CN106282108A - A kind of spleen mescenchymal stem cell with immunoloregulation function and preparation method and application - Google Patents
A kind of spleen mescenchymal stem cell with immunoloregulation function and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of spleen mescenchymal stem cell with immunoloregulation function and preparation method and application.Present invention employs removal hematopoietic cell digestion matrix organization acquisition Sp MSCs and pass on the strategy of purification, establish a kind of method from spleen separation and Culture MSCs, and separation obtains the Sp MSCs that a large amount of cell degree of purity is higher, it is further discovered that this crowd of Sp MSCs and there is powerful immunoregulation capability, non-specific antigen and the T lymphopoiesis of alloantigen activation and activation can be suppressed, and suppress T lymphocytic emiocytosis inflammatory cytokine.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of spleen mescenchymal stem cell with immunoloregulation function
And preparation method and application.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is that one has multi-lineage potential and height
The stem cell of self-renewal capacity.MSC is found in bone marrow by Friedenstein etc. the earliest, and research worker exists successively subsequently
The Various Tissues such as bone, umbilical cord, fat separate successfully.MSC can expand under appropriate conditions in a large number, and is divided into skeletonization
The cell of the Various Tissues types such as cell, adipose cell, endotheliocyte, neurocyte, is that current field of tissue engineering technology is the most frequently used
One of seed cell.Additionally, MSC also has hematopoiesis support ability, it is possible to regulation hematopoietic stem cell is divided into multiple pedigree
Hematopoietic cell, is the important component part of hematopoieticmicroenviron-ment.More meaningful, MSC can also regulate panimmunity cell
Grow and function, contribute to maintaining organism immune response and immune homeostasis.
Spleen is peripheral immune organ maximum in humans and animals body.Spleen be contained within panimmunity cell the hugest bite thin
Born of the same parents, T lymphocyte, bone-marrow-derived lymphocyte, dendritic cell and neutrophilic granulocyte etc., be that immunocyte is grown and ripe important device
One of official, especially the humoral immunization center of body and cellular immunization center.Wherein, dendritic cell can absorb swelling in body
Tumor antigen or the antigen of external source invasion, processing submission is given corresponding T lymphocyte, is promoted that relevant t lymphocyte subset group increases
Grow differentiation, and start the humoral immunization that bone-marrow-derived lymphocyte is master, it is achieved tumor is removed and cause of disease is removed.Monolconal antibody cell
Grow function to be regulated and controled by spleen microenvironment.The spleen matrix organization of multiple stromal cell mixing composition had both been immunocyte
Growth provides space and relies on, and expresses again various active molecular regulation immune cell function, is the important composition of spleen microenvironment
Part.Therefore, spleen matrix organization inner cell is grown for immunocyte and function regulates and controls to be always the heat of immunological investigation
One of point.But, the work of the adult cell such as spleen endothelium-derived stromal cell and fibroblast is paid close attention in conventional research more
With, for being in the spleen mescenchymal stem cell (spleen mesenchymal stem cells, Sp-MSCs) growing source
Whether participate in immunoregulation domestic and foreign literature report few.Therefore, the Sp-MSCs isolated culture method of a kind of high efficient and reliable is set up
Become the task of top priority.
It is unicellular that traditional MSCs training strategy mostly is tissue digestion, then cultivates unicellular and obtains MSC.But
It is that this strategy is applied to but encounter a difficult problem when Sp-MSCs cultivates, and reason is exactly rich in having substantial amounts of hematopoietic cell such as in spleen
Macrophages etc., they have the stronger ability being attached at culture bottle ware, it is difficult to removed by Secondary Culture.It is entrained in Sp-
Hematopoietic cell in MSCs usually can cause immunoreation, has had a strong impact on relevant scientific research and application.
Summary of the invention
It is an object of the present invention to provide the preparation method of a kind of spleen mescenchymal stem cell.
The preparation method of the spleen mescenchymal stem cell that the present invention provides comprises the steps:
(1) mill in vitro spleen, remove hematopoietic cell, obtain spleen matrix organization；
(2) with collagenase liquid, described spleen matrix organization is carried out digestion process, and after digestion process, remove described spleen
Hematopoietic cell in dirty matrix organization, obtains spleen matrix organization after digestion process；
(3) cultivate spleen matrix organization after described digestion process, i.e. can get spleen mescenchymal stem cell.
In said method,
Described mill as milling with syringe handle；Described mill after also include rinse step；The purpose of described flushing is
Removing the hematopoietic cell in spleen after milling, concrete grammar is spleen after milling with PBS flushing；The number of times of described flushing is 3-5
Secondary.
In said method,
It is that spleen matrix organization is soaked in glue that described spleen matrix organization is carried out digestion process by described collagenase liquid
In original enzyme liquid；
In described collagenase liquid, the mass fraction of collagenase is 0.1-0.15%.
In said method, described collagenase is type i collagen enzyme, II Collagenase Type or IV Collagenase Type.
In said method, described collagenase is specially II Collagenase Type.
