CN105886471B - A kind of preparation method and applications of novel regulatory Dendritic Cells - Google Patents
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Abstract
The present invention provides a kind of preparation methods of novel regulatory Dendritic Cells and joint DC-CIK to carry out immunotherapy of tumors method.Through separation mammalian mesenchymal stem cell and candidate stem cell；The mammalian mesenchymal stem cell obtained in co-incubation step under the conditions of suitable cell grows and is value-added and candidate stem cell；It after co-incubation 1 to 7 day, recycled from suspending nutrient solution, identify obtained novel regulatory Dendritic Cells.It is characterized in that, the novel regulatory Dendritic Cells is converted into mature Regulatory Dendritic Cells under the induction of CpGODN (CpG ODN) or the inducer with same inducing function, wherein the expression of irf4+ and irf8+ increases, and has immunoloregulation function.The novel regulatory Dendritic Cells is able to suppress tumour growth, and can combine DC-CIK and carry out immunotherapy of tumors.
Description
Technical field
The present invention relates to the fields for generating novel regulatory Dendritic Cells and its purposes in immunization therapy.Specifically,
Using mescenchymal stem cell induction of hematopoiesis stem/progenitor cells, to be divided into irf4+irf8+ modulability dendron shape thin the present invention relates to a kind of
The method of born of the same parents regDC, and its novel immune therapy of joint DC-CIK.
Background technique
Although modern medicine is quickly grown, malignant tumour is still a great problem that can not be captured.Currently, treatment is pernicious swollen
The method of tumor is mainly surgical operation, chemotherapy, radiotherapy and biologic treatment.First three treatment method is to treat big portion
Early stage limitation tumour and Concurrent Chemoradiotherapy Sensitivity tumour is divided to provide selection.But it is directed to recurrent and refractory tumour, residual tumor,
Tumor drug resistance, the advantage that biologic treatment has other treatment method irreplaceable.Wherein, tumor immunology treatment has special
Property it is strong, applicability is wide, side effect is light the advantages that, the tumour that can effectively kill remaining in vivo after patient is postoperative and chemicotherapy is thin
Born of the same parents achieve the purpose that treat tumour, prevention recurrence and transfer and final radical cure tumour.
Adoptive immunotherapy is straight to generating after patient by the way that a large amount of specific effector cells expanded in vitro are fed back
Connect the effect of killing tumor cell.CIK (Cytokine Induced Killer Cell), cytokine-induced killer cell is thin
Born of the same parents, are a kind of active heterogeneous populations of Efficient killing effect, and peripheral blood mononuclear cells is induced through cytokine profiles in vitro
Cultivate foreign cell of a group based on CD3+CD56+T generated, including CD4+ T helper cell, CD8+ killer T cell and
NK/T cell.With significant antiviral and antitumor activity, have the characteristics that efficient, wide spectrum killing, non-MHC limitation.Its mechanism
Are as follows: release granzyme and perforin direct killing tumour cell and virus infected cell, Fas/Fasl access induce tumour cell
Apoptosis and secrete cytokines (IFN-, TNF-, IL-6 etc.) inhibit tumour growth and virus replication, improve body's immunity.
DC-CIK is Dendritic Cells (the i.e. traditional Dendritic Cells that will have high expression MHC-I, II class molecule and costimulatory molecules
Conventional dendritic cell) load tumour antigen, offer to further enhance CIK immunization therapy to CIK cell
The method of function.Currently, the immune treatment technology of adopting of DC-CIK is more mature, at home and abroad widely applied.
Mescenchymal stem cell (MSC) is that be present in a kind of Asia with polyphyly differentiation potential in human multiple tissue all-round
Population of stem cells.MSCs is resistance to " niche " (microenvironment) of existence and to two side of immunological regulation of hematopoiesis function by constituting HSC
Face adjusts hemopoietic function of bone marrow.It has been had proven to amplification in vitro hematopoiesis in zoopery and some clinical researches report
The effect of stem/progenitor cells.Moreover, MSC has the function of adjusting panimmunity cell function.
Summary of the invention
Present invention aim to address subproblems present in existing treatment malignant tumour technology, provide a kind of novel tune
Section property Dendritic Cells and its production method.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of preparation method of novel regulatory Dendritic Cells, steps are as follows:
(1) mammalian mesenchymal stem cell and candidate stem cell are separated；
(2) under conditions of suitable cell is grown and is proliferated, the mammalian mesenchymal of acquisition in co-incubation step (1)
Stem cell and candidate stem cell；
(3) it after co-incubation 1 to 7 day, recycled from suspending nutrient solution, identify that obtained novel regulatory dendron shape is thin
Born of the same parents.
Preferably, the novel regulatory Dendritic Cells that recycling obtains in the step (3) is that (irf is irf4+irf8+
Interferon regulatory factor) immature Regulatory Dendritic Cells.
Preferably, wherein the mammalian mesenchymal stem cell is isolated from placenta, marrow, adipose tissue.
Preferably, wherein the mammalian mesenchymal stem cell be in vitro culture to the 1st to 10 generation mesenchyma it is dry thin
Born of the same parents.
Preferably, the novel regulatory Dendritic Cells obtained using the method is contained in the suspending nutrient solution
The novel regulatory Dendritic Cells of 1%-99%.
Preferably, the novel regulatory Dendritic Cells in CpG ODN (CpG ODN) or has same induction function
Mature Regulatory Dendritic Cells are converted under the induction of the inducer of energy, wherein the expression of irf4+ and irf8+ increases,
And there is immunoloregulation function.
Preferably, the novel regulatory Dendritic Cells can promote T cell and NK cell Proliferation.
Preferably, the novel regulatory Dendritic Cells can combine DC-CIK progress immunotherapy of tumors.
