Angiogenesiss.
Fig. 7 is shown that fluorescence micrograph, and Fig. 7 C-7F is shown that GFP, Oct4, DAPI and composite diagram, Fig. 7 G-7J
It is shown that GFP, nestin, DAPI and composite diagram.
Specific embodiment
The present invention can be conducted further description by the following examples, and under the scope of the present invention is not limited to
State embodiment.
The present invention carries out generality and/or specific description to used material in experiment and experimental technique.Although being
Realizing many materials that the object of the invention used and method of operating is it is known in the art that but the present invention still make to the greatest extent may be used by here
The detailed description of energy.
The extraction of embodiment 1. Cord blood regenerated particle and culture
In the present embodiment, extraction and the culture of Cord blood regenerated particle are illustrated.
Blood source：Umbilical cord blood collection in the blood bag containing anti-coagulants, anti-coagulants is original sodium citrate in blood bag, passes through
Cord blood is transported to company by cold chain transportation mode (temperature is maintained at 2 to 8 DEG C).
Condition：The processing procedure obtaining Cord blood regenerated particle needs whole sterile working.
(1) take Cord blood to carry out 200g centrifugation, centrifugation time is 10 minutes, is divided into two parts after centrifugation, upper solution A and
Lower sediment A.
(2) take off a layer precipitate A, by volume for 1:2-5 adds PBS slightly to wash, and carries out 200g centrifugation, centrifugation time is
10 minutes, after centrifugation, it is divided into two parts, upper solution B and lower sediment B.
(3) take off a layer precipitate B, by volume for 1:5-10 adds erythrocyte cracked liquid to be placed in horizontal shaker, shakes 20 points
Clock, carries out 2000g centrifugation afterwards, and centrifugation time is 10 minutes, is divided into two parts, upper solution C and lower sediment C after centrifugation,
Upper solution C discards.
(4) take off a layer precipitate C, by volume for 1:5-10 add PBS acutely rock, jitter time be 2 minutes, laggard
Row 200g is centrifuged, and centrifugation time is 10 minutes, is divided into two parts, upper solution D and lower sediment D, lower sediment D is abandoned after centrifugation
Fall.
(5) take upper solution A, upper solution B, upper solution D respectively, carry out 5000g centrifugation, centrifugation time is 10 points
Clock, after centrifugation, collects all precipitations.
(6) culture medium (the mixing serum of umbilical cord blood containing 20%) that the precipitation collected is placed in MEM is cultivated, culture
Condition is 37 DEG C, in 5%CO2 incubator, changes culture medium once within every 2-3 days.
(7) the culture culture medium of 10 days is collected, carry out 5000g centrifugation, to be enriched with Cord blood regenerated particle (regenerated particle
Form is shown in Fig. 1).
Embodiment 2. Cord blood regenerated particle promotes the function of myocardial damage Regeneration and Repair
The Cord blood regenerated particle (GFP mark) that experimental group passes through tail vein injection embodiment 1 acquisition arrives ischemic myocardial
The mouse damaging, control group injecting normal saline.The blood that after injection, every 24 hours collect every mouse, carries out AST detection.Note
After penetrating, collect mouse heart detection affected area of cutting into slices within continuous 9 weeks.
After Cord blood regenerated particle is transplanted to mouse, more cardiospheres in experimental group, are had to be gathered in cardiac muscle
Infarcted region (see Fig. 2, Fig. 3), shows that Cord blood regenerated particle defines cardiospheres.By to experimental group
Oct4 and CXCR4 detection (see Fig. 4) in cardiospheres region, finds that in nuclear area be in stained positive, shows
Cardiospheres region has Cord blood regenerated particle to assemble formation karyocyte or stem cell.For proving further newly to be formed
Cell whether be stem cell, and detect that Sox2 and DDX4 verifies the cell of Green Fluorescent Protein using Fluorescence Histochemical,
Result proves that the new cell being formed is stem cell (see Fig. 5).
Above embodiment explanation, Cord blood regenerated particle of the present invention has the function of promoting myocardial damage Regeneration and Repair.
Embodiment 3. Cord blood regenerated particle promotes the function of cerebrovascular regeneration
The regenerated particle (GFP mark) that experimental group passes through tail vein injection embodiment 1 acquisition arrives the little of ischemic brain damage
Mouse, control group injecting normal saline.
In the brains of 10 weeks check, find there is no GFP dyeing (Fig. 6 A) in control group, but special in the GFP of experimental group
Strong GFP is had to express (Fig. 6 B) in the capillary structure of xenoantibody reactive moieties.Further study show that, in capillary structure
GFP and Oct4 (Fig. 7 C-7F) of coexpression or nestin is also checked through (Fig. 7 G-7J).
