CN105535952A - Kit of high-efficiency novel DC autovaccine, method for preparing high-efficiency novel DC autovaccine and application of DC autovaccine - Google Patents
Kit of high-efficiency novel DC autovaccine, method for preparing high-efficiency novel DC autovaccine and application of DC autovaccine Download PDFInfo
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Abstract
The invention discloses a kit, method and application of a high-efficiency novel DC autovaccine, and belongs to the technical field of biology. The kit comprises a cytokine combination of GM-CSF, IL-4, FLT3-L and ATRA. According to the method, an existing GM-CSF+IL-4 cultivation technique is improved, is combined with FLT3-L to promote generation of pDC, mononuclear cells are increased to convert DC, generation of MDSC is inhibited by utilizing all-transretinoic acid (ATRA) to eliminate the negative adjustment effect of MDSC in preparation of DC vaccines, and an OK432 and CPH ODN co-stimulation mode is adopted to effectively promote mature of mDC and pDC and enhance the DC immunostimulation function.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of prepare efficient novel autologous DC vaccine test kit, method and application.
Background technology
" world's cancer report in 2014 " that the international cancer research institution of World Health Organization (WHO) subordinate delivers is pointed out, global cancer patient's quantity increases just with surprising rapidity.2012, the newly-increased cases of cancer in the whole world was estimated about to reach 1,400 ten thousand examples, estimated that in 20 years, this numeral will rise to 2,200 ten thousand.Number of cancer deaths also will soar to 1,300 ten thousand from annual 8200000 the same period.The current primary treatments of malignant tumor comprises operation, radiotherapy, chemotherapy and targeted therapy etc.Even but take multidisciplinary synthesis treatment pattern, the mortality rate of cancer still can be in any more.Novel Cancer Treatment Regimens urgently develops.
The immunization therapy of tumor is chosen as first of 2013 annual ten big sciences discoveries by " SCIENCE " magazine.Dendritic cell is as the unique known antigen-presenting cell (APC) that can activate Resting T cells ability in body, by the mode of MHCI molecule-antigen peptide complexes and MHCII molecule-antigen peptide complexes, activate corresponding T lymphocyte, play specific immune response antineoplastic action.Meanwhile, DC also by release interleukin-22 (IL-2), IFN-γ (interferon gamma), IFN-α (interferon-ALPHA), activation natural killer cell (NK), plays nonspecific immunity antitumor mechanism.The important function of DC in antineoplastic immune, makes it become one of focus in immunization therapy.
But the DC in tumor patient body, is subject to the impact of panimmunity restraining factors, be difficult to play its antitumor action.Immunoregulation cell (T suppression cell (MDSC), regulatory T cells (Treg) etc. of derived from bone marrow), the immune negative-feedback regu-lation factor (IL-10 (IL-10), transforming growth factor β (TGF-β) etc.), immune detection point (programmed death receptor 1 and part (PD-1/PD-L1), Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) etc.) all reduce the anti-tumor function of DC induction by number of ways.
Even cultivate tumor patient bone marrow in vitro, DC that PERIPHERAL BLOOD MONONUCLEAR CELL (PBMCs) is originated, be still subject to the impact of immunosuppression mechanism.The preparation method of current DC vaccine, the precursor of DC passes through immunological magnetic bead sorting or the adherent acquisition of mononuclearcell of CD14 monoclonal antibody, adopt again and (cultivate medullary system DC based on GM-CSF, mDC) or FLT3-L (cultivate Plasmacytoid DC, pDC) training method, after antigen load, toll-like receptor (TLR) agonist is utilized to stimulate it ripe.But in the preparation of tumor patient DC vaccine, GM-CSF or FLT3-L all can promote that DC precursor cell differentiation is MDSC.Part TLR agonist also can promote the generation of MDSC, plays immune negative-feedback regu-lation effect.MDSC can suppress the maturation of DC, reduces the functions such as its antigen presentation, migration, inducing T cell release IFN-γ.MDSC passes through propagation and the activation of the suppressor T cell such as arginase-1 (ARG-1), inductivity oxynitride synzyme (iNOS), reactive oxygen species (ROS), and can discharge the promotion Treg cell amplifications such as IL-10, TGF-β.How to eliminate the impact of MDSC on DC vaccine, become the key whether can preparing efficient DC vaccine.