In said method,
The culture medium that described collagenase liquid is uniformly mixed so as to obtain by II Collagenase Type, hyclone and α-MEM culture medium；
Described hyclone volume fraction in described collagenase liquid is 15-20%.
In said method, described II Collagenase Type mass fraction in described collagenase liquid is specially 0.1%, described tire
Ox blood serum volume fraction in described collagenase liquid is specially 15%.
In said method,
In step (2), the method for described removal is to draw the collagen in postdigestive described spleen matrix organization with suction pipe
The hematopoietic cell that enzyme liquid and digestion discharge.
In said method, the condition of described digestion process is 35-38 DEG C, 5%CO2Under the conditions of digest 0.5-4h.
In said method, the culture fluid used by described cultivation is glutamine, hyclone and α-MEM culture medium to be mixed
After the solution that obtains, described glutamine concentration in described culture fluid is 1-2mmol/L, and described hyclone is in described training
Volume fraction in nutrient solution is 10-15%.Described glutamine concentration in described culture fluid is specially 1mmol/L, described
Hyclone volume fraction in described culture fluid is specially 10%.
It is a further object to provide the spleen mescenchymal stem cell that said method prepares.
Final object of the present invention is to provide said method or the new application of above-mentioned spleen mescenchymal stem cell.
The invention provides said method or above-mentioned spleen mescenchymal stem cell following 1)-3) in any one should
With:
1) suppression T lymphopoiesis；
2) suppression T lymphocyte activation；
3) suppression T lymphocytic emiocytosis inflammatory factor.
In above-mentioned application, described inflammatory factor is IFN-γ and/or TNF-α and/or IL-17A.
In above-mentioned application, described T lymphocyte is T lymphocyte or the heterogenetic antigen of allogene antigen induction
The T lymphocyte of induction.
In said method or above-mentioned application, described spleen is normal for deriving from mice, rat, rabbit, Canis familiaris L., Cavia porcellus, sheep, monkey etc.
With the in vitro spleen of animal strains；The week old of described animal can be 2～20 weeks.
Advantage for present invention and beneficial effect: (1) the inventive method is simple to operate, convenient and practical；(2) present invention
The Sp-MSCs high purity obtained, hemopoietic is polluted few, is conducive to carrying out immunology correlational study and In vivo study；(3) this
The Sp-MSCs immunoregulation capability that bright method is obtained is strong, and the T that non-specific antigen and alloantigen can be suppressed to cause drenches
Bar cell proliferation, activation and inflammatory cytokine release.
The present invention removes hematopoietic cell in most spleen initially with grinding technique, reduces that hematopoietic cell is adherent to be caused
Pollution；Secondly, collagenase can be degraded the collagen in spleen matrix organization specifically, and organizational structure of loosening helps Sp-MSCs
Move in culture bottle；Again, after collagenase digesting, the hematopoietic cell of spleen matrix organization remained on surface discharges, by this
A little hematopoietic cells all abandon, and only cultivate postdigestive spleen matrix organization, can significantly improve the purity of Sp-MSCs.This
Invent and be found to be basis with above-mentioned, explore the strategy of removal hematopoietic cell-digestion matrix organization-pass on purification, establish one
Plant the method from spleen separation and Culture MSCs, and separation obtain the Sp-MSCs that a large amount of cell degree of purity is higher, it has further been found that
This crowd of Sp-MSCs have powerful immunoregulation capability, it is possible to suppression non-specific antigen and the T lymph of alloantigen activation
Cell proliferation and activation, and suppress T lymphocytic emiocytosis inflammatory cytokine.
Accompanying drawing explanation
Fig. 1 is separating digesting spleen matrix organization.Figure 1A is indigested spleen matrix organization, and Fig. 2 B is collagenase digesting
After spleen matrix organization.
Fig. 2 is original cuiture spleen mescenchymal stem cell.Fig. 2 A is that mescenchymal stem cell is climbed from spleen matrix organization block
Go out；Fig. 2 B is the spleen mescenchymal stem cell that propagation is good.
Fig. 3 is the streaming immunophenotype of spleen mescenchymal stem cell.Wherein, vertical coordinate represents cell quantity, and transverse axis represents
Positive rate.
Fig. 4 is spleen mescenchymal stem cell Multidirectional Differentiation.Fig. 4 A is alkalescence phosphorus after spleen mescenchymal stem cell Osteoblast Differentiation
Acid enzyme dyeing；Fig. 4 B is that spleen mescenchymal stem cell becomes oil red O stain after Adipose Differentiation.
Fig. 5 is that Sp-MSCs suppresses T lymphopoiesis.Fig. 5 A is Sp-MSCs suppression LTT experiment；Fig. 5 B is Sp-MSCs
Suppression MLR reaction.Wherein, abscissa represents fluorescence intensity, and vertical coordinate represents cell quantity, and what black peak represented does not breeds
T lymphocyte, what blue peak represented is add the stimulation of Con A or xenogeneic antigens after, the T lymph of notable propagation is thin
Born of the same parents, what red peak represented is that Sp-MSCs suppresses corresponding T lymphopoiesis.