Preferably, the novel regulatory Dendritic Cells can promote T cell and NK cell secretion of gamma-IFN.
Preferably, the novel regulatory Dendritic Cells is able to suppress tumour growth.
A kind of novel regulatory Dendritic Cells preparation method provided by the invention and its have enhancing T and NK cell secretion
IFN-γ and immune function adjustment effect is migrated to tumor area.Adopt in treatment in tumour immunity, using this new modulability tree
Prominent shape cell combines DC-CIK scheme, enhances CIK to the lethal effect of tumour, reaches remaining tumor cells in removing machine body, disappear
Except the factor of Preventive, increase a possibility that curing, extends life span, improve the quality of living.
Detailed description of the invention
Fig. 1: the form of in vitro culture human mesenchymal stem cell；
Fig. 2: the immunophenotype of human mesenchymal stem cell；
Fig. 3: the peripheral blood CD34+ hematopoietic stem/progenitor cells form obtained after sorting；
Fig. 4: the peripheral blood CD34+ hematopoietic stem/progenitor cells flow cytometer detection phenotype obtained after sorting；
Fig. 5: human mesenchymal stem cell and peripheral blood hematopoietic stem/progenitor cells co-cultured cell form, the 1st day；
Fig. 6: human mesenchymal stem cell and peripheral blood hematopoietic stem/progenitor cells co-cultured cell form, the 7th day；
Fig. 7: obtaining cell after flow cytometer detection mescenchymal stem cell and hematopoietic stem/progenitor cells co-cultivation is compared with single-population；
Fig. 8: co-culturing and obtaining cell is Dendritic Cells sample form；
Fig. 9: flow cytometer detection co-cultures the dendritic cell phenotypic obtained, prompts for Regulatory Dendritic Cells；
It is irf4, irf8 low expression that Figure 10: western blot detection, which co-cultures and obtains Dendritic Cells, by containing CpG
After 10 μ g/ml of ODN is incubated for 6h induced maturation stimulated in vitro, irf4, irf8 expression are remarkably reinforced, for mature modulability dendron shape
Cell；
Figure 11-12.irf4+irf8+regDC promotes CD4+T lymphocyte and CD8+T lymphopoiesis；
Figure 13 .irf4+irf8+regDC promotes CD4+T lymphocyte and CD8+T lymphocytic emiocytosis IFN-γ；
Proliferation of Figure 14 .irf4+irf8+regDC to NK cell；
Figure 15 .irf4+irf8+regDC promotes NK cell secretion of gamma-IFN；
Figure 16 .irf4+irf8+regDC and NK cell and CD8+T cell have synergistic antitumor effect；
Figure 17 .irf4+irf8+regDC promotes cDC, NK cell and CD8+T cell to migrate to tumor tissues；
Figure 18 .DC-CIK flow diagram.
Specific embodiment
In order to better illustrate the present invention, with reference to the attached drawing in the embodiment of the present invention, in the embodiment of the present invention
Technical solution is clearly and completely described.
In a first aspect, the method for the present invention is related to a kind of method for obtaining novel Dendritic Cells:
1. the acquisition of mescenchymal stem cell:
Suitable for meeting internationally recognized selected from separate sources health unrelated donor or relatives' (placenta, marrow, fat etc.)
Definition mescenchymal stem cell, conventional criteria cultural method, in vitro culture to 2-5 generation after carry out subsequent processing.
2. the acquisition of hematopoietic stem/progenitor cells:
(being obtained using specific ripeness standard method) is in human body: the peripheral blood mobilized by G-CSF standard method
Mononuclearcell mixture, or health donors marrow is collected, CD34+ hematopoietic stem/progenitor cells content is 80% or more；
3. mescenchymal stem cell and hematopoietic stem/progenitor cells co-culture:
1) opportunity co-cultured, 12h obtains conventional method first with the cell concentration of 2 × 104/ml before co-cultivation
3rd generation mescenchymal stem cell, which is inoculated in, is added 24 orifice plate cultures, so that its is adherent.The CD34+ hematopoiesis isolated and purified is added after 12h
Stem/progenitor cells are co-cultured.
2) two kinds of cell number ratios: the ratio between mescenchymal stem cell and hematopoietic stem/progenitor cells are 1: 5~1: 10.
3) co-cultivation condition: streptomysin containing 100U/ml, the RPMI-1640 complete medium of 100U/ml penicillin, 5%
CO2,37 DEG C of constant temperature incubations.
4) consumptive material: can according to experiment need 24 orifice plates, 6 orifice plates or other have identical function material.
5) cultivated days: 1~7 day.
6) harvest cellular processes: absorption accompanies feeding system suspension that it is to be identified to collect suspension cell after PBS washing altogether.
7) identification method: with the Regulatory Dendritic Cells of Flow cytometry and westernblot detection inductive formation
Phenotype and irf4+ and irf8+ protein expression.
Second aspect, of the invention is related to a kind of novel irf4+irf8+ Dendritic Cells:
The graft ingredient obtained after co-cultivation: contain 1%~99% Regulatory Dendritic Cells Hematopoietic Stem ancestral
Cell mixture.
The third aspect has the present invention relates to this novel irf4+irf8+ Dendritic Cells and adjusts panimmunity cell
Ability.
1. irf4+irf8+regDC promotes T cell and NK cell Proliferation after CpG ODN stimulation is mature；
2. maturation irf4+irf8+regDC can promote T cell and NK cell secretion of gamma-IFN；
3. maturation irf4+irf8+regDC can promote panimmunity cell to tumor tissues chemotactic；
4. maturation irf4+irf8+regDC can inhibit tumour growth.
