CN105535952B - Prepare kit, method and the application of efficient novel self DC vaccine - Google Patents
Prepare kit, method and the application of efficient novel self DC vaccine Download PDFInfo
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Abstract
The invention discloses kit, method and the applications of a kind of efficient novel self DC vaccine, belong to field of biotechnology.The kit of the invention includes following ingredient: the combination of cytokines of GM-CSF, IL-4, FLT3-L and ATRA.The present invention is by improving existing GM-CSF+IL-4 culture technique, use in conjunction FLT3-L promotes pDC to generate, increase mononuclearcell and converts DC, MDSC is inhibited to generate using all-trans retinoic acid (ATRA) simultaneously, eliminate negativity adjustment effect of the MDSC in DC vaccine preparation, and using OK432 and CPH ODN costimulation mode, mDC and pDC maturation is effectively facilitated, DC immunostimulation function is enhanced.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of kit for preparing efficient novel self DC vaccine,
Method and application.
Background technique
" world's cancer report in 2014 " that the international cancer research institution of World Health Organization subordinate delivers is pointed out, the whole world
Cancer patient's quantity just increases at an amazing speed.2012, the newly-increased cases of cancer in the whole world estimated about 14,000,000, it is contemplated that 20
This number will rise to 22,000,000 in year.Same period number of cancer deaths also will soar to 13,000,000 from annual 8200000.It is pernicious swollen
The current primary treatments of tumor include operation, radiotherapy, chemotherapy and targeted therapy etc..Even however taking multidisciplinary synthesis
The death rate of the mode for the treatment of, cancer still remains high.Novel Cancer Treatment Regimens urgently develop.
The immunization therapy of tumour is chosen as first of 2013 annual ten big sciences discoveries by " SCIENCE " magazine.Dendritic Cells
As the internal the only known antigen presenting cell (APC) that can activate Resting T cells ability, MHCI molecule-antigen can be passed through
The mode of peptide complexes and MHCII molecule-antigen peptide complexes activates corresponding T lymphocyte, plays specific immune response
Antineoplastic action.Meanwhile DC also can be by discharging interleukin-22 (IL-2), IFN-γ (interferon gamma), IFN-α (interferon
α), natural killer cells (NK) is activated, plays nospecific immunity antitumor mechanism.Important work of the DC in antineoplastic immune
With so that it becomes one of the hot spot in immunization therapy.
However, the intracorporal DC of tumor patient, is influenced by panimmunity restraining factors, it is difficult to play its antitumor work
With.Immunoregulation cell (the inhibition cell (MDSC) of derived from bone marrow, regulatory T cells (Treg) etc.), immune negative-feedback regu- lation
The factor (IL-10 (IL-10), transforming growth factor β (TGF-β) etc.), immune detection point (programmed death receptor 1 and its are matched
Body (PD-1/PD-L1), Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) etc.) DC induction can be reduced through a variety of ways
Anti-tumor function.
Even cultivating the DC of tumor patient marrow, the source peripheral blood mononuclear cells (PBMCs) in vitro, nevertheless suffer from
The influence of immunosuppression mechanism.The precursor of the preparation method of DC vaccine at present, DC passes through the immune of CD14 monoclonal antibody
Magnetic bead sorting or the adherent acquisition of mononuclearcell, then (culture medullary system DC, mDC) or FLT3-L are used based on GM-CSF
The training method of (culture Plasmacytoid DC, pDC), after waiting antigen loads, using toll-like receptor (TLR) agonist stimulate its at
It is ripe.However in tumor patient DC vaccine preparation, GM-CSF or FLT3-L can promote DC precursor cell differentiation for MDSC.Part
TLR agonist can also promote the generation of MDSC, play immune negative-feedback regu- lation effect.MDSC can inhibit the maturation of DC, reduce it
The functions such as antigen presentation, migration, inducing T cell release IFN-γ.MDSC passes through arginase -1 (ARG-1), inductivity nitrogen oxygen
The proliferation and activation of the inhibition T cell such as compound synthesis enzyme (iNOS), reactive oxygen species (ROS), and IL-10, TGF-β can be discharged etc.