Fig. 6 is the T lymphocyte activation of Sp-MSCs suppression non-specific antigen induction.Fig. 6 A is that Con A stimulates initiation T to drench
Bar cell activation；Fig. 6 B is the T lymphocyte activation of Sp-MSCs suppression CoA induction, (scale is equal to 200 microns).
Fig. 7 is the T lymphocyte activation of Sp-MSCs suppression allogene antigen induction.Fig. 7 A is allogeneic
Antigenic stimulus causes T lymphocyte activation；Fig. 7 B is the T lymphocyte activation of Sp-MSCs suppression allogene antigen induction,
(scale is equal to 200 microns).
Fig. 8 is the T Expressions In Lymphocytes inflammatory cytokine of Sp-MSCs suppression non-specific antigen activation.
Fig. 9 is the T Expressions In Lymphocytes inflammatory cytokine that Sp-MSCs suppression allogene is Antigen-activated.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Quantitative test in following embodiment, is respectively provided with three times and repeats experiment, results averaged.
The formula of the PBS in following embodiment is: NaCl (Beijing Chemical Works, cat.no.XK-13-
201-0001-218) 8.0g, Na2HPO4.12H2O (Beijing Chemical Works, cat.no.10020392) 2.9g,
KCl (Beijing Chemical Works, cat.no.XK-13-201-0024-217) 0.2g, KH2PO4(Beijing
Chemical Works, cat.no.XK-13-201-0024-113) 0.24g, it is dissolved in 1 liter of water.
At antibody used in following embodiment and purchase thereof and article No. is:
PE-labeled anti-CD11b(eBioscience,cat.no.12-0112)；
PE-labeled anti-CD29(eBioscience,cat.no.12-0291)；
FITC-labeled anti-CD34(eBioscience,cat.no.11-0341)；
PE-labeled anti-CD44(eBioscience,cat.no.12-0441)；
FITC-labeled anti-CD45(eBioscience,cat.no.11-0451)；
PE-labeled anti-CD105(eBioscience,cat.no.12-1051)；
PE-labeled anti-stem cell antigen-1(Sca-1)(eBioscience,cat.no.12-
5981)；
PE-labeled anti-Ia(eBioscience,cat.no.12-5321)；
FITC-labeled rat IgG2b isotype control(eBioscience,cat.no.11-4031)；
PE-labeled rat IgG2b isotype control(eBioscience,cat.no.12-4031)；
PE-labeled Armenian Hamster IgG isotype control(eBioscience,
cat.no.12-4888)；
FITC-labeled rat IgG2a isotype control(eBioscience,cat.no.11-4321)；
PE-labeled rat IgG2a isotype control(eBioscience,cat.no.12-4321)。
At stain in following embodiment and purchase thereof and article No. is:
Oil red O powder (Sigma-Aldrich, cat.no.O0625-25G)；
Isopropanol (Sigma-Aldrich, cat.no.I9516).
Allogeneic BALB/cl mice and C57BL/6 mice in following embodiment are Beijing dimension tonneau China public affairs
The product of department, SCXK (capital) 2012-0001.
Embodiment 1, a kind of method of separation and Culture Sp-MSCs
One, the separation of Sp-MSCs and cultivation
1, the separation of spleen
Taking 6～8 week old Healthy female C57 mices, de-neck is put to death, and 75% after alcohol-pickled 3-5 minute, lower point of aseptic condition
From spleen, remove surface texture with shears and tweezers, take out complete spleen with tweezers.
2, hematopoietic cell in spleen is removed
With syringe handle, it is fully milled 5 minutes in culture dish in super-clean bench, rush 3-5 time with PBS, remove big portion
Hematopoietic cell in the spleen divided, it is thus achieved that spleen matrix organization (Figure 1A).
3, collagenase digesting spleen matrix organization and original cuiture
The spleen matrix organization separating acquisition in step 2 is placed in collagenase liquid and carries out digesting (one milliliter of collagenase liquid
Put the spleen matrix organization of 3-5 mice), after 37 DEG C digest 0.5 hour, obtain postdigestive spleen matrix organization (Figure 1B),
The hematopoietic cell discharged with the collagenase liquid in the postdigestive spleen matrix organization of suction pipe absorption and digestion, abandons, and
Spleen matrix organization remaining after digestion is planted in culture fluid, carries out original cuiture.
Above-mentioned collagenase liquid be by II Collagenase Type (GIBCO, cat.no.17101015), hyclone (GIBCO,
Cat.no.12664-025) solution and obtained after α-MEM culture medium (Hyclone, cat.no.SH30265.01B) mixing, its
In, II Collagenase Type mass fraction in collagenase liquid is 0.1%, and hyclone volume fraction in collagenase liquid is
15%.
