CN106377547B - The extracting method and application thereof of Cord blood regenerated particle - Google Patents
The extracting method and application thereof of Cord blood regenerated particle Download PDFInfo
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- CN106377547B CN106377547B CN201610871433.3A CN201610871433A CN106377547B CN 106377547 B CN106377547 B CN 106377547B CN 201610871433 A CN201610871433 A CN 201610871433A CN 106377547 B CN106377547 B CN 106377547B
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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
A kind of extracting method of Cord blood regenerated particle is provided, steps are as follows: Cord blood being taken to carry out low-speed centrifugal, low-speed centrifugal after buffer washing is added to lower layer's precipitate A, medium-speed centrifuge after erythrocyte cracked liquid cracking is added to lower layer's precipitate B, buffer is added to lower layer's precipitate C acutely to shake, low-speed centrifugal.Upper solution A, upper solution B, upper solution D are taken respectively, carry out high speed centrifugation.All be deposited in MEM culture medium is collected to cultivate；The culture medium cultivated 10-30 days is collected, 3000-7000g centrifugation is carried out, to be enriched with Cord blood regenerated particle.Cord blood regenerated particle of the present invention can be used for treating the treatment of the ischemia myocardial damage of one of major disease.
Description
Technical field
The present invention relates to field of biomedicine, especially histocytologies, regenerative medicine field.More particularly to a kind of umbilical cord
Blood regenerated particle, its extracting method and application thereof.
Background technique
In the past few decades, the relevant medical research of various diseases is treated to have a great development.Scientists into
Various research and discussion are gone, chemically drug is to gene therapy, so that generally acknowledged stem-cell therapy at present.The reason of stem cell
It is just proposed by early in decades ago.In the past more than ten years, whole world various countries have very heavy in the scientific research of stem cell field
The discovery wanted.Firstly, the scientific research personnel of all stem cell fields recognizes that stem cell has very strong power of regeneration.That is exactly dry thin
Born of the same parents have the ability further to be divided into the cell or organ that human body is respectively organized.Such as stem cell can be divided into cardiac muscle cell to because of the heart
Downright bad cardiac muscle cell caused by muscular tissue ischemic regenerates.In addition stem cell can repair the liver due to caused by cirrhosis
Meronecrosis can reproduce liver cell.Have again is exactly that stem cell can reproduce marrow, blood vessel.To sum up, the function of stem cell
Have very much, they can almost rebuild all cell line.
Although stem cell has great clinical treatment potentiality, the quantity of adult stem cell is seldom, fresh bone marrow or blood
The stem cell that liquid extracts receives the limitation of quantity for treating, and the technology of amplification in vitro adult stem cell at present not ten
It is divided into ripe.Up to the present, the adult stem cell of marrow separation 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 by the cell cultivated, that is, lose dry thin
The ability they born of the same parents self amplification and broken up more.
In contrast, present invention discover that Cord blood regenerated particle be naturally occurring the activity in blood of human body and marrow
Ingredient, furthermore Cord blood regenerated particle of the present invention there is not yet external substances bring risk such as any rotaring redyeing gene etc..
Summary of the invention
The purpose of the present invention is intended to provide a kind of Cord blood regenerated particle, its extracting method and is obtained by this method
The purposes of Cord blood regenerated particle.The Cord blood regenerated particle that the present invention obtains can repair the heart because of caused by ischemic and damage
Wound regenerates Cardiac Stem Cells and cardiac muscular tissue, provides the method and road of a new treatment ischemia myocardial damage.The present invention
The regenerated particle ischemia myocardial damage that is specifically for use in one of major disease treatment among.Regenerated particle of the invention is also
It is specifically for use in and promotes among the regenerated treatment of the cerebrovascular.
The present invention provides a kind of extracting method of Cord blood regenerated particle, includes the following steps: that Cord blood is taken to carry out 50-
300g centrifugation, is divided into upper solution A and lower sediment A after centrifugation；Then lower layer's precipitate A is taken, buffer is added and slightly washs, into
Row 50-300g centrifugation, is divided into upper solution B and lower sediment B after centrifugation；Then lower layer's precipitate B is taken, erythrocyte cracked liquid is added
It shakes, 1000-3000g centrifugation is carried out after cracking, upper solution C and lower sediment C are divided into after centrifugation；Then lower sediment is taken
C is added buffer and acutely shakes, carries out 50-300g centrifugation later, upper solution D and lower sediment D are divided into after centrifugation；Then
Upper solution A, upper solution B, upper solution D are taken respectively, carries out 3000-7000g centrifugation, are collected all precipitatings and are placed in MEM's
It is cultivated in culture medium；The culture medium cultivated 10-30 days is finally collected, 3000-7000g centrifugation is carried out, to be enriched with Cord blood
Regenerated particle.Wherein the Cord blood is preferably fresh.
