A kind of tumor vaccine and application thereofTechnical field
The present invention relates to a kind of tumor vaccine and application thereof.
Background technology
In recent years, along with to the further investigation of tumor development molecular mechanism and developing rapidly of biotechnology, the tumor biotherapy based on immunization therapy is considered to be the 4th kind of tumor treatment model after operation, radiation and chemotherapy.Different from traditional Therapeutic Method, tumor biotherapy is not damaging under the prerequisite with body immune system and function, obtains antineoplastic effect by the material transferred tumor host defense mechanism or give specific aim targeting that natural (or genetic engineering) produce very strong.Current biotherapy mainly comprises cytokine therapy, immune cell therapy, gene therapy, molecular targeted therapy, Antybody therapy and tumor vaccine technology etc.Wherein, tumor vaccine therapy has developed into the more effective Therapeutic Method of one, in individual body, inject tumor vaccine, effectively can alleviate conditions of patients and reach the effect of preventing and treating high-risk individuals cancer return.
Because the vaccine therapy of tumor is without obvious toxic-side effects, thus development in recent years is very fast, and it is the tumor vaccine, monoclonal antibody tumor vaccine, tumor gene vaccine, tumour polypeptide vaccine etc. of carrier that current tumor vaccine mainly contains with cell.Wherein, tumor cell is a kind of important means of tumor vaccine as tumor biotherapy of carrier, is more and more subject to people and extensively payes attention to.But due to the less immunogenic of tumor cells expression antigen, surperficial MHC molecule, costimulatory molecules are expressed low or are lacked, and add the genetic background that tumor itself is complicated, original tumour-cell vaccine often can not induce very strong immunne response.For a change this is not enough, molecular modification technology can be adopted to change immunological characteristic or the genetic background of tumor cell, to improve its immunogenicity, induce stronger immunne response.Thus, the tumor vaccine of development good effect, applied range has become pressing issues of current treatment and prevention of tumour research.
Summary of the invention
The object of this invention is to provide a kind of tumor vaccine and application thereof.
Tumor vaccine provided by the invention, its preparation method in turn includes the following steps:
(1) encoding gene of special fusion rotein is imported in vitro tumor cell, obtain recombinant tumor cell;
Described special fusion rotein comprises polypeptide section and B7-1 peptide section from N end successively to C end; Described polypeptide section comprises poly-D-lysine from N end successively to C end (specifically can for form poly-D-lysine by 25-35 lysine residue; Preferably form poly-D-lysine by 30 lysine residues) and polyglutamic acid (can be specifically the polyglutamic acid that is made up of 25-35 glutaminic acid residue; The polyglutamic acid be preferably made up of 30 glutaminic acid residues);
(2) described recombinant tumor cell is carried out cell inactivation, obtain tumor vaccine.
For avoiding the space structure of described polypeptide section and described B7-1 peptide section to affect each other, between described polypeptide section and described B7-1 peptide section, also connection peptides can be had.The connection peptides that described connection peptides is preferably made up of 4 alanine residues.
Described special fusion rotein specifically can as shown in the sequence 1 of sequence table.
The encoding gene of described special fusion rotein specifically can as shown in the sequence 2 of sequence table.
In the preparation method of described tumor vaccine, also can comprise the steps: to get described recombinant tumor cell between step (1) and step (2), carry out processing (simulation clinical treatment) with chemotherapeutics.Described chemotherapeutics specifically can be cisplatin (Cisplatin, CDDP).The mode of described " processing with chemotherapeutics " is specific as follows: the cell suspension 1. getting described recombinant tumor cell, adds cisplatin and makes its concentration be 0.05mg/ml, cultivating collecting cell after 5 days, cultivates 30 days with after PBS buffer solution; 2. completing steps 1. after, collect living cells, suspend with after PBS buffer solution, obtain cell suspension; 3. get the cell suspension that 2. step obtains, add cisplatin and make its concentration be 0.05mg/ml, cultivating collecting cell after 5 days, cultivate 30 days with after PBS buffer solution; 4. completing steps 3. after, collect living cells, suspend with after PBS buffer solution, obtain cell suspension.
