CN110283787B - Preparation method of dendritic cell tumor vaccine - Google Patents
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Abstract
The invention relates to a preparation method of a dendritic cell tumor vaccine, which comprises the following steps: (1) Adding tumor cell bodies into a culture solution containing serum, adding a platycodin D culture solution, and placing into a carbon dioxide incubator for culturing for more than 2 hours; (2) Digesting the tumor cells cultured in the step (1) by trypsin, centrifuging, discarding the culture solution, adding DMEM for resuspension, then adding dendritic cells for co-culture for more than 2 hours, and adding lipopolysaccharide LPS for continuous culture to change the dendritic cells into a suspension state; (3) Sucking out the suspended dendritic cells, centrifuging, discarding the culture solution, adding DMEM for resuspension, and then separating and purifying to obtain mature dendritic cells; (4) Mature dendritic cells are taken and added with isotonic normal saline or phosphate buffer solution or serum-free DMEM to prepare the dendritic cell tumor vaccine. The tumor vaccine prepared by the method can promote the immune response of tumor antigens and has remarkable anti-tumor effect.
Description
Technical Field
The present invention relates to a pharmaceutical preparation containing immune cells, which contains dendritic cells from the immune system and can be used for preventing or treating tumors.
Background
There are various ways of loading Dendritic Cell (DC) tumor vaccines with antigens, but the objective is to present exogenous tumor antigens to dendritic cells, and then "cross-sensitize" cd8+ T cells by processing and presenting the dendritic cells with endogenous MHC class i molecules. From 43 clinical trials that have been reported, the antigen types used are whole tumor cell antigens (autologous tumor cells: freeze thawing, irradiation, apoptosis and fusion; apoptotic or necrotic tumor cell lines; about 34.8%), tumor polypeptide antigens (about 32.5%), tumor protein antigens (about 16.2%), tumor cell total RNA (electroporation; about 2.3%), mRNA encoding specific tumor antigens (electroporation; about 11.6%), DNA encoding specific tumor antigens (recombinant viral transduction, e.g., adenovirus; about 4.6%).
Sialylation modification on the surface of a tumor cell membrane is a main cause of immune escape of an organism, and excessive sialylation can cover specific antigens on the surface of the tumor membrane and block a plurality of immune recognition and killing process anomalies such as antigen presentation, NK cells, CTL cells and the like. Treatment of abnormal saccharification of tumor cell membranes is neglected, and the antigen-loaded dendritic cell vaccine immunotherapy of the above-mentioned various antigens is difficult to achieve the expected effect.
Sialic acid (Sia) is a nine-carbon monosaccharide that is widely found in the body and is negatively charged, usually in the form of short chain residues at the ends of cell surface glycoproteins and glycolipids. Because sialic acid is charged and localized at the end of the cell surface sugar chain, sialic acid plays an important role in mediating and regulating biological processes such as cell recognition, adhesion, signal transduction and the like. In a physiological state, sialic acid and adhesion molecules are identified, and the adhesion process between cells is regulated; the interaction between molecules is prevented or weakened through the action of charge repulsive force, the specific binding site of glycoprotein or glycolipid is shielded, and the degradation of glycoprotein or glycolipid is avoided. However, when sialylation modification of the sugar chains on the cell surface is abnormal, the biological processes inside, outside and between cells are changed accordingly.
Sialylation modification of the ends of protein chains is synthesized by the catalysis of sialyltransferases (sialyltransferases), which transfer N-acetylneuraminic acid (Neu 5 Ac) from cytidine monophosphate-beta-N-acetylneuraminic acid (CMP-beta-N-acetylneuro-amic acid), to the cell surface glycoprotein or glycolipid ends. Sialyltransferases can be divided into four major classes, 20 subtypes, ST3GALI-VI, ST6GALI-II, ST6GALNACI-VI and ST8SiaI-VI, respectively, based on the differences in glycosidic linkages formed during transfer of sialic acid to different glycoproteins or glycolipids.
The abnormal sialylation modification of the sugar chain on the surface of the tumor cell is closely related to the abnormal expression of sialyltransferase, the excessive increase of sialic acid on the surface of the tumor cell is caused by the high expression of sialyltransferase, and the sialic acid at the tail end of the sugar complex can shield the structures of glycoprotein and glycolipid, inhibit the combination of cells or molecules and specific recognition sites thereof, avoid the monitoring of an immune system and promote the occurrence of immune escape. Studies have shown that tumor cell malignancy is closely related to its surface sialic acid content: the higher the sialic acid content of the tumor cell surface, the more metastatic the tumor cell. Sialic acid in serum has been reported by related literature as a widely available tumor marker. Abnormally high expression of sialic acid on the surface of tumor cells inhibits the function of the relevant immune cells and immune molecules, thereby mediating tumor immune escape.