Promote the amplification of Treg cell.Influence of the MDSC to DC vaccine how is eliminated, the key that whether can prepare efficient DC vaccine is become.
Effect of the pDC in DC vaccine, also gradually causes to pay close attention to.Prematurity pDC mainly plays the work of inducing immune tolerance
With mature pDC can promote mDC maturation, induced NK cell activation, play antigen presentation effect by discharging a large amount of IFN-α
CTL and Th1 cell is stimulated to generate antineoplastic specificity immune response；The immunostimulation for how playing pDC needs further
Research.
In addition, the mechanism of action of programmed death receptor (PD-1) and its ligand (PD-L1) in DC vaccine also gradually obtains
To illustrating.On the one hand the activation of TLR enhances the ability of the antigen presentation inducing T cell specific immune response of DC, while
The expression that PD-L1 is resulted in DC increases.PDL-1 is combined with expression in the PD-1 of CTL, Th1 cell surface, causes these thin
Apoptosis, negative feedback inhibition T cell specific immune response occur for born of the same parents.In the preparation process of DC vaccine, PD-1/PD-L1 is blocked
Can signal path enhance the antitumor mechanism of DC vaccine, it is also desirable to further explore.
Therefore, it is necessary to provide a kind of negativity adjustment effect that can eliminate MDSC in DC vaccine preparation, enhancing mDC or
The efficient DC tumor vaccine of pDC immunostimulation function.
Summary of the invention
The technical problem to be solved in the present invention is to provide one kind can eliminate negativity adjusting of the MDSC in DC vaccine preparation
It effect, the kit that can prepare efficient novel self DC vaccine of enhancing mDC or pDC immunostimulation function, method and answers
With.
In order to solve the above technical problems, present invention offer technical solution is as follows:
On the one hand, a kind of kit for being used to prepare efficient novel self DC vaccine is provided, the kit includes such as
Lower ingredient: the combination of cytokines of GM-CSF, IL-4, FLT3-L and ATRA.
That kit of the invention can be used normal person source or autologous patient peripheral blood, Cord blood or marrow etc. come
The mononuclearcell in source carries out adhere-wall culture, obtains the attached cell of PBMCs, combination of cytokines induction through the invention
Immature DC is generated, then promotes DC mature using mixing TLRs agonist.The present invention is by cultivating skill to existing GM-CSF+IL-4
Art improves, and use in conjunction FLT3-L promotes pDC to generate, and increases mononuclearcell and converts DC, while utilizing total trans dimension first
Sour (ATRA) inhibits MDSC to generate, and eliminates negativity adjustment effect of the MDSC in DC vaccine preparation, effectively enhances pDC immunostimulation
Function.
Further, the kit further includes the TLRs agonist of OK432 and CPG ODN mixing.The present invention also combines
TLRs (OK432+CPG ODN) promotes DC (pDC, mDC) mature, increases DC transfer ability, enhances it and induces CTL and TH1 etc. thin
Born of the same parents generate specific immunity and kill antitumor ability.
Preferably, the kit include following ingredient: 500-1000U/ml GMCSF, 500-1000U/ml IL-4,
The combination of cytokines of 50-200ng/ml FLT3-L and 1-5 μM of ATRA.
Preferably, the kit further includes the TLRs excitement of 0.1-5KE/ml OK432 and 1-5 μM of CPG ODN mixing
Agent；Wherein, the CPG ODN is one kind of CPG ODN21798, CPG ODN2336, CPG ODN2006 or CPG ODN2395
Or it is a variety of；The CPG ODN is preferably CPG ODN21798.
