CN107937334A - A kind of cell factor extract and its application - Google Patents
A kind of cell factor extract and its application Download PDFInfo
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- CN107937334A CN107937334A CN201711077297.1A CN201711077297A CN107937334A CN 107937334 A CN107937334 A CN 107937334A CN 201711077297 A CN201711077297 A CN 201711077297A CN 107937334 A CN107937334 A CN 107937334A
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- 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
- C12N5/0655—Chondrocytes; Cartilage
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1369—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
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Abstract
The invention discloses a kind of cell factor extract and its application, the cell factor extract is prepared as follows：After whole blood and anti-coagulants are fully mixed, centrifugation, takes intermediate layer to add normal saline dilution, adds erythrocyte cracked liquid and/or menses derived stem cells liquid, stand, centrifugation, collects intermediate layer, the multigelation between 37 DEG C and 80 DEG C, centrifugation, takes supernatant, obtains cell factor extract；Or by menses derived stem cells normal saline into cell liquid, the then multigelation between 37 DEG C and 80 DEG C, centrifugation, takes supernatant, acquisition cell factor extract.Cell factor extract provided by the invention can effectively induce stem cells hyperplasia in vitro and into cartilage differentiation, and preparation method is easy, easily obtains, effective and safe, has great application potential in cartilage tissue engineered field, easy to large-scale promotion application.
Description
(1) technical field
The present invention relates to one kind into cartilage vitro differentiation method, more particularly to a kind of cell factor extract and its in vitro
Induce menses derived stem cells propagation and into the application in cartilage differentiation.
(2) background technology
Osteoarthritis (Osteoarthritis, OA) is clinical most commonly seen degenerative osteoarthropathy, soft with joint
Bone regression is main feature, and occur together the symptoms such as serious arthralgia, deformity, dysfunction, influences people's normal life
(Lancet,2011.377(9783):2115–26).With the arrival of aging society, OA incidence rises year by year, the whole world 60
Year, the OA incidence of above the elderly was up to 80%, and patient populations account for the 15% of population in the world, was the master for causing adult disabled
Want reason, serious threat human health (Ann Intern Med, 2000.133 (8):635-46).However, the pass due to regression
Section cartilage lacks itself repair ability, and existing medicine can not really realize repair of cartilage, be a great problem that Orthopedic Clinical faces
(Tissue Eng Part B Rev,2010.16(6):617-27)。
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs), which has, to be derived from a wealth of sources, is easy to extraction culture, is low
The features such as immunogenicity, multi-lineage potential, powerful multiplication capacity, immunoregulation and anti-inflammatory, specified conditions can induce in vitro
Under be divided into cartilage cell, and then form cartilaginous tissue, cartilage degeneration can be directed to and cartilage damage carries out effective prevention, be OA's
Treat open up a new way (Circulation, 2007.116 (18):2053-61).Common MSCs is filled between marrow
Matter stem cell, but it has certain intrusion traumatic and easily cause patient's discomfort in separation process.Navel blood stem cell is also
The important sources of MSCs, but limited to since its acquisition depends on maternal gestational.Other MSCs such as fat stem cells,
The problems such as amniotic fluid stem cell, umbilical cord stem cells etc., and Regenerated energy force difference relatively low there are accessibility (J Obstet Gynaecol
Res,2012.38(5):804-9).Therefore, there is an urgent need for find a kind of noninvasive and safe and reliable MSCs sources.2007 by Meng
Deng having isolated a kind of exuberant MSCs from menstrual blood, subsequent this kind of cell is referred to as menses derived stem cells (J
Transl Med,2007.5:57).Menses derived stem cells possess the advantages of other multinomial stem cells can not reach, including materials
Easily, without invasion, low immunogenicity, without Tumor formation etc., there are great clinical practice potentiality.It there is no and done through haematogenous at present
Cell induces the correlative study into cartilage differentiation to report.
In addition, the clinical practice of MSCs faces many common problems：1. MSCs vigor in vitro and differentiation potential are weaker
(Arthritis Rheum,2002.46(3):704-13)；2. being in gradual aging trend in MSCs Process of in vitro, breed energy
Power weakens (Tissue Eng, 2002.8 (6) by generation:901-10)；3. the characteristic such as the in-vitro multiplication of MSCs, differentiation, immune is trained
Supporting many factors such as condition, cell density, growth factor influences (Stem Cells, 2006.24 (2):462-71).It is existing
Although MSCs derivants can promote it to need to add the component such as hormone and growth factor, can not directly apply into cartilage differentiation
In clinic.Therefore, except carrying out source problem, the clinical practice of MSCs also needs to solve the problems, such as growth and differentiation etc..