The effect of pDC in DC vaccine, also causes concern gradually.Immaturity pDC mainly plays the effect of inducing immune tolerance, and ripe pDC is by discharging a large amount of IFN-α, and promote that mDC is ripe, induced NK cell activation, playing antigen presentation effect stimulates CTL and Th1 cell to produce antineoplastic specificity immunne response; How to play the immunostimulation of pDC, need further research badly.
In addition, programmed death receptor (PD-1) and part (PD-L1) mechanism of action in DC vaccine thereof are also elucidated gradually.The activation of TLR, enhances the ability of the antigen presentation inducing T cell specific immune response of DC on the one hand, result also in PD-L1 simultaneously and raise in the expression of DC.PDL-1 combines at the PD-1 of CTL, Th1 cell surface with expression, causes these apoptosis, negative feedback inhibition T cell specific immune response.In the preparation process of DC vaccine, block PD-1/PD-L1 signal path, can strengthen the antitumor mechanism of DC vaccine, also need further exploration.
Therefore, be necessary to provide a kind of efficient DC tumor vaccine can eliminated the negativity regulating action of MDSC in the preparation of DC vaccine, strengthen mDC or pDC immunostimulation function.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind ofly can eliminate the negativity regulating action of MDSC in the preparation of DC vaccine, the test kit can preparing efficient novel autologous DC vaccine strengthening mDC or pDC immunostimulation function, method and application.
For solving the problems of the technologies described above, the invention provides technical scheme as follows:
On the one hand, provide a kind of test kit for the preparation of efficient novel autologous DC vaccine, described test kit comprises following composition: the combination of cytokines of GM-CSF, IL-4, FLT3-L and ATRA.
Test kit of the present invention can use the mononuclearcell in the sources such as that normal person originates or autologous patient peripheral blood, Cord blood or bone marrow, carry out adhere-wall culture, obtain the attached cell of PBMCs, by combination of cytokines inductive formation immature DC of the present invention, then mixing TLRs agonist is adopted to promote that DC is ripe.The present invention is by improving existing GM-CSF+IL-4 culture technique, use in conjunction FLT3-L promotes that pDC generates, increase mononuclearcell and transform DC, utilize all-trans-retinoic acid (ATRA) to suppress MDSC to generate simultaneously, eliminate the negativity regulating action of MDSC in the preparation of DC vaccine, effectively strengthen pDC immunostimulation function.
Further, described test kit also comprises the TLRs agonist of OK432 and CPGODN mixing.The present invention also combines TLRs (OK432+CPGODN), promotes that DC (pDC, mDC) is ripe, increases DC transfer ability, strengthens the cell generation specific immunitys such as its induction CTL and TH1 and kills and wounds antineoplastic ability.
Preferably, described test kit comprises following composition: the combination of cytokines of 500-1000U/mlGMCSF, 500-1000U/mlIL-4,50-200ng/mlFLT3-L and 1-5 μM of ATRA.
Preferably, described test kit also comprises the TLRs agonist of 0.1-5KE/mlOK432 and 1-5 μM of CPGODN mixing; Wherein, described CPGODN is one or more of CPGODN21798, CPGODN2336, CPGODN2006 or CPGODN2395; Described CPGODN is preferably CPGODN21798.
On the other hand, a kind of method utilizing mentioned reagent box to prepare efficient novel autologous DC vaccine is provided, comprises the steps:
1) preparation of mononuclearcell: under aseptic condition, obtains mononuclearcell from peripheral blood, Cord blood or bone marrow, then washs with normal saline or Hanks liquid, centrifugal 10 minutes of 1000rpm, and inhale and abandon supernatant, precipitation is PBMCs;
2) preparation of DC precursor: PBMCs is in culture bottle, 37 DEG C, 5%CO
2hatch 1-2h under condition, inhale and abandon supernatant, half adherent PBMCs is the cell subsets comprising DC precursor;
3) cultivation of immature DC: the culture medium culturing cultivated by half adherent PBMCs GM-CSF+IL-4+FLT3-L+ATRA+ autologous plasma obtains immature DC.