In the inventive solutions, lower layer's precipitate A is taken, buffer is added for 1:2-5 by volume.
In the inventive solutions, lower layer's precipitate B is taken, erythrocyte cracked liquid is added for 1:5-10 by volume and is placed in
Horizontal shaker shakes 20 minutes, carries out 1000-3000g centrifugation later, and centrifugation time is 5-20 minutes, and two are divided into after centrifugation
Point, upper solution C and lower sediment C, upper solution C are discarded.
In the inventive solutions, lower layer's precipitate C is taken, buffer is added for 1:5-10 by volume and acutely shakes,
Jitter time is 1-2 minutes, carries out 50-300g centrifugation later, and centrifugation time is 5-20 minutes, and two parts are divided into after centrifugation, on
Layer solution D and lower sediment D, lower sediment D are discarded.
In the inventive solutions, upper solution A, upper solution B, upper solution D are taken respectively, carry out 3000-
7000g centrifugation, centrifugation time are 5-20 minutes, after centrifugation, collect precipitating.
In the inventive solutions, the precipitating being collected into is placed in the culture medium of MEM (containing 20% mixing Cord blood
Serum) it is cultivated, condition of culture is 37 DEG C, and in 5%CO2 incubator, replacement culture medium is primary 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.
The present invention also provides the Cord bloods that the extracting method of Cord blood regenerated particle through the invention obtains to regenerate grain
Son, the extracting method include the following steps: to take Cord blood to carry out 50-300g centrifugation, upper solution A and lower layer are divided into after centrifugation
Precipitate A；Then lower layer's precipitate A is taken, buffer is added and slightly washs, carries out 50-300g centrifugation, upper solution B is divided into after centrifugation
With lower sediment B；Then lower layer's precipitate B is taken, erythrocyte cracked liquid is added and shakes, carries out 1000-3000g centrifugation, centrifugation later
After be divided into upper solution C and lower sediment C；Then lower layer's precipitate C is taken, buffer is added and acutely shakes, carries out 50-300g later
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,
3000-7000g centrifugation is carried out, all precipitatings of collection, which 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 the Cord blood is preferably fresh.
In the inventive solutions, lower layer's precipitate A is taken, buffer is added for 1:2-5 by volume.
In the inventive solutions, lower layer's precipitate B is taken, erythrocyte cracked liquid is added for 1:5-10 by volume and is placed in
Horizontal shaker shakes 20 minutes, carries out 1000-3000g centrifugation later, and centrifugation time is 5-20 minutes, and two are divided into after centrifugation
Point, upper solution C and lower sediment C, upper solution C are discarded.
In the inventive solutions, lower layer's precipitate C is taken, buffer is added for 1:5-10 by volume and acutely shakes,
Jitter time is 1-2 minutes, carries out 50-300g centrifugation later, and centrifugation time is 5-20 minutes, and two parts are divided into after centrifugation, on
Layer solution D and lower sediment D, lower sediment D are discarded.
In the inventive solutions, upper solution A, upper solution B, upper solution D are taken respectively, carry out 3000-
7000g centrifugation, centrifugation time are 5-20 minutes, after centrifugation, collect precipitating.
In the inventive solutions, the precipitating being collected into is placed in the culture medium of MEM (containing 20% mixing Cord blood
Serum) it is cultivated, condition of culture is 37 DEG C, and in 5%CO2 incubator, replacement culture medium is primary 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, includes the following steps:
1) Cord blood is taken to carry out 50-300g centrifugation, centrifugation time is 5-20 minutes, two parts is divided into after centrifugation, upper layer is molten
Liquid A and lower sediment A.
2) take lower sediment A in step 1, by volume for 1:2-5 be added buffer slightly wash, carry out 50-300g from
The heart, centrifugation time are 5-20 minutes, and two parts, upper solution B and lower sediment B are divided into after centrifugation.