Above embodiment explanation, Cord blood regenerated particle of the present invention has the function of promoting brain damage revascularization.
Claims (10)
1. a kind of extracting method of Cord blood regenerated particle, is characterised by, comprises the steps：Cord blood is taken to carry out 50-300g
Centrifugation, is divided into upper solution A and lower sediment A after centrifugation；Then take off a layer precipitate A, add buffer solution slightly to wash, carry out
50-300g is centrifuged, and is divided into upper solution B and lower sediment B after centrifugation；Then take off a layer precipitate B, add erythrocyte cracked liquid to put
In horizontal shaker shake, carry out 1000-3000g centrifugation after cracking, after centrifugation, be divided into upper solution C and lower sediment C；Then
Take off a layer precipitate C, add buffer solution acutely to rock, carry out 50-300g centrifugation afterwards, after centrifugation, be divided into upper solution D and lower floor
Precipitation D；Then take upper solution A, upper solution B, upper solution D respectively, carry out 3000-7000g centrifugation, collect all precipitations
It is placed in the culture medium of MEM and cultivated；Finally collect the culture culture medium of 10-30 days, carry out 3000-7000g centrifugation, with richness
Collection Cord blood regenerated particle.
2. extracting method as claimed in claim 1 it is characterised in that：Cord blood is taken to carry out 50-300g centrifugation, centrifugation time is
5-20 minute, is divided into two parts, upper solution A and lower sediment A after centrifugation；Take off a layer precipitate A, by volume for 1:2-5 adds
Enter buffer solution slightly to wash, carry out 50-300g centrifugation, centrifugation time is 5-20 minute, is divided into two parts, upper solution after centrifugation
B and lower sediment B；Take off a layer precipitate B, by volume for 1:5-10 adds erythrocyte cracked liquid to be placed in horizontal shaker, cracks it
After carry out 1000-3000g centrifugation, centrifugation time be 5-20 minute, be divided into two parts, upper solution C and lower sediment after centrifugation
C, upper solution C discards；Take off a layer precipitate C, by volume for 1:5-10 adds buffer solution acutely to rock, and jitter time is 1-2
Minute, carry out 50-300g centrifugation afterwards, centrifugation time is 5-20 minute, is divided into two parts, upper solution D and lower floor after centrifugation
Precipitation D, lower sediment D discards；Take upper solution A, upper solution B, upper solution D respectively, carry out 3000-7000g centrifugation, from
The heart time is 5-20 minute.
3. extracting method as claimed in claim 1 it is characterised in that：Take upper solution A, upper solution B, upper solution respectively
D, carries out 3000-7000g centrifugation, and centrifugation time is 5-20 minute, after centrifugation, collects all precipitations and is placed in the mixing navel containing 20%
Cultivated in the culture medium of the MEM with blood serum, condition of culture is 37 DEG C, in 5%CO2 incubator, changed culture within every 2-3 days
Base is once.
4. a kind of Cord blood regenerated particle, is characterised by, it is to be prepared by the extracting method of any one of claim 1-3.
5. a kind of Cord blood regenerated particle biologic product, is characterised by, it comprises the Cord blood regenerated particle of claim 4.
6. a kind of composition of Cord blood regenerated particle, is characterised by, it contains the Cord blood regenerated particle of claim 4.
7. a kind of kit of Cord blood regenerated particle, is characterised by, it includes the Cord blood regenerated particle of claim 4.
8. Cord blood regenerated particle according to claim 4 is used in the medicine treating myocardial damage, cerebrovascular trauma in preparation
Purposes.
9. purposes according to claim 8, is characterised by, wherein said myocardial damage is ischemia myocardial damage.
10. Cord blood regenerated particle according to claim 4 is used for regenerating cardiac muscular tissue, regeneration vessel, regeneration skin in preparation
And the purposes in hair follicle, regeneration liver histocyte, the medicine of regeneration renal tissue cell.
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| CN108531454A (en) * | 2018-04-09 | 2018-09-14 | 孔五 | The purposes of Cord blood regenerated particle and composition in treating brain degenerative disease |
| CN108635376A (en) * | 2018-04-09 | 2018-10-12 | 孔五 | Purposes of the Cord blood regenerated particle and combinations thereof in treating skin wound healing |
| CN109777772A (en) * | 2018-09-29 | 2019-05-21 | 孔五一 | A kind of Cord blood regenerated particle and the preparation method and application thereof |
| CN109771443A (en) * | 2018-09-29 | 2019-05-21 | 孔五一 | A kind of application of Cord blood regenerated particle in preparation treatment acute kidney injury drug |
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| CN109771443A (en) * | 2018-09-29 | 2019-05-21 | 孔五一 | A kind of application of Cord blood regenerated particle in preparation treatment acute kidney injury drug |
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