4) acquisition immature DC is carried out antigen load, the TLRs agonist utilizing OK432 and CPGODN to mix stimulates 24h to make DC ripe.
Wherein, described CPGODN is one or more of CPGODN21798, CPGODN2336, CPGODN2006 or CPGODN2395; Described CPGODN is preferably CPGODN21798.
Test kit of the present invention can use the mononuclearcell in the sources such as that normal person originates or autologous patient peripheral blood, Cord blood or bone marrow, carry out adhere-wall culture, obtain the attached cell of PBMCs, by combination of cytokines inductive formation immature DC of the present invention, then mixing TLRs agonist is adopted to promote that DC is ripe.The present invention is by improving existing GM-CSF+IL-4 culture technique, use in conjunction FLT3-L promotes that pDC generates, increase mononuclearcell and transform DC, utilize all-trans-retinoic acid (ATRA) to suppress MDSC to generate simultaneously, eliminate the negativity regulating action of MDSC in the preparation of DC vaccine, effectively strengthen pDC immunostimulation function; The present invention also combines TLRs (OK432+CPGODN), promotes that DC (pDC, mDC) is ripe, increases DC transfer ability, strengthens the cell generation specific immunitys such as its induction CTL and TH1 and kills and wounds antineoplastic ability.。
Preferably, described TLRs agonist is the TLRs agonist comprising 0.1-5KE/mlOK432 and 1-5 μM of CPGODN mixing; Or, described TLRs agonist is the TLRs agonist comprising 0.1-5KE/mlOK432 and 1-5 μM of CPGODN mixing, and also comprises POLY (IC), TNF-α, IFN-γ, other Type C PGODN etc. one or more in the TLRs agonist of described OK432 and CPGODN mixing.With on the basis of both OK432 and CPGODN use in conjunction, the use in conjunction of other stimulus object as POLY (IC), other Type C PGODN of TNF-α, IFN-γ etc. also can be added.
Further, culture medium in described step 3 is the culture medium comprising 500-1000U/mlGMCSF+500-1000U/mlIL-4+50-200ng/mlFLT3-L+1-5 uMATRA+1-5% autologous plasma, after DC precursor subgroup being cultivated 4-5 days by described culture medium, 100ug/ml antigen load.
Further, described step 1) in the preparation method of mononuclearcell be under aseptic condition, by peripheral blood, Cord blood or medullary cell 50-100ml, or singly adopt PERIPHERAL BLOOD MONONUCLEAR CELL subgroup 80-100ml, proceed in centrifuge tube, add equal-volume normal saline dilution, then gone to above lymphocyte separation medium by 2:1 volume or 1:1 volume, 2000 turns, slow liter falls slowly, centrifugal 20-30 minute, draws tunica albuginea layer, is mononuclearcell.The separation of mononuclearcell can carry out centrifugalize with the centrifuge tube of 50ml.
Again on the one hand, a kind of efficient novel autologous DC vaccine utilizing said method to prepare is provided.
Finally, a kind of application of efficient novel autologous DC vaccine in the medicine preparing anti tumor immune response utilizing said method to prepare is provided.
In sum, beneficial effect of the present invention shows as:
Test kit of the present invention can use the mononuclearcell in the sources such as that normal person originates or autologous patient peripheral blood, Cord blood or bone marrow, carry out adhere-wall culture, obtain the attached cell of PBMCs, by combination of cytokines inductive formation immature DC of the present invention, then mixing TLRs agonist is adopted to promote that DC is ripe.The present invention is by improving existing GM-CSF+IL-4 culture technique, use in conjunction FLT3-L promotes that pDC generates, increase mononuclearcell and transform DC, utilize all-trans-retinoic acid (ATRA) to suppress MDSC to generate simultaneously, eliminate the negativity regulating action of MDSC in the preparation of DC vaccine, and adopt OK432 and CPHODN stimulation mode altogether, effectively promote that mDC and pDC is ripe, strengthens DC immunostimulation function.