On the other hand, a kind of method for preparing efficient novel self DC vaccine using mentioned reagent box is provided, including such as
Lower step:
1) preparation of mononuclearcell: under aseptic condition, obtaining mononuclearcell from peripheral blood, Cord blood or marrow,
It is washed again with physiological saline or Hanks liquid, 1000rpm is centrifuged 10 minutes, is inhaled and is abandoned supernatant, and precipitating is PBMCs；
2) preparation of DC precursor: PBMCs is in culture bottle, and 37 DEG C, 5%CO2Under the conditions of be incubated for 1-2h, inhale on abandoning
Clearly, half adherent PBMCs is the cell subsets for including DC precursor；
3) culture of immature DC: half adherent PBMCs GM-CSF+IL-4+FLT3-L+ATRA+ autologous plasma is trained
Feeding culture medium culture obtains immature DC.
4) immature DC will be obtained and carries out antigen load, stimulated using the TLRs agonist of OK432 and CPG ODN mixing
Make DC mature for 24 hours.
Wherein, the CPG ODN is CPG ODN21798, CPG ODN2336, CPG ODN2006 or CPG ODN2395
It is one or more；The CPG ODN is preferably CPG ODN21798.
That kit of the invention can be used normal person source or autologous patient peripheral blood, Cord blood or marrow etc. come
The mononuclearcell in source carries out adhere-wall culture, obtains the attached cell of PBMCs, combination of cytokines induction through the invention
Immature DC is generated, then promotes DC mature using mixing TLRs agonist.The present invention is by cultivating skill to existing GM-CSF+IL-4
Art improves, and use in conjunction FLT3-L promotes pDC to generate, and increases mononuclearcell and converts DC, while utilizing total trans dimension first
Sour (ATRA) inhibits MDSC to generate, and eliminates negativity adjustment effect of the MDSC in DC vaccine preparation, effectively enhances pDC immunostimulation
Function；The present invention also combines TLRs (OK432+CPG ODN), promotes DC (pDC, mDC) mature, increases DC transfer ability, enhancing
It induces the cells such as CTL and TH1 to generate specific immunity and kills antitumor ability.
Preferably, the TLRs agonist is the TLRs for including 0.1-5KE/ml OK432 and 1-5 μM of CPG ODN mixing
Agonist；Alternatively, the TLRs agonist is the TLRs excitement for including 0.1-5KE/ml OK432 and 1-5 μM of CPG ODN mixing
Agent, and further include POLY (IC), TNF-α, IFN-γ, other types in the TLRs agonist of OK432 and CPG ODN mixing
CPG ODN's etc. is one or more.On the basis of both OK432 and CPG ODN use in conjunction, other stimulations can also be added
The use in conjunction of the other Type C PG ODN of object such as POLY (IC), TNF-α, IFN-γ etc..
Further, the culture medium in the step 3 is to include 500-1000U/ml GMCSF+500-1000U/ml IL-
The culture medium of 4+50-200ng/ml FLT3-L+1-5uM ATRA+1-5% autologous plasma is thin by DC precursor with the culture medium
After born of the same parents' subgroup culture 4-5 days, 100ug/ml antigen load.
Further, the preparation method of mononuclearcell is under aseptic condition, by peripheral blood, Cord blood in the step 1)
Or bone marrow cell 50-100ml, or peripheral blood mononuclear cells subgroup 80-100ml is singly adopted, it is transferred in centrifuge tube, is added isometric
Normal saline dilution, then gone to above lymphocyte separation medium by 2:1 volume or 1:1 volume, 2000 turns, it is slow to rise slowly
Drop is centrifuged 20-30 minutes, draws tunica albuginea layer, as mononuclearcell.The separation of mononuclearcell can use the centrifuge tube of 50ml
It is centrifuged.
In another aspect, providing a kind of efficient novel self DC vaccine prepared using the above method.
Finally, providing a kind of efficient novel self DC vaccine using above method preparation to prepare antineoplastic immune anti-
The application in drug answered.
