CN102154199B - Method for in-vitro preparation of semimature dendritic cells - Google Patents
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Abstract
The invention discloses a method for in-vitro preparation of semi-mature dendritic cells, which comprises: collecting suspension of mouse bone marrow cells, centrifuging, lysing, suspending cells in a Roswell Park Memorial Institute (RPMI) 1610 complete culture medium, adding recombinant murine granulocyte macrophage colony stimulating factor (rmGM-CSF) at a concentration of 10 to 20 mu g/L, recombinant murine interleukin 4 (rmIL-4) at a concentration of 2.5 to 10 mu g/L and Jagged-1/Fc at a concentration of 1 to 5mg/L, incubating, collecting loose anchorage-dependent cells, resuspending cells in a complete culture medium, adding the rmGM-CSF, rmIL-4 and Jagged-1/Fc in the same amount, collecting loose anchorage-dependent cells, adding the rmGM-CSF and rmIL-4 in half amount and Jagged-1/Fc in the same amount in the 6th to 8th day, and collecting loose anchorage-dependent cells, namely semi-mature DCsJagged-1. The cells are only treated by Jagged-1/Fc, and can reduce the immune response capability of allogeneic lymphocytes and help to induce immune tolerance.
Description
Technical field
The invention belongs to biology and medicine technology field, particularly a kind of preparation method of half ripe dendritic cell.
Background technology
Dendritic cell (DCs) is one of two large cores that form Cellular Immunity, different Dendritic cells subsets instruct the differentiation of different effect T cell, thereby are determining body immune system enforcement immunne response or two kinds of distinct functions of immunological tolerance in the identification exotic antigen.Large quantity research has proved immature dendritic cell (iDCs), and fully matured dendritic cell (mDCs) and half ripe dendritic cell (semi-mature DCs) play diverse decisive role in the mediation body immune system is exercised immunological tolerance or immunne response.From solving the purpose of tissue, organ-graft refection and treatment autoimmune disorder, people are making great efforts to find untiringly has the half ripe dendritic cell of inducing the body immunological tolerance.Therefore, its theory significance and clinical value are apparent.
Latest report, adopt half ripe dendritic cell prepared by different methods to have different characteristics, thereby have sufficient cause to infer that they induce also difference of the effect of body immunological tolerance and mechanism thereof.Up to now, reported the half ripe dendritic cell of 3 kinds of different qualities, i.e. semi-mature DCs
iL-10, semi-matureDCs
iL-6with semi-mature DCs
tNF-αand preparation method thereof.
Sato K etc., by separating people's peripheral blood mononuclear cell, cultivate 7d in vitro with GM-CSF (50ng/ml) and IL-4 (50ng/ml), then add TNF-a (50ng/ml) or LPS (100ng/ml) to continue to cultivate 3d; Also available IL-10 (50ng/ml) culturing cell 3d, then add TNF-a (50ng/ml) orLPS (100ng/ml) to continue to cultivate 3d, obtains semi-mature DCs
iL-10.Semi-mature DCs
iL-10main characteristic be that TNF-α, IL-6 and IFN-γ reduce, MHC-II, CD80 and CD86 also reduce.
Park SJ etc. cultivate bone marrow cells in mice with the perfect medium containing 10ng/ml GM-CSF or 0.5%GM-CSF supernatant in vitro, every 2d changes liquid with the DC substratum, abandon non-adherent cell, cultivate 6d, be divided into derived from bone marrow dendritic cell (BMDCs), stimulate with 50ng/ml IL-6 afterwards, then add 100ng/mlLPS, 40ng/ml TNF-α or 40 μ g/ml anti-CD40 antibody, obtain semi-mature DCs
iL-6.Semi-mature DCs
iL-6main characteristic be that TNF-α and IL-12 reduce, CD40, CD80 and CCR7 also reduce.
Menges M etc. uses the 10%GM-CSF supernatant inducing culture bone marrow cells in mice that contains of GM-CSF (200U/ml) or clone secretion to be divided into BMDCs in vitro, then add TNF-α (500U/ml), LPS (1 μ g/ml) or LPS associating anti-CD40 antibody (5 μ g/ml) are cultivated 24h, obtain semi-mature DCs
tNF-α.Semi-mature DCs
tNF-αmain characteristic be that TNF-α, IL-6 and IFN-γ reduce, MHC-II, CD80 and CD86 raise.