Fourth aspect, the present invention relates to this novel irf4+irf8+ Regulatory Dendritic Cells of application combine DC-CIK into
Row immunotherapy of tumors.
The application time of 1.irf4+irf8+regDC
It carries out feeding back the irf4+irf8+ that above-mentioned induction obtains on the day before beginning DC-CIK treats each course for the treatment of
RegDC, second day starts, and carries out induction, culture, amplification and the feedback of DC-CIK cell (according to routine clinical scheme).
The application dose of 2.irf4+irf8+regDC
The hematopoietic stem/progenitor cells mixture of infusion irf4+irf8+regDC of the 106/kg containing 1%-99% is given through vein.
3.irf4+irf8+regDC feedback mode:
It is identical that mode is fed back with DC-CIK.Venoclysis.
The arrangement that DC-CIK is fed back: (can flexible arrangement according to clinical setting)
1) effector T cell is fed back: 2~3 courses for the treatment of, and 8 times/course for the treatment of, 1 × 10^9/ times.Cell is expanded to the 4-5 days
Afterwards, freeze-stored cell, recovery before feeding back.
2) DC-CIK is fed back: 1 × 10^9/ times, 2~4 times/course for the treatment of.It takes out part cell every time to feed back, remaining cell addition
Culture medium continues to cultivate.
Co-culturing, inducing method of the invention is suitable for various 2~5 tissue-derived generation mescenchymal stem cells.The induction
The virus-free importing of cultural method, easy to operate, efficiency is higher.The present invention also utilizes the irf4+irf8+ modulability tree for co-culturing harvest
Prominent shape cell has important immunoloregulation function to body panimmunity cell.It is thin by mescenchymal stem cell and Hematopoietic Stem ancestral
The method that born of the same parents graft co-cultures, induction the latter, which generates, is rich in irf4+irf8+ Regulatory Dendritic Cells graft, and combines
Clinical DC-CIK cell therapy is adopted the process for the treatment of for tumour immunity, can be sketched are as follows: by adipose tissue or marrow with standard
The mescenchymal stem cell of method harvest, the 3rd generation mescenchymal stem cell that standard method culture obtains are inoculated in training with certain density
Bottle is supported, after it is covered with, is added with 1: 5~1: 10 ratios and is co-cultured 1~7 day from marrow or peripheral blood hematopoietic stem/progenitor cells
Afterwards, the suspension cell that separation co-culture system generates, identification account for 1%~99% irf4+irf8+ tune of harvest co-cultured cell
The generation of section property Dendritic Cells, and the specifc immunity function of the cell is verified.That is: 1. by CpG ODN stimulate at
After ripe, irf4+irf8+regDC promotes T cell and NK cell Proliferation；2. maturation irf4+irf8+regDC can promote T cell and
NK cell secretion of gamma-IFN；3. maturation irf4+irf8+regDC can promote panimmunity cell to tumor tissues chemotactic；4. mature
Irf4+irf8+regDC can inhibit tumour growth.Finally, by the regDC of this novel irf4+irf8+ and the clinical side DC-CIK
Case joint carries out anti-tumor immunotherapy.
Those skilled in the art are known, and the method and step of above-mentioned culture listed by the present invention and induction is only the mesh of illustration
, it may not realize the preferred plan of the method for the invention, those skilled in the art can make above method step
It is appropriate change and remain to achieve the object of the present invention, such change also falls into the scope of protection of present invention.Certainly, originally
Mescenchymal stem cell used in invention the method is not limited to the 3rd generation mescenchymal stem cell, and marrow, rouge may be selected in source
The source for mesenchymal stem cells tissue of fat tissue, umbilical cord and other international endorsements.It is adjusted using irf4+irf8+ of the invention
Property Dendritic Cells carry out joint DC-CIK treatment method, dosage and application time do not limit to the solution of the present invention.
The mescenchymal stem cell induction donor hematopoietic stem/progenitor cells that the method for the present invention obtains are divided into rich in irf4+irf8+
It is pernicious to combine DC-CIK regimen on tumor patient's body residual using the immunomodulatory properties of the latter for Regulatory Dendritic Cells
Cell is killed, to achieve the purpose that further increase tumour immunity curative effect.For disease after clinically clear operation and chemicotherapy
Malignant cell is remained in human body, reduces recurrence rate, is improved cure rate and is provided new adoptive immunotherapy.
1 major experimental reagent of embodiment and consumptive material
(1) major experimental reagent
Penicillin, streptomysin and trypsase-EDTA are purchased from GIBCO company.
Human lymphocyte separating liquid (Ficoll, the Tianjin ocean Hao biological products company)
Cell factor rhIL-2, rhIL-12, IL-4, IL-7, GM-CSF, TNF- σ (Peprotech company)；
Human recombination solubility CD40 Ligand (rh sCD40-L；Bender company)；
Antibody: mouse anti human CD11c, CD80, CD40, CD86, CD29, CD34, CD44, CD73, CD90, CD105,
CD106, CD117, Flk-1, HLA-ABC, CD3 monoclonal antibody, CD28 monoclonal antibody and HLA-DR antibody, anti-mouse NK1.1mAb, anti-mouse
CD8+T cell mAb, rabbit anti-mouse CD16/CD56, CD8, CD3, CD19 streaming antibody (Becton Dickinson company,
Biolegend company)；
Magnetic bead sorting kit: CD34, CD4, CD8, NK cell (MiltenyiBiotec company)；
ELISA kit: IFN-γ (BD Pharmingen company)；
CFSE dyes liquid kit (green skies company)
Cell culture fluid ingredient: MCDB (Sigma company), DMEM/F-12 (GibcoBRL company), RPMI-1640
(GibcoBRL company), X-VIVO-15, gt-t551 serum free medium (BioWhittaker company)；D-Hanks balances salt
Solution, Ficoll (1.077g/cm3) (Pharmacia company)；Fetal calf serum (Hyclone company)；
Trypsase (GibcoBRL company), EDTA (Tianjin Chemical Reagents Factory No.1)；(GibcoBRL is public for glutamine
Department), dexamethasone, sodium β-glycerophosphate, ascorbic acid, bovine serum albumin(BSA) (Sigma company)；
(2) major experimental consumables associated therewith
15ml, 50ml centrifuge tube, T-25cm2, T-75cm2 Tissue Culture Flask (costar company), 24 orifice plates, 96 hole concave bottoms
Culture plate (Corning company)
The external evoked method of embodiment 2, irf4+irf8+ Regulatory Dendritic Cells (irf4+irf8+regDC)
Separation, culture and the identification (Fig. 1-2) of the third generation mescenchymal stem cell of step 1. human bone marrow-derived
(1) it separates
1. the marrow 5-10ml of aseptic collection healthy volunteer is in sterile calparine pipe.