Above-mentioned culture fluid is by glutamine (GIBCO, cat.no.35050-038), hyclone and α-MEM culture medium
The solution obtained after mixing, wherein, glutamine concentration in culture fluid is that 1mmol/L, hyclone are in culture fluid
Volume fraction is 10%.
4, the MSCs (Sp-MSCs) in amplification spleen source
By spleen matrix organization postdigestive in step 3 at 35～38 DEG C, 5%CO2Under the conditions of cultivate after 72 hours with training
Nutrient solution full dose first changes liquid, removes spleen matrix organization, retains the Sp-MSCs migrated out from spleen matrix organization, i.e. obtains
Sp-MSCs.Liquid is changed the most every three days by culture fluid half amount.Pass when Sp-MSCs covers with 70～80% digestion after culture bottle area
Generation, continuous passage, take the 4th generation Sp-MSCs and be further used for following experimentation.
Two, the morphologic detection of Sp-MSCs
The 4th generation Sp-MSCs quiescent culture about 3-5 days in culture fluid step one obtained, under inverted microscope
Observe that cell climbs out of (Fig. 2 A) from piece of tissue.These cells are fusiformis adherent growth, after transmission is cultivated, grow fast
Speed, and MSC form and the growth characteristic (Fig. 2 B) of classics in swirl shape or fusiform dense growth, can be met.
Three, the immunophenotype detection of Sp-MSCs
The 4th generation Sp-MSCs step one obtained carries out surface markers detection, detects the 4th generation Sp-MSCs's respectively
CD11b, CD29, CD34, CD44, CD45, CD105, the expression of Sca-1 and Ia.Specifically comprising the following steps that of flow cytometer detection
1, cell is prepared
The 4th generation Sp-MSCs obtained by pancreatin (GIBCO, cat.no.27250-018) digestion step one, is prepared as slender
Born of the same parents' suspension, adjusts cell quantity to 1 × 106Cell/streaming pipe, resuspended to 100 μ LPBS/ streaming pipes after cleaning.
2, antibody labeling
Requirement to specifications, adds following fluorescently-labeled antibody and corresponding Isotype control respectively in streaming pipe:
CD11b、CD45、Sca-1、CD29、CD31、CD34、CD44、CD86、CD105、Ia.4 DEG C of lucifuges hatch 30 minutes.MSCs immunity
Phenotype analytical associated antibodies and concentration thereof are as shown in table 1 with corresponding isotype control Ab.
Table 1, MSCs Immunophenotype analysis associated antibodies and application
| Antigen | Fluorescence | Application concentration | Corresponding Isotype control |
| CD11b | PE | 0.5μg/million cells.mL-1 | PE-Rat IgG2b |
| CD29 | PE | 1μg/million cells.mL-1 | PE-Armenian Hamster IgG |
| CD34 | FITC | 1μg/million cells.mL-1 | FITC-Rat IgG2a |
| CD44 | PE | 0.125μg/million cells.mL-1 | PE-Rat IgG2b |
| CD45 | FITC | 0.25μg/million cells.mL-1 | FITC-Rat IgG2b |
| CD105 | PE | 0.5μg/million cells.mL-1 | PE-Rat IgG2a |
| Sca-1 | PE | 0.5μg/million cells.mL-1 | FITC-Rat IgG2a |
| Ia | PE | 0.02μg/million cells.mL-1 | PE-Rat IgG2b |
| Rat IgG2a isotype antibody | PE | 1μg/million cells.mL-1 | |
| Rat IgG2a isotype antibody | FITC | 0.5μg/million cells.mL-1 | |
| Rat IgG2b isotype antibody | PE | 0.125μg/million cells.mL-1 | |
| Rat IgG2b isotype antibody | FITC | 0.5μg/million cells.mL-1 |
3, flow cytometer showed
After washing twice of cell with PBS after adding fluorescently-labeled antibody, 4 DEG C, 300g is centrifuged 8 minutes, abandons supernatant, and it is heavy to take
Form sediment, upper machine analysis.
Result is as shown in Figure 3.It can be seen that labelling CD11b, the CD34 of Sp-MSCs low expression hematopoietic cell,
CD45, low expression costimulatory molecules Ia, high expressed stroma labelling CD29 and CD44, high expressed stem cell labeling CD105 and
Sca-1.Illustrate that step one separates the non-hematopoietic stem cell that the Sp-MSCs obtained is a group mesenchymal derivation, and there is relatively low exempting from
Epidemic focus, meets the MSC immunophenotypic characteristics of classics.
Four, the Multidirectional Differentiation ability detection of Sp-MSCs
1, Osteoblast Differentiation
Sp-MSCs step one obtained carries out Osteoblast Differentiation, with alkaline phosphatase staining after Osteoblast Differentiation, under microscope
Photograph, observed result.Specifically comprise the following steps that
(1) osteogenic induction
The 4th generation Sp-MSCs that results step one obtains, according to 1 × 104/ hole kind is in 24 well culture plates, respectively in comparison
Culture medium and Osteogenic Induction Medium carry out inducing culture, within every 2-3 days, changes liquid.