In described step (2), described cell inactivation specifically adopts ametycin to carry out.The actual conditions of described cell inactivation can be: cultivate 1 hour under the ametycin concentration of 100 μ g/ml.
The preparation method of described tumor vaccine is specific as follows:
(I) encoding gene of described special fusion rotein is imported in vitro tumor cell, obtain recombinant tumor cell;
(II) to suspend described recombinant tumor cell with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml;
(III) get the cell suspension that step (II) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid;
(IV) after completing steps (III), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml;
(V) get the cell suspension that step (IV) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid;
(VI) after completing steps (V), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml;
(VII) get the cell suspension that step (VI) obtains, add ametycin and make its concentration be 100 μ g/ml, at 37 DEG C, 5%CO2environment in quiescent culture 1 hour;
(VIII) after completing steps (VII), collect by the cell of ametycin deactivation, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining cell concentration is 1 × 106the cell suspension of individual/ml, is tumor vaccine.
Described in vitro tumor cell can be melanoma cell or lung adenocarcinoma cell.
Described melanoma cell refers to from melanomatous tumor cells ex vivo, can be obtained by primary separation, also can obtain by being purchased approach.Melanoma cell specifically can be B16-F10 cell.
Described lung adenocarcinoma cell refers to the tumor cells ex vivo from adenocarcinoma of lung.Described lung adenocarcinoma cell can be obtained by primary separation LA795 adenocarcinoma of lung mice with tumor (animal housing provides by Cancer Hospital of Chinese Academy of Medical Sciences, qualified number: SCXK11-00-0005) tumor tissues, also can obtain by being purchased approach.Lung adenocarcinoma cell specifically can be LA795 cell (Shanghai Yan Sheng Industrial Co., Ltd., article No. is YS2359).
Can by adding the stronger immunne response of immunological adjuvant induction on the basis of tumor vaccine provided by the invention.
Described special fusion rotein cross-film is expressed, and poly-D-lysine embeds the cell membrane of tumor cell, and B7-1 peptide section is free in outside the cell membrane of tumor cell.B7-1 peptide section provides costimulatory signal, inducer T lymphocyte propagation and secrete cytokines, thus strengthens the activation of T cell and the specific killing action of tumor.Compared with existing tumor vaccine (tumor vaccine that direct killing tumor cells obtains), the immunogenicity of tumor vaccine provided by the invention is stronger, Graft Versus Tumor is higher, more effective for the tumor cell of transfer or drug resistance after chemotherapy, to the prevention and therapy of tumor, there is significant application value.
Compared with existing tumor vaccine (tumor vaccine that direct killing tumor cells obtains), tumor vaccine provided by the invention can effectively prevention and therapy kinds of tumors occur, and has the curative effect of highly significant.The present invention has substantial worth for the treatment of tumor and the treatment of prevention, particularly melanoma and adenocarcinoma of lung and prevention.
Accompanying drawing explanation
Fig. 1 is the structural representation of carrier pDisplay.
Fig. 2 is the characterization result of reconstitution cell first and reconstitution cell second.
Detailed description of the invention
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Carrier pDisplay (structural representation is shown in Fig. 1): Invitrogen company, USA.B16-F10 cell:.LA795 cell: Shanghai Yan Sheng Industrial Co., Ltd., product article No. is YS2359.RPMI-1640 culture fluid: HyClone, article number is SH30809.01B.Mouse-anti people B7-1 monoclonal antibody: Bioscience company of the U.S..Cisplatin (Cisplatin, CDDP): Sigma Co., USA, catalog number is P4394.Ametycin (MitomycinC, MMC): purchased from Sigma, catalog number is M0503.C57BL/J6 mice: Beijing company of dimension tonneau China, male, body weight 18-26g.T739 mice: Cancer Hospital of Chinese Academy of Medical Sciences animal housing, male, body weight 18-26g.
Embodiment 1, prepare tumor vaccine
One, recombinant tumor cell is prepared
1, by between SmaI and the SalI restriction enzyme site of the double chain DNA molecule forward insertion vector pDisplay shown in the sequence 2 of sequence table, recombiant plasmid is obtained.
