CN104353067B - A kind of anti-malignant mela noma vaccine combination and its application - Google Patents
A kind of anti-malignant mela noma vaccine combination and its application Download PDFInfo
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Abstract
The present invention relates to a kind of anti-malignant mela noma vaccine combination, it is made up of antigen mixture with BMDC culture mixed culture, wherein the antigen mixture includes malignant melanoma antigen and tuberculosis antigen and/or bar-shaped corpusculum.The vaccine combination can preferably excite autologous internal specific T cell to breed, and there is extremely strong specificity to kill function to target malignant MC, and the vaccine combination is easy to use, and therapeutic effect is good, with good application prospect.
Description
Technical field
The present invention relates to biological pharmacy technical field, it is related to a kind of anti-malignant mela noma vaccine combination and its application.
Background technology
Chromoma is a kind of high grade malignancy, easy transfer, poor prognosis, case fatality rate insensitive to chemotherapy radiotherapy
High malignant tumour.This disease occurs mainly in skin, accounts for the 3rd of malignant tumour of skin.With modern tumor immunology
Develop rapidly, the immunization therapy carried out for chromoma generation, development and metastasis has turned into the heat of current research
Point.Although chromoma is insensitive to chemicotherapy, it is immunogenicity tumour higher, thus tumor vaccine and it is immune because
Son treatment is widely used.But apply the tumour cell of inactivation or be used as knurl seedling treatment chromoma seedling after being crushed and control
When treating chromoma, because the immunogenicity of tumour is too low, it is not enough to induction body and produces effective antitumour immune response,
It can be seen that the specificity of chromoma and immune response are weak, it is impossible to effectively induce or activate the immune response of patient.
Therefore, the problem that presently, there are be how to increase maligna element tumor vaccine specificity antineoplastic immunity it is anti-
Should.
The content of the invention
The technical problems to be solved by the invention are directed to the deficiencies in the prior art, there is provided a kind of anti-malignant mela noma epidemic disease
Seedling composition, the vaccine combination can preferably excite autologous internal specific T cell to breed, and to target malignant melanoma
There is cell extremely strong specificity to kill function, and the vaccine combination is easy to use, and therapeutic effect is good, with well using preceding
Scape.
Therefore, first face of the present invention provides a kind of anti-malignant mela noma vaccine combination, it is by antigen mixture
It is made with BMDC culture mixed culture, wherein the antigen mixture includes malignant melanoma antigen and tuberculosis
Sick antigen and/or bar-shaped corpusculum.
According to the present invention, the antigen mixture is 2-1 with the volume ratio of BMDC culture：1；Preferred antigens are mixed
Compound is 1 with the volume ratio of BMDC culture：1.
In certain embodiments of the present invention, in the antigen mixture, the malignant melanoma antigen contains
It is 80%-100% (volume) to measure, and the content of tuberculosis antigen is 0-20% (volume), and the content of bar-shaped corpusculum is 0-20% (bodies
Product).It is preferred that in the antigen mixture, the content of the malignant melanoma antigen is 80%-98% (volume), tuberculosis
The content of antigen is 2%-10% (volume), and the content of bar-shaped corpusculum is 2%-10% (volume).Further preferably in the epidemic disease
In seedling composition, the content of the malignant melanoma antigen is 93% (volume), and the content of tuberculosis antigen is 5% (body
Product), the content of bar-shaped corpusculum is 2% (volume).
In the present invention, tuberculosis antigen and bar-shaped corpusculum can be while and malignant mela nomas in the antigen mixture
Antigen forms mixture and can also form mixture with malignant melanoma antigen respectively.It is preferred that simultaneously anti-with malignant mela noma
Original shape resulting mixture.
According to the present invention, the malignant melanoma antigen includes malignant mela noma attenuated cell and/or its is pernicious black
Melanoma attenuated cell modified body.
In the present invention, the malignant mela noma attenuated cell modified body is through 2,4- dinitros by malignant melanoma cell
Base benzene is modified and obtained after inactivation or break process.The malignant mela noma attenuated cell is passed through by malignant melanoma cell
Obtained after inactivation or break process.
In some embodiments of the invention, the malignant melanoma cell includes B16 cells or M3 cells.