Matched group culture medium is the culture medium FBS with α-MEM culture medium being uniformly mixed so as to obtain, and wherein, FBS cultivates at matched group
Volume fraction in base is 10%；
Osteogenic Induction Medium is by dexamethasone (Sigma-Aldrich, cat.no.D2915), β-phosphoglycerol
(Sigma-Aldrich, cat.no.G6251), vitamin C phosphate (Sigma-Aldrich, cat.no.A8960) and α-MEM
The culture medium that culture medium is uniformly mixed so as to obtain, wherein, dexamethasone concentration in Osteogenic Induction Medium is 10-7Mol/L, β-phosphoric acid
Glycerol concentration in Osteogenic Induction Medium is 10mM, and vitamin C phosphate concentration in Osteogenic Induction Medium is 50 μ
M。
(2) alkaline phosphatase staining
The 14th day of inducing culture, removes culture medium；Use the alkaline phosphatase staining test kit (Alkaline of commercialization
Phosphatase Kit, Sigma-Aldrich, cat.no.85L3R) dyeing: what in culture plate, addition test kit carried consolidates
Determine liquid (citric acid 40 μ L+ deionized water 2mL+ acetone 3mL) and fix 30 seconds；The dyeing that test kit carries is added again in culture plate
Liquid (strong blue RR 62.5 μ L+ deionized water 3mL+As-Mx 125 μ L)；Lucifuge is reacted 30 minutes；Outwell dye liquor, add a small amount of
PBS, takes a picture under microscope.
2, fat differentiation is become
The 4th generation Sp-MSCs that results step one obtains carries out adipogenic induction differentiation, uses oil red O after adipogenic induction differentiation
(Oil-red-O) dyeing, takes a picture under microscope, observed result.
(1) adipogenic induction differentiation
The 4th generation Sp-MSCs that results step one obtains, according to 2 × 104/ hole kind is in 24 well culture plates, respectively in comparison
Inducing culture in culture medium and Adipogenic induction culture medium, changes liquid in every 2-3 days.
Matched group culture medium is the culture medium FBS with α-MEM culture medium being uniformly mixed so as to obtain, and wherein, FBS cultivates at matched group
Volume fraction in system is 10%；
Adipogenic induction culture medium is by dexamethasone (Sigma-Aldrich cat no D2915), IBMX (Sigma-
Aldrich, cat.no.I5879), insulin (Sigma-Aldrich, cat.no.I6634) and α-MEM culture medium are uniformly mixed so as to obtain
Culture medium, wherein, dexamethasone concentration in Adipogenic induction culture medium is 10-6Mol/L, IBMX train at Adipogenic induction
The concentration supported in base is 0.5 μM, and insulin concentration in Adipogenic induction culture medium is 10ng/mL.
(2) oil red O stain
The 14th day of inducing culture, removes culture medium；Dyeing liquor (oil red O powder 0.5g+ isopropanol is added in culture plate
100mL)；Lucifuge is reacted 15 minutes；Outwell dye liquor, add a small amount of PBS, take a picture under microscope.
As shown in Figure 4 A, the mice Sp-MSCs alkaline phosphatase staining cultivated in Osteogenic Induction Medium is positive for result,
This result shows that Sp-MSCs prepared by the present invention has Osteoblast Differentiation potential.Oil red O has preferably fat binding ability.Little
Mus Sp-MSCs Adipogenic induction, after 7 days, i.e. engenders in visible cell that significant quantities of fat is dripped, after adding oil red O stain liquid,
Fat drop be can be observed and contaminated for redness, this result shows that mouse spleen MSC has good one-tenth Adipose Differentiation ability (Fig. 4 B).
Five, the immunoregulation capability detection of Sp-MSCs
Whether the Sp-MSCs prepared to detect the present invention has immunoloregulation function, by separation obtain T lymphocyte with
The 4th generation Sp-MSCs that step one obtains co-cultures, and observes the T lymphopoiesis after cultivation, activates and secrete inflammatory thin
The situation of intracellular cytokine.
1, lymphoblastic transformation experiment (lymphocyte transformation test, LTT)
(1) separation of mouse T lymphocyte
Mill Mus spleen (deriving from C57BL/6 mice) with syringe handle under aseptic condition, the Crude cell of acquisition is hanged
Liquid is crossed 200 mesh steel sieves and is removed agglomerate, then separates liquid (Hao ocean, Tianjin is biological, 20120218) with Ficoll and is centrifuged with 800g
20min, abandons supernatant, obtains spleen immunocyte；Clean with PBS again, spleen immunocyte is crossed the 50ml being filled with nylon hair
Syringe, the cell not attached is T lymphocyte, and flow cytometer detection CD3 cell proportion is used for next step after being higher than 80%
Experiment.