2. taking a sterile centrifuge tube, marrow is suitably diluted with D-Hanks liquid.
3. take a new centrifuge tube, it is separately added into recovery to the marrow after the Ficoll of room temperature and above-mentioned dilution, when addition
Carefully operation does not destroy interface, and the ratio of the two is 1: 1.
4. 20 DEG C are centrifuged being put into room temperature desk centrifuge after above-mentioned centrifuge tube trim with 1800 revs/min of speed
20 minutes.After taking out centrifuge tube, sterile working carefully draws tunica albuginea layer and obtains mononuclearcell, by mononuclearcell D-
Hanks liquid is washed twice and is counted.
(2) it cultivates
1. above-mentioned mononuclearcell is inoculated in the culture bottle of 25cm2 with the density of 1 × 106/cm2, cell culture body
System is containing 58%DMEM/F12+40%MCDB-201,2% fetal calf serum (FCS), 10ng/ml EGF, 10ng/ml PDGF 1
× Insulin-Transferrin-selenous acid (Insulin-Transferrin-Selenium, ITS), 1 × linoleic acid-ox blood are pure
Albumen (linoleic acid-bovine serum albumin, LA-BSA), 50 μM of β mercaptoethanols, 2mM L-Glutamine,
The culture solution of 100 μ g/ml penicillin and 100U/ml streptomycin sulphate, 37 DEG C, the incubator culture of 5%CO2,95% humidity.
After 2.24 hours, suspension cell, supplementing culture medium are removed, cell was changed the liquid once every three days, long to 70- to cell
80% when converging, with 0.125% trypsase+0.01%EDTA had digestive transfer culture.The mesenchymal stem cell cryopreserving in 3-4 generation is in liquid nitrogen
Tank is spare.
Certainly, the isolated culture method of other mescenchymal stem cells well known in the art can be used also to obtain in the present invention
3rd generation mescenchymal stem cell is directly obtained using commercially available 3rd generation mescenchymal stem cell without being voluntarily separately cultured
?.
(3) it identifies
1. the cellular morphology for the 3rd generation mescenchymal stem cell that first two steps obtain is shown in Fig. 1.
2. the phenotype of the 3rd generation mescenchymal stem cell is detected with indirect immunofluorescence, with fluorescein isothiocynate (FITC)
Mouse anti human CD29, CD34, CD44, CD73, CD90, CD105, CD106 of label, CD117, Flk-1, HLA-ABC and HLA-
Flow cytometer detection is carried out after DR antibody (antibody is purchased from BD company) label, flow cytometer is ACCURI C6 (Becton
Dickinson)。
As a result see that Fig. 2, abscissa indicate that cell fluorescence intensity, ordinate indicate cell number；P1 indicates selected cell colony.
Phenotypic results show that CD29, CD44, CD73, CD90, CD105, Flk-1 and HLA-ABC of the 3rd generation mescenchymal stem cell are
The positive, CD34 and HLA-DR molecule are feminine gender.
The extraction and identification (Fig. 3-4) of step 2. human peripheral blood source hemopoietic stem/progenitor cells
(1) it extracts
1. continuous 5 days, mobilizing hematopoietic stem/progenitor cells are subcutaneously injected to peripheral blood using 5 μ g/kg/d of rhG-CSF.
2. anticoagulant heparin blood sampling tube 10ml is taken, with one times of 1: 1 volume dilution of PBS containing 0.6%ACD-A.
3. Ficoll and peripheral blood are placed in centrifuge tube with the layering of 1: 2 volume, carefully blood is slowly added on Ficoll,
It is sure not to obscure interface, is placed in centrifugation (1800rpm, 20min) in 4 DEG C of horizontal centrifuges.Centrifuge brake is closed, to delay automatically
After slow shutdown, centrifuge tube is taken out, it is seen that pipe inner boundary understands, is divided into 4 layers, from top to bottom successively are as follows: blood plasma, mononuclearcell
Layer, Ficoll liquid, granulocyte and red blood cell layer.
4. be gently inserted into capillary syring on mononuclearcell layer (tunica albuginea layer), carefully by tunica albuginea confluent monolayer cells suction line,
It moves into another test tube, as far as possible the Ficoll liquid of Wu Xi lower layer.PBS centrifuge washing 3 times containing 0.6%ACD-A are added
(1000rpm, 10min).
5. abandoning supernatant, the 300 μ l of PBS containing 0.6%ACD-A is added, prepares row immuno magnetic cell separation and purifies CD34+ cell.
6. checking cell viability, 1 drop cell suspension is added into 1 drop, 2% trypan blue liquid, checks have in 200 cells after 5min
How much core contaminates navy blue cell (dead cell), finds out percentage.Live cell fraction is required 95% or more.