Above visible, the half ripe dendritic cell that adopts different methods to prepare has different cell phenotype characteristics and produces the cytokine of different levels, thereby effect and the mechanism thereof of pointing out them to induce the body immunological tolerance are totally different, and exact function still remains to be confirmed.Wherein, have need to be respectively with inducing ripe and suppressing ripe two kinds of factors or many factors removes to process iDCs, so that technology of preparing is more numerous, because depending on two kinds of antipodal factors of effect, cause the half ripe degree of dendritic cell to be difficult to control again, the mechanism of DCs differentiation and function are also just different.Therefore, we explore the technology of preparing of the novel half ripe dendritic cell subgroup of single controlling factors.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art, with not enough, provides a kind of external preparation method of half ripe dendritic cell, comprises the following steps:
(1) collect bone marrow cells in mice suspension, the centrifugal supernatant of abandoning on ice bath;
(2) the centrifugal cell obtained of step (1) is resuspended in phosphoric acid buffer (PBS), adds cell suspension volume 15-20 erythrocyte cracked liquid doubly, 37.0 ℃ of lucifuge effect 8-10min are then centrifugal;
(3) the centrifugal cell obtained of step (2) is resuspended in containing in RPMI 1640 perfect mediums of 10% foetal calf serum;
(4) add 10-20 μ g/L recombined small-mouse granulocyte-macrophage colony stimutaing factor (rmGM-CSF), 2.5-10 μ g/L recombined small-mouse interleukin-4 (rmIL-4) and 1-5mg/L restructuring chimeric protein (Jagged-1/Fc) in the cell suspension obtained in step (3);
(5) being placed in incubator hatches;
(6) every other day 1 time, repeat following operation: remove the upper strata suspension cell, collect loose attached cell, the centrifugal supernatant of abandoning, then with the resuspended centrifugal cell obtained of RPMI 1640 perfect medium containing 10% foetal calf serum, add the isodose rmGM-CSF of same step (4), rmIL-4 and Jagged-1/Fc, then be placed in incubator and continue to hatch;
(7) 6-8d start, and add the rmGM-CSF and the rmIL-4 that compare half-value dose with step (4), and Jagged-1/Fc dosage is constant, all the other same steps (6);
(8) 10-12d abandon the firm attached cell of bottom, the loose attached cell of results, half ripe dendritic cell (the semi-mature DCs that Jagged-1 induces
jagged-1).
In described step (1), (6), centrifugal condition is 4 ℃, 300 * g, centrifugal 3-5min.
The minimum volume that in described step (2), the add-on of phosphoric acid buffer is the energy re-suspended cell.
When centrifugal in described step (2) with cold PBS as washing soln, centrifuge washing 3 times, centrifugal condition is 4 ℃, 300 * g, centrifugal 3-5min.
In described step (3), the resuspended rear cell density of cell is 2-5 * 10
9/ L.
In described step (5), (6), (7), the interior condition of incubator is 37.0 ℃, 5%CO
2.
Mechanism of the present invention: already having proved that Jagged-1 was one of main part be present in mammalian cell Notch acceptor, is the single transmembrane glycoprotein, marrow, fetal livers stroma cell, thymic epithelial cells etc., expression is all arranged.The Jagged-1-Notch signal is maintaining hematopoietic stem/progenitor and thymocyte to the very important effect of performance in the differentiation of T, B and NK cell development.All there are the expression of Notch acceptor and Jagged-1 part in another discovery dendritic cell (DCs) and T lymphocytic cell surface, infer that the Jagged-1-Notch signal may regulate and control the differentiation and maturation of DCs, again because of this two kinds of cell surface mutual Notch acceptors and Jagged-1 part, imagine mutually combining of two kinds of iuntercellular parts and acceptor and realize intercellular information interchange for DCs and T lymphocytes interactions, thereby for instructing the T cytodifferentiation that basic substance is provided.Characteristic and DCs surface that the present invention just is being based on above-mentioned Jagged-1 regulating cell Development And Differentiation exist the discovery of Jagged-1 acceptor to design.
The present invention compared with prior art has following advantage and beneficial effect:
(1) preparation method of the present invention relies on Jagged-1/Fc and processes cell, and semi-mature DCs
iL-6, semi-mature DCs
iL-10with semi-mature DCs
tNF-αrely on respectively interleukin 6 (IL-6), interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-α) and process cell.