In conclusion beneficial effects of the present invention show themselves in that
That kit of the invention can be used normal person source or autologous patient peripheral blood, Cord blood or marrow etc. come
The mononuclearcell in source carries out adhere-wall culture, obtains the attached cell of PBMCs, combination of cytokines induction through the invention
Immature DC is generated, then promotes DC mature using mixing TLRs agonist.The present invention is by cultivating skill to existing GM-CSF+IL-4
Art improves, and use in conjunction FLT3-L promotes pDC to generate, and increases mononuclearcell and converts DC, while utilizing total trans dimension first
Sour (ATRA) inhibits MDSC to generate, and eliminates negativity adjustment effect of the MDSC in DC vaccine preparation, and use OK432 and CPH
ODN costimulation mode effectively facilitates mDC and pDC maturation, enhances DC immunostimulation function.
Detailed description of the invention
Fig. 1 is mDC, pDC co-culture system based on GM-CSF and FLT-3 of one each group of the embodiment of the present invention
The percentage of mDC, pDC and MDCS cell subsets；
Fig. 2 is each group DC80 maturation phenotype with Flow cytometry of two each group of the embodiment of the present invention；
Fig. 3 is each group DC83 maturation phenotype with Flow cytometry of two each group of the embodiment of the present invention；
Fig. 4 is each group DC86 maturation phenotype with Flow cytometry of two each group of the embodiment of the present invention；
Fig. 5 is the expression of the PD-L1 of each group DC with Flow cytometry of two each group of the embodiment of the present invention；
Fig. 6 is the CCR-7 expression of each group DC of three Western Blot method of embodiment of the present invention detection；
Fig. 7 is each subgroup percentage after the vaccine-induced T lymphocyte of DC of three each group of the embodiment of the present invention is Proliferative Activated.
Specific embodiment
To keep the embodiment of the present invention technical problems to be solved, technical solution and advantage clearer, below in conjunction with
Drawings and the specific embodiments are described in detail.But the present invention is limited to absolutely not these examples.As described below is only that the present invention is preferable
Embodiment, be only used to explain the present invention, it cannot be understood as the limitations of the invention patent range.It should be understood that
It is that any modifications, equivalent replacements and improvementsmade within the spirit and principles of the invention, etc. should be included in the present invention
Protection scope within.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Embodiment one
DC is cultivated using GM-CSF+IL-4+FLT3-L+ATRA combination of cytokines
It is prepared as follows efficient novel self DC vaccine, is included the following steps:
1) preparation of mononuclearcell:, which extracting tumor patient 50-100ml peripheral blood, adopts under aseptic condition or singly patient peripheral
The about 80-100ml such as blood mononuclear cell, can be transferred in 50ml centrifuge tube, isometric normal saline dilution be added, then press 2:1
Volume or 1:1 volume are gone to above lymphocyte separation medium, 2000 turns, slow to rise slow drop (rising 1 drop 0), and 20-30 points of centrifugation
Clock is drawn tunica albuginea layer, as mononuclearcell, then is washed 2 times with physiological saline or Hanks liquid, and 1000 turns are centrifuged 10 minutes,
It inhales and abandons supernatant, precipitating is PBMCs；
2) preparation of DC precursor: PBMCs is in culture bottle, and 37 DEG C, 5%CO2Under the conditions of be incubated for 1-2h, inhale on abandoning
Clearly, half adherent PBMCs is the cell subsets for including DC precursor；
3) it is 3 groups by obtained in step 2 half adherent PBMCs points:
Group 1: the culture of 500-1000U/ml GM-CSF+500-1000U/ml IL-4+1-5% autologous plasma culture is utilized
Immature DC is obtained after base 4-5 days；
Group 2: 500-1000U/ml GM-CSF+500-1000U/ml IL-4+50-200ng/ml FLT3-L+1- is utilized
Immature DC is obtained behind culture medium 4-5 days of 5% autologous plasma culture；
3 (immature DCs of the invention) of group: 500-1000U/ml GM-CSF+500-1000U/ml IL-4+50- is utilized
Immature DC is obtained behind culture medium 4-5 days of 200ng/ml FLT3-L+1-5uM ATRA+1-5% autologous plasma culture；
4) immature DC of group 1, group 2, group 3 is used into 100ug/ml antigen load for 24 hours respectively, 500-2000U/ml TNF-α
Stimulation is for 24 hours.