3) lower sediment B in step 2 is taken, erythrocyte cracked liquid is added for 1:5-10 by volume and is placed in horizontal shaker, shakes
It is 20 minutes dynamic, 1000-3000g centrifugation is carried out later, and centrifugation time is 5-20 minutes, and two parts, upper solution C are divided into after centrifugation
It is discarded with lower sediment C, upper solution C.
4) lower sediment C in step 3 is taken, buffer is added for 1:5-10 by volume and acutely shakes, jitter time 1-2
Minute, 50-300g centrifugation is carried out later, and centrifugation time is 5-20 minutes, and two parts, upper solution D and lower layer are divided into after centrifugation
D is precipitated, lower sediment D is discarded.
5) 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 is taken respectively, carries out 3000-
7000g centrifugation, centrifugation time are 5-20 minutes, after centrifugation, collect all precipitatings.
6) culture medium (the mixing serum of umbilical cord blood containing 20%) that the precipitating for being collected into step 5 is placed in MEM is cultivated,
Condition of culture is 37 DEG C, and in 5%CO2 incubator, replacement culture medium is primary within every 2-3 days.
7) culture medium that step 6 is cultivated 10-30 days is collected, carries out 3000-7000g centrifugation, to be enriched with Cord blood regeneration
Particle.
The fresh Cord blood original of acquisition described in the extracting method of above-mentioned Cord blood regenerated particle is stored in blood preseration bag
In, contain anti-coagulants.
Cord blood used meets Decree of the State Council of the People's Republic of China the 208th " blood product management rules " in step 1,
Production technology and the test basis screening of Cord blood meet state food pharmaceuticals administration general bureau and sent out on 01 25th, 2007
Cloth " how screening blood donor? ", screening process is carried out by the delivery room office worker of chain hospital: firstly, by inquiry medical history and body
Inspection excludes unsuitable blood donor, especially exclusion people at highest risk, all kinds of hepatitis and AIDS, syphilitic.Secondly, to blood donor liver
Dirty function [including alanine aminotransferase (ALT) and bilirubin], hepatitis B surface antibody (HBsAg), HIV-1/HIV-2
Antibody, HCV antibody and syphilis are detected.Again, blood donor is by signing " blood donor's informed consent form " after screening.
The qualified Cord blood cold chain transportation receipt position acquired in step 1, temperature are maintained at 2 to 8 DEG C.
Buffer used in step 2 and step 4 can be any one known in the art, preferably PBS, be
Life Invitrogen, lot number 1747277, PBS, PH7.4,10X, 500ML.
Erythrocyte cracked liquid can be any one known in the art, preferably 155mM NH in step 34CL
(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, lot number 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/
The ratio that ml maltonic acid calcium (Sigma-Aldrich, CAS 299-28-5) is 10:1 mixes, and is placed in 56 DEG C and inactivates 30 points
Clock mixes later, 4000rpm/ minutes, is centrifuged 5 minutes, abandons precipitating, -80 DEG C of supernatant are saved backup, and this method is sterile behaviour
Make.
The all operationss of step 1 to step 7 should be sterile working.
On the other hand, the present invention provides the Cord blood regenerated particle obtained by following extracting methods, the method packets
Include following steps:
1) Cord blood is taken to carry out 50-300g centrifugation, centrifugation time is 5-20 minutes, two parts is divided into after centrifugation, upper layer is molten
Liquid A and lower sediment A.
2) take lower sediment A in step 1, by volume for 1:2-5 be added buffer slightly wash, carry out 50-300g from
The heart, centrifugation time are 5-20 minutes, and two parts, upper solution B and lower sediment B are divided into after centrifugation.
3) lower sediment B in step 2 is taken, erythrocyte cracked liquid is added for 1:5-10 by volume and is placed in horizontal shaker, shakes
It is 20 minutes dynamic, 1000-3000g centrifugation is carried out later, and centrifugation time is 5-20 minutes, and two parts, upper solution C are divided into after centrifugation
It is discarded with lower sediment C, upper solution C.
4) lower sediment C in step 3 is taken, PBS is added for 1:5-10 by volume and acutely shakes, jitter time is 1-2 points
Clock carries out 50-300g centrifugation later, and centrifugation time is 5-20 minutes, and two parts are divided into after centrifugation, and upper solution D and lower layer are heavy
Shallow lake D, lower sediment D are discarded.