Accompanying drawing explanation
Fig. 1 is the percentage ratio of mDC, pDC and MDCS cell subsets of mDC, pDC co-culture system based on GM-CSF and FLT-3 that the embodiment of the present invention one is respectively organized;
Fig. 2 be the embodiment of the present invention two respectively group with the ripe phenotype of each group of DC80 of Flow cytometry;
Fig. 3 be the embodiment of the present invention two respectively group with the ripe phenotype of each group of DC83 of Flow cytometry;
Fig. 4 be the embodiment of the present invention two respectively group with the ripe phenotype of each group of DC86 of Flow cytometry;
Fig. 5 be the embodiment of the present invention two respectively group with the expression of the PD-L1 of each group of DC of Flow cytometry;
Fig. 6 is the CCR-7 expression of each group of DC that the embodiment of the present invention three WesternBlot method detects;
Fig. 7 is the rear each subgroup percentage ratio of DC vaccine-induced T lymphopoiesis activation that the embodiment of the present invention three is respectively organized.
Detailed description of the invention
For embodiments of the invention will be solved technical problem, technical scheme and advantage clearly, be described in detail below in conjunction with the accompanying drawings and the specific embodiments.But the present invention is limited to absolutely not these examples.The following stated is only the good embodiment of the present invention, only in order to explain the present invention, therefore can not be interpreted as the restriction of the scope of the claims of the present invention.It should be pointed out that all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Embodiment one
GM-CSF+IL-4+FLT3-L+ATRA combination of cytokines is utilized to cultivate DC
Prepare efficient novel autologous DC vaccine as follows, comprise the steps:
1) preparation of mononuclearcell: extract tumor patient 50-100ml peripheral blood under aseptic condition or singly adopt the about 80-100ml such as peripheral blood mononuclear cells, can proceed in 50ml centrifuge tube, add equal-volume normal saline dilution, gone to above lymphocyte separation medium by 2:1 volume or 1:1 volume again, 2000 turns, slow liter falls slowly (rise 1 and fall 0), centrifugal 20-30 minute, draw tunica albuginea layer, be mononuclearcell, then wash 2 times with normal saline or Hanks liquid, 1000 leave the heart 10 minutes, supernatant is abandoned in suction, and precipitation is PBMCs;
2) preparation of DC precursor: PBMCs is in culture bottle, 37 DEG C, 5%CO
2hatch 1-2h under condition, inhale and abandon supernatant, half adherent PBMCs is the cell subsets comprising DC precursor;
3) the half adherent PBMCs obtained in step 2 is divided into 3 groups:
Group 1: obtain immature DC after culture medium 4-5 days that utilize 500-1000U/mlGM-CSF+500-1000U/mlIL-4+1-5% autologous plasma to cultivate;
Group 2: obtain immature DC after culture medium 4-5 days that utilize 500-1000U/mlGM-CSF+500-1000U/mlIL-4+50-200ng/mlFLT3-L+1-5% autologous plasma to cultivate;
Group 3 (immature DC of the present invention): obtain immature DC after culture medium 4-5 days that utilize 500-1000U/mlGM-CSF+500-1000U/mlIL-4+50-200ng/mlFLT3-L+1-5uMATRA+1-5% autologous plasma to cultivate;
4) immature DC of group 1, group 2, group 3 is used 100ug/ml antigen load 24h respectively, 500-2000U/mlTNF-α stimulates 24h.
Utilize Flow cytometry step 4) in group 1, group 2, organize 3 immature DC, MDSC cell quantity and phenotype; The results are shown in Figure shown in 1: group is 2 compared with group 1, and the percentage ratio of the output increased of the immaturity mDC that GM-CSF and FLT3 use in conjunction can be higher, and can significantly improve the output of immaturity pDC, immaturity pDC about 15%, MDSC does not have significant change; Group 3 is compared with group 2, GM-CSF and FLT3 use in conjunction also not only significantly can reduce the percentage ratio of MDSC under the effect of ATRA, MDSC is almost eliminated, and can improve the output of immaturity mDC and pDC, and immaturity pDC is especially obvious, improve 10%; Further, group 3 is compared with group 1, the use in conjunction of GM-CSF, FLT3 and ATRA, can significantly improve immature DC, immaturity mDC improves 10%, and immaturity pDC improves 25%, organize 2 times more than that 3 immaturity pDC subgroup percentage ratios are group 1, greatly improve the immunostimulation function of pDCd, and make MDSC secretory volume extremely low, largely eliminate the negativity regulating action of MDSC in the preparation of DC vaccine.Further illustrate and adopt GM-CSF+IL-4+FLTL-3+ATRA combination of cytokines to cultivate DC, more immature DC can be obtained, and MDSC generates decline.