X=prepares the total number of nucleated cell (ml) of cultivating system total volume (ml) × 4/4 big lattice
As a result: its purity is greater than 96%, and platform expects that blue dyeing observation mononuclearcell activity, survivaling cell rate are greater than 95%.
7. 1 × 108 mononuclearcell layer to be suspended to the PBS buffer solution of 300 μ l 0.5%BSA and 0.6%ACD-A
In, 100 μ l of Fc receptor blocking pharmacon is added, to block the receptor-mediated non-specific binding of Fc.
100 μ l AntiCD3 McAb, 4 immunomagnetic beads are added, in 4 DEG C of incubation 30min after mixing.Centrifuge washing 2 times (1000rpm,
10min), 500 μ l buffer solutions is added to suspend.
8. splitter is fixed in the magnetic field MACS, label cell is slow transitted through into MiniMACS splitter, elution removal
CD34- cell.
9. splitter is removed magnetic field, add 1ml MACS buffer pressurization elution splitter, collection group is divided into CD34+ cell
And it counts.
(2) it identifies
The CD34+ hematopoietic stem/progenitor cells that light harvests under the microscope are in small circular.After flow cytometry analysis magnetic bead sorting
CD34+ cell purity, purity about 98.9%.
Step 3. mesenchymal stem cell and hematopoietic stem/progenitor cells co-culture method (Fig. 5-6)
1. 24 orifice plate cultures are added with the cell concentration of 2 × 104/ml in mesenchymal stem cell, keep its adherent.
2. sucking the original culture medium of mescenchymal stem cell, the CD34+ Hematopoietic Stem ancestral thin (two of immunological magnetic bead sorting harvest is added
Person's ratio is 5: 1) and RPMI-1640 complete medium continues to cultivate.
3. condition of culture: streptomysin containing 100U/ml, the RPMI-1640 complete medium of 100U/ml penicillin, 5%CO2,
37 DEG C of constant temperature incubations.
4. in incubation, to prevent Derived from Mesenchymal Stem Cells and apoptosis, being set with new mescenchymal stem cell within every 3-4 days
It changes.Specific method:
(1) suspension cell is first sucked out, is placed in centrifuge tube.
(2) cell being attached on mescenchymal stem cell is digested with 0.125% trypsase+0.01%EDTA.
(3) dead cell and mescenchymal stem cell are removed with Ficoll.
(4) suspension cell in the cell of acquisition and (1) is mixed, is counted after washing, and addition is posted new mesenchyma and done carefully
24 orifice plates of born of the same parents continue to cultivate.
5. cell uses flow cytomery cell phenotype after PBS is washed immediately at the end of culture.
The form and identification of embodiment 3, irf4+irf8+ Regulatory Dendritic Cells (irf4+irf8+regDC)
(1) form (Fig. 7)
It is seen under light microscopic by the Regulatory Dendritic Cells (regDC) that mescenchymal stem cell induction of hematopoiesis stem/progenitor cells generate
It examines, it is seen that cell membrane has cynapse generation.
(2) surface marker factor identification (Fig. 8)
The expression in relation to the cell surface marker factor and costimulatory molecules is detected, the novel regulatory tree of inductive formation is prompted
Prominent shape cell (regDC) low expression CD11c, weak expression CD80, CD86, CD40.
(3) inductive formation Regulatory Dendritic Cells quantity (Fig. 9)
After mescenchymal stem cell and hematopoietic stem/progenitor cells co-culture 7 days, suspension cell is harvested.Flow cytometer detection prompt obtains
More single group's cell is obtained, the 56.9% of whole suspension cells is accounted for about.
(4) the induced maturation method of Regulatory Dendritic Cells
It co-cultures the regDC obtained to be inoculated in 96 orifice plates with every hole 5-10 × 104 cell concentration, with ODN containing CpG
The X-VIVO-15 serum free medium that 10 μ g/ml are incubated for 6h continues culture 3 days.I.e. maturation regDC after harvest suspension cell.
(5) after Westernblot detection co-cultures after immature and induced maturation regDC characteristic protein irf4,
The expression (Figure 10) of irf8
Irf4, irf8 are inflammation regulation GAP-associated protein GAP, while being also the developmental regulation molecule of Dendritic Cells.It is co-culturing
After 1~7 day, two albumen are low expression, the i.e. regDC of irf4+irf8+ low expression.And works as and CpG ODN is used to induce regDC
After maturation, two protein expressions of irf4, irf8 are remarkably reinforced, i.e. the regDC of irf4+irf8+ strongly expressed.And as control
GAPDH albumen is constant.Specific method is referring to westernblot routine experiment step.
The immunoloregulation function of embodiment 4, irf4+irf8+ Regulatory Dendritic Cells (irf4+irf8+regDC)
(1) promote CD4+T lymphocyte and CD8+T lymphopoiesis (see Figure 11-12)
It is high after the immature regDC and induced maturation of the low expression irf4+irf8+ that this experiment detection co-culturing, inducing obtains
Express rush breeder reaction of the regDC of irf4+irf8+ to CD4+T lymphocyte.See Figure 11,12.Prompt is by activation maturation
Irf4+irf8+regDC, which has, promotes T cell proliferation function.Specific step is as follows:
1. collecting irf4+irf8+regDC after co-culturing and being incubated for 6h induced maturation using 10 μ g/ml of CpG ODN, it is added
Into 96 well culture plates, every 1 × 10^5 of hole.
2. the healthy volunteer peripheral blood 5ml of anticoagulant heparin is taken, it is molten with the PBS buffering of EDTA containing 2mM and 0.5%BSA
1: 1 volume dilution of liquid.Tunica albuginea layer mononuclearcell is separated with Ficoll liquid.