Dendritic cells are an active professional antigen presenting cell. Mature dendritic cells induce immune response of exogenous antigen, can promote T cell mediated immune response by presenting tumor antigen, high expression co-stimulatory molecules and adhesion molecules, and tumor cell surface sialylation structures such as mucin and ganglioside can inhibit activation and maturation of dendritic cells, thereby preventing T cell anti-tumor immune response.
Dendritic cells, which are the most powerful professional antigen presenting cells, are critical for eliciting a strong immune response to tumor antigens. However, the tumor host has less infiltration of tumor dendritic cells and impaired functions, so that the dendritic cells of the host carrying tumor antigens are cultured in vitro to prepare the dendritic cell tumor vaccine, which is an effective strategy for obtaining strong immune response of the tumor host.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a dendritic cell tumor vaccine, and the tumor vaccine prepared by the method can promote the immune response of tumor antigens and has remarkable anti-tumor effect.
The scheme for solving the technical problems is as follows:
a method for preparing a dendritic cell tumor vaccine, the method comprising the steps of:
(1) Adding tumor cell bodies into a DMEM or RPMI-1640 cell culture solution containing serum, adding a culture solution with the concentration of platycodin D of 1-30 uM, placing the culture solution into an incubator with the concentration of carbon dioxide of 5%, and culturing for more than 2 hours at 37 ℃;
(2) Digesting the tumor cells cultured in the step (1) by trypsin, adding dendritic cells according to the ratio of the number of the tumor cells to the number of the dendritic cells=1 to 5 to 1, co-culturing for more than 2 hours, and adding LPS with the lipopolysaccharide concentration of 100 to 300ng/ml for continuous culture to change the dendritic cells from an adherent state to a suspension state;
(3) Sucking out the suspended dendritic cells, centrifuging at 1000g, discarding the culture solution, adding the DMEM cell culture solution for resuspension, and then separating and purifying by using a magnetic cell separation technique or a flow separation technique to obtain mature dendritic cells;
(4) And adding the mature dendritic cells into isotonic DMEM cell culture solution, so that the concentration of the mature dendritic cells is 50-500 ten thousand/ml, and obtaining the dendritic cell tumor vaccine.
In the step (1) of the method, the concentration of the platycodin is adjusted according to different tumor cells, so that the survival of the tumor cells is ensured to be not less than 80 percent, and the sufficient tumor cells are ensured when the tumor cells and the dendritic cells are co-cultured.
In the above method, the dendritic cells are obtained by the following method:
taking marrow-derived or spleen-derived or blood-derived dendritic cells for in vitro culture, and amplifying by using granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to obtain the dendritic cells.
The invention firstly uses platycodin D to reduce sialic acid expression of tumor cells, so that antigens can be fully exposed to facilitate dendritic cell recognition, and then the dendritic cell number is added for co-culture, thereby further promoting dendritic cell recognition and endocytosis of tumor antigens and obviously improving the treatment effect of tumors. In addition, the method has the advantages of simple preparation process and low cost.
Detailed Description
Example 1 (preparation of tumor vaccine)
The tumor vaccine in this example consists of the following steps:
1. adding murine liver cancer cell body into serum-containing DMEM (or RPMI-1640) culture solution, adding 5uM culture solution of platycodin D, placing into 5% carbon dioxide incubator, and culturing at 37deg.C for more than 2 hr. The purpose of this step is to reduce sialic acid expression in the tumor cells, but not to cause death of the tumor cells, so that the sialic acid-capped antigen on the surface of the tumor cells is exposed and recognized by dendritic cells.
2. Digesting the tumor cells cultured in the step (1) by trypsin, adding dendritic cells according to the ratio of the number of the tumor cells to the number of the dendritic cells=0.5 to 1, co-culturing for more than 2 hours, and adding LPS with the lipopolysaccharide concentration of 100-300 ng/ml for continuous culture to change the dendritic cells from an adherent state to a suspension state; wherein, the dendritic cells are obtained by the following method: taking marrow-derived or spleen-derived or blood-derived dendritic cells for in vitro culture, and amplifying by using granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to obtain the dendritic cells.