Material, system the present invention relates to a kind of promotion menses derived stem cells propagation being never disclosed and into cartilage differentiation
Preparation Method and its application, there is larger difference compared with the existing methods：1. the material is autologous source property, heterologous thing is avoided that
Risk existing for matter, can be quickly applied to clinic；2. the preparation method is easy, easy to operate, independent of specific apparatus, it is adapted to push away
Extensively.By the menses derived stem cells that handle of the present invention by limit the problems such as making artificial cartilage thoroughly break away from source and bioactivity
System, is expected to applied to the structure research of tissue engineering bone/cartilage and the clinical treatment of orthopaedic disease, be it is a kind of it is really easy, economical,
Safely, effectively and easily large-scale promotion application method.
(3) content of the invention
It is an object of the present invention to provide a kind of enrichment from human peripheral and menses derived stem cells bioactive substance
Method, the bioactivity of the Porcine HGF containing high-purity and activated protein is obtained by the methods of centrifugation and multigelation
Material, i.e. cell factor extract, the material can promote menses derived stem cells proliferation activity, and induce it into cartilage differentiation,
And heterologous contact scar equivalent risk is not present, both safe and effective stem cell can be provided for clinic and promote to rise in value and into cartilage differentiation
Material and preparation method, it is intended to solve the problems, such as clinical repair of cartilage：Lack applicable tissue engineering bone/cartilage structure side
Method.
The technical solution adopted by the present invention is：
The present invention provides a kind of cell factor extract, and the cell factor extract is prepared one of as follows：(1)
After whole blood and anti-coagulants are fully mixed, (150~1500 × g is centrifuged 6~20 minutes, preferably 1200 × g centrifugations for centrifugation for the first time
6 minutes), take middle white batt layer to add normal saline dilution (preferred cell concentration 1 × 106~1 × 108A/ml), then add
Enter erythrocyte cracked liquid, 30 minutes are stood after mixing, and second of centrifugation (150~1500 × g centrifugations 6~20 minutes, preferably 1000
× g is centrifuged 6 minutes), middle white batt layer is collected, multigelation 3-5 times is (cold i.e. at -80 DEG C between 37 DEG C and -80 DEG C
Freeze, melt at 37 DEG C), third time centrifugation (150~1500 × g is centrifuged 6~20 minutes, and preferably 1500 × g is centrifuged 10 minutes), takes
Supernatant, obtains cell factor extract；(2) by menses derived stem cells normal saline into cell liquid (preferably 1 ×
106~1 × 108A/ml), then multigelation 3-5 times between 37 DEG C and -80 DEG C, centrifugation (150~1500 × g centrifugations 6~
20 minutes, preferably 1500 × g was centrifuged 10 minutes), supernatant is taken, obtains cell factor extract；(3) whole blood and anti-coagulants are filled
Divide after mixing, centrifugation for the first time (150~1500 × g speed centrifuges 6~20 minutes, preferably 1200 × g centrifugations 6 minutes), in taking
Between white flock layer add normal saline dilution, add erythrocyte cracked liquid, 30 minutes stood after mixing, second centrifugation
(150~1500 × g speed centrifuges 6~20 minutes, and preferably 1000 × g is centrifuged 6 minutes), collects middle white batt layer physiology
Brine is diluted to middle white batt layer dilution (cell concentration 1 × 106A/ml~1 × 108A/ml), then will be through haematogenous
Stem cell is with normal saline into 1 × 106A/ml~1 × 108A/ml cell liquid, then by middle white batt layer physiology
Salt water diluent is mixed with cell liquid, multigelation 3-5 times between 37 DEG C and -80 DEG C, and third time centrifuges (150~1500 × g
Speed centrifuges 6~20 minutes, and preferably 1500 × g is centrifuged 10 minutes), supernatant is taken, obtains cell factor extract.
Further, anti-coagulants is sodium citrate, heparin or hirudin in the method (1), is preferably sodium citrate.It is described
Anti-coagulants can be added in whole blood with the mode of the vacuum blood collection tube containing anti-coagulants, this is that well known to a person skilled in the art blood sampling
Anticoagulant methods.