IL-4 cytokine also can replace with other cytokines, as IFN-α.
Embodiment two
Step 2 by embodiment one) in half adherent PBMCs 500-1000U/mlGM-CSF+500-1000U/mlIL-4+50-200ng/mlFLT3-L+1-5uMATRA+1-5% autologous plasma obtain immature DC after cultivate culture medium 4-5 days, obtain ripe DC in the following manner:
1) the immature cell 100ug/ml antigen load 24h will obtained;
2) by above-mentioned steps 1) in cell be divided into 2 groups:
Group 1: stimulate 24h with 500-2000U/mlTNF-α;
Group 2: stimulate 24h with the TLRs agonist of 0.1-5KE/mlOK432+1-5uMCPGODN2336 mixing;
Group 3: stimulate 24h with the TLRs agonist of 0.1-5KE/mlOK432+1-5uMCPGODN2006 mixing;
Group 4: stimulate 24h with the TLRs agonist of 0.1-5KE/mlOK432+1-5uMCPGODN2395 mixing;
Group 5:(DC vaccine of the present invention) stimulate 24h with the TLRs agonist of 0.1-5KE/mlOK432+1-5uMCPGODN21798 mixing;
Utilize Flow cytometry step 2) in the DC maturity symbol thing of each group, namely the ripe DC phenotype of each group is detected, mainly the expression that ripe DC phenotype has CD83, CD80, CD86 of directive significance is detected, as shown in Figure 2,3, 4: group 2,3,4,5 is compared with group 1, and the percentage ratio of ripe DC phenotype is all significantly increased.OK432 and CPGODN stimulates the ripe Be very effective of DC, stimulates gained DC apparently higher than traditional TNF-α that utilizes merely;
Simultaneously by detecting the inducer T lymphocyte apoptosis capacity of the expression reflection DC of PD-L1 part, as shown in Figure 5: group 2-5 all decreases with the expression of PD-L1 on the ripe DC of group 1, wherein organize 5 (use in conjunction of OK432 and CPGODN21798) the most obvious, reduce (47%) over half, expression only 38%, illustrate that technical scheme that the present invention prepares DC significantly reduces the expression of PD-L1, obviously can reduce the immunosuppressive action of PD-1/PD-L1.
Meanwhile, detect the various levels of cytokine secretion of ripe DC by Elisa, as shown in table 1.As can be seen from Table 1, DC prepared by the present invention's (group 5) can secrete the cytokine that more can play nonspecific immunity antitumor mechanism, DC cell IFN-α secretion level as organized 5 improves 5 times, IL-12p70 improves 4 times, the immunosuppressant cytokine that has of DC secretion prepared by the present invention simultaneously (group 5) obviously reduces, secretory volume as IL-10 (compares with group 1 lower than 20pg/ml, reduce nearly 80%), the secretory volume of TGF-β is lower than 10pg/ml (with group 1 ratio, reducing nearly 70%).
Table 1Elisa detects the levels of cytokine secretion of the ripe DC of each group
In sum, DC vaccine prepared by the present invention has higher cellular maturity, secretes the cytokine of more nonspecific immunity antitumor mechanism and secrete less immunosuppressive factor, and this is significant for the anti-tumor function increasing DC.
Also can add following one or more stimulus object POLY (IC), TNF-α, IFN-γ again in the group 2-5 of above-described embodiment two, for the Maturity improving DC, also there is certain contribution.