Utilize group 1, group 2,3 immature DCs of group, MDSC cell quantity and the phenotype in Flow cytometry step 4)；Knot
Fruit is as shown in Figure 1: for group 2 compared with group 1, GM-CSF and FLT3 use in conjunction can be with higher prematurity mDC, and can significantly mention
The percentage of the yield of high prematurity pDC, the output increased of prematurity pDC about 15%, MDSC does not have significant change；Group 3 with
Group 2 is compared, and GM-CSF and FLT3 use in conjunction and the percentage that MDSC not only can be significantly reduced under the action of ATRA make
MDSC is almost eliminated, and can be improved the yield of prematurity mDC and pDC, and prematurity pDC is especially apparent, and is improved
10%；Further, for group 3 compared with group 1, the use in conjunction of GM-CSF, FLT3 and ATRA can significantly improve immature DC,
Prematurity mDC improves 10%, and prematurity pDC improves 25%, and 3 prematurity pDC subgroup percentages of group are as many as 2 times of group 1,
The immunostimulation function of pDCd is greatly improved, and keeps MDSC secretory volume extremely low, largely eliminates MDSC in DC vaccine system
Negativity adjustment effect in standby.It further illustrates and uses GM-CSF+IL-4+FLTL-3+ATRA combination of cytokines culture DC, energy
More immature DCs are enough obtained, and MDSC generates decline.
IL-4 cell factor also could alternatively be other cell factors, such as IFN-α.
Embodiment two
By half adherent PBMCs 500-1000U/ml GM-CSF+500-1000U/ml in the step 2) of embodiment one
Prematurity is obtained behind culture medium 4-5 days of IL-4+50-200ng/ml FLT3-L+1-5uM ATRA+1-5% autologous plasma culture
DC obtains maturation DC as follows:
1) for 24 hours by the immature cell 100ug/ml antigen load of acquisition；
2) by above-mentioned steps 1) in cell be divided into 2 groups:
Group 1: for 24 hours with the stimulation of 500-2000U/ml TNF-α；
Group 2: it is stimulated for 24 hours with the TLRs agonist of 0.1-5KE/ml OK432+1-5uM CPG ODN2336 mixing；
Group 3: it is stimulated for 24 hours with the TLRs agonist of 0.1-5KE/ml OK432+1-5uM CPG ODN2006 mixing；
Group 4: it is stimulated for 24 hours with the TLRs agonist of 0.1-5KE/ml OK432+1-5uM CPG ODN2395 mixing；
Group 5:(DC vaccine of the present invention) it is exciting with the TLRs of 0.1-5KE/ml OK432+1-5uM CPG ODN21798 mixing
Agent stimulates for 24 hours；
Using the DC maturity symbol object of each group in Flow cytometry step 2), that is, the mature DC phenotype of each group is detected,
Mainly there is the expression quantity of CD83, CD80, CD86 of directive significance to be detected to mature DC phenotype, as shown in Figure 2,3, 4:
Compared with group 1, the percentage of mature DC phenotype is significantly increased group 2,3,4,5.OK432 and CPG ODN stimulates DC maturation to imitate
Fruit is significant, hence it is evident that stimulates gained DC higher than traditional simple utilization TNF-α；
The inducer T lymphocyte apoptosis capacity for reflecting DC by the expression of detection PD-L1 ligand simultaneously, such as Fig. 5 institute
Show: the expression quantity of PD-L1 decreases on the group 2-5 and mature DC of group 1, wherein organizing 5 (OK432 and CPG ODN21798's
Use in conjunction) it is the most obvious, it reduces more than half (47%), expression quantity only 38% illustrates that the present invention prepares the technical side of DC
Case significantly reduces the expression quantity of PD-L1, can be substantially reduced the immunosuppressive action of PD-1/PD-L1.