5) 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 is taken respectively, carries out 3000-
7000g centrifugation, centrifugation time are 5-20 minutes, after centrifugation, collect all precipitatings.
6) culture medium (the mixing serum of umbilical cord blood containing 20%) that the precipitating for being collected into step 5 is placed in MEM is cultivated,
Condition of culture is 37 DEG C, and in 5%CO2 incubator, replacement culture medium is primary within every 2-3 days.
7) culture medium that step 6 is cultivated 10-30 days is collected, carries out 3000-7000g centrifugation, to be enriched with Cord blood regeneration
Particle.
The fresh Cord blood original of acquisition described in the extracting method of above-mentioned Cord blood regenerated particle is stored in blood preseration bag
In, contain anti-coagulants.
Cord blood used meets Decree of the State Council of the People's Republic of China the 208th " blood product management rules " in step 1,
Production technology and the test basis screening of Cord blood meet state food pharmaceuticals administration general bureau and sent out on 01 25th, 2007
Cloth " how screening blood donor? ", screening process is carried out by the delivery room office worker of chain hospital: firstly, by inquiry medical history and body
Inspection excludes unsuitable blood donor, especially exclusion people at highest risk, all kinds of hepatitis and AIDS, syphilitic.Secondly, to blood donor liver
Dirty function [including alanine aminotransferase (ALT) and bilirubin], hepatitis B surface antibody (HBsAg), HIV-1/HIV-2
Antibody, HCV antibody and syphilis are detected.Again, blood donor is by signing " blood donor's informed consent form " after screening.
The qualified Cord blood cold chain transportation receipt position acquired in step 1, temperature are maintained at 2 to 8 DEG C.
Buffer used in step 2 and step 4 can be any one known in the art, preferably PBS, be
Life Invitrogen, lot number 1747277, PBS, PH7.4,10X, 500ML.
Erythrocyte cracked liquid can be any one known in the art, preferably 155mM NH4CL in step 3
(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, lot number 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/
The ratio that ml maltonic acid calcium (Sigma-Aldrich, CAS 299-28-5) is 10:1 mixes, and is placed in 56 DEG C and inactivates 30 points
Clock mixes later, 4000rpm/ minutes, is centrifuged 5 minutes, abandons precipitating, -80 DEG C of supernatant are saved backup, and this method is sterile behaviour
Make.
The all operationss of step 1 to step 7 should be sterile working.
Further, the present invention provides the biological agents for containing Cord blood regenerated particle of the present invention, include navel of the present invention
Composition with blood regenerated particle, and the kit including Cord blood regenerated particle of the present invention.
The present invention also provides Cord blood regenerated particle biological agent of the invention in preparation for treating myocardial damage, brain
Purposes in the drug of injury of blood vessel, and in preparation for regenerating cardiac muscular tissue, regeneration vessel, regeneration skin and hair follicle, again
Raw liver organization cell regenerates purposes in the drug of renal tissue cell, especially promotes myocardial damage regeneration to repair in preparation
Purposes in the multiple or promotion regenerated drug of the cerebrovascular.Cord blood regenerated particle of the present invention is especially possible for treatment 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 treating myocardial damage, cerebrovascular trauma etc. in cell therapy.
Cord blood regenerated particle of the present invention is in regenerative medicine, regeneration cardiac muscular tissue, to regenerate skin and hair at regeneration vessel
Capsule, regeneration liver histocyte, regeneration renal tissue cell.
In embodiments of the present invention, making cell or tissue regeneration method includes giving to this hair of patient effective amounts
Bright Cord blood regenerated particle or composition or drug containing Cord blood regenerated particle of the present invention, regenerate cell or tissue.
In a preferred embodiment, it is giving to patient, optionally in vitro culture and amplification Cord blood regenerated particle of the present invention.?
In one embodiment, Cord blood regenerated particle of the present invention can be used to regenerate cardiac muscle cell (cardiomyocyte) with life
Repair the damaged tissues of heart injury (such as from acute or chronic heart disease) in reason ground.Can by transplant, be implanted into (such as this
The implantation of invention Cord blood regenerated particle itself or the part as stroma cell composition), (such as direct injection is to disease for injection
At the site of disease or the patient's condition, such as ischemic site or brain of the direct injection into the heart of the individual with myocardial damage
The ischemic site of injury of blood vessel), infusion, by catheter delivery or any other is well known by persons skilled in the art for mentioning
It completes for the method for the treatment of by Cord blood regenerated particle of the present invention or Therapeutic combinations comprising Cord blood regenerated particle of the present invention
Object is given to needing its individual.