(2) preparation of CSFE method Labeled T-Lymphocytes
CFSE is a kind of fluorescent dye that can mix cell, and along with cell proliferation divides, the fluorescence entrained by cell is strong
Degree is gradually lowered.The speed of cell proliferation is the fastest, and its fluorescence-intensity decay ground is the fastest.By fluorescein based dye CFSE markers step 1 point
From the T lymphocyte obtained.Specifically comprise the following steps that mouse T lymphocyte being 2% hyclone containing volume fraction
It is 1 × 10 that PBS adjusts cell density7Individual/ml, is subsequently adding CFSE (Sigma, catalog number is 21888-25MG-F), makes
Its concentration is 10 μm, and lucifuge dyes 20 minutes, with a large amount of resuspended containing the PBS that volume fraction is 2% hyclone, and washing, eventually
Only dyeing, obtains the T lymphocyte of CFSE labelling, for follow-up experiment.
(3) Con A stimulates T lymphocyte
To adjust the density of the T lymphocyte of CFSE labelling containing the PBS that volume fraction is 2% hyclone for 1 × 106
Individual/ml, is subsequently adding Con A (Sigma, catalog number is c2010-25MG) so that it is concentration is 2 μ g/ml, obtains T lymph
(density of T lymphocyte is 1 × 10 to cell culture system6Individual/2mL).
(4) cultivation of T lymphocyte
The 4th generation Sp-MSCs that step one obtains is added according to whether the T lymphocyte cultivating system obtained to step (3)
It is divided into following two groups:
1) LTT experimental group: the T lymphocyte cultivating system obtained to step (3) adds the 4th generation Sp-that step one obtains
MSCs, the density making Sp-MSCs cell is 1 × 105Individual/2mL, co-cultures 48h；
2) LTT matched group: without Sp-MSCs, cultivates T lymphocyte cultivating system 48h.
Collect the T lymphocyte suspended in the cultivating system of above-mentioned experimental group and matched group, for following experimental analysis.
(5) interpretation of result
1) suppression T lymphopoiesis
Use in the cultivating system of Beckman streaming instrument LTT experimental group that step (4) is obtained and LTT matched group and suspend
T lymphocyte carry out flow cytometer showed, often 50000 cells collected by pipe.
In LTT matched group, after T lymphocyte is stimulated by Con A, rapidly, fluorescence-intensity decay is obvious for propagation, and
In LTT experimental group in the presence of Sp-MSCs, the speed of T lymphocyte decay substantially slows down, and shows that T lymphopoiesis receives
Suppression (Fig. 5 A).
2) the T lymphocyte activation of Sp-MSCs suppression heterogenetic antigen induction
Use inverted phase contrast microscope (Nikon, 21560) LTT experimental group that step (4) is obtained and LTT matched group
The T lymphocyte suspended in cultivating system is observed.
Con A is a kind of non-specific antigen, has powerful activated T lymphocytes ability.As shown in Figure 6A, LTT comparison
In group, after Con A stimulates, T lymphocyte significantly activates, and is gathered into huge cell mass, and has Sp-in LTT experimental group
In the presence of MSCs, this activation is significantly inhibited, and most of T lymphocytes are loose individual cells or minicell
Group (Fig. 6 B).Result above shows the T lymphocyte activation that mice Sp-MSC can suppress heterogenetic antigen to induce.
3) the T lymphocytic emiocytosis inflammatory factor of suppression non-specific antigen activation
For further looking at the Sp-MSCs Immune Function to T lymphocyte, by quantitative PCR detection in step (4)
The level of inflammatory cytokine IFN-γ, TNF-α and the IL-17A of each group T Expressions In Lymphocytes.Primer sequence is:
Mouse interferon gamma (IFN-γ): upstream sequence-5'-CTCATGGCTGTTTCTGGCTGTTA-3', downstream sequence
5'-GACGCTTATGTTGTTGC TGATGG-3'；
Murine tumor necrosis factor α (TNF-α): upstream sequence 5 '-CACTTGGTGGTTTGCTACGA-3, downstream sequence
5′-GCCTCCCTCTCATCAGTTCTA-3′；
Mouse interleukin 17A (IL-17A): upstream sequence 5'-TCCAGAAGGCCCTCAGACTA-3', downstream sequence
5'-AGCA TCTTCTCGACCCTGAA-3'；
Internal reference GADPH upstream sequence 5'-AAACCCATCACCATCTTCCA-3', downstream sequence 5'-GTGGTT
CACACCCATCACAA-3'。
Above-mentioned primer is by the raw work biosynthesis in Shanghai.
Result as shown in Figure 8, is compared with being not added with Sp-MSCs, can substantially suppress T in LLT experimental group to drench after adding Sp-MSCs
The intracellular IFN-γ of bar, TNF-α and the expression of IL-17A.