In some embodiments of the invention, the BMDC culture by BMDC containing GM-CSF,
In the RPMI1640 of IL-4 and hyclone, in 5%CO2, under the conditions of 37 culture obtain within 6 days.
In other embodiments of the invention, the condition of culture of the mixed culture be containing GM-CSF, IL-4 and
In the RPMI1640 of hyclone, in 5%CO2, cultivate 1 day under the conditions of 37.
In one embodiment of the invention, the tuberculosis antigen of stating is for BCG vaccine.
Second aspect of the present invention provides a kind of vaccine combination according to present invention one side and is preparing
Prevent and treat the application in the medicine of malignant mela noma.
In certain specific embodiments of the invention, vaccine combination described in the first aspect of the invention according to
Under type is prepared：
Step A, adds containing GM-CSF (800U/mL), IL-4 (500U/mL) and 5% hyclone in DC
RPMI1640, in 5%CO2, under the conditions of 37 DEG C culture the nutrient solution containing DC cultures is obtained within 6 days；
Step B, tuberculosis antigen, malignant melanoma antigen and rod are contained to being added in the nutrient solution containing DC cultures
The antigen mixture of shape corpusculum continues to cultivate 24 hours, and the nutrient solution containing anti-malignant mela noma vaccine combination is obtained；
Step C, collects anti-malignant mela noma vaccine combination cell, and use flow cytometry cell phenotype.
In recent years it is found that haptens can induce the immune response of very strong toleragen-tumour, immunogenicity is improved, is promoted
Enter immune system identification and reinforced immunological is attacked, break the immune tolerance of tumour.2,4- dinitro benzenes (DNP) etc. are to be used for earliest
The classical haptens of research contact delayed allergy (DTH).DTH is that body receives allergen (such as haptens) again
Stimulate the immune response based on the T cell caused by the haptens mediation occurred after 24~48h, haptens makes T cell activation, promotees
Make the T to form sensitizationDWith Tc cells, when body contacts identical haptens again, TDCell is to discharge the panimmunity factor,
And with the common killing tumor cells of Tc.The polypeptide (antigen) that this haptens adheres to MHC is combined for forming immunogenicity
It is extremely important step.
Due to many molecules of tumour antigen developed by molecule, the cell of DNP modifications will produce the albumen of a large amount of haptens inductions,
Processed by antigen presenting cell (APC, including DC), with MHC I and class Ⅱmolecule formation compound, produced in cell surface huge
Big groove, considerably increases the binding site with t cell responses, therefore, it is changed into original weak tumour antigen epitope strong anti-
Former epitope；Secondly, the malignant melanoma cell of DNP modifications is more easy to offer signal by APC captures, can more effectively induce swollen
Knurl specific immune response.Thirdly, specific for tumour antigen can be amplified and passed to by DC as a kind of antigen presenting cell
T lymphocytes, play specificity antineoplastic effect.
The present inventor's research discovery, the malignant melanoma cell modified with simple DNP and BMDC
(DC) combine and compare, tuberculosis antigen and malignant melanoma antigen and bar-shaped corpusculum antigen mixture and dendron shape will be contained
The culture of cell (DC) anti-malignant mela noma vaccine combination obtained after being mixed 24 hours, it is formed with DC
Compound produce synergy from each other, can preferably excite DC, promote the propagation of specific T-cells, and then produce more
Strong SC lymphocyte immunity reaction.
Anti- malignant mela noma vaccine combination of the invention can preferably excite autologous internal specific T cell to increase
Grow, and there is extremely strong specificity to kill function to target malignant MC, the vaccine combination is easy to use, treatment
Effect is good, with good application prospect.
Brief description of the drawings
The present invention is illustrated below in conjunction with accompanying drawing.
During Fig. 1 is embodiment 13H-TdR methods determine proliferation function result of each group DC stimulations to T cell；Accompanying drawing mark in figure
The implication of note is as follows：1DNP-M3+ BCG vaccines+bar-shaped corpusculum+DC groups；2 M3+ BCG vaccines+bar-shaped corpusculum+DC groups；3 DNP-M3+
BCG vaccine+DC groups；4 M3+ BCG vaccine+DC groups；Bar-shaped corpusculum+DC the groups of 5 DNP-M3+；Bar-shaped corpusculum+DC the groups of 6 M3+；7 DC
Group.