In some embodiments, the therapeutic combination comprising Cord blood regenerated particle of the present invention optionally include it is a kind of or
A variety of other cell types, for example, mature cell (such as fibroblast or endothelial cell) or stem cell or progenitor cells.
This kind of therapeutic agent and/or one or more other cells can combine individually or with two or more this kind of compounds or reagent
It gives to their individual of needs.
In some embodiments, individual to be treated is mammal.In a particular embodiment, to be treated
Body is people.In some specific embodiments, individual is farming animals or domestic animal.In other specific embodiments, to
The individual for the treatment of is horse, sheep, ox, pig, dog or cat.
Although various embodiments of the invention are described above, it should be understood that only with example rather than limitation
Mode proposes.Various changes can be made to disclosed embodiment according to the present invention without departing from spirit or model of the invention
It encloses.Therefore, width of the invention and range should not be limited by any implementations described above.
Cord blood regenerated particle of the present invention is a new discovery, solves the problems, such as adult stem cell.First, Cord blood is again
Raw particle is internal existing normal cell, without any processing or infection.Second, we have invented Cord blood regenerated particle bodies
Outer amplification technique, the Cord blood regenerated particle after amplification still maintain its function, do not break up voluntarily.Third is transplanted in vivo
Cord blood regenerated particle does not have any side effect.In hundreds of zooperies, we do not have found an example tumour.Due to this hair
Bright Cord blood regenerated particle can be extracted from blood, in this way it is avoided that using embryonic tissue, also just without religion and ethics
Problem.In addition Cord blood regenerated particle does not cause immunological rejection.
Unless otherwise defined, all terms (including technical and scientific term) used herein all have and art technology
The normally understood identical meaning of personnel.Those of moreover, it is to be understood that term, limited such as in universaling dictionary, it should be solved
It is interpreted as having and their consistent meanings of meaning in the background of related fields, and is not able to idealize or over formalization
Meaning explain, unless specifically stated so limit.
Detailed description of the invention
The form of Cord blood regenerated particle is shown in Fig. 1, and Figure 1A is form when Cord blood regenerated particle is just cultivated, figure
1B is the form after the culture of Cord blood regenerated particle 1 week.
After regenerated particle transplanting is shown in Fig. 2, many cells chondritic in the region MI is located in Mice Body.Regenerated particle
The ventricle wall of the heart of the mouse (Fig. 2A, 2C, 2E) of transplanting and the mouse compared with salt water (Fig. 2 B, 2D, 2F) passes through 2.5
×, 10 × compared 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
Be be implanted into regenerated particle mouse cardiac ventricles wall overall thickness it is thicker.
Fig. 3 show be another regenerated particle transplanting mouse heart image, Fig. 3 G to 3J respectively 2.5 ×, 10 ×, 40
× and 100 × times lower image, the results showed that the region MI regenerated particle, which transplants mouse heart, more Cardiospheres.
Fig. 4 show be regenerated particle transplanting mouse heart in Oct4 and CXCR4 expression, as a result, it has been found that Oct4 and
CXCR4 is expressed in nucleus, does not detect positive staining in control group, and finds Oct4 intensive small by image
The cell expressed in particulate matter and non-fully broken up.
The new stained photographs for forming cell are shown in Fig. 5.Fig. 5 A is the heart sections for injecting 2 weeks regenerated particles；Fig. 5 B
It is the heart sections for injecting 8 weeks regenerated particles；Fig. 5 C is the expression of Sox2+GFP in the heart for inject 2 weeks regenerated particles；
Fig. 5 D is the expression of DDX4+GFP in the heart for inject 2 weeks regenerated particles；Fig. 5 E is the heart for injecting 8 weeks regenerated particles
In, the expression of Sox2+GFP；Fig. 5 F is the expression of DDX4+GFP in the heart for inject 8 weeks regenerated particles.
The brain that Fig. 6 is shown 10 weeks is partially sliced, and Fig. 6 A is control group, no angiogenesis；Fig. 6 B is experimental group, is had
Angiogenesis.