2, Transfected Recombinant Plasmid B16-F10 cell step 1 obtained, obtains reconstitution cell first.
3, by carrier pDisplay transfection B16-F10 cell, compared with control cells first is obtained.
4, Transfected Recombinant Plasmid LA795 cell step 1 obtained, obtains reconstitution cell second.
5, by carrier pDisplay transfection LA795 cell, compared with control cells second is obtained.
Two, the sign of reconstitution cell
1, the sign of reconstitution cell first
(1) with RPMI-1640 culture fluid suspension reconstitution cell first, compared with control cells first or B16-F10 cell, obtaining cell concentration is 1 × 106the cell suspension of individual/ml;
(2) cell suspension that 100 μ l steps one obtain is got, add 2 μ L mouse-anti people's B7-1 monoclonal antibodies and make its concentration be 200 μ g/ml, room temperature stationary incubation 1h, then suspend with the PBS buffer solution of pH7.4,0.01M, then GoatantimouseIgG/FITC is added and 4 DEG C of stationary incubation 1h, then suspend with the PBS buffer solution of pH7.4,0.01M, obtain 1 × 106the cell suspension of individual/ml, fine-still 1 cell suspension, on microscope slide, carries out immunofluorescence confocal laser scanning microscope.
Fig. 2 is shown in by photo, and A is reconstitution cell first, and B is compared with control cells first.B16-F10 cell is consistent with the phenotype of compared with control cells first.Can observe, reconstitution cell first surface display fluorescence (namely having B7-1), compared with control cells first and B16-F10 cell surface all do not show fluorescence.
2, the sign of reconstitution cell second
(1) with RPMI-1640 culture fluid suspension reconstitution cell second, compared with control cells second or LA795 cell, obtaining cell concentration is 1 × 106the cell suspension of individual/ml;
(2) with (2) of step 1.
Fig. 2 is shown in by photo, and A is reconstitution cell second, and B is compared with control cells second, and C is LA795 cell.LA795 cell is consistent with the phenotype of compared with control cells second.Can observe, reconstitution cell second surface display fluorescence (namely having B7-1), compared with control cells second and LA795 cell surface all do not show fluorescence.
Three, tumor vaccine is prepared
1, tumor vaccine first-I is prepared
(1) by RPMI-1640 culture fluid suspension reconstitution cell first, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(6) get the cell suspension that step (5) obtains, add ametycin (effect is as killed cells) and make its concentration be 100 μ g/ml, at 37 DEG C, 5%CO2environment in quiescent culture 1 hour.
(7) after completing steps (6), collect by the cell of ametycin deactivation, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining cell concentration is 1 × 106the cell suspension of individual/ml, is tumor vaccine first-I.
2, tumor vaccine first-II is prepared
(1) by RPMI-1640 culture fluid suspension reconstitution cell first, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, add ametycin (effect is as killed cells) and make its concentration be 100 μ g/ml, at 37 DEG C, 5%CO2environment in quiescent culture 1 hour.
(3) after completing steps (2), collect by the cell of ametycin deactivation, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining cell concentration is 1 × 106the cell suspension of individual/ml, is tumor vaccine first-II.
3, tumor vaccine second-I is prepared
Replace reconstitution cell first by reconstitution cell second, other is with step 1, obtains tumor vaccine second-I.
4, tumor vaccine second-II is prepared
Replace reconstitution cell first by reconstitution cell second, other is with step 2, obtains tumor vaccine second-II.
5, reference substance first-I is prepared
With compared with control cells methyl for reconstitution cell first, other is with step 1, obtains reference substance first-I.
6, reference substance first-II is prepared
With compared with control cells methyl for reconstitution cell first, other is with step 2, obtains reference substance first-II.
7, reference substance first-III is prepared
Replace reconstitution cell first with B16-F10 cell, other is with step 1, obtains reference substance first-III.
8, reference substance first-IV is prepared
Replace reconstitution cell first with B16-F10 cell, other is with step 2, obtains reference substance first-IV.