The purpose of this step is to facilitate dendritic cell recognition and endocytosis of the tumor antigen.
3. Sucking out the suspended dendritic cells, centrifuging at 1000g, discarding the culture solution, adding the DMEM cell culture solution for resuspension, and then separating and purifying by using a magnetic cell separation technique or a flow separation technique to obtain mature dendritic cells;
4. and adding the mature dendritic cells into an isotonic serum-free DMEM cell culture solution, so that the concentration of the mature dendritic cells is 500 ten thousand/ml, and obtaining the dendritic cell tumor vaccine.
Example 2 (experiment of platycodin D inhibiting sialic acid in murine breast cancer cells, enhancing the effect of dendritic vaccine against Breast carcinogenesis)
(1) Action of Chinese medicine platycodin D on five high-expression sialyltransferase genes of breast cancer cells
The experiment was divided into tumor model group, 5 μm platycodon group.
Breast cancer cells were inoculated into 6cm dishes of 5mL each, and then placed in an incubator for culture. 5mL of complete culture solution is added into the blank control group, 5mL of PD drug-containing culture solution with the concentration of 5 mu M is added into the experimental group respectively, and the culture is continued in an incubator for 48 hours. When the two groups of cells grew to a density of 80%, the cells were collected separately, and total cellular RNA was extracted. The cDNA was reverse transcribed using a reverse transcription kit.
The cDNA obtained after reverse transcription is used as a reaction template, a reaction system is prepared according to the instruction provided by the kit, and then the reaction system is placed in a fluorescence PCR instrument, and experiments are carried out according to the reaction program provided by the instruction. The obtained data adopts instrument attached software to calculate the Ct value of the threshold cycle number, calculates the difference between the Ct value of the target gene and the Ct value of the reference gene GAPDH, uses 2-delta Ct to represent the relative expression times of the mRNA of each group of genes, and uses each group to carry out statistical analysis.
Experimental results showed that ST3GAL1, ST6GALNAC2, ST8SIA3, ST8SIA5, ST3GAL6mRNA was down-regulated in the platycodon set compared to the tumor model set, see table 1 below.
Table 1 PD effect on breast cancer cell sialyltransferase mRNA (x±s, n=3)
| Group of | ST3GAL1 | ST3GAL6 | ST6GALNAC2 | ST8SIA3 | ST8SIA5 |
| Tumor model group | 1.001±0.081 | 1.003±0.070 | 1.002±0.081 | 1.003±0.117 | 1.005±.0.112 |
| 5 mu M platycodon grandiflorum group | 0.627±0.037* | 0.683±0.057* | 0.673±0.125* | 0.153±0.078* | 0.575±0.043* |
Note that: compared with model group, P <0.05
(2) Animal experiment
The experiments were divided into three groups, tumor model group: subcutaneous injection of 200 ten thousand murine breast cancer cells; control group: subcutaneous injection of breast cancer cells loaded with dendritic cells (50 ten thousand), and one week later injection of 200 ten thousand murine breast cancer cells; experimental group: after reducing sialic acid in tumor cells by subcutaneous injection of platycodin D, dendritic cells (50 ten thousand) were reloaded, and after one week 200 ten thousand murine breast cancer cells were injected.
Dendritic cells were obtained from C57 mouse bone marrow, cultured in vitro, purified by adherence, and granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were amplified.
And (3) in vitro culturing murine breast cancer cells, dividing the cells into three groups when the tumor cells grow to 80% of the area of a culture dish, wherein the tumor model group, the control group and the experimental group are not treated, and the tumor cells of the first two groups are treated by adding 5 mu M platycodin D for 24 hours.
The control group and the experimental group are added into a dendritic cell culture container according to the ratio of the tumor cells to the dendritic cells (1:1), so that the tumor cells and the dendritic cells are co-cultured for 24 hours, and the dendritic cells are promoted to recognize and endocytose tumor antigens;
in the co-culture system, LPS of 200ng/ml is added to promote dendritic cells to be in a mature state, the function of the mature dendritic cells for stimulating T cells is enhanced, the cells are changed from an adherent state to a suspension state, and breast cancer cells still maintain the adherent state, so that the differentiation and collection are facilitated;
the suspension of dendritic cells was pipetted, centrifuged at 1000g, the culture medium was discarded, and the dendritic cells were purified by DMEM resuspension, magnetic cell sorting or flow sorting. CD11C is expressed on the surface of all known C57 mouse dendritic cells, so that after incubating the magnetic bead coated CD11C antibody with a suspension of co-cultured cell system and placing in a magnetic field, the CD11C antibody-bound cells will remain in the sorting column and after disengaging from the magnetic field, CD11C positive dendritic cells can be isolated.
