CN104353067A - Malignant melanoma resisting vaccine composition and application thereof - Google Patents
Malignant melanoma resisting vaccine composition and application thereof Download PDFInfo
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Abstract
The invention relates to malignant melanoma resisting vaccine composition which is prepared through mixed culture of an antigen mixture and dendritic cell culture, wherein the antigen mixture comprises malignant melanoma antigens and tuberculosis antigens and/or Auer bodies. The vaccine composition can better stimulate specific T cell proliferation inside the body, has very high specificity killing function on targeted malignant melanoma cells, is convenient to use and good in treatment effect and has good application prospect.
Description
Technical field
The present invention relates to biological pharmacy technical field, relate to a kind of anti-malignant melanoma vaccine combination and application thereof.
Background technology
Malignant melanoma is high, the easy transfer of a kind of grade malignancy, poor prognosis, the malignant tumor that all insensitive to chemotherapy radiotherapy, case fatality rate is high.Primary disease mainly occurs in skin, accounts for the 3rd of malignant tumour of skin.Along with developing rapidly of modern tumor immunology, the immunization therapy carried out for malignant melanoma generation, development and metastasis has become the focus studied at present.Although malignant melanoma is insensitive to chemicotherapy, it is the tumor that immunogenicity is higher, and therefore tumor vaccine and immune factor treatment are widely used.But the tumor cell of application deactivation or it is broken after when being used as tumor vaccine treatment malignant melanoma Seedling treatment malignant melanoma, because the immunogenicity of tumor is too low, be not enough to induction body and produce effective antitumour immunoreation, specificity and the immunoreation of visible malignant melanoma are weak, can not effectively induce or activate the immunoreation of patient.
Therefore, current Problems existing is the specificity antineoplastic immunity reaction how increasing maligna element tumor vaccine.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, a kind of anti-malignant melanoma vaccine combination is provided, this vaccine combination can excite autologous internal specific T cell to breed preferably, and to target malignant melanoma cell, there is extremely strong specificity and kill function, this vaccine combination is easy to use, therapeutic effect is good, has good application prospect.
For this reason, the present invention first face provides a kind of anti-malignant melanoma vaccine combination, it is made up of antigen mixture and dendritic cell culture Mixed culture, and wherein said antigen mixture comprises malignant melanoma antigen and tuberculosis antigen and/or bar-shaped corpusculum.
According to the present invention, the volume ratio of described antigen mixture and dendritic cell culture is 2-1:1; The volume ratio of preferred antigens mixture and dendritic cell culture is 1:1.
In certain embodiments of the present invention, in described antigen mixture, the content of described malignant melanoma antigen is 80%-100% (volume), and the content of tuberculosis antigen is 0-20% (volume), and the content of bar-shaped corpusculum is 0-20% (volume).Preferably in described antigen mixture, the content of described malignant melanoma antigen is 80%-98% (volume), and the content of tuberculosis antigen is 2%-10% (volume), and the content of bar-shaped corpusculum is 2%-10% (volume).Preferred in described vaccine combination further, the content of described malignant melanoma antigen is 93% (volume), and the content of tuberculosis antigen is 5% (volume), and the content of bar-shaped corpusculum is 2% (volume).
In the present invention, in described antigen mixture, tuberculosis antigen and bar-shaped corpusculum can forming mixture with malignant melanoma antigen also can form mixture with malignant melanoma antigen respectively simultaneously.Mixture is formed with malignant melanoma antigen while of preferred.
According to the present invention, described malignant melanoma antigen comprises malignant melanoma attenuated cell and/or its malignant melanoma attenuated cell modifies body.
In the present invention, it is to be modified through 2,4-dinitro benzene by malignant melanoma cell and obtain after deactivation or break process that described malignant melanoma attenuated cell modifies body.Described malignant melanoma attenuated cell is obtained after deactivation or break process by malignant melanoma cell.
In some embodiments of the invention, described malignant melanoma cell comprises B16 cell or M3 cell.
In some embodiments of the invention, described dendritic cell culture by dendritic cell in the RPMI1640 containing GM-CSF, IL-4 and hyclone, at 5%CO
2, cultivate under 37 conditions and obtain for 6 days.