2, mixed lymphocyte reaction (mixed lymphocytes reaction, MLR)
(1) preparation of heart xenotransplantaion system
According to the method in step 1, isolated T lymphocyte from allogeneic BALB/cl mice；By irradiation
(cobalt-60 of 10Gy dosage irradiates) the T lymphocyte of allogeneic BALB/cl mice crossed, the most illuminated C57BL/6
The T lymphocyte of mice and culture medium (α-MEM culture medium containing the FBS that volume fraction the is 10%) mixing of 2mL, wherein,
The number of cells of the T lymphocyte of allogeneic BALB/cl mice and the T lymphocyte of C57BL/6 mice than for 1:10,
Obtain co-culture system；
(2) cultivation of heart xenotransplantaion system
It is divided into following two groups according to whether the 4th generation Sp-MSCs adding step one acquisition to co-culture system:
1) MLR experimental group: add the 4th generation Sp-MSCs that step one obtains in co-culture system, allogeneic
BALB/cl mouse T lymphocyte, the T lymphocyte of C57BL/6 mice and the number of Sp-MSCs cell ratio is for 1:10:1, altogether training
Support 48h；
2) MLR matched group: without Sp-MSCs, cultivates co-culture system 48h.
Collect the T lymphocyte suspended in the cultivating system of above-mentioned experimental group and matched group, for following experimental analysis.
(3) interpretation of result
1) suppression T lymphopoiesis
Use in the cultivating system of Beckman streaming instrument MLR experimental group that step (2) is obtained and MLR matched group and suspend
T lymphocyte carry out flow cytometer showed, often 50000 cells collected by pipe.
In MLR matched group, allogene antigen also promotes T lymphocyte significantly to breed, similar to LLT result, MLR
In experimental group, Sp-MSCs shows suppression T lymphopoiesis ability (Fig. 5 B).
2) the T lymphocyte activation of Sp-MSCs suppression allogene antigen induction
Use inverted phase contrast microscope (Nikon, 21560) MLR experimental group that step (2) is obtained and MLR matched group
The T lymphocyte suspended in cultivating system is observed.
In addition to heterogenetic antigen, allogene antigen equally activated T lymphocytes.As shown in Figure 7 A,
In MLR matched group, after allogeneic T lymphocyte mixes, T lymphocyte significantly activates, can in culture dish
See multiple huge cell mass, and in MLR experimental group, Sp-MSCs then can significantly inhibit this activation.In culture dish
T lymphocyte cannot be polymerized to maxicell agglomerate, cash as loose small cell cluster (Fig. 7 B) more.Result above shows little
Mus Sp-MSCs can suppress the T lymphocyte activation of allogene antigen induction.
3) the T lymphocytic emiocytosis inflammatory factor that suppression allogene is Antigen-activated
For further looking at the Sp-MSCs Immune Function to T lymphocyte, by quantitative PCR detection in step (2)
The level of inflammatory cytokine IFN-γ, TNF-α and the IL-17A of each group T Expressions In Lymphocytes.Primer sequence is ibid.
Result, as it is shown in figure 9, compare with being not added with Sp-MSCs, can substantially suppress T in MLR experimental group to drench after adding Sp-MSCs
The intracellular IFN-γ of bar, TNF-α and the expression of IL-17A.
Claims (10)
1. a preparation method for spleen mescenchymal stem cell, comprises the steps:
(1) mill in vitro spleen, remove hematopoietic cell, obtain spleen matrix organization；
(2) with collagenase liquid, described spleen matrix organization carried out digestion process, and after digestion process, remove described spleen base
Hematopoietic cell in matter tissue, obtains spleen matrix organization after digestion process；
(3) cultivate spleen matrix organization after described digestion process, obtain spleen mescenchymal stem cell.
Method the most according to claim 1, it is characterised in that:
Described mill after also include rinse step；
It is that spleen matrix organization is soaked in collagenase that described spleen matrix organization is carried out digestion process by described collagenase liquid
In liquid；
In described collagenase liquid, the mass fraction of collagenase is 0.1-0.15%.
Method the most according to claim 2, it is characterised in that:
Described collagenase is type i collagen enzyme, II Collagenase Type or IV Collagenase Type；
And/or, described collagenase is specially II Collagenase Type.
The most according to the method in claim 2 or 3, it is characterised in that:
The culture medium that described collagenase liquid is uniformly mixed so as to obtain by II Collagenase Type, hyclone and α-MEM culture medium；
Described hyclone volume fraction in described collagenase liquid is 15-20%.
Method the most according to claim 4, it is characterised in that:
Described II Collagenase Type mass fraction in described collagenase liquid is 0.1%, and described hyclone is at described collagenase
Volume fraction in liquid is 15%.
6. according to described method arbitrary in claim 1-5, it is characterised in that: the condition of described digestion process is 35-38 DEG C,
5%CO2Under the conditions of digest 0.5-4h.
7. the spleen mescenchymal stem cell that in claim 1-6, arbitrary described method prepares.
8. in claim 1-6 arbitrary described method or the spleen mescenchymal stem cell described in claim 7 following 1)-3)
In application in any one:
1) suppression T lymphopoiesis；
2) suppression T lymphocyte activation；
3) suppression T lymphocytic emiocytosis inflammatory factor.