(2) above-mentioned semi-mature DCs be take inducing as basis of GM-CSF and/or IL-4, but semi-mature DCs
iL-6with semi-mature DCs
iL-10the processing of two kinds of factors of derived need, first with IL-6 or IL-10, process cell, then use TNF-α or bacteria lipopolysaccharide (LPS) etc. to process cell, namely need two or more factor associating.And semi-mature DCs
jagged-1only with Jagged-1/Fc, process cell.
(3) the semi-mature DCs that preparation method of the present invention obtains
jagged-1characteristic, comprise and the cytokine of cell phenotype and generation be different from semi-mature DCs
iL-6, semi-mature DCs
iL-10with semi-mature DCs
tNF-α.The full ripe DCs induced with classical way LPS compares, and proves semi-mature DCs
jagged-1in the half ripe state, its main characteristic is that the expression of major histocompatibility complex class I I quasi-molecule (MHC-II) and CD40 slightly raises, CD80 obviously raises, and the CD86 variation is not obvious, and the full ripe mDCs (Figure 1A, 1B) that significantly induces lower than classical way LPS of the expression level of these costimulatory moleculeses; The level of TNF-α obviously reduces, and interleukin 12 (IL-12) is slight to raise, and IL-4 significantly increases, and IL-6 and IFN-γ (IFN-γ) change not obvious.The Change Pattern of this cytokine is conducive to CD4
+t cells is to Th2 cells (Th2) and regulatory T cells (Treg) the direction differentiation (Fig. 2) of Promote immunity adjusting and immunological tolerance; Semi-mature DCs
jagged-1cause the ability of allogeneic lymphocyte immunne response to weaken (Fig. 3), be conducive to inducing immune tolerance.
(4) semi-mature DCs
jagged-1characteristic be different from the semi-mature DCs reported
iL-6, semi-mature DCs
iL-10with semi-mature DCs
tNF-α, depend primarily on and adopt different induction methods, be i.e. Jagged-1/Fc mediation.
The accompanying drawing explanation
Fig. 1 is the Lymphocytic phenotype figure that Jagged 1/Fc induces semi-mature DCs.A is that two positive cell per-cent changes, and B is that average fluorescent strength changes; * P<0.05, * * P<0.01, with corresponding control group relatively.
Fig. 2 is the cytokine variation diagram that Jagged 1/Fc induces semi-mature DCs.* P<0.05, compare with control group.
Fig. 3 is that Jagged 1/Fc induces the affect figure of semi-mature DCs on allogeneic lymphocyte immunne response ability.* P<0.05, * * P<0.01, with corresponding control group relatively.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but the working of an invention mode is not limited to this.
Embodiment mono-semi-mature DCs
jagged-1preparation
Balb/c (being provided by Guangdong Medical Lab Animal Center) is provided the dislocation of neck marrow, and the femur that aseptic separation is complete and shin bone are removed muscle and reticular tissue, put into 75% alcohol and soak 2min, and PBS rinses.Cut off the bone two ends, with filling the irrigation with syringe medullary space of cold PBS, piping and druming is placed in the bone marrow cell suspension of plate on ice bath gently, and 200 order stainless steel mesh screens filter, and collects bone marrow cell suspension centrifugal (4 ℃, 300 * g, 3min).Add 5ml erythrocyte cracked liquid (Sigma-Aldrich) after resuspended with the cold PBS of 300 μ l, 37.0 ℃ of lucifuge effect 8min, cold PBS washes (4 ℃, 300 * g, 3min) 3 times.Finally, cell is resuspended in containing in 10% foetal calf serum RPMI1640 perfect medium (GibcoBRL), and adjusting cell density is 2 * 10
9/ L.The every hole 2mL of cell suspension is inoculated in 6 orifice plates, adds respectively 20 μ g/L rmGM-CSF (Peprotech), 10 μ g/LrmIL-4 (Peprotech) and 5mg/L Jagged 1/Fc (R& D Systems), be placed in 37.0 ℃, 5%CO
2in incubator, hatch.1 time every other day, remove the upper strata suspension cell, collect loose attached cell, centrifugal (4 ℃, 300 * g, 3min) abandon supernatant.Use the perfect medium re-suspended cell, add respectively rmGM-CSF, rmIL-4 and the Jagged 1/Fc of Isodose.8d adds half-value dose rmGM-CSF and rmIL-4 after beginning to change liquid, and Jagged1/Fc dosage is constant.12d abandons the firm attached cell of bottom, results loose attached cell, i.e. semi-matureDCs
jagged-1.
