CN103638517A - Special-purpose kit for preparing human dendritic cell vaccines - Google Patents
Special-purpose kit for preparing human dendritic cell vaccines Download PDFInfo
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Abstract
The invention belongs to the technical field of cellular immunology and specifically discloses a special-purpose kit for preparing human dendritic cell vaccines capable of largely secreting IL(Interleukin)-12. The kit consists of a monocyte separating-obtaining culture medium, a culture medium for promoting DC (Dendritic Cell) to induce and differentiate, an agent for promoting the DC to mature and a tumor antigen. The DC prepared by the special-purpose kit disclosed by the invention is mainly applied to treating cancer patients or preventing cancer high-risk population. The special-purpose kit disclosed by the invention has the advantage that the prepared DC can secrete a great deal of IL-12, so that T cells are promoted to differentiate towards Th1 type immune response direction; and meanwhile, the special-purpose kit has the advantages of being simple in preparation process, low in cost, easy for large-scale production, and the like.
Description
Technical field
The present invention relates to dedicated kit prepared by a kind of human dendritic cell vaccine, particularly a kind of dedicated kit that can secrete in a large number the dendritic cell vaccine of IL-12.
Background technology
Dendritic cell (dendritic cell, DC) be at present known to the strongest antigen presenting cell (antigen-presenting cell, APC) of function, maximum feature be can stimulate primary tape T cell (
t cell) activation and increment, be the person that makes of specific immune response.Therefore, DC has a wide range of applications in tumour immunity cell therapy.Yet the ratio of DC in human peripheral is very low, and the function of the interior DC of tumor patient body is mostly in inhibitory state.Therefore, how to obtain sufficient amount and there is inducing T cell and to Th1 type immunne response direction, be divided into the DC of cytotoxic T lymphocyte (cytotoxic T lymphocytes, CTL), become the key of clinical practice.
At present, the amplification scheme of DC routine is a lot, as classical GM-CSF, IL-4 induction DC differentiation, then use TNF-α or combination cytokine IL-1 β, IL-6, TNF-α, PGE-2 etc., or associating polyinosinic acid (polyinosinic:polycytidylic acid, poly-I:C), bacteria lipopolysaccharide (bacterial lipopolysaccharide, LPS) etc. promote its maturation, yet above-mentioned short DC maturing agent only can make the low-level secretion of ripe DC IL-12, and IL-12 is that DC promotion T cell is to the key molecule of Th1 type immunne response direction differentiation.Therefore, how to induce the ripe also IL-12 of secreting high levels of DC, immunocyte is implemented to killing tumor cell and play central role.
Summary of the invention
The object of this invention is to provide and can secrete in a large number dedicated kit prepared by the human dendritic cell vaccine of IL-12 a kind of preparation.
Dedicated kit prepared by a kind of human dendritic cell vaccine that can secrete in a large number IL-12 provided by the present invention obtains culture medium, short DC inductive differentiation medium, short DC maturing agent and tumor antigen by mononuclear cell separation and forms.
As preferably, it is PAA that culture medium is obtained in described mononuclear cell separation
tMculture medium, GT-T551
tMculture medium, Opti-MEM
tMany one in culture medium, RPMI-1640 culture medium.
As preferably, the solvent of described short DC inductive differentiation medium is X-VIVO-15
tMculture medium or AIM-V
tMin culture medium any one, solute is GM-CSF and IL-4, wherein the concentration of GM-CSF is 800-1000IU/ml, the concentration of IL-4 is 800-1000IU/ml.
As preferably, the solvent of described short DC maturing agent is X-VIVO-15
tMculture medium or AIM-V
tMin culture medium any one, solute is IL-1 β, IL-6, TNF-α, or a kind of in IL-1 β, IL-6, TNF-α and IFN-γ, Mtb-Hag or two kinds, this short DC maturing agent joins after cultivating system, IL-1 β final concentration is 8-15ng/mL, and IL-6 final concentration is 50-150ng/mL, and TNF-α final concentration is 8-15ng/mL, Mtb-Hag final concentration is 5～10 μ g/ml, and IFN-γ final concentration is 100～1000IU/ml.
As preferably, described tumor antigen is one or more in following antigen: CD19, CD20, WT-1, MUC1, LMP2, HPV E6E7, EGFRvIII, HER-2/neu, Idiotype, MAGEA3, p53, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53mutant, Proteinase3, bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, it is 1～20 μ g/ml that this antigen adds concentration after cultivating system.
As preferably, the PH that culture medium, short DC inductive differentiation medium, short DC induction maturation medium are obtained in described mononuclear cell separation is 7.2～7.4.
Another object of the present invention is to provide a kind of method of cultivating human dendritic cell vaccine.