In other embodiments of the present invention, the condition of culture of described Mixed culture is in the RPMI1640 containing GM-CSF, IL-4 and hyclone, at 5%CO
2, cultivate 1 day under 37 conditions.
In one embodiment of the invention, stating tuberculosis antigen described in is bacillus calmette-guerin vaccine.
The present invention second aspect provides a kind of application of vaccine combination in the medicine preparing prevention and therapy malignant melanoma according to the present invention first aspect.
In detailed description of the invention more of the present invention, the vaccine combination described in first aspect of the present invention is prepared in such a way:
Steps A, adds the RPMI1640 containing GM-CSF (800U/mL), IL-4 (500U/mL) and 5% hyclone, at 5%CO in DC
2, cultivate 6 days obtained culture fluid containing DC culture under 37 DEG C of conditions;
Step B, the antigen mixture added in the culture fluid containing DC culture containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum continues cultivation 24 hours, the obtained culture fluid containing anti-malignant melanoma vaccine combination;
Step C, collects anti-malignant melanoma vaccine combination cell, and uses flow cytometry cell phenotype.
In recent years it is found that hapten can induce the immunne response of very strong toleragen-tumor, improve immunogenicity, Promote immunity system identification reinforced immunological are attacked, and break the immunologic tolerance of tumor.2,4-dinitro benzene (DNP) etc. is the earliest for studying the hapten of the classics of contact delayed hypersensitivity (DTH).DTH is the T cell caused by the hapten mediation that body accepts to occur after allergen (as hapten) stimulates 24 ~ 48h is again main immunoreation, and hapten makes T cell activation, impels the T forming sensitization
dwith Tc cell, when body contacts identical hapten again, T
dnamely cell discharges the panimmunity factor, and killing tumor cell common with Tc.It is very important step that the polypeptide (antigen) that this hapten adheres to MHC combines for forming immunogenicity.
Due to the many molecules of tumor antigen developed by molecule, the albumen that a large amount of hapten of generation brings out by the cell that DNP modifies, through antigen presenting cell (APC, comprise DC) processing, form complex with MHC I and class Ⅱmolecule, produce huge groove at cell surface, considerably increase the binding site with t cell responses, therefore, original weak tumor antigen epi-position is made to change strong antigen epi-position into; Secondly, the malignant melanoma cell that DNP modifies more easily is caught by APC and is offered signal, can more effectively induced tumor specific immune response.Thirdly, specific for tumour antigen, as a kind of antigen presenting cell, can amplify and passes to T lymphocyte, play specificity antineoplastic effect by DC.
The present inventor studies discovery, compared with the malignant melanoma cell modified with simple DNP is combined with dendritic cell (DC), by anti-malignant melanoma vaccine combination obtained after 24 hours for the culture Mixed culture containing tuberculosis antigen and malignant melanoma antigen and bar-shaped corpusculum antigen mixture and dendritic cell (DC), synergism is produced between the complex that itself and DC are formed is mutual, DC can be excited better, promote the propagation of specific T-cells, and then produce stronger specific cytotoxicity lymphocyte immunity reaction.
Anti-malignant melanoma vaccine combination according to the present invention can excite autologous internal specific T cell to breed preferably, and to target malignant melanoma cell, there is extremely strong specificity and kill function, this vaccine combination is easy to use, and therapeutic effect is good, has good application prospect.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is described.
Fig. 1 is in embodiment 1
3h-TdR method measures the proliferation function result of each group of DC stimulation to T cell; In figure, the implication of Reference numeral is as follows: 1DNP-M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group; 2 M3+ bacillus calmette-guerin vaccines+bar-shaped corpusculum+DC group; 3 DNP-M3+ bacillus calmette-guerin vaccine+DC groups; 4 M3+ bacillus calmette-guerin vaccine+DC groups; The bar-shaped corpusculum of 5DNP-M3++DC group; The bar-shaped corpusculum of 6 M3++DC group; 7 DC groups.