Embodiment bis-semi-mature DCs
jagged-1preparation
The dislocation of neck marrow is provided by C57BL/6 mouse (being provided by Guangdong Medical Lab Animal Center), and the femur that aseptic separation is complete and shin bone are removed muscle and reticular tissue, put into 75% alcohol and soak 2min, and PBS rinses.Cut off the bone two ends, with filling the irrigation with syringe medullary space of cold PBS, piping and druming is placed in the bone marrow cell suspension of plate on ice bath gently, and 200 order stainless steel mesh screens filter, and collects bone marrow cell suspension centrifugal (4 ℃, 300 * g, 3min).Add 6ml erythrocyte cracked liquid (Sigma-Aldrich) after resuspended with the cold PBS of 300 μ l, 37.0 ℃ of lucifuge effect 9min, cold PBS washes (4 ℃, 300 * g, 3min) 3 times.Finally, cell is resuspended in containing in 10% foetal calf serum RPMI1640 perfect medium (GibcoBRL), and adjusting cell density is 2 * 10
9/ L.The every hole 2mL of cell suspension is inoculated in 6 orifice plates, adds respectively 10 μ g/L rmGM-CSF (Peprotech), 2.5 μ g/L rmIL-4 (Peprotech) and 1mg/L Jagged 1/Fc (R& D Systems), be placed in 37.0 ℃, 5%CO
2in incubator, hatch.1 time every other day, remove the upper strata suspension cell, collect loose attached cell, centrifugal (4 ℃, 300 * g, 3min) abandon supernatant.Use the perfect medium re-suspended cell, add respectively rmGM-CSF, rmIL-4 and the Jagged 1/Fc of Isodose.8d adds half-value dose rmGM-CSF and rmIL-4 after beginning to change liquid, and Jagged 1/Fc dosage is constant.12d abandons the firm attached cell of bottom, results loose attached cell, i.e. semi-mature DCs
jagged-1.
Embodiment tri-semi-mature DCs
jagged-1preparation
The dislocation of neck marrow is provided by C57BL/6 mouse (being provided by Guangdong Medical Lab Animal Center), and the femur that aseptic separation is complete and shin bone are removed muscle and reticular tissue, put into 75% alcohol and soak 2min, and PBS rinses.Cut off the bone two ends, with filling the irrigation with syringe medullary space of cold PBS, piping and druming is placed in the bone marrow cell suspension of plate on ice bath gently, and 200 order stainless steel mesh screens filter, and collects bone marrow cell suspension centrifugal (4 ℃, 300 * g, 3min).Add 4.5ml erythrocyte cracked liquid (Sigma-Aldrich) after resuspended with the cold PBS of 300 μ l, 37.0 ℃ of lucifuge effect 9min, cold PBS washes (4 ℃, 300 * g, 3min) 3 times.Finally, cell is resuspended in containing in 10% foetal calf serum RPMI1640 perfect medium (GibcoBRL), and adjusting cell density is 2 * 10
9/ L.The every hole 2mL of cell suspension is inoculated in 6 orifice plates, adds respectively 15 μ g/L rmGM-CSF (Peprotech), 7 μ g/L rmIL-4 (Peprotech) and 3mg/L Jagged 1/Fc (R& D Systems), be placed in 37.0 ℃, 5%CO
2in incubator, hatch.1 time every other day, remove the upper strata suspension cell, collect loose attached cell, centrifugal (4 ℃, 300 * g, 3min) abandon supernatant.Use the perfect medium re-suspended cell, add respectively rmGM-CSF, rmIL-4 and the Jagged 1/Fc of Isodose.8d adds half-value dose rmGM-CSF and rmIL-4 after beginning to change liquid, and Jagged 1/Fc dosage is constant.12d abandons the firm attached cell of bottom, results loose attached cell, i.e. semi-mature DCs
jagged-1.