Further, anticoagulant and volume of whole blood ratio are 1 in the method (1):9, preferably anti-coagulants is mass concentration
3.2%~3.8% sodium citrate aqueous solution.
Further, in the method (1) erythrocyte cracked liquid be aqueous ammonium chloride solution, lymphocyte separation medium or other
Commercialization or the erythrocyte cracked liquid voluntarily prepared, 1.0% aqueous ammonium chloride solution of preferred mass concentration, aqueous ammonium chloride solution and institute
The volume ratio for obtaining sample is preferably 1:2, remaining erythrocyte cracked liquid is operated according to description of commodity.
Further, in the method (1) centrifugation for the first time, second of centrifugation and third time centrifugal condition be 150~
1500 × g is centrifuged 6~10 minutes；First time centrifugal condition centrifuges 6 minutes for 1200 × g more preferably in the method (1)；Second
Secondary centrifugal condition centrifuges 6 minutes for 1000 × g；Third time centrifugal condition centrifuges 10 minutes for 1500 × g.Has been carried out to whole blood
Once centrifuge, it is therefore an objective to other active materials such as blood platelet etc. in separating red corpuscle, leucocyte and whole blood, by removing supernatant
Liquid, takes middle white batt layer, can avoid most of red blood cell and leucocyte in whole blood.The sample size that different centrifugation rates obtain
It is different.The purpose for adding erythrocyte cracked liquid is to remove remaining red blood cell, after being centrifuged by second, can remove residue blood
The fragment residual such as slurry and red blood cell, further concentrates target sample.
Configure different cell concentrations according to demand, the purpose for carrying out multigelation be crack whole blood in competent cell and
Menses derived stem cells, discharge the various types of cells factor, and cell fragment is removed by centrifuging, and finally obtain cell factor and active egg
White mixture.3~5 obtained cell factors of exploration discovery early period freeze thawing and protein content highest of the invention.
Further, cell liquid concentration is 10 in the method (2)3~109A/ml.
Further, centrifugal condition centrifuges 6~20 minutes for 150~1500 × g in the method (2), more preferably with 1500
× g speed centrifuges 10 minutes.
Further, in the method (3) centrifugation for the first time, second of centrifugation and third time centrifuge equal condition for 150~
1500 × g is centrifuged 6~20 minutes；First time centrifugal condition is to centrifuge 6 points with 1200 × g of rotating speed more preferably in the method (3)
Clock；Second of centrifugal condition is to be centrifuged 6 minutes with 1000 × g of rotating speed；Third time centrifugal condition is to centrifuge 10 with 1500 × g of rotating speed
Minute.
Cell factor extract of the present invention can also be according to being actually needed to acquisition without cell and cell fragment
Solution is concentrated and/or dried, so that preparation that be made concentration or solid.
The present invention also provides a kind of cell factor extract to induce menses derived stem cells to be divided into cartilage in vitro
Application in cell and propagation, the specific application are：Menses derived stem cells (stem cell of human or animal) are seeded to
In DMEM culture mediums (culture medium that can also be other commercializations), 24h is cultivated under the conditions of 37 DEG C, takes out partial medium
(preferably 1/4 volume), adds the cell factor extract of equivalent, continues culture 7~21 days, done carefully through haematogenous so as to induce
Born of the same parents are divided into cartilage cell and breed；The addition volume of the cell factor extract is with former culture volume ratio with 1/4 for most
Good, stem cell starts to breed after cultivating 1 day, 7 days cell differentiations that hop to it, and differentiation in 21 days is complete.
Compared with existing method of inducing differentiation, the invention has the advantages that：
1) general differentiation derivant needs to add hormone and recombinant growth factors TGF-β etc., and all cells of the present invention because
Seed extract is autologous source property, the risk that immunogenicity and exogenous material can be avoided to produce.
2) general differentiation derivant does not promote the function of stem cells hyperplasia vigor, and cell factor extract energy of the present invention
Significantly improve the activity of stem cell, promote propagation.
To sum up, cell factor extract provided by the invention can effectively induce stem cells hyperplasia in vitro and divide into cartilage
Change, and preparation method is easy, easily obtains, effective and safe, has great application potential in cartilage tissue engineered field, easy to big
Scale promotes and applies.
(4) illustrate
Fig. 1, menses derived stem cells streaming Identification of the antibodies figure.
Cell factor extract composition figure prepared by Fig. 2, ELISA method detection embodiment 1.