Fig. 2 is in embodiment 2
3h-TdR method measures the proliferation function result of each group of DC stimulation to T cell; In figure, the implication of Reference numeral is as follows: 1 DNP-B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group; 2 B16+ bacillus calmette-guerin vaccines+bar-shaped corpusculum+DC group; 3 DNP-B16+ bacillus calmette-guerin vaccine+DC groups; 4 B16+ bacillus calmette-guerin vaccine+DC groups; The bar-shaped corpusculum of 5 DNP-B16++DC group; The bar-shaped corpusculum of 6 B16++DC group; 7 DC groups.
Detailed description of the invention
For making the present invention easier to understand, describe the present invention in detail below in conjunction with embodiment and accompanying drawing, these embodiments only play illustrative effect, are not limited to range of application of the present invention.
1, reagent and instrument
Dinitrofluorobenzene is purchased from Chemical Reagent Co., Ltd., Sinopharm Group;
CD8-FITC, CD4-FITC, IFN-PE monoclonal antibody and different methyllanthionine fluorescein (FITC) purchased from American R & D company;
RPMI1640 culture fluid, collagenase and DNA enzymatic are Sigma product;
Mice dislikes black cell line B16 (H-2
b), M3 cell (H-2
ddislike black cell strain) preserve for this laboratory;
FACS/Calibur flow cytometer purchased from American Becton-Dickinson company;
Optical microscope is purchased from Japanese Olympus company;
WD-9405B type horizontal shaker is purchased from Beijing Liuyi Instrument Factory.
2, laboratory animal
BALB/c mouse (H-2
d), C57BL/6 mice (H-2
b) by Military Medical Science Institute animal is provided, 6 ~ 8 week age, female, body weight (22 ± 2) g, all raises under SPF level condition.
Embodiment
Embodiment 1:
1, the preparation of malignant melanoma antigen
(1) preparation of malignant melanoma attenuated cell
Get M3 malignant melanoma cell system (H-2
d) (2xl0
6/ mL) add in 6 orifice plates, then warp
60it is for subsequent use that Co irradiates 30Gy.
(2) DNP modifies the preparation of malignant melanoma attenuated cell
Get M3 malignant melanoma cell system (H-2
d) (2xl0
6/ mL) add in 6 orifice plates, then to add DNP solution 100L to final concentration be 0.2% (5mmol/L, pH7.2), hatches 10min for 37 DEG C, then warp
60it is for subsequent use that Co irradiates 30Gy.
The preparation of the antigen mixture 2, containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum
Measure malignant melanoma antigen respectively according to 80%-100% (volume), measure tuberculosis antigen according to 0%-20% (volume), according to 0%-20% (volume) measure bar-shaped corpusculum mix homogeneously after for subsequent use.
3, the preparation of DC and activation
(1) preparation of DC
Get BALB/c mouse (H-2
d) peripheral blood and medullary cell, be separated with Ficoll and obtain mononuclearcell, wash 2 times with RPMI1640, containing 10% hyclone RPMI1640 in 5%CO
2, cultivate 2 hours under 37 DEG C of conditions, wash out suspension cell gently for subsequent use.