Fluorescence micrograph is shown in Fig. 7, and GFP, Oct4, DAPI and composite diagram, Fig. 7 G-7J is shown in Fig. 7 C-7F
GFP, nestin, DAPI and composite diagram is shown.
Specific embodiment
The present invention can be further described by the following examples, and under the scope of the present invention is not limited to
State embodiment.
The present invention carries out general and/or specific description to material and experimental method used in experiment.Although being
It realizes many materials used in the object of the invention and operating method is it is known in the art that still the present invention still makees to the greatest extent may be used herein
It can detailed description.
The extraction and culture of 1. Cord blood regenerated particle of embodiment
In the present embodiment, the extraction and culture of Cord blood regenerated particle are illustrated.
Blood source: umbilical cord blood collection into the blood bag containing anti-coagulants, anti-coagulants is original sodium citrate in blood bag, is passed through
Cord blood is transported to company by cold chain transportation mode (temperature is maintained at 2 to 8 DEG C).
Condition: the treatment process for obtaining Cord blood regenerated particle needs whole sterile working.
(1) Cord blood is taken to carry out 200g centrifugation, centrifugation time is 10 minutes, be divided into two parts after centrifugation, upper solution A and
Lower sediment A.
(2) lower layer's precipitate A is taken, PBS is added for 1:2-5 by volume and slightly washs, carries out 200g centrifugation, centrifugation time is
10 minutes, two parts, upper solution B and lower sediment B are divided into after centrifugation.
(3) lower layer's precipitate B is taken, erythrocyte cracked liquid is added for 1:5-10 by volume and is placed in horizontal shaker, shakes 20 points
Clock carries out 2000g centrifugation later, and centrifugation time is 10 minutes, and two parts, upper solution C and lower sediment C are divided into after centrifugation,
Upper solution C is discarded.
(4) take lower layer's precipitate C, by volume for 1:5-10 be added PBS acutely shake, jitter time be 2 minutes, it is laggard
Row 200g centrifugation, centrifugation time are 10 minutes, and two parts, upper solution D and lower sediment D are divided into after centrifugation, and lower sediment D is abandoned
Fall.
(5) upper solution A, upper solution B, upper solution D are taken respectively, carries out 5000g centrifugation, and centrifugation time is 10 points
Clock after centrifugation, collects all precipitatings.
(6) culture medium (the mixing serum of umbilical cord blood containing 20%) that the precipitating being collected into is placed in MEM is cultivated, is cultivated
Condition is 37 DEG C, and in 5%CO2 incubator, replacement culture medium is primary within every 2-3 days.
(7) culture medium cultivated 10 days is collected, 5000g centrifugation is carried out, to be enriched with Cord blood regenerated particle (regenerated particle
Form is shown in Fig. 1).
The function of 2. Cord blood regenerated particle of embodiment promotion myocardial damage Regeneration and Repair
Experimental group arrives ischemic myocardial by the Cord blood regenerated particle (GFP label) that tail vein injection embodiment 1 obtains
The mouse of damage, control group injecting normal saline.The blood for collecting every mouse after injection for every 24 hours, carries out AST detection.Note
After penetrating, continuous 9 weeks collection mouse hearts are simultaneously sliced detection affected area.
There are more cardiospheres to be gathered in cardiac muscle after Cord blood regenerated particle is transplanted to mouse, in experimental group
Infarcted region (see Fig. 2, Fig. 3), shows that Cord blood regenerated particle forms cardiospheres.By to experimental group
The Oct4 and CXCR4 in the region cardiospheres detect (see Fig. 4), and discovery is in stained positive in nuclear area, show
The region cardiospheres has Cord blood regenerated particle to assemble to form karyocyte or stem cell.Further to prove newly to be formed
Cell whether be stem cell, and using Fluorescence Histochemical detect Sox2 and DDX4 to verify the cell of Green Fluorescent Protein,
As a result prove that the cell newly formed is stem cell (see Fig. 5).
Above embodiment explanation, Cord blood regenerated particle of the present invention have the function of promoting myocardial damage Regeneration and Repair.
3. Cord blood regenerated particle of embodiment promotes the regenerated function of the cerebrovascular
Experimental group arrives the small of ischemic brain damage by the regenerated particle (GFP label) that tail vein injection embodiment 1 obtains
Mouse, control group injecting normal saline.