Fig. 3, CCK-8 method detect the cell viability of menses derived stem cells, and numerical value is higher, and to represent cell viability stronger；* generations
Table P compared with blank control (Ctr) group<0.01, there is significant difference；A is result in embodiment 1；B is 2 result of embodiment；
C is 3 result of embodiment.
Fig. 4, menses derived stem cells A Li Xinlan dye micro- sem observation；Control group：Untreated stem cell；
Treatment group：By the cell factor extract-treated stem cell of 14 days of the present invention；A is result in embodiment 1；B is 2 knot of embodiment
Fruit；C is 3 result of embodiment.
II Collagen Type VIs (left side) and proteoglycans (right side) mRNA tables in Fig. 5, Real time PCR detection menses derived stem cells
Up to level；Ctr：Control group；2×107：Material 2 × 10 of the present invention7/ ml concentration treatment groups；* represents P compared with the control group<
0.01, there is significant difference.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
The present inventor is by research extensively and profoundly, the different composition forms of discovery cell factor extract of the present invention
Promote menses derived stem cells propagation and into cartilage differentiation, invention will be further described by the following examples
And explanation, however, protection scope of the present invention is not limited to following embodiments.
Embodiment 1：
1) whole blood is collected in the vacuum blood collection tube containing sodium citrate aqueous solution, sodium citrate aqueous solution mass concentration
For 3.2%~3.8%.The volume ratio of sodium citrate aqueous solution and whole blood is 1:9.
2) centrifuge tube is assigned to after fully mixing whole blood in step 1) and sodium citrate aqueous solution, with 1200 × g of rotating speed
Centrifugation 6 minutes, suctions out the superiors using pipettor after centrifugation and discards, then takes middle white batt layer and be transferred to new centrifugation
Guan Zhong, avoids extracting red blood cell as far as possible, adds physiological saline and is configured to 1ml volumes.
3) erythrocyte cracked liquid is added in step 2) centrifuge tube, the erythrocyte cracked liquid is mass fraction 1.0%
Aqueous ammonium chloride solution, the volume ratio of aqueous ammonium chloride solution and material in pipe is 1:2,30 minutes are stood after mixing, with rotating speed 1000
× g is centrifuged 6 minutes, is collected middle white batt layer using pipettor, is placed in new centrifuge tube and carries out microscopic count.
4) multigelation 5 times between 37 DEG C and -80 DEG C by centrifuge tube, are then centrifuged 10 minutes with 1500 × g speed, taken
Supernatant, obtains cell factor extract, is that of the present invention can induce menses derived stem cells propagation and into cartilage differentiation
Material.The composition of ELISA method detection cell factor extract, as a result as shown in Figure 2, each cytokine content is above 10 μ
The content of g/ml, wherein IGF-1 are close to 50 μ g/ml.
5) by menses derived stem cells (streaming Identification of the antibodies as shown in Figure 1, it was demonstrated that it is CD73+、CD90+、CD105+、
CD45-Cell, meets mescenchymal stem cell feature) it is placed in Tissue Culture Flask and cultivates, nutrient solution is DMEM culture mediums, at 37 DEG C
Under the conditions of cultivate 24h, add 1/4 debulking step 4) the cell factor extract for preparing, after co-culturing 21 days, it is possible to find stem cell
Major part is divided into cartilage cell, using non-refinement intracellular cytokine extract as control (A in Fig. 4), and is bred (in Fig. 3
A)。
Embodiment 2：
1) menses derived stem cells are placed in sterile centrifugation tube, 10 is configured to physiological saline6/ ml concentrations of cells hangs
Liquid.
2) multigelation 5 times between 37 DEG C and -80 DEG C by centrifuge tube, are then centrifuged 10 minutes with 1500 × g speed, taken
Supernatant, obtains cell factor extract, is that can induce menses derived stem cells propagation in the present embodiment and into cartilage differentiation
Material.
3) menses derived stem cells are placed in Tissue Culture Flask and cultivated, nutrient solution is DMEM culture mediums, in 37 DEG C of conditions
Lower culture 24h, adds 1/4 debulking step 2) cell factor extract, after co-culturing 21 days, it is possible to find stem cell largely breaks up
For cartilage cell, using non-refinement intracellular cytokine extract as control (B in such as Fig. 4), and bred (B in Fig. 3).