Meanwhile the various levels of cytokine secretion of maturation DC are detected by Elisa, as shown in table 1.As can be seen from Table 1,
The DC of the present invention (group 5) preparation can secrete the cell factor that can more play nospecific immunity antitumor mechanism, such as group 5
DC cell IFN-α secretion level improve 5 times, IL-12p70 improves 4 times, while the DC secretion of the present invention (group 5) preparation
It is significantly reduced with immunosuppressive cell factor, as the secretory volume of IL-10 (compares with group 1, reduces close lower than 20pg/ml
80%), the secretory volume of TGF-β lower than 10pg/ml (with group 1 compare, reduce it is nearly 70%).
1 Elisa of table detects the levels of cytokine secretion of the mature DC of each group
In conclusion DC vaccine prepared by the present invention has higher cellular maturity, the more nospecific immunities of secretion
The cell factor and the less immunosuppressive factor of secretion of antitumor mechanism, this has great meaning for the anti-tumor function for increasing DC
Justice.
Can also be added again in the group 2-5 of above-described embodiment two following one or more stimulant POLY (IC), TNF-α,
IFN-γ, the maturity for improving DC also have certain contribution.
Embodiment three
Selection secretion IL-12p70 ability is most strong, PD-L1 expresses minimum OK432+CPGODN21798 combined stimulation method and obtains
To DC vaccine compared with the DC vaccine that TNF-α method obtains, detect maturation DC transfer ability and inducing T cell proliferation activation energy
Power.
It is 2 groups by the adherent PBMCs of half in the step 2) of embodiment one points:
1 (traditional DC vaccine) of group: after GM-CSF+IL-4 culture 4-5 days, for 24 hours, TNF-α stimulation is mature for antigen load
24h；
2 (DC vaccine of the present invention, DC vaccines identical with the group 5 in embodiment two) of group: with GM-CSF+IL-4+FLT3-L+
After ATRA is cultivated 4-5 days, 100ug/ml antigen load for 24 hours, then with OK432+CPG ODN21798 stimulates maturation；
The DC vaccine prepared and T cell are mixed for 24 hours.
Western Blot detects the expression quantity of CCR-7, as shown in fig. 6, the expression quantity of the CCR-7 of group 2 is apparently higher than group
1, illustrate that the DC maturity of 2 preparation of group is higher, further illustrates that DC prepared by the present invention has higher maturity and stronger
Transfer ability.
Flow cytometry T cell subgroup percentage, as shown in Figure 7: DC vaccine prepared by the present invention can be better
Inducing T cell proliferation activation after obtain have more preferable specific immune response cell subsets, as CTL cell subsets ratio with
Group 1 remains basically stable, and the percentage of Th1 cell subsets improves nearly 2 times, and nearly 40%；And drop the percentage of Th2 cell subsets
It is half lower, while DC vaccine prepared by the present invention can effectively reduce the ratio of immunosuppressant cell subgroup, such as Treg cell
Subgroup is almost 0 (compared with group 1, hence it is evident that reduce), can effectively reduce the effect of the immune negative feedback mechanism such as Treg cell.
Mtt assay detects T cell to the killing rejection ability of tumor cell line, and the cell poisoning intensity of traditional DC vaccine is only
5.6%, and the cell of DC vaccine prepared by the present invention kills intensity up to 20.5%, makes 4 times of traditional DC vaccine, present invention preparation
DC vaccine compared with traditional DC vaccine have stronger cell kill ability.
The self DC vaccine preparation technology DC obtained of new high-efficiency of the present invention, can preferably inducing T cell to tumour
Specific immune response, reduce the effect of the immune negative feedback mechanism such as Treg cell.
It should be pointed out that being used to prepare DC vaccine with the mononuclearcell of tumor patient is to reduce foreign cell
Immunological rejection, from prevent, treat or other purposes can also with the cell in normal cell people source prepare DC vaccine.