(2) activation of DC
The RPMI1640 containing GM-CSF (800U/mL), IL-4 (500U/mL) and 5% hyclone is added, at 5%CO in washed out suspension cell
2, cultivate under 37 DEG C of conditions, every 2d half amount changes liquid 1 time.Basis of microscopic observation cellular morphology.Above-mentioned cell was divided into 7 groups in 6th day:
1st group is that DNP modifies evil black cell M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group (DNP-M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC), cultivates the antigen mixture adding evil black cell M3 (after irradiating), bacillus calmette-guerin vaccine and the bar-shaped corpusculum modified containing DNP on the 6th day continue cultivation 24h at DC;
For disliking black cell M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group (M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC), cultivating at DC the antigen mixture adding the black cell M3 of evil (after irradiating), bacillus calmette-guerin vaccine and bar-shaped corpusculum on the 6th day and continuing to cultivate 24h for 2nd group;
3rd group is that DNP modifies the black cell M3+ bacillus calmette-guerin vaccine+DC group (DNP-M3+ bacillus calmette-guerin vaccine+DC) of evil, cultivates the antigen mixture adding the black cell M3 of evil (after irradiating) and the bacillus calmette-guerin vaccine modified containing DNP on the 6th day continue cultivation 24h at DC;
For disliking black cell M3+ bacillus calmette-guerin vaccine+DC group (M3+ bacillus calmette-guerin vaccine+DC), cultivating at DC the antigen mixture adding the black cell M3 of evil (after irradiating) and bacillus calmette-guerin vaccine on the 6th day and continuing to cultivate 24h for 4th group;
5th group is that DNP modifies evil black cell M3+ bar-shaped corpusculum+DC group (the bar-shaped corpusculum of DNP-M3++DC), cultivates the antigen mixture adding evil black cell M3 (after irradiating) and the bar-shaped corpusculum modified containing DNP on the 6th day continue cultivation 24h at DC;
For disliking black cell M3+ bar-shaped corpusculum+DC group (the bar-shaped corpusculum of M3++DC), cultivating at DC the antigen mixture adding the black cell M3 of evil (after irradiating) and bar-shaped corpusculum on the 6th day and continuing to cultivate 24h for 6th group;
7th group is simple DC group, cultivates 7d at DC.
For subsequent use after collecting each group of cell flow cytometry cell phenotype.
4, each group DC is to the excitation experiment of T cell
Utilize the CD3 cell (total T cell) of MACS magnetic bead sorting instrument and CD3 monoclonal antibody positive sorting BALB/c mouse peripheral blood, by the CD that previous step obtains
3+the RPMI1640 of cell containing IL-2 (100U/mL) and 10% hyclone is diluted to 3 × 10
6/ mL, adds 24 well culture plates (1mL/ hole) and 96 well culture plates (100L/ hole) respectively, adds 1-7 group respectively, at 5%CO by 1/10 of T cell
2, cultivate 72h under 37 DEG C of conditions.Collect the cell in 24 orifice plates, wash 2 times with RPMI1640, action effect cell for subsequent use.Add in 96 orifice plates
3h-TdR (37kBq/ hole) continues to cultivate 12h, measures radionuclide flicker numeration per minute (CPM) value, calculates stimulation index (SI) according to formula I.
SI=experimental group CPM/ feminine gender group CPM (I)
5, T cell is tested the specific killing of malignant melanoma cell
Respectively with difference group DC activate T cell for effector lymphocyte, with
3the M3 cell of the exponential phase of H-TdR labelling is target cell, it is matched group that non-MHC mates cell B16, by effect target than for 50:1, effect target altogether incubation time be 24h, carry out cellulotoxic experiment, establish maximum release group (target cell+1%TritonX100) and spontaneous release group (target cell+culture fluid) simultaneously, measure and often organize supernatant CPM value.Before adding T cell, first add cold B16 cell with the amount of 10 times of target cells, get rid of the activity of NKT (NK) cell.T cell specific killing activity calculates according to formula II.
6, statistical procedures data represent with ± S, and adopt variance analysis and t inspection, with SPSS11.0 software statistics, P<0.05 is that difference has significance.
7, interpretation of result
7.1, the result of T cell propagation
(1) antigen+DC of variable concentrations stimulates the proliferation function to T cell
With the volume ratio of antigen and DC for antigen+DC that 1:1 investigates variable concentrations respectively stimulates proliferation function to T cell, wherein malignant melanoma antigen is that DNP modifies malignant melanoma attenuated cell.Adopt
3h-TdR method measures the proliferation function of antigen+DC stimulation to T cell of variable concentrations, the results are shown in Table 1.
As can be seen from Table 1, the bar-shaped corpusculum of 93% concentration malignant melanoma antigen, 5% concentration bacillus calmette-guerin vaccine and 2% concentration, to the effect of stimulating proliferation of T cell comparatively strong (P<0.05), the results are shown in Table 1.