Embodiment three
Choose that secretion IL-12p70 ability is the strongest, PD-L1 expresses the DC vaccine that minimum OK432+CPGODN21798 combined stimulation method obtains and compare with the DC vaccine that TNF-α method obtains, detect ripe DC transfer ability and inducing T cell proliferation activation capacity.
Step 2 by embodiment one) in half adherent PBMCs be divided into 2 groups:
Group 1 (traditional DC vaccine): after cultivating 4-5 days with GM-CSF+IL-4, antigen load 24h, TNF-α stimulates ripe 24h;
Group 2 (DC vaccine of the present invention, the DC vaccines identical with the group 5 in embodiment two): after cultivating 4-5 days with GM-CSF+IL-4+FLT3-L+ATRA, 100ug/ml antigen load 24h, then stimulate ripe with OK432+CPGODN21798;
By the DC vaccine for preparing and T cell Mixed culture 24h.
WesternBlot detects the expression of CCR-7, and as shown in Figure 6, the expression of the CCR-7 of group 2 is apparently higher than group 1, and DC Maturity prepared by explanation group 2 is higher, further illustrates DC prepared by the present invention and has higher Maturity and stronger transfer ability.
Flow cytometry T cell subgroup percentage ratio, as shown in Figure 7: DC vaccine prepared by the present invention better the rear acquisition of inducing T cell proliferation activation can have the cell subsets of better specific immune response, ratio as CTL cell subsets remains basically stable with group 1, the percentage ratio of Th1 cell subsets improves 2 times nearly, nearly 40%; And make the percentage ratio of Th2 cell subsets reduce half, DC vaccine prepared by the present invention simultaneously effectively can reduce the ratio of immunosuppressant cell subgroup, as Treg cell subsets is almost 0 (compared with group 1, obvious reduction), effectively can reduce the effect of the immune negative feedback mechanisms such as Treg cell.
Mtt assay detects T cell and kills and wounds rejection ability to tumor cell line, the cell poisoning intensity of tradition DC vaccine only 5.6%, and the cell poisoning intensity of DC vaccine prepared by the present invention reaches 20.5%, make 4 times of traditional DC vaccine, DC vaccine prepared by the present invention has stronger cell and kills ability compared with traditional DC vaccine.
The DC that the autologous DC vaccine preparation technology of new high-efficiency of the present invention obtains, better to the specific immune response of tumor, can reduce the effect of the immune negative feedback mechanisms such as Treg cell by inducing T cell.
It is pointed out that with the mononuclearcell of tumor patient for the preparation of DC vaccine it is immunological rejection in order to reduce foreign cell, also can prepare DC vaccine with the cell in normal cell people source from prevention, treatment or other objects.
In sum, the present invention adopts the large cultivating system of GM-CSF and FLT3-L two to prepare immature DC, and MDSC is converted into DC to adopt ATRA to promote, thus effectively increases immature DC quantity, reduces MDSC content; Recycling OK432 and CPGODN stimulates DC ripe altogether.Technology of preparing of the present invention facilitates maturation and the migration of DC, add the secretion that DC discharges IL-12p70, IFN-α simultaneously, reduce the secretion of the negativity regulatory factors such as IL-10, TGF-β, reduce the generation of Treg cell, enhance the ability of DC inducer T lymphocyte specific immune response, the PD-L1 reducing ripe DC expresses.
Test kit of the present invention can use the mononuclearcell in the sources such as that normal person originates or autologous patient peripheral blood, Cord blood or bone marrow, carry out adhere-wall culture, obtain the attached cell of PBMCs, by combination of cytokines inductive formation immature DC of the present invention, then mixing TLRs agonist is adopted to promote that DC is ripe.The present invention is by improving existing GM-CSF+IL-4 culture technique, use in conjunction FLT3-L promotes that pDC generates, increase mononuclearcell and transform DC, utilize all-trans-retinoic acid (ATRA) to suppress MDSC to generate simultaneously, eliminate the negativity regulating action of MDSC in the preparation of DC vaccine, effectively strengthen pDC immunostimulation function.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1., for the preparation of a test kit for efficient novel autologous DC vaccine, it is characterized in that, described test kit comprises following composition: the combination of cytokines of GM-CSF, IL-4, FLT3-L and ATRA.