3. going out CD4+T lymphocyte using anti-CD4 and CD8 immunological magnetic bead sorting according to Miltenyi MACS illustration method
And CD8+T lymphocyte.With two kinds of cell purities of flow cytometry analysis.
4. being washed after sorting through PBS, adds equivalent CFSE dilution (5 μM) to enter in lymphocyte suspension, mix well, 37 DEG C
It gently swinging down after ten minutes, the ice-cold RPMI1640 of equivalent (10% fetal calf serum) stopped reaction is added, PBS is washed twice after 1 minute,
It is spare to be suspended in complete medium；
5.2 × 105 human peripheral CD4+T cells and each 100 μ l of CD8+T cell, what addition was obtained containing co-culturing, inducing
After prematurity and induced maturation in the 96 hole U-shaped bottom culture plates of irf4+irf8+regDC.37C, 5%CO2 and saturated humidity condition
Lower culture 7 days.
6. collecting the lymphocyte of culture, flow cytometer detects CFSE positive cell number after PBS washing.
(2) promote CD4+T lymphocyte and CD8+T lymphocytic emiocytosis IFN-γ (Figure 13)
D4+T lymphocyte that above-mentioned experiment is obtained and CD8+T lymphocyte ELISA method detection its secretion of gamma-IFN
Ability.Compared to immature rf4+irf8+regDC, the rf4+irf8+regDC of CpG ODN activation, which has, promotes two kinds of lymphs thin
The ability of the more IFN-γ of intracrine.(co-culturing 3 days)
(3) to the proliferation of NK cell and promotion NK cell secretion of gamma-IFN (Figure 14-15) after stimulating
This experiment detect co-culturing, inducing obtain immature regDC and induced maturation after regDC proliferation and
Promote NK cell secretion of gamma-IFN.See Figure 13,14.Specific step is as follows:
1. it is thin to use Miltenyi MACS magnetic bead sorting to go out NK first from healthy volunteer's peripheral blood mononuclear cells
Born of the same parents, method are the same.
2. the NK cell that sorting obtains is co-cultured in RPMI-1640 with irf4+irf8+regDC with 1: 1 ratio respectively
7 days.
3. collecting cell conditioned medium, the concentration of cell factor IFN-γ is surveyed with ELISA.
4. application CFSE dyeing (specific method is same as above) method detects NK ability of cell proliferation.
(4) irf4+irf8+regDC and NK cell and CD8+T cell have synergistic antitumor effect (Figure 16)
1. constructing B16 subcutaneous transplantation tumor mouse model: every group of 6 C57BL/6 mouse.5 × 10^5B16 is subcutaneously injected in oxter
Tumour cell continuous 6 days；
2. the 7th day intratumor injection is unactivated or CpG handles 2 × 10^6 of regDC after 6h, detection tumor size variation；
3.PBS control group is blank control；
4. other two groups of control groups processing:
1) CD8+T cell in removing body: -2~0 day, using 200 μ g/ of anti-mouse CD8+T cell mAb intravenous injection only/
It, every 6 days repeated doses intraperitoneal injection is primary during furthermore testing.Cooperate with the regDC processing of CpG activation；
2) it NK cell in removing body: -2, -1,0,2 day, is activated using anti-mouse NK1.1mAb100 μ g/ pcs/day collaboration CpG
RegDC processing；
5. putting to death animal, tumor tissues are taken out, calculate tumour the widest part cross sectional area to assess size；
6. the regDC of visible CpG activation has the function of neoplasm growth, and has with NK cell and CD8+T cell
The effect of synergistic antitumor.
(5) cDC, NK cell and CD8+T cell is promoted to migrate (Figure 17) to tumor tissues
1. above-mentioned condition is intervened the 14th day, B16 tumor-bearing mice is put to death, it is thin to extract single core for collagenase digesting tumor tissues
Born of the same parents, cDC (traditional Dendritic Cells expresses CD11+, CD3-, CD19-) in airflow classification tumor tissues, NK cell (CD3-,
) and the number of CD8+T cell (volumetric tumor tissue per cubic centimeter) CD16+/CD56+；
2. PBS and unactivated regDC group, regDC processing group induction cDC, NK cell and CD8+ of CpG activation are compared in prompt
T cell is migrated to tumor tissues area.
Embodiment 5, irf4+irf8+ Regulatory Dendritic Cells joint DC-CIK carry out immunotherapy of tumors.(Figure 18)
The application dose of step 1.irf4+irf8+regDC and time
At first day of beginning DC-CIK treatment, infusion 106/kg swashing through CpG ODN containing 1%-99% was given through vein
The hematopoietic stem/progenitor cells mixture of irf4+irf8+regDC living；Start within second day, carry out the induction of DC-CIK cell, culture,
Amplification and feedback (according to routine clinical scheme).
The preparation of step 2. progress DC-CIK treatment
It acquires peripheral blood in patients 70ml (can also be acquired by blood separator), it is conventional to separate tunica albuginea layer mononuclearcell.
Physiological saline is divided into three parts after cell is resuspended, each 50ml.DC-CIK (the first course for the treatment of culture), DC-CIK (second course for the treatment of are done respectively
Culture, first freezes), effector T cell Fiber differentiation (two courses for the treatment of).It is centrifuged 2300rpm, 10min, after abandoning supernatant, with 2ml's
DC-CIK is resuspended in gt-t551 culture medium, and the X-15 culture medium of 40ml is added in effector T cell.