50 ten thousand cell dendritic cells (0.5 ml) are subcutaneously injected into the right armpit part of a C57 mouse and used as a vaccine, and after the dendritic cells enter the body of the C57 mouse, antigen information of tumor cells is presented to T, B cells of the C57 mouse, so that an immune mechanism aiming at the tumor cells is established in the body of the C57 mouse.
200 ten thousand murine breast cancer cells were subcutaneously injected in the left underarm of C57 mice after one week, and whether dendritic cells injected one week ago could exert the key effect of the vaccine was observed. After one week, the difference in tumor incidence among the three groups was found to be very significant, statistically significant, as shown in table 2.
Table 2: three-component tumor comparison table
| Number of examples | Subcutaneous number of neoplasia cases | Rate of tumor formation | |
| Tumor model group | 10 | 10 | 100％ |
| Control group | 10 | 5 | 50 |
| Experimental group | 10 | 0 | 0 |
Example 3 (experiment of platycodin D inhibiting sialic acid in murine colon cancer cells, enhancing the protective Effect of dendritic vaccines against colon cancer production)
(1) Function of traditional Chinese medicine platycodin D on sialyltransferase gene highly expressed in colon cancer cells
The experiment was divided into tumor model group, 10 μm platycodon group.
Colon cancer cells were inoculated into 6cm dishes of 5mL each and then placed in an incubator for culture. 5mL of complete culture solution is added into the blank control group, 5mL of PD drug-containing culture solution with the concentration of 10 mu M is added into the experimental group respectively, and the culture is continued in an incubator for 48 hours. When the two groups of cells grew to a density of 80%, the cells were collected separately, and total cellular RNA was extracted. The cDNA was reverse transcribed using a reverse transcription kit.
The cDNA obtained after reverse transcription is used as a reaction template, a reaction system is prepared according to the instruction provided by the kit, and then the reaction system is placed in a fluorescence PCR instrument, and experiments are carried out according to the reaction program provided by the instruction. The obtained data adopts instrument attached software to calculate the Ct value of the threshold cycle number, calculates the difference between the Ct value of the target gene and the Ct value of the reference gene GAPDH, uses 2-delta Ct to represent the relative expression times of the mRNA of each group of genes, and uses each group to carry out statistical analysis.
Experimental results showed that ST3GAL1, ST6GALNAC2, ST8SIA3, ST8SIA5, ST3GAL6mRNA was down-regulated in the platycodon set compared to the tumor model set, see table 3 below.
Table 3 PD effect on colon cancer cell sialyltransferase mRNA (x±s, n=3)
| Group of | ST3GAL1 | ST3GAL6 | ST6GALNAC2 | ST8SIA3 | ST8SIA5 |
| Tumor model group | 1.000±0.008 | 1.003±0.090 | 1.003±0.089 | 1.006±0.127 | 1.007±.142 |
| 10 mu M platycodon grandiflorum group | 0.517±0.027* | 0.699±0.055* | 0.543±0.159* | 0.164±0.098* | 0.473±0.033* |
Note that: compared with model group, P <0.05
(2) Animal experiment
The experiments were divided into three groups, tumor model group: subcutaneous injection of 200 ten thousand murine colon cancer cells; control group: tumor cells loaded with dendritic cells (50 ten thousand) were subcutaneously injected, and 200 ten thousand murine colon cancer cells were injected after one week; experimental group: after the tumor cells were reduced in sialic acid by the platycodin D by subcutaneous injection, dendritic cells (50 ten thousand) were reloaded, and 200 ten thousand murine colon cancer cells were injected after one week.
Dendritic cells were obtained from C57 mouse bone marrow, cultured in vitro, purified by adherence, and granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were amplified.
And (3) culturing the murine colon cancer cells in vitro, and dividing the murine colon cancer cells into three groups when the tumor cells grow to 80% of the area of a culture dish, wherein the tumor model group, the control group and the experimental group are not treated, and the tumor cells of the experimental group are treated for 24 hours by adding 10 mu M platycodin D.
