CN102600462A - Human dendritic cell tumor vaccine, preparation and application thereof - Google Patents
Human dendritic cell tumor vaccine, preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a human dendritic cell tumor vaccine, and preparation and application thereof. The preparation method comprises collecting CD14<+> peripheral blood mononuclear cells and tumor antigen; culturing CD14<+> peripheral blood mononuclear cells with culture medium containing GM-CSF (granulocyte-macrophage colony-stimulating factor) and IL-4 (interleukin-4) for 100-140 h to obtain immature human dendritic cell; and inducing loading of tumor antigen onto the immature human dendritic cell through beta-dextran with concentration of 80-120mug/mL, to obtain human dendritic cell tumor vaccine. According to the invention, the human dendritic cell tumor vaccine is obtained by inducing loading of tumor antigen onto the immature human dendritic cell through beta-dextran. The vaccine can improve immunizing potency of human dendritic cell, promote multiplication capacity of T-cells, especially can externally stimulate the T-cells to achieve specific tumor killing ability, thereby treating various malignant solid tumors.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of human dendritic cell tumor vaccine.
Background technology
BMDC (Dendritic cells, be called for short DC) accounts for 0.3% of whole cell colony in the blood greatly, is a kind of full-time antigen presenting cell, and it can excite and regulate and control the immunne response of body.The following characteristics of BMDC make its effectively immunne response of submission antigen stimulation body: phagocytosis, pinocytosis and receptor mediated endocytosis can be passed through and capture antigen (like apoptosis and the downright bad cell microorganism and the foreign protein of solubility etc.) efficiently in (1); (2) have strong mobility, can the antigen that capture be transported to the primary and secondary lymphatic organ from peripheral tissues; (3) can high level expression MHC-I, MHC-II molecule, forming complex and be expressed in the BMDC surface with the antigenic determinant that is processed to form in the cell provides the first essential signal for the special lymphocyte of activation antigen; (4) can high level expression costimulatory molecules and adhesion molecule, the T lymphocyte special for activation antigen provides essential secondary signal; (5) can synthesize important cytokine (like IL-12 and IFN-γ etc.), activate the panimmunity relevant cell.So BMDC is effective antigens presenting cell, its antigen presentation ability is better than other antigen presenting cell (like B cell and macrophage) far away.
Immunotherapy of tumors is the method for the another kind treatment tumor outside radiotherapy, chemotherapy and the operative treatment, though present this method also only is in I phase and clinical research stage II phase, it has been acknowledged as the tumor therapeuticing method of tool prospect.The key of immunotherapy of tumors is to remove tumor cell through the immune system that activates body, and wherein crucial is to produce TS cytotoxic T lymphocyte (CTLs).Extensive studies shows: effective antitumour CTLs replys that the full-time antigen presenting cell of needs---BMDC is activated the T lymphocyte.BMDC is at the special T lymphocyte of activation antigen and keep on the T lymphocyte activity and have important function.At present dendritic cell tumor vaccine is by rapid, extensive studies, and in zoopery and early stage clinical experiment, obtained result highly significant.These results of study show: dendritic cell tumor vaccine not only can bring out the immunne response to primary tumo(u)r, and can bring out the immunne response to metastatic tumour.
Summary of the invention
One of the object of the invention is to address the above problem, and a kind of human dendritic cell tumor vaccine is provided.
Two of the object of the invention provides the method for preparing of above-mentioned human dendritic cell tumor vaccine.
Three of the object of the invention provides the application of above-mentioned human dendritic cell tumor vaccine.
The technical scheme that realizes one of the object of the invention is: a kind of human dendritic cell tumor vaccine, it is to induce acquisition by beta glucan.
Two the technical scheme that realizes the object of the invention is: a kind of method for preparing of human dendritic cell tumor vaccine has following steps: 1. obtain CD14
+PMBC and tumor antigen; 2. the CD14 that 1. obtains with the culture medium culturing step that contains GM-CSF and IL-4
+PMBC 100h～140h obtains the immaturity human dendritic cell; The concentration of GM-CSF is 80ng/mL～120ng/mL in the culture medium of said GM-CSF of containing and IL-4, and the concentration of IL-4 is 30ng/mL～70ng/mL; The tumor antigen of 3. inducing step 1. to obtain with beta glucan loads in the immaturity human dendritic cell that 2. step obtain, and obtains the human dendritic cell tumor vaccine; The concentration of said beta glucan is 80 μ g/mL～120 μ g/mL.