Induction, culture and the amplification of step 3. responsiveness T lymphocyte；
1) CD3 is coated with the preparation of bottle: the CD3 monoclonal antibody (1mg/ml) of 192ul being taken to be added to PBS (the PH 8.6- of 96ml
9.2) it, is added to after mixing in 8 T75 culture bottles, every bottle of 12ml, 4 ° of refrigerators is flat on after shaking up, rear can be used (one for 24 hours
A course for the treatment of needs 4 coating bottles).Before use, every bottle of 15ml salt water stands two minutes, wash twice；
2) cell suspension of 20ml X-15 culture medium+2.5ml is respectively added into coating bottle, is put into incubator culture；It is remaining
20ml cell suspension be placed in super-clean bench and (not covered tightly from lid greatly), used after 6h；
3) anti-CD28 (being below final concentration) 2 μ g/mi, IL-2100U/ml, IL-1210ng/ml is added after 3h,
IL-410μg/ml。
4) 20ml cell suspension remaining in super-clean bench is averagely added in 8 culture bottles after 6h, after being put into incubator
Continue to cultivate；
5) culture medium is collected after 22h and bottom of bottle cell, salt water wash twice, cultivate 8 after 2300rpm, 10min centrifugation
Bottle inner cell merges in 1 centrifuge tube；
6) supernatant is removed into cell suspension centrifugation, 20ml X-15 is added, cell is resuspended, and 1 μ g/ml of CD3 monoclonal antibody is added,
Anti-CD281 μ g/ml, IL-2 (10 μ g/L), IL-7 (10 μ g/L), to (each culture bottle respectively contains 80- in 8 T175 bottles
100ml X-15+8ml blood plasma), it is placed in incubator and cultivates；
7) growing state of cell, the 4-5 days freeze-stored cells are observed.
The preparation of step 4.DC-CIK；
1) prepare 3 T75 bottle, respectively label DC, CIK, CIK, to being added 19ml1640 in DC bottles, in CIK bottles of another two
It is each that 19ml T551 culture medium+1ml blood plasma is added；
2) by the DC-CIK (2ml cell suspension) of first course for the treatment of of separator well with 700ul, 600ul's, 600ul adds respectively
Enter into DC bottles and two CIK bottles；
3) to being put into incubator culture after being separately added into the rhIFN- γ 1000u/ml of 20ul in two CIK bottles；
Induction, culture and the amplification of step 5.DC (DC here refers to traditional Dendritic Cells cDC)
1) it is put into after incubator 1.5h and changes liquid for cell suspension DC bottles, 14ml gt-t551 (containing 300U/LIL-2) culture is added
Base+1ml blood plasma+15ul 1000U/ml IL-4+10.65ul1000U/L GM-CSF (cell face is not blown and beaten when addition,
DC is attached cell)；
2) the 3rd day, 1ml blood plasma+15ul 1000U/ml IL-4+15ul 1000U/L GM-CSF is added；
3) the 5th day, immature DC harvest (vertical culture):
Cell face is blown and beaten with the culture medium in culture bottle, salt water wash bottle 2 times, is centrifuged 1500rpm, 6min removes supernatant, draws
10ml culture medium, 8ml are added in 1 T75 bottles, and 2ml is added in centrifuge tube, and cell is resuspended；
4) 10ul 1000U/ml IL-4+10ul 1000U/L GM-CSF+10ul10ng/ is separately added into centrifuge tube
Ml TNF-α+500ul blood plasma, is transferred in T75 bottles and cultivates vertically in incubator；
5) the 7th day, maturation DC is harvested:
Culture medium blows and beats bottom of bottle, with 10ml salt water wash bottle bottom 2 times, by media transfer into 50ml centrifuge tube, is transferred to
In centrifuge tube, centrifugation, 1500rpm, 8min remove supernatant, are added 2ml gt-t551 (IL-2 containing 300U/L), mix, are infused with 5ml
Emitter is transferred in DC-CIK culture bag.
Induction, culture and the amplification of step 6.CIK cell:
1) 4.-3 is met on), for 24 hours after complement factor: 20ul 300U/L IL-2,60ul100U/L IL-1 α, 20ul
Anti-CD3 antibody 50ng/L；
2) the 4th day, 19ml gt-t551 (IL-2 containing 300U/L) and 1ml blood plasma is added, is placed in and continues to train in incubator
It supports；
3) the 6th day, turn bag (final system is 240ml): by the media transfer in 2 bottles CIK bottles into culture bag, by suitable
Following liquid are added in sequence: 15ml GT-T551 (IL-2 containing 300U/L) (after wash bottle), 8ml blood plasma (5%), 152ml T551 (contain
300U/L IL-2), final system is 240ml in culture bag.Nut cap steps up pipe, is placed in incubator and cultivates；
4) the 8th day, liquid 480ml (5% blood plasma+gt-t551 culture medium (containing i1-2)) is filled out, final system is 720ml；
5) the 10th day, liquid 720ml (5% blood plasma+gt-t551 culture medium (containing i1-2)) is filled out, final system is that 1440ml (takes out
5ml in bag taking keeps sample, while feeding back in the first time that this day carries out effector T cell)
The feedback of step 7.DC-CIK:
1) DC-CIK is fed back: 1 × 10^9/ times, 4 times/course for the treatment of.It takes out part every time to feed back, remainder addition culture medium continues
Culture.