The control group and the experimental group are added into a dendritic cell culture container according to the ratio of the tumor cells to the dendritic cells (0.5:1), so that the tumor cells and the dendritic cells are cultured together for 24 hours, and the dendritic cells are promoted to recognize and endocytose tumor antigens;
in the co-culture system, LPS with 200ng/ml lipopolysaccharide is added to promote dendritic cells to be in a mature state, the function of the mature dendritic cells for stimulating T cells is enhanced, the cells are changed from an adherent state to a suspension state, and colon cancer cells still maintain the adherent state, so that the differentiation and collection are facilitated;
the suspension of dendritic cells was pipetted, centrifuged at 1000g, the culture broth was discarded, resuspended in DMEM, and the dendritic cells were isolated and purified by magnetic cell sorting or flow sorting. 80 ten thousand cell dendritic cells (0.5 ml) are subcutaneously injected into the right armpit part of a C57 mouse and used as a vaccine, and after the dendritic cells enter the body of the C57 mouse, antigen information of tumor cells is presented to T, B cells of the C57 mouse, so that an immune mechanism aiming at the tumor cells is established in the body of the C57 mouse.
200 ten thousand murine colon cancer cells were subcutaneously injected in the left underarm of C57 mice after one week, and whether dendritic cells injected one week ago could exert the key effect of the vaccine was observed. After one week of tumor cell injection, the difference in tumor incidence among the three groups was found to be very significant, statistically significant, as shown in table 4 below.
Table 4 comparative table of three component tumors
| Number of examples | Subcutaneous number of neoplasia cases | Rate of tumor formation | |
| Tumor model group | 10 | 9 | 90％ |
| Control group | 10 | 5 | 50 |
| Experimental group | 10 | 0 | 0 |
Example 4 (Effect experiment of dendritic cell tumor vaccine)
4.1 establishment of mouse liver cancer cell subcutaneous transplantation tumor model
Will be 30The C57 mice were divided into 3 groups according to the random number table method, model group, experimental group and control group. Adjusting breast cancer cell concentration to 5x10 with phosphate buffer 6 Per ml, 0.5ml was injected subcutaneously in the right anterior axilla of each group of mice to establish a subcutaneous graft model. After one week, all mice developed tumors. The model group was not injected, and the experimental group and the control group were treated subcutaneously with the dendritic cell tumor vaccine.
4.2 vaccine preparation and injection
Dendritic cells were obtained from C57 mouse bone marrow, cultured in vitro, purified by adherence, and granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) were amplified.
And (3) in vitro culturing murine liver cancer cells, and dividing the cells into three groups, namely a tumor model group, a control group and an experimental group when the tumor cells grow to 80% of the area of a culture dish, wherein the first two groups of tumor cells are not treated, and the experimental group of tumor cells are treated for 24 hours by adding 10 mu M platycodin D.
The experimental group and the control group are added into a dendritic cell culture container according to the ratio of the tumor cells to the dendritic cells (1:1), so that the tumor cells and the dendritic cells are cultured together for 24 hours, and the dendritic cells are promoted to recognize and endocytose tumor antigens;
in the co-culture system, LPS with 200ng/ml lipopolysaccharide is added to promote dendritic cells to be in a mature state, the function of the mature dendritic cells for stimulating T cells is enhanced, the cells are changed from an adherent state to a suspension state, and colon cancer cells still maintain the adherent state, so that the differentiation and collection are facilitated;
the suspension of dendritic cells was pipetted, centrifuged at 1000g, the culture broth was discarded, resuspended in DMEM, and the dendritic cells were isolated and purified by magnetic cell sorting or flow sorting.
100 ten thousand cell dendritic cells (0.5 ml) are subcutaneously injected into the right armpit part of a C57 mouse and used as a vaccine, and after the dendritic cells enter the body of the C57 mouse, antigen information of tumor cells is presented to T, B cells of the C57 mouse, so that the immune mechanism of the C57 mouse against the tumor cells is enhanced.
4.3 vaccine immunological Effect detection
Two weeks after injection, all mice were sacrificed, subcutaneous tumors were removed, tumor sizes were measured, paraffin-embedded tumor tissue sections were analyzed by immunohistochemistry, and the numbers of cd4+ and cd8+ cells were counted. The vaccine stimulates the activation and proliferation of T cells, and through secreting cytokines and chemotactic factors, the number of effector T cells at a tumor part is increased through vascular endothelial barrier, CD4+ is polarized into Th 1 cells, cytokines such as IL-2, IFN and the like are generated, and the proliferation of the Th 1 cells is promoted, so that the immune effect is amplified. Cd8+ CTL cells directly kill tumor cells by cytolysis. The results are shown in tables 5 and 6 below.