In conclusion the present invention prepares immature DC using GM-CSF and the big cultivating system of FLT3-L two, promoted using ATRA
It is converted into DC into MDSC, to effectively increase immature DC quantity, reduces MDSC content；Recycle OK432 and CPG ODN total
Stimulate DC mature.Technology of preparing of the invention promotes the maturation and migration of DC, while increasing DC release IL-12p70, IFN-
The secretion of α reduces the secretion of the negativity regulatory factor such as IL-10, TGF-β, reduces the generation of Treg cell, enhances DC induction
The ability of T lymphocyte specific immune response reduces the PD-L1 expression of maturation DC.
That kit of the invention can be used normal person source or autologous patient peripheral blood, Cord blood or marrow etc. come
The mononuclearcell in source carries out adhere-wall culture, obtains the attached cell of PBMCs, combination of cytokines induction through the invention
Immature DC is generated, then promotes DC mature using mixing TLRs agonist.The present invention is by cultivating skill to existing GM-CSF+IL-4
Art improves, and use in conjunction FLT3-L promotes pDC to generate, and increases mononuclearcell and converts DC, while utilizing total trans dimension first
Sour (ATRA) inhibits MDSC to generate, and eliminates negativity adjustment effect of the MDSC in DC vaccine preparation, effectively enhances pDC immunostimulation
Function.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of kit for being used to prepare self DC vaccine, which is characterized in that the kit includes culture medium, the culture
Base includes the cell of 500-1000U/mlGM-CSF, 500-1000U/mlIL-4,50-200ng/mlFLT3-L and 1-5 μM of ATRA
Combinations of factors and 1-5% autologous plasma.
2. kit according to claim 1, which is characterized in that the kit further includes OK432 and CPG ODN mixing
TLRs agonist.
3. kit according to claim 1 or 2, which is characterized in that the kit further includes 0.1-5KE/ml
The TLRs agonist of OK432 and 1-5 μM of CPG ODN mixing；Wherein, the CPG ODN is CPG ODN21798, CPG
ODN2336, CPG ODN2006 or CPG ODN2395's is one or more.
4. a kind of method for preparing self DC vaccine using any kit of claim 1-3, which is characterized in that including
Following steps:
1) preparation of mononuclearcell: under aseptic condition, mononuclearcell is obtained from peripheral blood, Cord blood or marrow, then use
Physiological saline or the washing of Hanks liquid, 1000rpm are centrifuged 10 minutes, are inhaled and are abandoned supernatant, and precipitating is PBMCs；
2) preparation of DC precursor: PBMCs is in culture bottle, and 37 DEG C, 5%CO2Under the conditions of be incubated for 1-2h, inhale and abandon supernatant, half pastes
The PBMCs of wall is the cell subsets for including DC precursor；
3) culture of immature DC: by half adherent PBMCs with including 500-1000U/ml GM-CSF+500-1000U/ml
The culture medium culture of IL-4+50-200ng/ml FLT3-L+1-5 μM ATRA+1-5% autologous plasma obtains immature DC；
4) immature DC will be obtained and carries out antigen load, stimulated using the TLRs agonist of OK432 and CPG ODN21798 mixing
Make DC mature for 24 hours, obtains the DC vaccine.
5. the method according to claim 4 for preparing self DC vaccine, which is characterized in that the TLRs agonist is to include
The TLRs agonist of 0.1-5KE/ml OK432 and 1-5 μM of CPG ODN21798 mixing；
Alternatively, the TLRs agonist be include 0.1-5KE/ml OK432 and 1-5 μM of CPG ODN21798 mixing TLRs swash
Dynamic agent, and further include POLY (IC), TNF-α, IFN-γ one in the TLRs agonist of OK432 and CPG ODN21798 mixing
Kind is a variety of.
6. the method according to claim 4 or 5 for preparing self DC vaccine, which is characterized in that the training in the step 3)
Supporting base is to include 500-1000U/ml GMCSF+500-1000U/ml IL-4+50-200ng/ml FLT3-L+1-5uM ATRA+
The culture medium of 1-5% autologous plasma is used in step 4) with the culture medium by after DC precursor subgroup culture 4-5 days
100ug/ml antigen load.
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