Antigen+the DC of table 1 variable concentrations stimulates the proliferation function to T cell
(2) each group DC stimulates the proliferation function to T cell
Proliferation function to T cell is stimulated for 1:1 investigates each group of DC respectively with the volume ratio of antigen and DC, wherein DNP-M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC organizes and in M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC, the content of malignant melanoma antigen is 93% (volume), the content of tuberculosis antigen is 5% (volume), and the content of bar-shaped corpusculum is 2% (volume); DNP-M3+ bacillus calmette-guerin vaccine+DC organizes and in M3+ bacillus calmette-guerin vaccine+DC, the content of malignant melanoma antigen is 80% (volume), and the content of tuberculosis antigen is 20% (volume); The bar-shaped corpusculum of DNP-M3++DC organizes and in the bar-shaped corpusculum of M3++DC group, the content of malignant melanoma antigen is 80% (volume), and the content of bar-shaped corpusculum is 20% (volume).
Adopt
3h-TdR method measures the proliferation function of each group of DC stimulation to T cell, the results are shown in Figure 1.As can be seen from Figure 1, add simultaneously DNP, M3, bacillus calmette-guerin vaccine, bar-shaped corpusculum and DC combination T cell proliferation function comparatively without DNP modification group, single hapten bacillus calmette-guerin vaccine or bar-shaped corpusculum and simple DC group by force, the DC that activates of combination of the M3 cell namely after DNP modifies and hapten bacillus calmette-guerin vaccine, bar-shaped corpusculum, the T cell multiplication capacity that it brings out, apparently higher than other groups, illustrates that the DC that the antigen mixture containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum activates can induce the black specific T-cells effect of stronger evil.
(3) not synantigen-DC comparison T cell propagation impact
Investigate the impact of not synantigen-DC comparison T cell propagation using DNP-M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group as antigen, adopt
3h-TdR method measures the proliferation function of antigen+DC stimulation to T cell of different proportion, the results are shown in Table 2
The proliferation function of table 2 different proportion antigen mixture and DC culture volume comparison T cell
| Antigen mixture: DC (v/v) | Stimulation index SI value |
| 1:1 | 63 |
| 1.5:1 | 50 |
| 2:1 | 41 |
As can be seen from Table 2, when antigen composition (DNP-M3, bacillus calmette-guerin vaccine, bar-shaped corpusculum) is 1:1 with the volume ratio of dendritic cell culture, its proliferation function produced T cell is the strongest.
7.2, T cell is to the killing activity of tumor cell
Under adopting radionuclide method to measure the effect of DNP-M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group, M3-DC group and DC group, T cell is to the lethal effect of tumor cell (M3 cell), contrasts, the results are shown in Table 3 with normal saline group.
As can be seen from Table 3, DNP-M3+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC organizes the T cell of activation imitating target than during for 50:1, obviously can kill and wound M3 cell, but faint to the lethal effect of B16 cell; The T cell that the T cell of M3-DC group activation and simple DC activate is all very low to the lethal effect of 2 kinds of tumor cells.
The cytotoxicity of table 3T cell (
)
Embodiment 2:
1, the preparation of malignant melanoma antigen
(1) preparation of malignant melanoma attenuated cell
Take the logarithm the malignant melanoma cell system B16 (H-2 grown
b) (1xl0
7/ mL) add in 6 orifice plates, then warp
60it is for subsequent use that Co irradiates 30Gy.
(2) DNP modifies the preparation of malignant melanoma attenuated cell
Take the logarithm the malignant melanoma cell system B16 (H-2 grown
b) (1xl0
7/ mL) add in 6 orifice plates, then to add DNP solution 100 μ L be 0.2% (5mmol/L, pH7.2) to final concentration, hatches 10min for 37 DEG C, then warp
60it is for subsequent use that Co irradiates 30Gy, close rate 1.88Gy/min.
The preparation of the antigen mixture 2, containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum
Malignant melanoma antigen B16 attenuated cell is measured respectively according to 80%-98% (volume), measure tuberculosis antigen bacillus calmette-guerin vaccine according to 2%-10% (volume), according to 2-10% (volume) measure bar-shaped corpusculum mix homogeneously after for subsequent use.