The CD14 of above-mentioned steps described in 1.
+PMBC is to be separated earlier from human peripheral by blood cell separator to obtain PMNC, obtains through the sorting from PMNC of magnetic bead sorting system again.
The tumor antigen of above-mentioned steps described in 1. is to obtain through bioanalysis and/or physics method inducing apoptosis of tumour cell.Promptly can pass through the bioanalysis inducing apoptosis of tumour cell separately, also can pass through physics method inducing apoptosis of tumour cell separately, can also pass through physics method inducing apoptosis of tumour cell again through bioanalysis earlier.Described bioanalysis is to adopt antineoplastic chemotherapy medicine to handle tumor cell to make it apoptosis; Said physics method is to adopt isotope roentgenization tumor cell to make it apoptosis, perhaps adopts the ultraviolet radiation tumor cell to make it apoptosis, perhaps tumor cell is carried out multigelation and makes it apoptosis.
Above-mentioned tumor is various malignant solid tumors, comprises hepatocarcinoma, gastric cancer, esophageal carcinoma, breast carcinoma, bladder cancer, carcinoma of prostate etc.
Three the technical scheme that realizes the object of the invention is: the application of above-mentioned human dendritic cell tumor vaccine in the preparation antitumor drug.
The good effect that the present invention has is: the present invention adopts beta glucan induced tumor antigen load in immature human dendritic cell; Thereby obtain the human dendritic cell tumor vaccine; This vaccine can improve the immunizing potency of human dendritic cell; Promote the multiplication capacity of T cell, can have the specific tumour kill capability by stimulated in vitro T cell especially, thereby be used to treat various malignant solid tumors.
Description of drawings
Fig. 1 is the structural formula of beta glucan.
Fig. 2 engulfs the sketch map of beta glucan for the immaturity human dendritic cell.
Fig. 3 influences the sketch map that costimulatory molecules is expressed for beta glucan.
Fig. 4 influences the sketch map of cytokine concentrations for beta glucan.
Fig. 5 is the detection sketch map of the specific tumour kill capability of T cell.
The specific embodiment
(embodiment 1)
The human dendritic cell tumor vaccine of present embodiment is obtained by following method:
1. use Fresenius blood cell separator and the disposable consumption pipeline supporting with it; Select crosspointer mononuclearcell program, people's relevant parameters (sex, height, body weight, hematocrit etc.) is gathered in input, is 50mL/min～80mL/min according to gathering people's concrete condition adjustment velocity of blood flow; Anticoagulant and whole blood ratio are 1: 10; Mononuclearcell is separated from human peripheral, handled botal blood volume and be no more than 2 times of self blood volume, obtain PMNC.The PMNC that obtains is placed the centrifuge tube of 15mL earlier, get 10 μ L cells and count, add anti-people CD14 then
+Immunomagnetic beads antibody is 10
8Cells/100 μ L, 4 ℃ of lucifuges are cultivated 15min, then with PBS (phosphate buffer) the washed cell 5min of 2mL, remove supernatant, add the PBS of 0.5mL again, obtain CD14 with the positive sorting of automatic magnetic bead sorting system at last
+PMBC.
It is (commercially available that tumor cell (tumor of present embodiment derives from gastric cancer) is inoculated in serum-free medium; Down together), after adding antineoplastic chemotherapy medicine 5-fluorouracil continues to cultivate 36h, collect the tumor cell of apoptosis; Then with PBS washing 3 times; Then the tumor cell of apoptosis is collected and is suspended in the serum-free medium, multigelation is 5 times between 37 ℃ and liquid nitrogen, obtains tumor antigen.
2. the CD14 that 1. step is obtained
+The PMBC washing afterwards prepares the cell suspension of 1mL, cell counting, adjustment cell density to 3 * 10 with serum-free medium
6/ mL adds in 6 well culture plates, behind the adherent 2h, collects suspension cell, and-80 ℃ of preservations are subsequent use.
In above-mentioned 6 well culture plates, add the serum-free medium (GM-CSF concentration is 100ng/mL, and IL-4 concentration is 50ng/mL) contain GM-CSF and IL-4, at 37 ℃, 5% CO
2Condition under cultivate CD14
+PMBC changes liquid (assurance GM-CSF concentration is 100ng/mL, and IL-4 concentration is 50ng/mL, down together) and once, after cultivating 72h, changes liquid more once after cultivating 24h, the cell of cultivating 100h～140h acquisition is the immaturity human dendritic cell.