Application the most according to claim 8, it is characterised in that: described inflammatory factor be IFN-γ and/or TNF-α and/or
IL-17A。
Application the most according to claim 8 or claim 9, it is characterised in that: described T lymphocyte is that allogene antigen lures
The T lymphocyte led or the T lymphocyte of heterogenetic antigen induction.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439620A (en) * | 2018-11-02 | 2019-03-08 | 杜玲娟 | A kind of method and apparatus for activating stem cells |
| WO2019022386A3 (en) * | 2017-07-28 | 2019-03-21 | 가톨릭대학교 산학협력단 | Pharmaceutical composition for preventing or treating rheumatoid arthritis including inferior nasal concha-derived mesenchymal stem cell as active ingredient |
| CN109609584A (en) * | 2018-12-10 | 2019-04-12 | 天津长和生物技术有限公司 | The detection method of mescenchymal stem cell immunoregulation capability |
| CN114686427A (en) * | 2022-05-23 | 2022-07-01 | 中国人民解放军总医院第一医学中心 | Spleen-regulating B lymphocyte and preparation method and application thereof |
| US11612620B2 (en) | 2017-07-28 | 2023-03-28 | Catholic University Industry Academic Cooperation Foundation | Pharmaceutical composition for preventing or treating rheumatoid arthritis comprising nasal inferior turbinate-derived mesenchymal stem cells as an active ingredient |
| CN117343892A (en) * | 2023-09-28 | 2024-01-05 | 中山大学孙逸仙纪念医院 | Construction method and application of artificial lymph node primordium |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298606A (en) * | 2008-02-14 | 2008-11-05 | 天津环宇商桥商务信息咨询有限公司 | Preparation, storage and use of umbilical cord and placenta mesenchymal stem cell for clinical therapy |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
| CN102965338A (en) * | 2012-12-04 | 2013-03-13 | 东南大学 | Extraction and culture method of human umbilical cord mesenchymal stem cells |
| CN103263440A (en) * | 2013-02-08 | 2013-08-28 | 周胜利 | Method for extracting and preparing homology mesenchymal stem cell injection from placenta and umbilical cord |
-
2016
- 2016-09-14 CN CN201610827426.3A patent/CN106282108A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298606A (en) * | 2008-02-14 | 2008-11-05 | 天津环宇商桥商务信息咨询有限公司 | Preparation, storage and use of umbilical cord and placenta mesenchymal stem cell for clinical therapy |
| CN102676451A (en) * | 2011-09-05 | 2012-09-19 | 博雅干细胞科技有限公司 | Method for separating mesenchymal stem cells from placenta |
| CN102965338A (en) * | 2012-12-04 | 2013-03-13 | 东南大学 | Extraction and culture method of human umbilical cord mesenchymal stem cells |
| CN103263440A (en) * | 2013-02-08 | 2013-08-28 | 周胜利 | Method for extracting and preparing homology mesenchymal stem cell injection from placenta and umbilical cord |
Non-Patent Citations (5)
| Title |
|---|
| KRAMPERA M ET AL: "Immune regulation by mesenchymal stem cells derived from adult spleen and thymus", 《STEM CELLS DEV》 * |
| YA-CHIN HOU ET AL: "Therapeutic efficacy of spleen-derived mesenchymal stem cells in mice with acute pancreatitis", 《J STEM CELL RES THER》 * |
| ZHU H ET AL: "A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone", 《NATURE PROTOCOLS》 * |
| 李铎: "人脐带间充质干细胞分离培养方法的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑 E059-11》 * |
| 杨中萌 等: "大鼠脂肪和骨髓来源间充质干细胞的生物学特性比较", 《中国组织工程研究与临床康复》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019022386A3 (en) * | 2017-07-28 | 2019-03-21 | 가톨릭대학교 산학협력단 | Pharmaceutical composition for preventing or treating rheumatoid arthritis including inferior nasal concha-derived mesenchymal stem cell as active ingredient |
| US11612620B2 (en) | 2017-07-28 | 2023-03-28 | Catholic University Industry Academic Cooperation Foundation | Pharmaceutical composition for preventing or treating rheumatoid arthritis comprising nasal inferior turbinate-derived mesenchymal stem cells as an active ingredient |
| CN109439620A (en) * | 2018-11-02 | 2019-03-08 | 杜玲娟 | A kind of method and apparatus for activating stem cells |
| CN109609584A (en) * | 2018-12-10 | 2019-04-12 | 天津长和生物技术有限公司 | The detection method of mescenchymal stem cell immunoregulation capability |
| CN114686427A (en) * | 2022-05-23 | 2022-07-01 | 中国人民解放军总医院第一医学中心 | Spleen-regulating B lymphocyte and preparation method and application thereof |
| CN114686427B (en) * | 2022-05-23 | 2022-07-29 | 中国人民解放军总医院第一医学中心 | Spleen-regulating B lymphocyte and preparation method and application thereof |
| CN117343892A (en) * | 2023-09-28 | 2024-01-05 | 中山大学孙逸仙纪念医院 | Construction method and application of artificial lymph node primordium |
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