The method is according to specific reagent box description operation requirements and adds under the condition of 10% autologous plasma, energy high expressed CD80, CD86, HLA-DR, low expression CD83, the preparation that can secrete the human dendritic cell vaccine of a large amount of IL-12 simultaneously.
Dedicated kit selectivity prepared by human dendritic cell vaccine of the present invention is strong, can from person monocytic cell, induce the DC vaccine that can secrete in a large number IL-12.The people DC vaccine that the dedicated kit of preparing with this human dendritic cell vaccine obtains can directly coordinate the therapeutic modalities such as traditional operation, chemotherapy and radiation, or inducing specific cytotoxic T cell (cytotoxic T lymphocyte in vitro, CTL) carry out feeding back in body, can reach in routine treatment and remove after massive tumor cell, remove the tumor cell of a small amount of residual or diffusion, to improve, to consolidate oncotherapy effect, the object of reduce recurrence, improving the quality of living; Or cancer high-risk group is carried out to the object that direct feedback reaches prevention.
Accompanying drawing explanation
1. ripe the 7th day Maturity of the short maturing agent induction of Fig. 1 Flow cytometry various combination DC DC detects (n=8); First row to the four row represent respectively DC surface molecular CD80, CD83, CD86 and HLA-DR, the first row to fourth line represents respectively IL-1 β, IL-6, TNF-α, PGE2, IL-1 β, IL-6, TNF-α, IFN-γ, the various combination DC such as IL-1 β, IL-6, TNF-α, Mtb-HAg and IL-1 β, IL-6, TNF-α, IFN-γ, Mtb-HAg urge maturing agent.
2. the short maturing agent of Fig. 2 various combination DC produces the impact (n=8) of IL-12 on DC; IFN-γ and Mtb-HAg produce IL-12 to DC all facilitation, and particularly both combines with IL-1 β, IL-6 and TNF-α amount that induction promotes DC secretion IL-12 and are not less than IL-1 β, IL-6, TNF-α and PGE2 and combine 1000 times that induce.
The specific embodiment
With example, illustrate the present invention below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses in example, is the operating instruction execution that the method for observing a usual practice and producer provide below.
Embodiment 1
The first step: further separated and obtain mononuclear cell from the PBMCs that separation obtains.Comprise the following steps:
1. gather detection in peripheral blood of patients underwent 50ml, through ficoll-general shadow glycosamine density gradient centrifugation, obtain mononuclearcell.Concrete steps are: 1500 revs/min, centrifugal 10 minutes, draw upper plasma layer, 56 ℃ of deactivations are centrifugal standby after 30 minutes, and with the hemocyte of normal saline two-fold dilution precipitation, human lymphocyte separating medium and dilute blood add in centrifuge tube in the ratio of 1:2,2000 revs/min, centrifugal 20 minutes, carefully draw tunica albuginea layer, with normal saline washing 2 times, rotating speed is respectively 1600 revs/min, 1300 revs/min, all centrifugal 7 minutes, obtain PERIPHERAL BLOOD MONONUCLEAR CELL.
2. the PBMCs of above-mentioned separation is resuspended in to mononuclear cell separation and obtains in culture medium, adjusting cell density is (5～10) * 10
6/ ml, cumulative volume is that 10ml adds in the Tissue Culture Flask of T75ml, adherent 2h in the incubator of 37 ℃, 5%CO2 and saturated humidity.
3. rock gently culture bottle, make not attached cell Eddy diffusion; Then inhale the not attached cell that abandons suspension, the attached cell staying is mononuclear cell.
Second step: induction mononuclear cell breaks up to DC.Comprise the following steps:
1. the short DC inductive differentiation medium 20ml that is added with 10% autologous plasma is added in the culture bottle of above-mentioned T75, be then placed in the incubator of 37 ℃, 5%CO2 and saturated humidity and cultivate.
2. after the 3rd day (72h), inhale and abandon the culture medium in 10ml culture bottle, and add the short DC inductive differentiation medium that contains 10% autologous plasma that 10ml is fresh; Add tumor antigen, making its final concentration is 10 μ g/ml simultaneously.
The 3rd step: induction becomes ripe DC to the mononuclear cell of DC differentiation, and carries out the detection of Maturity and IL-12 secretory volume.Comprise the following steps:
1. at the 5th day of cell culture, add short DC maturing agent.
2. 6th～7 of cell culture days, Maturity and the IL-12 secretory volume of by flow cytometry and ELISA, carrying out DC respectively detected.
In the situation of the not high and IL-12 hyposecretion of DC Maturity that the relatively original culture medium culturing of the present invention goes out, the secretion of its Maturity and IL-12 is all higher.Referring to Fig. 1 and Fig. 2.
Claims (8)
1. the dedicated kit that prepared by human dendritic cell vaccine, is characterized in that comprising:
(1) culture medium is obtained in mononuclear cell separation;
(2) short DC inductive differentiation medium;
(3) short DC maturing agent;
(4) tumor antigen.