Table 5: tumor size comparison table for three groups of mice
Table 6: immunohistochemical detection of tumor tissue CD4+, CD8+ cell count (400 x observation under the microscope)
| Model group | Control group | Experimental group | |
| Cd4+ cell count | 15.41±3.25 | 21.38±2.85 | 36.86±4.56 |
| Cd8+ cell count | 16.36±4.56 | 25.75±6.45 | 45.75±4.16 |
1.1 establishment of mouse liver cancer cell subcutaneous transplantation tumor model
30C 57 mice were divided into 3 groups according to the random number table method, a model group, an experimental group and a control group. Adjusting the concentration of liver cancer cells to 5x10 with phosphate buffer solution 6 Per ml, 0.5ml was injected subcutaneously in the right anterior axilla of each group of mice to establish a subcutaneous graft model. After one week, all mice developed tumors. The model group was not injected, and the experimental group and the control group were treated subcutaneously with the dendritic cell tumor vaccine.
1.2 vaccine preparation and injection
The vaccine prepared in example 1 was used for subcutaneous injection in the axilla of mice, 0.5ml each time, against the experimental group. The control vaccine and experimental vaccine only differed in that the tumor cells in step 1 were not treated with platycodin D, and the other steps were the same as in example 1.
1.3 vaccine immunological Effect detection
Two weeks after injection, all mice were sacrificed, subcutaneous tumors were removed, tumor sizes were measured, paraffin-embedded tumor tissue sections were analyzed by immunohistochemistry, and the numbers of cd4+ and cd8+ cells were counted. The vaccine stimulates the activation and proliferation of T cells, and through secreting cytokines and chemotactic factors to chemotactic T cells, the T cells enter a tumor part through vascular endothelium, CD4+ is polarized into Th 1 cells, cytokines such as IL-2, IFN and the like are generated, and the proliferation of the Th 1 cells is promoted, so that the immune effect is amplified. Cd8+ CTL cells directly kill tumor cells by cytolysis. The results are shown in tables 7 and 8 below.
Table 7: tumor size comparison table for three groups of mice
Table 8: immunohistochemical detection of tumor tissue CD4+, CD8+ cell count (400 x observation under the microscope)
| Model group | Control group | Experimental group | |
| Cd4+ cell count | 15.41±3.25 | 21.38±2.85 | 36.86±4.56 |
| Cd8+ cell count | 16.36±4.56 | 25.75±6.45 | 45.75±4.16 |
Claims (2)
1. A method for preparing a dendritic cell tumor vaccine, the method comprising the steps of:
(1) Adding tumor cell bodies into a DMEM or RPMI-1640 cell culture solution containing serum, adding an equal volume of a culture solution with the concentration of platycodin D of 5-10 uM, placing the culture solution into an incubator with the concentration of carbon dioxide of 5%, and culturing for more than 2 hours at 37 ℃;
(2) Digesting the tumor cells cultured in the step (1) by trypsin, adding dendritic cells subjected to combined induction by granulocyte-macrophage colony stimulating factor and interleukin-4 according to the ratio of the number of the tumor cells to the number of the dendritic cells=1:5-5:1, co-culturing for more than 2 hours, adding LPS with the lipopolysaccharide concentration of 100-300 ng/ml, and continuously culturing to change the dendritic cells from an adherent state to a suspension state;
(3) Sucking out the suspended dendritic cells, centrifuging at 1000g, discarding the culture solution, adding DMEM for resuspension, and then separating and purifying by using a magnetic cell separation technique or a flow separation technique to obtain mature dendritic cells;
(4) Adding isotonic physiological saline or phosphate buffer solution or serum-free DMEM cell culture solution into mature dendritic cells to make the concentration of the mature dendritic cells be 50-500 ten thousand/ml, so as to obtain the dendritic cell tumor vaccine.
2. The method for preparing a dendritic cell tumor vaccine according to claim 1, wherein the dendritic cells are obtained by the following method: taking marrow-derived or spleen-derived or blood-derived dendritic cells for in vitro culture, and amplifying by using granulocyte-macrophage colony stimulating factor and interleukin-4 to obtain the dendritic cells.
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