Embodiment tetra-semi-mature DCs
jagged-1detection
(1) semi-mature DCs
jagged-1the semi-mature DCs that collects with cold PBS centrifuge washing embodiment mono-of phenotype analytical
jagged-13 times (4 ℃, 300 * g, 3min), the cold PBS re-suspended cell (5 * 10 of 100 μ L
5-1 * 10
6).Add respectively 0.25 μ g anti-CD11c-FITC (eBioscience) and 1.0 μ ganti-MHC-II-PE (eBioscience), 0.25 μ g anti-CD11c-FITC (eBioscience) and 0.125 μ g anti-CD86-FITC (eBioscience), 0.25 μ g anti-CD11c-FITC (eBioscience) and 0.06 μ g anti-CD80-PE-CY5 (eBioscience), 0.25 μ g anti-CD11c-FITC (eBioscience) and 0.25 μ g anti-CD40-APC (eBioscience) dyeing, establish 3 multiple holes for every group, mix gently rear 4.0 ℃ of lower lucifuges and place 30min.Through (4 ℃ of PBS washings 2 times, 300 * g, 3min), 100 μ L PBS are resuspended, slowly add 4% paraformaldehyde PBS solution 100 μ L on vibrator, FACSCalibur flow cytometer (Becton Dickinson) upper detect CD11c respectively with CD40, CD80, the two positive cell per-cent (Figure 1A) of CD86 and MHC-II and average fluorescent strength (Figure 1B).10000 cells of every pipe sample detection, institute's data that obtain are analyzed with CELLQuest software (Becton Dickinson), separately with classical way LPS, induce full ripe mDCs to make comparisons.From Figure 1A, with control group, compare, except CD86, semi-matureDCs
jagged-1the costimulatory molecules CD40 of cell surface reflection cell maturation degree and the expression of CD86 raise to some extent, the MHC-II molecule also raises to some extent, but the per-cent of CD40, CD86 costimulatory molecules and MHC-II molecule is all significantly lower than full ripe mDCs, the overall average fluorescence intensity of this and these molecules of cell surface expression match (Figure 1B), therefore, the variation of the phenotype of cell shows semi-mature DCs
jagged-1really in the half ripe state.
(2) semi-mature DCs
jagged-1cytokine analysis
Collect semi-mature DCs
jagged-1the cultured cell in vitro supernatant, induce the cells and supernatant of full ripe mDCs to make comparisons with classical way LPS, adopts mouse double antibodies sandwich ELISA test kit (JINGMEI BIOTECH) to detect semi-mature DCs
jagged-1the level of IL-4, IL-6, IL-12, TNF-α and IFN-γ in culture supernatant, concrete operations are undertaken by the test kit specification sheets, read absorbance finally by BIO-RAD 680 type microplate reader at the 450nm place, and calculate its concentration through typical curve.Fig. 2 demonstration, the level of TNF-α obviously reduces, and interleukin 12 (IL-12) is slight to raise, and IL-4 significantly increases, and IL-6 and IFN-γ (IFN-γ) change not obvious; Wherein, the DCs that the slight rising explanation Jagged-1 of IL-12 induces is in the half ripe state, and the level of TNF-α or IFN-γ reduces or be unchanged, is unfavorable for that the DCs that Jagged-1 induces instructs CD4
+the Th1 direction differentiation that T cells is replied to Promote immunity occurs that IL-4 significantly increases simultaneously, and the Change Pattern of this cytokine is conducive to CD4
+t cells is to Th2 and the differentiation of Treg direction of Promote immunity adjusting and immunological tolerance; The full ripe mDCs that the general layout of the DCs generation cytokine that Jagged-1 induces is induced from LPS produces the general layout fully different (Fig. 2) of cytokine.Generally acknowledged that the full ripe mDCs that LPS induces instructs CD4
+helper T cell 1 (Th1) the direction differentiation that T cells is replied to Promote immunity, therefore, semi-mature DCs
jagged-1the cytokine Change Pattern illustrate that its function is also in the half ripe state, is conducive to inducing immune tolerance.