9, reference substance second-I is prepared
Replace reconstitution cell first by compared with control cells second, other is with step 1, obtains reference substance second-I.
10, reference substance second-II is prepared
Replace reconstitution cell first by compared with control cells second, other is with step 2, obtains reference substance second-II.
11, reference substance second-III is prepared
Replace reconstitution cell first with LA795 cell, other is with step 1, obtains reference substance second-III.
12, reference substance second-IV is prepared
Replace reconstitution cell first with LA795 cell, other is with step 2, obtains reference substance second-IV.
The immunological effect of embodiment 2, tumor vaccine first
One, lymphopoiesis is promoted
1, get C57BL/J6 mice, get spleen, be separated and obtain lymphocyte.
2, with the lymphocyte that RPMI-1640 culture fluid resuspending step 1 obtains, obtaining cell concentration is 1 × 106the lymphocyte suspension of individual/ml.
3, the lymphocyte suspension that 50 μ l products to be tested and 50 μ l steps 2 obtain is mixed, at 37 DEG C, 5%CO2environment in quiescent culture 3 days, then adopt MTT colorimetric method for determining light absorption value (A570nm), calculate proliferation index (ProliferationIndex, PI).Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The process replacing product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Often kind of product to be tested arranges 5 reprocessings, and blank arranges 5 reprocessings.
The light absorption value of PI=(light absorption value of the light absorption value-blank control wells of test hole)/blank control wells.
The PI adding the test group of tumor vaccine first-I is 3.95 ± 0.6.
The PI adding the test group of tumor vaccine first-II is 3.93 ± 0.3.
The PI adding the test group of reference substance first-I is 1.71 ± 0.2.
The PI adding the test group of reference substance first-II is 1.74 ± 0.3.
The PI adding the test group of reference substance first-III is 1.70 ± 0.4.
The PI adding the test group of reference substance first-IV is 1.73 ± 0.7.
Result shows: compared with each reference substance, and tumor vaccine first-I and tumor vaccine first-II can stimulate lymphopoiesis significantly, and the effect of each reference substance does not have significant difference.
Two, lymphocytic emiocytosis cytokine is promoted
1, the pretreatment of tumor cell
(1) with RPMI-1640 culture fluid suspension B16-F10 cell, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
2, mouse model is prepared
Get C57BL/J6 mice, (namely date of injection is respectively experiment the 1st day, the 8th day and the 15th day to the cell suspension obtained in right fore subcutaneous injection step 1 weekly; Containing 2 × 10 in the cell suspension of every injected in mice at every turn6individual living cells).Test the 5th day, the 12nd day and the 19th day, the tumor in situ size of mice is followed successively by 1.4cm3, 2.1cm3and 3.2cm3, obtain model mice.
3, test the 36th day, get the spleen of the mice of model mice, be separated and obtain lymphocyte.
4, with the lymphocyte that RPMI-1640 culture fluid resuspending step 3 obtains, obtaining cell concentration is 1 × 106the lymphocyte suspension of individual/ml.
5, the lymphocyte suspension that 50 μ l products to be tested and 50 μ l steps 4 obtain is mixed, at 37 DEG C, 5%CO2environment in quiescent culture 48 hours, the then centrifugal 4min of 250g, collects supernatant.Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The process replacing product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Often kind of product to be tested arranges 5 reprocessings, and blank arranges 5 reprocessings.
6, the supernatant that obtains of detecting step 5, detects the wherein concentration of IFN-γ and the concentration of IL-2.Be MouseIFN-gammaQuantikineELISAKit (RD company of the U.S., catalog number: MIF00) for detecting the test kit of IFN-γ.Be MouseIL-2QuantikineELISAKit (RD company of the U.S., catalog number: M2000) for detecting the test kit of IL-2.
The results are shown in Table 1.