2) after shaking up culture bag, the culture solution of 500ml is poured out in 10 50ml centrifuge tubes, and leave and take 200ul and be used for
It counts, centrifugation, 2000rpm, 8min；
3) after being centrifuged, supernatant is removed, cell, 10 pipes and 1 pipe is resuspended with 10ml salt water, then washed pipe 2 times with 10ml salt,
Salt water is supplemented to 50ml；
4) it is centrifuged, 2000rpm, 8min remove supernatant, draw 3ml salt water with liquid-transfering gun and blow even cell, then draw 2ml salt water
Wash pipette tips, supplement salt water to 50ml；
5) it is centrifuged, 1800rpm, 8min, supernatant keep sample (endotoxin detection), take 3ml salt water from centrifuge tube with liquid-transfering gun
Even cell is blown, then 1ml salt is taken to wash pipette tips (2 times), 1ml blood plasma is added, 200ul 300U/L IL-2 (salt water mixing) is mixed,
Cell suspension is transferred in 100ml transfering bag with 20ml syringe；
6) 10ml salt washing pipe is taken, is then transferred in 100ml transfering bag, supplements 80ml salt water into 100ml transfering bag, heat
Close pipe, sample presentation；
7) suitable GT-T551 (IL-2 containing 300U/L) (first time 280ml is added into culture bag；It adds for 2nd time
180ml, adds 100ml the 3rd time) it mixes, 5ml culture solution bacterial examination is taken, nut cap is put in incubator and cultivates.
Step 8. effector T cell is fed back
1) effector T cell is fed back: totally three courses for the treatment of, and 8 times/course for the treatment of, 1 × 10^9/ times.Cell is expanded to the 4-5 days
Afterwards, freeze-stored cell, recovery before feeding back；The effector T cell to thaw is transferred to greatly from, is mixed, centrifugation, 1200rpm,
6min；
2) supernatant is removed, 20ml salt water is added and mixes, centrifugation, 1200rpm, 6min；
3) supernatant keeps sample (endotoxin detection), takes 2ml salt water that cell is resuspended, then 7ml salt is taken to wash pipette, and 1ml7 is added
Number liquid；Cell suspension is transferred in 100ml transfering bag with syringe；Leave and take 20ul cell suspension for count and vigor；
4) it to pipe is washed from middle addition 10ml salt greatly, is transferred in 100ml transfering bag with syringe, then mended into transfering bag
Salt water 40ml is filled, heat seal pipe prepares to feed back.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Within the technical scope of the present disclosure, any changes or substitutions that can be easily thought of by anyone skilled in the art,
It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection of claims
Subject to range.
Claims (4)
1. a kind of preparation method of Regulatory Dendritic Cells, which is characterized in that steps are as follows:
(1) mammalian mesenchymal stem cell and candidate stem cell are separated；
(2) under conditions of suitable cell is grown and is proliferated, the mammalian mesenchymal obtained in co-incubation step (1) is dry thin
Born of the same parents and candidate stem cell；
The method of co-cultivation is as follows:
1) opportunity co-cultured, 12h is first with 2 × 10 before co-cultivation4The cell concentration of/ml, the 3rd generation that conventional method is obtained
Mescenchymal stem cell is inoculated in 24 orifice plate cultures of addition and the CD34+ Hematopoietic Stem ancestral isolated and purified is added after 12h so that its is adherent
Cell is co-cultured；
2) two kinds of cell number ratios: the ratio between mescenchymal stem cell and hematopoietic stem/progenitor cells are 1: 5~1: 10；
3) condition: streptomysin containing 100U/ml, the RPMI-1640 complete medium of 100U/ml penicillin, 5%CO is co-cultured2, 37
DEG C constant temperature incubation；
In incubation, to prevent Derived from Mesenchymal Stem Cells and apoptosis, replaced with new mescenchymal stem cell within every 3-4 days, tool
Body method are as follows: suspension cell is first sucked out, is placed in centrifuge tube；It is filled between being attached to 0.125% trypsase+0.01%EDTA digestion
Cell on matter stem cell；Dead cell and mescenchymal stem cell are removed with Ficoll；The cell of acquisition and suspension cell are mixed,
It is counted after washing, and is added and posts 24 orifice plates of new mescenchymal stem cell and continue to cultivate；
4) it after co-incubation 7 days, recycled from suspending nutrient solution, identify obtained Regulatory Dendritic Cells；
The Regulatory Dendritic Cells that recycling obtains in the step 4) are that the immature modulability dendron shape of irf4+irf8+ is thin
Born of the same parents；
The Regulatory Dendritic Cells are converted into mature Regulatory Dendritic Cells under the induction of CpG ODN, wherein irf4+
And the expression of irf8+ increases, and has immunoloregulation function；
The induced maturation method of Regulatory Dendritic Cells are as follows: co-culture the regDC of acquisition with every hole 5-10 × 104A cell is dense
Degree is inoculated in 96 orifice plates, continues culture 3 days with the X-VIVO-15 serum free medium that 10 μ g/ml of ODN containing CpG is incubated for 6h,
I.e. maturation regDC after harvest suspension cell；
Regulatory Dendritic Cells through CpG ODN induced maturation can promote T cell and NK cell Proliferation；
Regulatory Dendritic Cells through CpG ODN induced maturation can promote T cell and NK cell secretion of gamma-IFN；
Regulatory Dendritic Cells through CpG ODN induced maturation are able to suppress tumour growth.
2. preparation method according to claim 1, which is characterized in that the mammalian mesenchymal stem cell is isolated from tire
Disk, marrow, adipose tissue.
3. preparation method according to claim 2, which is characterized in that the mammalian mesenchymal stem cell is to train in vitro
It supports to the mescenchymal stem cell in the 1st to 10 generation.
4. the Regulatory Dendritic Cells obtained using any preparation method of claim 1-3, which is characterized in that described
Contain the Regulatory Dendritic Cells of 1%-99% in suspending nutrient solution.
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| "CpG ODN促进树突状细胞成熟的实验研究";李岩 等;《四川省理科学杂志》;20031231;第25卷(第1期);第21页 * |
| "MEF来源的间充质干细胞诱导生成的调节型树突状细胞的基因表达谱分析";葛超卓 等;《基础医学与临床》;20140430;第34卷(第4期);第433-438页 * |
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