In 10 weeks brains check, discovery does not have GFP to dye (Fig. 6 A) in control group, but in the GFP of experimental group spy
There is strong GFP expression (Fig. 6 B) in the capillary structure of xenoantibody reactive moieties.Further study show that in capillary structure
The GFP and Oct4 (Fig. 7 C-7F) or nestin of coexpression are also checked through (Fig. 7 G-7J).
Above embodiment explanation, Cord blood regenerated particle of the present invention have the function of promoting cerebral injury revascularization.
Claims (10)
1. a kind of extracting method of Cord blood regenerated particle, is characterized in that, include the following steps: that Cord blood is taken to carry out 50-300g
Centrifugation, is divided into upper solution A and lower sediment A after centrifugation；Then lower layer's precipitate A is taken, buffer is added and slightly washs, carries out
50-300g centrifugation, is divided into upper solution B and lower sediment B after centrifugation；Then lower layer's precipitate B is taken, erythrocyte cracked liquid is added and sets
It is shaken in horizontal shaker, 1000-3000g centrifugation is carried out after cracking, upper solution C and lower sediment C are divided into after centrifugation；Then
Lower layer's precipitate C is taken, buffer is added and acutely shakes, carries out 50-300g centrifugation later, upper solution D and lower layer is divided into after centrifugation
Precipitate D；Then upper solution A, upper solution B, upper solution D are taken respectively, are carried out 3000-7000g centrifugation, are collected all precipitatings
It is placed in the culture medium of MEM and is cultivated；The culture medium cultivated 10-30 days is finally collected, 3000-7000g centrifugation is carried out, with richness
Collect Cord blood regenerated particle.
2. extracting method as described in claim 1, it is characterised in that: take Cord blood to carry out 50-300g centrifugation, centrifugation time is
5-20 minutes, two parts, upper solution A and lower sediment A are divided into after centrifugation；Lower layer's precipitate A is taken, is added by volume for 1:2-5
Enter buffer slightly to wash, carry out 50-300g centrifugation, centrifugation time is 5-20 minutes, and two parts, upper solution are divided into after centrifugation
B and lower sediment B；Lower layer's precipitate B is taken, erythrocyte cracked liquid is added for 1:5-10 by volume and is placed in horizontal shaker, cracks it
1000-3000g centrifugation is carried out afterwards, and centrifugation time is 5-20 minutes, and two parts, upper solution C and lower sediment are divided into after centrifugation
C, upper solution C are discarded；Lower layer's precipitate C is taken, buffer is added for 1:5-10 by volume and acutely shakes, jitter time 1-2
Minute, 50-300g centrifugation is carried out later, and centrifugation time is 5-20 minutes, and two parts, upper solution D and lower layer are divided into after centrifugation
D is precipitated, lower sediment D is discarded；Upper solution A, upper solution B, upper solution D are taken respectively, carry out 3000-7000g centrifugation, from
The heart time is 5-20 minutes.
3. extracting method as described 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 minutes, after centrifugation, collects all precipitatings and is placed in the mixing navel containing 20%
It is cultivated in the culture medium of MEM with blood serum, condition of culture is 37 DEG C, in 5%CO2 incubator, the culture of replacement in every 2-3 days
Base is primary.
It 4. a kind of Cord blood regenerated particle, is characterized in that, is prepared by the extracting method of any one of claim 1-3.
5. a kind of Cord blood regenerated particle biological agent, is characterized in that, it includes the Cord blood regenerated particles of claim 4.
6. a kind of composition of Cord blood regenerated particle, is characterized in that, the Cord blood regenerated particle containing claim 4.
7. a kind of kit of Cord blood regenerated particle, is characterized in that comprising the Cord blood regenerated particle of claim 4.
8. Cord blood regenerated particle according to claim 4 is in preparing the drug for treating myocardial damage, cerebrovascular trauma
Purposes.
9. purposes according to claim 8, is characterized in that, wherein the myocardial damage is ischemia myocardial damage.
10. Cord blood regenerated particle according to claim 4 is in preparation for regenerating cardiac muscular tissue, regeneration vessel, regeneration skin
And the purposes in the drug of hair follicle, regeneration liver histocyte, 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 |
| 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 |
| 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 |
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| CN1553951A (en) * | 2000-11-03 | 2004-12-08 | ���ﰲҽ�ƹɷ�����˾ | Human cord blood derived unrestricted somatic stem cells (USSC) |
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