3, the preparation of DC and suddenly work
(1) preparation of DC
Get C57BL/6 mice (H-2
b) peripheral blood and medullary cell, be separated with Ficoll and obtain mononuclearcell, wash 2 times with RPMI1640, containing 10% hyclone RPMI1640 in 5%CO
2, cultivate 2 hours under 37 DEG C of conditions, wash out suspension cell gently for subsequent use.
(2) activation of DC
The RPMI1640 containing GM-CSF (800U/mL), IL-4 (500U/mL) and 5% hyclone is added, at 5%CO in washed out suspension cell
2, cultivate under 37 DEG C of conditions, every 2d half amount changes liquid 1 time.Basis of microscopic observation cellular morphology.Above-mentioned cell was divided into 7 groups in 6th day:
1st group is that DNP modifies evil black cell B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group (DNP-B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC), cultivates the antigen mixture adding evil black cell M3 (after irradiating), bacillus calmette-guerin vaccine and the bar-shaped corpusculum modified containing DNP on the 6th day continue cultivation 24h at DC;
For disliking black cell B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group (B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC), cultivating at DC the antigen mixture adding the black cell B16 of evil (after irradiating), bacillus calmette-guerin vaccine and bar-shaped corpusculum on the 6th day and continuing to cultivate 24h for 2nd group;
3rd group is that DNP modifies the black cell B16+ bacillus calmette-guerin vaccine+DC group (DNP-B16+ bacillus calmette-guerin vaccine+DC) of evil, cultivates the antigen mixture adding the black cell B16 of evil (after irradiating) and the bacillus calmette-guerin vaccine modified containing DNP on the 6th day continue cultivation 24h at DC;
For disliking black cell B16+ bacillus calmette-guerin vaccine+DC group (B16+ bacillus calmette-guerin vaccine+DC), cultivating at DC the antigen mixture adding the black cell B16 of evil (after irradiating) and bacillus calmette-guerin vaccine on the 6th day and continuing to cultivate 24h for 4th group;
5th group is that DNP modifies evil black cell B16 bar-shaped corpusculum+DC group (the bar-shaped corpusculum of DNP-B16++DC), cultivates the antigen mixture adding evil black cell B16 (after irradiating) and the bar-shaped corpusculum modified containing DNP on the 6th day continue cultivation 24h at DC;
For disliking black cell B16+ bar-shaped corpusculum+DC group (the bar-shaped corpusculum of B16++DC), cultivating at DC the antigen mixture adding the black cell B16 of evil (after irradiating) and bar-shaped corpusculum on the 6th day and continuing to cultivate 24h for 6th group;
7th group is simple DC group, cultivates 7d at DC.
For subsequent use after collecting each group of cell flow cytometry cell phenotype.
4, each group DC is to the excitation experiment of T cell
Utilize the CD3 cell (total T cell) of MACS magnetic bead sorting instrument and CD3 monoclonal antibody positive sorting C57BL/6 mouse peripheral blood, by the CD that previous step obtains
3+the RPMI1640 of cell containing IL-2 (100U/mL) and 10% hyclone is diluted to 3 × 10
6/ mL, adds 24 well culture plates (1mL/ hole) and 96 well culture plates (100L/ hole) respectively, adds 1-7 group respectively, at 5%CO by 1/10 of T cell
2, cultivate 72h under 37 DEG C of conditions.Collect the cell in 24 orifice plates, wash 2 times with RPMI1640, action effect cell for subsequent use.Add in 96 orifice plates
3h-TdR (37kBq/ hole) continues to cultivate 12h, measures radionuclide flicker numeration per minute (CPM) value, calculates stimulation index (SI) according to formula I.
SI=experimental group CPM/ feminine gender group CPM (I)
5, T cell is tested the specific killing of malignant melanoma cell
Respectively with difference group activate T cell for effector lymphocyte, with
3the B16 cell of the exponential phase of H-TdR labelling is target cell, it is matched group that non-MHC mates cell M3, by effect target than for 50:1, effect target altogether incubation time be 24h, carry out cellulotoxic experiment, establish maximum release group (target cell+1%TritonX100) and spontaneous release group (target cell+culture fluid) simultaneously, measure and often organize supernatant CPM value.Before adding T cell, first add cold M3 cell with the amount of 10 times of target cells, get rid of the activity of NKT (NK) cell.T cell specific killing activity calculates according to formula II.