2. the dedicated kit that according to claim 1 prepared by human dendritic cell vaccine, is characterized in that, it is PAA that culture medium is obtained in described mononuclear cell separation
tMculture medium, GT-T551
tMculture medium, Opti-MEM
tMany one in culture medium, RPMI-1640 culture medium.
3. according to the dedicated kit that described in claim 1 or 2 prepared by human dendritic cell vaccine, it is characterized in that, the solvent of described short DC inductive differentiation medium is X-VIVO-15
tMculture medium or AIM-V
tMin culture medium any one, solute is GM-CSF and IL-4, wherein the concentration of GM-CSF is 800-1000IU/ml, the concentration of IL-4 is 800-1000IU/ml.
4. the dedicated kit that according to claim 3 prepared by human dendritic cell vaccine, is characterized in that, the solvent of described short DC maturing agent is X-VIVO-15
tMculture medium or AIM-V
tMin culture medium any one, solute is IL-1 β, IL-6, TNF-α, or a kind of in IL-1 β, IL-6, TNF-α and IFN-γ, Mtb-Hag or two kinds, this short DC maturing agent joins after cultivating system, IL-1 β final concentration is 8-15ng/mL, and IL-6 final concentration is 50-150ng/mL, and TNF-α final concentration is 8-15ng/mL, Mtb-Hag final concentration is 5～10 μ g/ml, and IFN-γ final concentration is 100～1000IU/m1.
5. the dedicated kit of preparing according to human dendritic cell vaccine described in claim 1 or 2 or 4, it is characterized in that, described tumor antigen is one or more in following antigen: CD19, CD20, WT-1, MUC1, LMP2, HPVE6E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53mutant, Proteinase3, bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, it is 1～20 μ g/ml that this antigen adds concentration after cultivating system.
6. the dedicated kit that according to claim 3 prepared by human dendritic cell vaccine, it is characterized in that, described tumor antigen is one or more in following antigen: CD19, CD20, WT-1, MUC1, LMP2, HPV E6E7, EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53, NY-ESO-1, PSMA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53mutant, Proteinase3, bcr-abl, Tyrosinase, Survivin, PSA, hTERT, Sarcoma, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG, NA17, PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGEA1, sLe (animal), CYP1B1, PLAC1, GM3, BORIS, Tn, it is 1～20 μ g/ml that this antigen adds concentration after cultivating system.
7. according to the dedicated kit that described in claim 1 or 2 prepared by human dendritic cell vaccine, it is characterized in that, the PH that culture medium, short DC inductive differentiation medium, short DC induction maturation medium are obtained in described mononuclear cell separation is 7.2～7.4.
8. according to dedicated kit described in claim 1-7 any one, cultivate the method for human dendritic cell vaccine, it is characterized in that: described dedicated kit need to be used in conjunction with 10% autologous plasma, energy high expressed CD80, CD86, HLA-DR, low expression CD83, the while can be secreted the human dendritic cell vaccine of a large amount of IL-12.
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| CN201310638797.3A CN103638517A (en) | 2013-12-04 | 2013-12-04 | Special-purpose kit for preparing human dendritic cell vaccines |
| PCT/CN2014/086279 WO2015081741A1 (en) | 2013-12-04 | 2014-09-11 | Method and kit for preparing immunological cells |
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| CN201310638797.3A CN103638517A (en) | 2013-12-04 | 2013-12-04 | Special-purpose kit for preparing human dendritic cell vaccines |
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| CN103933558A (en) * | 2014-05-13 | 2014-07-23 | 无锡伊琳生物技术有限公司 | Preparation and use method of novel broad-spectrum therapeutic tumour vaccine |
| CN103948917A (en) * | 2014-04-02 | 2014-07-30 | 江苏和泽生物科技有限公司 | Method for preparing dendritic cell vaccine |
| WO2015081741A1 (en) * | 2013-12-04 | 2015-06-11 | 深圳市合一康生物科技有限公司 | Method and kit for preparing immunological cells |
| CN104830787A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on AFP antigen, targeting immune cell population, preparation method and applications thereof |
| CN104830803A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on P53 antigen, targeting immune cell population, preparation method and applications thereof |
| CN104830791A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on PAP antigen, targeting immune cell population, preparation method and applications thereof |
| CN104830795A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on MAGE-3 antigen, targeting immune cell population, preparation method and applications thereof |