(3) semi-mature DCs
jagged-1impact on lymph-node cell immunne response ability
BALB/C mice (Guangdong Medical Lab Animal Center provides) is put to death in the dislocation of neck marrow, under aseptic separation bilateral axillary regions, clavicle, inguinal region superficial lymph knot and mesenteric lymph nodes, remove tunicle, mechanical mill on ice bath, filter with 200 order stainless steel mesh screens.The collecting cell suspension, cold PBS centrifugal (4 ℃, 300 * g, 3min) washed cell 2 times, containing 10% foetal calf serum RPMI 1640 (GibcoBRL) perfect medium re-suspended cell, adjusting cell density is 2 * 10
9/ L, as reacting cells.Separately get semi-mature DCs prepared by the C57BL/6 bone marrow cells in mice by above-mentioned steps (1) item
jagged-1as irritation cell, irritation cell and reacting cells are pressed cell count 1: 5, and 1: 10, the ratio of 1: 20 was inoculated in 96 orifice plates, and cumulative volume 200 μ l, be placed in 37.0 ℃, 5%CO
2mixed culture 72h in incubator, add tetramethyl-azo azoles blue (MTT) (Sigma-Aldrich), making final concentration is 5mg/L, centrifugal supernatant (300 * the g that abandons after 4h, 5min), add 100 μ l DMSOs (DMSO), lucifuge is positioned over slight concussion 10min on micro oscillator, detects absorbancy (OD) value at the 540nm place by 680 type microplate reader (BIO-RAD).As shown in Figure 3, compare semi-mature DCs with control group
jagged-1cause the immunne response ability of allogeneic lymphocyte obviously to weaken, the fully matured DCs that LPS induces causes the immunne response ability of allogeneic lymphocyte to strengthen.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (6)
1. the external preparation method of a half ripe dendritic cell is characterized in that comprising the following steps:
(1) collect bone marrow cells in mice suspension, the centrifugal supernatant of abandoning on ice bath;
(2) the centrifugal cell obtained of step (1) is resuspended in phosphoric acid buffer, adds cell suspension volume 15-20 erythrocyte cracked liquid doubly, 37.0 ℃ of lucifuge effect 8-10min are then centrifugal;
(3) the centrifugal cell obtained of step (2) is resuspended in containing in RPMI 1640 perfect mediums of 10% foetal calf serum;
(4) add 10-20 μ g/L recombined small-mouse granulocyte-macrophage colony stimutaing factor rmGM-CSF, 2.5-10 μ g/L recombined small-mouse interleukin-4 rmIL-4 and 1-5mg/L restructuring chimeric protein Jagged-1/Fc in the cell suspension obtained in step (3);
(5) being placed in incubator hatches;
(6) every other day 1 time, repeat following operation: remove the upper strata suspension cell, collect loose attached cell, the centrifugal supernatant of abandoning, with the resuspended centrifugal cell obtained of RPMI 1640 perfect medium containing 10% foetal calf serum, add the isodose rmGM-CSF of same step (4), rmIL-4 and Jagged-1/Fc, then be placed in incubator and continue to hatch;
(7) 6-8 days start, and add the rmGM-CSF and the rmIL-4 that compare half-value dose with step (4), and Jagged-1/Fc dosage is constant, all the other same steps (6);
(8) 10-12 days abandon the firm attached cell of bottom, the loose attached cell of results, the half ripe dendritic cell that Jagged-1 induces.
2. a kind of external preparation method of half ripe dendritic cell according to claim 1, it is characterized in that: in described step (1), (6), centrifugal condition is 4 ℃, 300 * g, centrifugal 3-5min.
3. a kind of external preparation method of half ripe dendritic cell according to claim 1 is characterized in that: in described step (2), the add-on of phosphoric acid buffer is minimum volume that can re-suspended cell.
4. a kind of external preparation method of half ripe dendritic cell according to claim 1, it is characterized in that: when centrifugal in described step (2) with cold phosphoric acid buffer as washing soln, centrifuge washing 3 times, centrifugal condition is 4 ℃, 300 * g, centrifugal 3-5min.
5. a kind of external preparation method of half ripe dendritic cell according to claim 1, it is characterized in that: in described step (3), the resuspended rear cell density of cell is (2-5) * 10
9/ L.
6. a kind of external preparation method of half ripe dendritic cell according to claim 1 is characterized in that: in described step (5), (6), (7) in incubator condition be 37.0 ℃, 5%CO
2.
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| Title |
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| Kostianovsky A M,ET AL.UP-REGULATION OF GENE RELATED TO ANERGY IN LYMPHOCYTES IS ASSOCIATED WITH NOTCH-MEDIATED HUMAN T CELL SUPPRESSION.《J IMMUNOL》.2007,第178卷6158-6163. * |
| 邢飞跃,等.可溶性Jagged 1/Fc 嵌合蛋白诱导髓系树突状细胞的分化和成熟.《科学通报》.2008,第53卷(第1期),89-95. * |
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