6, the immunization therapy that mice dislikes black model and hapten transformation is set up
Get C57BL/ mice (H-2b) totally 30, black cell line B16 (H-2 will be disliked
b) 1 × 10
7individual cell seeding, in mouse back, can touch subcutaneous tumors tuberosity after 10d.Tumor-bearing mice is divided into 9 groups at random, often organizes 6, respectively at compositions and the normal saline (normal saline group) of mice left dorsal injection difference group, all animal periodic evaluation tumor sizes.Every 3d vernier caliper measurement tumor body major diameter (a), minor axis (b), calculate the volume (V) of tumor body: V=ab
2/ 2; After inoculation 28d, peel off each group of tumor and weigh, after 4 weeks, calculating tumour inhibiting rate according to formula III:
Tumour inhibiting rate=each experimental group tumor weight-normal saline group tumor weight/normal saline group tumor weight × 100% (III)
7, statistical procedures data represent with ± S, and adopt variance analysis and t inspection, with SPSS11.0 software statistics, P<0.05 is that difference has significance.
8, interpretation of result
(1) each group DC stimulates the proliferation function to T cell
Proliferation function to T cell is stimulated for 1:1 investigates each group of DC respectively with the volume ratio of antigen and DC, wherein DNP-B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC organizes and in B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC, the content of malignant melanoma antigen is 93% (volume), the content of tuberculosis antigen is 5% (volume), and the content of bar-shaped corpusculum is 2% (volume); DNP-B16+ bacillus calmette-guerin vaccine+DC organizes and in B16+ bacillus calmette-guerin vaccine+DC, the content of malignant melanoma antigen is 80% (volume), and the content of tuberculosis antigen is 20% (volume); The bar-shaped corpusculum of DNP-B16++DC organizes and in the bar-shaped corpusculum of B16++DC group, the content of malignant melanoma antigen is 80% (volume), and the content of bar-shaped corpusculum is 20% (volume).
Adopt
3h-TdR method measures the proliferation function of each group of DC stimulation to T cell, the results are shown in Figure 2.As can be seen from Figure 2, add simultaneously DNP, B16, bacillus calmette-guerin vaccine, bar-shaped corpusculum and DC combination T cell proliferation function comparatively without DNP modification group, single hapten bacillus calmette-guerin vaccine or bar-shaped corpusculum and simple DC group by force, the DC that activates of combination of the B16 cell namely after DNP modifies and hapten bacillus calmette-guerin vaccine, bar-shaped corpusculum, the T cell multiplication capacity that it brings out, apparently higher than other groups, illustrates that the DC that the antigen mixture containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum activates can induce the black specific T-cells effect of stronger evil.
(2) T cell is to the killing activity of tumor cell
Under adopting radionuclide method to measure DNP-B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC group, B16-DC group and the effect of DC group, T cell is to the lethal effect of tumor cell (B16 cell), compares group, the results are shown in Table 4 with normal saline group.
Isotope method is adopted to measure T cell to the lethal effect of B16 cell, result shows, DNP-B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+DC organizes the T cell of activation imitating target than during for 50:1, obviously can kill and wound B16 cell, but faint to M3 cell (H-2d dislikes black cell strain) lethal effect; The T cell that the T cell of B16-DC group activation and simple DC activate is all very low to the lethal effect of two kinds of tumor cells, in table 4.