The testing result (pg/ml) of table 1 mouse boosting cell secretion of gamma-IFN and IL-2
| IFN-γ | IL-2 |
| Blank | 39.0±2.5 | 15.6±0.5 |
| Reference substance first-IV | 46.2±3.5 | 26.9±1.3 |
| Reference substance first-III | 45.6±2.8 | 26.3±1.5 |
| Reference substance first-II | 46.4±3.1 | 26.7±1.2 |
| Reference substance first-I | 45.9±3.3 | 25.9±1.6 |
| Tumor vaccine first-II | 131.6±98 | 101.9±5.9 |
| Tumor vaccine first-I | 132.9±10.2 | 101.3±6.4 |
Three, zoopery
1, the pretreatment of tumor cell
(1) with RPMI-1640 culture fluid suspension B16-F10 cell, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
2, mouse model is prepared
Get C57BL/J6 mice, (namely date of injection is respectively experiment the 1st day, the 8th day and the 15th day to the cell suspension obtained in right fore subcutaneous injection step 1 weekly; Containing 2 × 10 in the cell suspension of every injected in mice at every turn6individual living cells).Test the 5th day, the 12nd day and the 19th day, the tumor in situ size of mice is followed successively by 1.4 ± 0.5cm3, 2.1 ± 0.1cm3with 3.2 ± 1.2cm3, obtain model mice.
3, grouping administration
Get model mice, be divided into 7 groups at random, often organize 10, give different products to be tested as follows: (namely date of injection is respectively experiment the 22nd day, the 29th day and the 36th day at right fore subcutaneous injection product to be tested weekly, natural law is counted continuously with step 2), every injected in mice 0.2ml product to be tested at every turn.Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The process replacing product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.The size variation of continuous observation tumor in situ, respectively at experiment the 26th day, the 33rd day and the 40th day statistics tumor size of mice and survival rate, test the survival rate that the 45th day adds up mice.The results are shown in Table 2.Only survival mice is added up during statistics tumor size.
Table 2 tumor size and survival results
The immunological effect of embodiment 3, tumor vaccine second
1, the pretreatment of tumor cell
(1) with RPMI-1640 culture fluid suspension LA795 cell, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(2) get the cell suspension that step (1) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(3) after completing steps (2), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106the cell suspension of individual/ml.
(4) get the cell suspension that step (3) obtains, add cisplatin and make its concentration be 0.05mg/ml, at 37 DEG C, 5%CO2environment in quiescent culture 5 days, then collecting cell, with the PBS buffer solution of pH7.4,0.01M, then cultivates 30 days in RPMI-1640 culture fluid.
(5) after completing steps (4), collect living cells, with the PBS buffer solution of pH7.4,0.01M, then suspend with RPMI-1640 culture fluid, obtaining viable cell concentrations is 1 × 106cell suspension.
2, mouse model is prepared
Get T739 mice, (namely date of injection is respectively experiment the 1st day, the 8th day and the 15th day to the cell suspension obtained in right fore subcutaneous injection step 1 weekly; Containing 2 × 10 in the cell suspension of every injected in mice at every turn6individual living cells).Test the 19th day, the metastatic nodules of each mouse lung is 8.5 ± 1.2, and the weight of the tumor in situ (tumor that the original position of namely injecting tumor cell occurs) of each mice is 3.3 ± 0.9g, obtains model mice.
3, grouping administration
Get model mice, be divided into 7 groups at random, often organize 10, give different products to be tested as follows: (namely date of injection is respectively experiment the 22nd day, the 29th day and the 36th day at right fore subcutaneous injection product to be tested weekly, natural law is counted continuously with step 1), every injected in mice 0.2ml product to be tested at every turn.Product to be tested is respectively tumor vaccine first-I, tumor vaccine first-II, reference substance first-I, reference substance first-II, reference substance first-III or reference substance first-IV prepared by embodiment 1.The process replacing product to be tested with equal-volume RPMI-1640 culture fluid is set, as blank.Test the 40th day, add up the metastatic nodules of each mouse lung, add up the weight of the tumor in situ (tumor that the original position of namely injecting tumor cell occurs) of each mice, the survival rate of statistics mice.The results are shown in Table 3.Only survival mice is added up when the metastatic nodules of statistics mouse lung and tumor in situ weight.
The metastatic nodules of table 3 mouse lung, tumor in situ weight and survival rate