2. test kit according to claim 1, is characterized in that, described test kit also comprises the TLRs agonist of OK432 and CPGODN mixing.
3. test kit according to claim 2, is characterized in that, described test kit comprises following composition: the combination of cytokines of 500-1000U/mlGM-CSF, 500-1000U/mlIL-4,50-200ng/mlFLT3-L and 1-5 μM of ATRA.
4. according to the arbitrary described test kit of claims 1 to 3, it is characterized in that, described test kit also comprises the TLRs agonist of 0.1-5KE/mlOK432 and 1-5 μM of CPGODN mixing; Wherein, described CPGODN is one or more of CPGODN21798, CPGODN2336, CPGODN2006 or CPGODN2395.
5. utilize the arbitrary described test kit of claim 1-4 to prepare a method for efficient novel autologous DC vaccine, it is characterized in that, comprise the steps:
1) preparation of mononuclearcell: under aseptic condition, obtains mononuclearcell from peripheral blood, Cord blood or bone marrow, then washs with normal saline or Hanks liquid, centrifugal 10 minutes of 1000rpm, and inhale and abandon supernatant, precipitation is PBMCs;
2) preparation of DC precursor: PBMCs is in culture bottle, 37 DEG C, 5%CO
2hatch 1-2h under condition, inhale and abandon supernatant, half adherent PBMCs is the cell subsets comprising DC precursor;
3) cultivation of immature DC: the culture medium culturing cultivated by half adherent PBMCs GM-CSF+IL-4+FLT3-L+ATRA+ autologous plasma obtains immature DC;
4) acquisition immature DC is carried out antigen load, the TLRs agonist utilizing OK432 and CPGODN21798 to mix stimulates 24h to make DC ripe, obtains described DC vaccine.
6. the method for the efficient novel autologous DC vaccine of preparation according to claim 5, is characterized in that, described TLRs agonist is the TLRs agonist comprising 0.1-5KE/mlOK432 and 1-5 μM of CPGODN mixing;
Or described TLRs agonist is the TLRs agonist comprising 0.1-5KE/mlOK432 and 1-5 μM of CPGODN mixing, and also comprise in the TLRs agonist that mixes of described OK432 and CPGODN POLY (IC), TNF-α, IFN-γ one or more.
7. the method for the efficient novel autologous DC vaccine of the preparation according to claim 5 or 6, it is characterized in that, described step 3) in culture medium be the culture medium comprising 500-1000U/mlGMCSF+500-1000U/mlIL-4+50-200ng/mlFLT3-L+1-5 uMATRA+1-5% autologous plasma, after DC precursor subgroup being cultivated 4-5 days by described culture medium, 100ug/ml antigen load.
8. the method for the efficient novel autologous DC vaccine of preparation according to claim 5, it is characterized in that, described step 1) in the preparation method of mononuclearcell be under aseptic condition, by peripheral blood, Cord blood or medullary cell 50-100ml, or singly adopt PERIPHERAL BLOOD MONONUCLEAR CELL subgroup 80-100ml, proceed in centrifuge tube, add equal-volume normal saline dilution, gone to above lymphocyte separation medium by 2:1 volume or 1:1 volume again, 2000 turns, slow liter falls slowly, centrifugal 20-30 minute, draw tunica albuginea layer, be mononuclearcell.
9. the efficient novel autologous DC vaccine utilizing the arbitrary described method of claim 5-8 to prepare.
10. the application of efficient novel autologous DC vaccine in the medicine preparing anti tumor immune response prepared of the arbitrary described method of claim 5-8.
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| DIVYA SEKAR等: "Technical Advance: Generation of human pDC equivalents from primary monocytes using Flt3-L and their functional validation under hypoxia", 《JOURNAL OF LEUKOCYTE BIOLOGY》 * |
| MELISSA BRANHAM-O"CONNOR等: "Fusion induced reversal of dendritic cell maturation:An altered expression of inflammatory chemokine and chemokine receptors in dendritomas", 《ONCOLOGY REPORTS》 * |
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