The cytotoxicity of table 4T cell (
)
(4) anti-malignant melanoma vaccine combination is to the immunization therapy of tumor-bearing mice
After B16 cell implantation in vivo, 8 ~ 12d all can touch subcutaneous tumors tuberosity.20d unexpected death after 1 the mice medication of hapten transformation group.Normal saline group and DC group tumor volume increase gradually, and B16-DC group tumor volume after the 17th day starts to reduce, and after 4 weeks, tumour inhibiting rate is 50.11%; There is analog result, tumour inhibiting rate 57.79 in DNP+ bacillus calmette-guerin vaccine+bar-shaped corpusculum group; Namely DNP-B16+ bacillus calmette-guerin vaccine+bar-shaped corpusculum+-DC group has from 10th ~ 12 days tumor bodies and obviously reduces, obviously early than B16-DC group, and inhibitory rate 98.96% during 28d, apparently higher than other each group (P<0.01), tumor is close to and suppresses completely, this is very inaccessible in the prior art, in table 5.
The anti-malignant melanoma vaccine combination of table 5 is on the impact of 28d tumor-bearing mice
As can be seen from above-described embodiment, compared with the malignant melanoma cell modified with simple DNP is combined with dendritic cell (DC), by anti-malignant melanoma vaccine combination obtained after 24 hours for the culture Mixed culture containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum antigen mixture and dendritic cell (DC), synergism is produced between the complex that itself and DC are formed is mutual, DC can be excited better, promote the propagation of specific T-cells, and then produce extremely strong specific cytotoxicity lymphocyte immunity reaction.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. an anti-malignant melanoma vaccine combination, it is made up of antigen mixture and dendritic cell culture Mixed culture, and wherein said antigen mixture comprises malignant melanoma antigen and tuberculosis antigen and/or bar-shaped corpusculum.
2. vaccine combination according to claim 1, is characterized in that, the volume ratio of described antigen mixture and dendritic cell culture is 2-1:1; The volume ratio of preferred antigens mixture and dendritic cell culture is 1:1.
3. vaccine combination according to claim 1 and 2, it is characterized in that, in described antigen mixture, the content of described malignant melanoma antigen is 80%-100% (volume), the content of tuberculosis antigen is 0-20% (volume), and the content of bar-shaped corpusculum is 0-20% (volume); Preferably in described antigen mixture, the content of described malignant melanoma antigen is 80%-98% (volume), and the content of tuberculosis antigen is 2%-10% (volume), and the content of bar-shaped corpusculum is 2%-10% (volume); Preferred in described vaccine combination further, the content of described malignant melanoma antigen is 93% (volume), and the content of tuberculosis antigen is 5% (volume), and the content of bar-shaped corpusculum is 2% (volume).
4. according to the vaccine combination in claim 1-3 described in any one, it is characterized in that, described malignant melanoma antigen comprises malignant melanoma attenuated cell and/or its malignant melanoma attenuated cell modifies body.
5. vaccine combination according to claim 4, is characterized in that, it is to be modified through 2,4-dinitro benzene by malignant melanoma cell and obtain after deactivation or break process that described malignant melanoma attenuated cell modifies body; Described malignant melanoma attenuated cell is obtained after deactivation or break process by malignant melanoma cell.
6. vaccine combination according to claim 5, is characterized in that, described malignant melanoma cell comprises B16 cell or M3 cell.
7., according to the vaccine combination in claim 1-6 described in any one, it is characterized in that, described dendritic cell culture by dendritic cell in the RPMI1640 containing GM-CSF, IL-4 and hyclone, at 5%CO
2, cultivate under 37 conditions and obtain for 6 days.
8. according to the vaccine combination in claim 1-7 described in any one, it is characterized in that, the condition of culture of described Mixed culture is in the RPMI1640 containing GM-CSF, IL-4 and hyclone, at 5%CO
2, cultivate 1 day under 37 conditions.
9. according to the vaccine combination in claim 1-8 described in any one, it is characterized in that, described tuberculosis antigen is bacillus calmette-guerin vaccine.
10. one kind according to the application of the vaccine combination in claim 1-9 described in any one in the medicine preparing prevention and therapy malignant melanoma.
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| MASARU UDAGAWA等: "Enhancement of Immunologic Tumor Regression by Intratumoral Administration of Dendritic Cells in Combination with Cryoablative Tumor Pretreatment and Bacillus Calmette-Guerin Cell Wall Skeleton Stimulation", 《CLINICAL CANCER RESEARCH》 * |
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