3. with step 2. cultivate immaturity human dendritic cell that 120h obtains according to 1: 3 cell quantity than joining in the tumor antigen that 1. step obtain; Add beta glucan (concentration of beta glucan is 100 μ g/mL) simultaneously; Induce 48h, tumor antigen is loaded in the immaturity human dendritic cell, last conventional centrifuging washing; Collect load tumor antigen human dendritic cell and be suspended in the normal saline, obtain the human dendritic cell tumor vaccine.
(experimental example 1, immaturity human dendritic cell can be engulfed beta glucan)
Get 5 * 10
51. the step of individual embodiment 1 is cultivated the immaturity human dendritic cell that 120h obtains and is cultivated 1h with the beta glucan (concentration is 20 μ g/mL) of fluorescein DTAF labelling at 37 ℃, places on ice, washs 3 times with the PBS of pre-cooling, carries out the fluidic cell detection at once.Experimental result is seen Fig. 2.
Can be known by Fig. 2: the immaturity human dendritic cell can be engulfed beta glucan.
(experimental example 2, beta glucan are expressed costimulatory molecules and the influence of cytokine concentrations)
This experimental example adopts three groups of human dendritic cells to carry out CD83, CD86, CD40, HLA-DR, the detection of CCR7 surface marker, judges Maturity.Wherein first group is that 1. the step of the embodiment 1 of centrifugal collection cultivates the immaturity human dendritic cell that 120h obtains.Second group for being excited first group of human dendritic cell behind the immaturity human dendritic cell 48h that obtains by beta glucan (concentration is 100 μ g/ml).The 3rd group is the human dendritic cell behind the immaturity human dendritic cell 48h that is excited first group of acquisition by TNF-α (concentration is 20ng/mL), and experimental result is seen Fig. 3.
Can be known by Fig. 3: the expression of second group costimulatory molecules after being excited by beta glucan is obviously risen.
In addition, collect the culture supernatant of above-mentioned three groups of human dendritic cells, detect its cytokine concentrations, the result sees Fig. 4.
Can be known by Fig. 4: second group cytokine concentrations after being excited by beta glucan obviously rises.
(the specific tumour kill capability of experimental example 3, T cell)
Human dendritic cell tumor vaccine that embodiment 1 is obtained and T cell according to 1: 10 quantity than Mixed culture 24h; Add IL-2 (concentration is 500U/mL); Collecting the T cell, and mixes with target cell, is that 30: 1,20: 1,10: 1,5: 1 ratio joins in the culture plate according to effect target ratio respectively; Maximum release aperture, effector lymphocyte nature release aperture, target cell nature release aperture are set simultaneously, at 37 ℃, 5% CO
2Condition under cultivate 4h after, add the LDH reactant liquor and detect.Experimental result is seen Fig. 5.
Can be known by Fig. 5: the T cell of the human dendritic cell tumor vaccine stimulated in vitro that is obtained by embodiment 1 has the specific tumour kill capability.
Claims (10)
1. human dendritic cell tumor vaccine, it is characterized in that: it is to induce acquisition by beta glucan.
2. human dendritic cell tumor vaccine according to claim 1 is characterized in that it is the method acquisition by following steps:
1. obtain CD14
+PMBC and tumor antigen;
2. the CD14 that 1. obtains with the culture medium culturing step that contains GM-CSF and IL-4
+PMBC 100h～140h obtains the immaturity human dendritic cell; The concentration of GM-CSF is 80ng/mL～120ng/mL in the culture medium of said GM-CSF of containing and IL-4, and the concentration of IL-4 is 30ng/mL～70ng/mL;
The tumor antigen of 3. inducing step 1. to obtain with beta glucan loads in the immaturity human dendritic cell that 2. step obtain, and obtains the human dendritic cell tumor vaccine; The concentration of said beta glucan is 80 μ g/mL～120 μ g/mL.
3. human dendritic cell tumor vaccine according to claim 2 is characterized in that: the CD14 of step described in 1.
+PMBC is to be separated earlier from human peripheral by blood cell separator to obtain PMNC, obtains through the sorting from PMNC of magnetic bead sorting system again.