Embodiment 3：
1) step 1) in embodiment 1 to the middle white batt layer 3) prepared is prepared with step 1) in embodiment 2 thin
Born of the same parents' suspension by volume 1:1 mixing, is placed in sterile centrifugation tube.
2) multigelation 5 times between 37 DEG C and -80 DEG C by centrifuge tube, are then centrifuged 10 minutes with 1500 × g speed, taken
Supernatant, obtains cell factor extract, is that can induce menses derived stem cells propagation in the present embodiment and into cartilage differentiation
Material.
3) menses derived stem cells are placed in Tissue Culture Flask and cultivated, nutrient solution is DMEM culture mediums, in 37 DEG C of conditions
Lower culture 24h, takes out 1/4 volume nutrient solution, adds 1/4 debulking step 2) cell factor extract, can after co-culturing 21 days
It was found that stem cell is largely divided into cartilage cell, using non-refinement intracellular cytokine extract as control (C in such as Fig. 4), and obtain
Propagation (C in such as Fig. 3).The gene expression dose of menses derived stem cells is detected by real time PCR methods, is sent out
The mRNA expressions of II Collagen Type VIs and proteoglycans significantly improve (such as after the stimulation of above-mentioned cell factor extract in existing cell
Fig. 5), prove menses derived stem cells into the trend of cartilage differentiation from molecular level.
Claims (10)
1. a kind of cell factor extract, it is characterised in that the cell factor extract is prepared one of as follows：(1) will
After whole blood is fully mixed with anti-coagulants, centrifuge for the first time, take middle white batt layer to add normal saline dilution, add red thin
Cellular lysate liquid, 30 minutes are stood after mixing, and second of centrifugation, collects middle white batt layer, between 37 DEG C and -80 DEG C repeatedly
Freeze thawing, third time centrifuge, and take supernatant, obtain cell factor extract；(2) by menses derived stem cells normal saline
Into cell liquid, the then multigelation between 37 DEG C and -80 DEG C, centrifugation, takes supernatant, acquisition cell factor extract；(3) will
After whole blood is fully mixed with anti-coagulants, centrifuge for the first time, take middle white batt layer to add normal saline dilution, add red thin
Cellular lysate liquid, 30 minutes are stood after mixing, and second centrifuges, and collect middle white batt layer and with normal saline dilution into centre
White flock layer dilution, then by menses derived stem cells normal saline into cell liquid, it is then that middle white is cotton-shaped
Layer dilution mix with cell liquid, the multigelation between 37 DEG C and -80 DEG C, and third time centrifugation, takes supernatant, acquisition cell because
Seed extract.
2. cell factor extract as claimed in claim 1, it is characterised in that in the method (1) anti-coagulants for sodium citrate,
Heparin or hirudin, the anticoagulant are 1 with volume of whole blood ratio:9.
3. cell factor extract as claimed in claim 1, it is characterised in that erythrocyte cracked liquid is quality in the method (1)
1.0% aqueous ammonium chloride solution of concentration.
4. cell factor extract as claimed in claim 1, it is characterised in that centrifuged for the first time in the method (1), second
Centrifugation and third time centrifugal condition are that 150~1500 × g is centrifuged 6~10 minutes.
5. cell factor extract as claimed in claim 1, it is characterised in that cell liquid concentration is 10 in the method (2)3~
109A/ml.
6. cell factor extract as claimed in claim 1, it is characterised in that in the method (2) centrifugal condition for 150~
1500 × g is centrifuged 6~20 minutes.
7. cell factor extract as claimed in claim 1, it is characterised in that centrifuged for the first time in the method (3), second
Centrifugation and third time centrifuge equal condition and are centrifuged 6~20 minutes for 150~1500 × g.
8. cell factor extract as claimed in claim 1, it is characterised in that method (3) the middle white batt layer dilution
It is 1 × 10 with cell liquid concentration6A/ml~1 × 108A/ml, the middle white batt layer dilution and cell liquid volume
Than for 1:1.
9. cell factor extract described in a kind of claim 1 induce in vitro menses derived stem cells be divided into cartilage cell and
Application in propagation.
10. application as claimed in claim 9, it is characterised in that the application is：Menses derived stem cells are seeded to DMEM trainings
Support in base, 24h is cultivated under the conditions of 37 DEG C, take out partial medium, add the cell factor extract of equivalent, continue to cultivate
7~21 days, so as to induce menses derived stem cells to be divided into cartilage cell and breed.
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