| CN104998260A (en) * | 2015-07-08 | 2015-10-28 | 深圳爱生再生医学科技有限公司 | DC cell-based HPV virus vaccine preparation method |
| CN105238753A (en) * | 2015-11-18 | 2016-01-13 | 成都百赛泰科生物科技有限公司 | Kit used for preparing CTL and application thereof |
| CN105647970A (en) * | 2016-02-22 | 2016-06-08 | 河北利同康生物科技有限公司 | Prostate-cancer-specific PAP (prostatic acid phosphatase)-GM-CSF (granulocyte-macrophage colony-stimulating factor)-IL-6 (interluekin-6) genetic recombinant fusion protein and preparation method thereof |
| CN107119016A (en) * | 2017-04-06 | 2017-09-01 | 暨南大学 | A kind of induction PC 12 cell differentiations are the method for neuron |
| CN108291204A (en) * | 2015-09-15 | 2018-07-17 | 西北生物治疗药物公司 | The method for being related to handling for the activated Dendritic Cells composition of the subject with advanced cancer and immunization therapy |
| CN111330000A (en) * | 2020-04-20 | 2020-06-26 | 北京瀚海拓新生物技术有限公司 | Novel method for treating liver cancer by loading legumain protein on dendritic cells |
| CN111434674A (en) * | 2018-12-25 | 2020-07-21 | 上海细胞治疗集团有限公司 | Polypeptide compositions and their use in cancer immunotherapy |
| CN112402596A (en) * | 2019-08-22 | 2021-02-26 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN113444688A (en) * | 2021-06-24 | 2021-09-28 | 广西中医药大学 | Human dendritic cell induction method and composition for resisting virus and tumor |
-
2013
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| 买月琴: "结核患者DC亚群的变化和结核杆菌抗原对DC成熟及调节γδT细胞功能的影响", 《中国优秀硕士学位论文全文数据库（电子期刊）》, no. 1, 15 January 2013 (2013-01-15), pages 061 - 72 * |
Cited By (19)
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| WO2015081741A1 (en) * | 2013-12-04 | 2015-06-11 | 深圳市合一康生物科技有限公司 | Method and kit for preparing immunological cells |
| CN103948917A (en) * | 2014-04-02 | 2014-07-30 | 江苏和泽生物科技有限公司 | Method for preparing dendritic cell vaccine |
| CN103933558A (en) * | 2014-05-13 | 2014-07-23 | 无锡伊琳生物技术有限公司 | Preparation and use method of novel broad-spectrum therapeutic tumour vaccine |
| CN103933558B (en) * | 2014-05-13 | 2015-11-18 | 无锡伊琳生物技术有限公司 | A kind of preparation and application of therapeutic tumor vaccine of novel, wide spectrum |
| CN104830803A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on P53 antigen, targeting immune cell population, preparation method and applications thereof |
| CN104830795A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on MAGE-3 antigen, targeting immune cell population, preparation method and applications thereof |
| CN104830787A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on AFP antigen, targeting immune cell population, preparation method and applications thereof |
| CN104830791A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | DC cell based on PAP antigen, targeting immune cell population, preparation method and applications thereof |
| CN104998260A (en) * | 2015-07-08 | 2015-10-28 | 深圳爱生再生医学科技有限公司 | DC cell-based HPV virus vaccine preparation method |
| CN108291204A (en) * | 2015-09-15 | 2018-07-17 | 西北生物治疗药物公司 | The method for being related to handling for the activated Dendritic Cells composition of the subject with advanced cancer and immunization therapy |
| CN105238753A (en) * | 2015-11-18 | 2016-01-13 | 成都百赛泰科生物科技有限公司 | Kit used for preparing CTL and application thereof |
| CN105647970A (en) * | 2016-02-22 | 2016-06-08 | 河北利同康生物科技有限公司 | Prostate-cancer-specific PAP (prostatic acid phosphatase)-GM-CSF (granulocyte-macrophage colony-stimulating factor)-IL-6 (interluekin-6) genetic recombinant fusion protein and preparation method thereof |
| CN107119016A (en) * | 2017-04-06 | 2017-09-01 | 暨南大学 | A kind of induction PC 12 cell differentiations are the method for neuron |
| CN111434674A (en) * | 2018-12-25 | 2020-07-21 | 上海细胞治疗集团有限公司 | Polypeptide compositions and their use in cancer immunotherapy |
| CN111434674B (en) * | 2018-12-25 | 2024-02-09 | 上海细胞治疗集团有限公司 | Polypeptide composition and use thereof in cancer immunotherapy |
| CN112402596A (en) * | 2019-08-22 | 2021-02-26 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN112402596B (en) * | 2019-08-22 | 2023-12-08 | 上海细胞治疗集团有限公司 | Polypeptide composition and vaccine |
| CN111330000A (en) * | 2020-04-20 | 2020-06-26 | 北京瀚海拓新生物技术有限公司 | Novel method for treating liver cancer by loading legumain protein on dendritic cells |
| CN113444688A (en) * | 2021-06-24 | 2021-09-28 | 广西中医药大学 | Human dendritic cell induction method and composition for resisting virus and tumor |
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Application publication date: 20140319 |