4. human dendritic cell tumor vaccine according to claim 2 is characterized in that: the tumor antigen of step described in 1. is to obtain through bioanalysis and/or physics method inducing apoptosis of tumour cell.
5. human dendritic cell tumor vaccine according to claim 4 is characterized in that: described bioanalysis is to adopt antineoplastic chemotherapy medicine to handle tumor cell to make it apoptosis; Said physics method is to adopt isotope roentgenization tumor cell to make it apoptosis, perhaps adopts the ultraviolet radiation tumor cell to make it apoptosis, perhaps tumor cell is carried out multigelation and makes it apoptosis.
6. the method for preparing of the described human dendritic cell tumor vaccine of claim 1 is characterized in that having following steps:
1. obtain CD14
+PMBC and tumor antigen;
2. the CD14 that 1. obtains with the culture medium culturing step that contains GM-CSF and IL-4
+PMBC 100h～140h obtains the immaturity human dendritic cell; The concentration of GM-CSF is 80ng/mL～120ng/mL in the culture medium of said GM-CSF of containing and IL-4, and the concentration of IL-4 is 30ng/mL～70ng/mL;
The tumor antigen of 3. inducing step 1. to obtain with beta glucan loads in the immaturity human dendritic cell that 2. step obtain, and obtains the human dendritic cell tumor vaccine; The concentration of said beta glucan is 80 μ g/mL～120 μ g/mL.
7. the method for preparing of human dendritic cell tumor vaccine according to claim 6 is characterized in that: the CD14 of step described in 1.
+PMBC is to be separated earlier from human peripheral by blood cell separator to obtain PMNC, obtains through the sorting from PMNC of magnetic bead sorting system again.
8. the method for preparing of human dendritic cell tumor vaccine according to claim 6 is characterized in that: the tumor antigen of step described in 1. is to obtain through bioanalysis and/or physics method inducing apoptosis of tumour cell.
9. the method for preparing of human dendritic cell tumor vaccine according to claim 8 is characterized in that: described bioanalysis is to adopt antineoplastic chemotherapy medicine to handle tumor cell to make it apoptosis; Said physics method is to adopt isotope roentgenization tumor cell to make it apoptosis, perhaps adopts the ultraviolet radiation tumor cell to make it apoptosis, perhaps tumor cell is carried out multigelation and makes it apoptosis.
10. claim 1 or the 2 described human dendritic cell tumor vaccines application in the preparation antitumor drug.
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| CN103599528A (en) * | 2013-12-04 | 2014-02-26 | 深圳市合一康生物科技有限公司 | Method for preparing human dendritic cell vaccine |
| CN104491853A (en) * | 2014-12-29 | 2015-04-08 | 深圳市赛欧细胞生物科技有限公司 | Preparation method of human dendritic cell tumour vaccine |
| WO2015132806A1 (en) * | 2014-03-04 | 2015-09-11 | Shakti Upadhyay | A composition comprising ex-vivo generated dendritic cells |
| CN105154403A (en) * | 2015-07-09 | 2015-12-16 | 深圳爱生再生医学科技有限公司 | Dendritic cell antigen loading method |
| CN105316291A (en) * | 2015-12-04 | 2016-02-10 | 广州赛莱拉干细胞科技股份有限公司 | Prostate specific antigen-loaded DC cell and DC cell tumor vaccine |
| CN105670994A (en) * | 2016-02-28 | 2016-06-15 | 深圳爱生再生医学科技有限公司 | DC (dendritic cell) inducer and application thereof |
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| CN111000867A (en) * | 2020-01-07 | 2020-04-14 | 河北医科大学 | Application of tumor cell supernatant after chemotherapy drug action |
| CN111375054A (en) * | 2018-12-27 | 2020-07-07 | 复旦大学附属肿瘤医院 | Tumor vaccine composition, and preparation method and application thereof |
| CN111587288A (en) * | 2017-07-14 | 2020-08-25 | 生物克隆专利有限公司 | Maturation of dendritic cells |
| CN111789940A (en) * | 2020-07-31 | 2020-10-20 | 张家港市中医医院 | Dendritic cell tumor vaccine and preparation method and application thereof |
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| CN111789940A (en) * | 2020-07-31 | 2020-10-20 | 张家港市中医医院 | Dendritic cell tumor vaccine and preparation method and application thereof |
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