During Fig. 2 is embodiment 23H-TdR methods determine proliferation function result of each group DC stimulations to T cell；Accompanying drawing mark in figure
The implication of note is as follows：1 DNP-B16+ BCG vaccines+bar-shaped corpusculum+DC groups；2 B16+ BCG vaccines+bar-shaped corpusculum+DC groups；3 DNP-
B16+ BCG vaccine+DC groups；4 B16+ BCG vaccine+DC groups；Bar-shaped corpusculum+DC the groups of 5 DNP-B16+；Bar-shaped corpusculum+the DC of 6 B16+
Group；7 DC groups.
Specific embodiment
To make the present invention easier to understand, the present invention is described in detail below in conjunction with embodiment and accompanying drawing, these realities
Apply example only serve it is illustrative, it is not limited to range of application of the invention.
1st, 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) are public purchased from U.S. R＆D
Department；
RPMI1640 nutrient solutions, clostridiopetidase A and DNA enzymatic are Sigma products；
Mouse dislikes black cell line B16 (H-2b), M3 cells (H-2dDislike black cell line) for this laboratory preserves；
FACS/Calibur flow cytometers are purchased from Becton-Dickinson companies of the U.S.；
Light microscope is purchased from Olympus companies of Japan；
WD-9405B types horizontal shaker is purchased from Beijing Liuyi Instrument Factory.
2nd, experimental animal
BALB/c mouse (H-2d), C57BL/6 mouse (H-2b) provided by Military Medical Science Institute animal, 6~8 week old,
Female, body weight (22 ± 2) g is raised under the conditions of SPF grades.
Embodiment
Embodiment 1：
1st, the preparation of malignant melanoma antigen
(1) preparation of malignant mela noma attenuated cell
Take M3 malignant melanoma cells system (H-2d)(2xl06/ mL) add 6 orifice plates in, Ran Houjing6030Gy is standby for Co irradiations
With.
(2) DNP modifies the preparation of malignant mela noma attenuated cell
Take M3 malignant melanoma cells system (H-2d)(2xl06/ mL) add 6 orifice plates, then add DNP solution 100L to end
Concentration is 0.2% (5mmol/L, pH7.2), and 37 DEG C are incubated 10min, Ran Houjing6030Gy is standby for Co irradiations.
The preparation of the antigen mixture the 2nd, containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum
Malignant melanoma antigen is measured according to 80%-100% (volume) respectively, according to the amount of 0%-20% (volume)
Tuberculosis antigen is taken, it is standby after bar-shaped corpusculum is well mixed according to measuring for 0%-20% (volume).
3rd, the preparation and activation of DC
(1) preparation of DC
Take BALB/c mouse (H-2d) peripheral blood and bone marrow cell, separated with Ficoll and obtain mononuclearcell, use
RPMI1640 is washed 2 times, in 5%CO in the RPMI1640 containing 10% hyclone2, cultivate 2 hours under the conditions of 37 DEG C, gently
Rinse out suspension cell standby.
(2) activation of DC
Contain GM-CSF (800U/mL), IL-4 (500U/mL) and 5% hyclone to being added in the suspension cell for being washed out
RPMI1640, in 5%CO2, cultivate under the conditions of 37 DEG C, per 2d, half measures and changes liquid 1 time.Basis of microscopic observation cellular morphology.6th day
Above-mentioned cell is divided into 7 groups：
1st group be DNP modifications dislike black cell M3+ BCG vaccines+bar-shaped corpusculum+DC groups (DNP-M3+ BCG vaccines+bar-shaped corpusculum+
DC), being cultivated in DC adds the antigen of the black cell M3 (after irradiation) of evil, BCG vaccine and bar-shaped corpusculum containing DNP modifications to mix on the 6th day
Compound persistently cultivates 24h；
2nd group, to dislike black cell M3+ BCG vaccines+bar-shaped corpusculum+DC groups (M3+ BCG vaccines+bar-shaped corpusculum+DC), is trained in DC
Support and add within the 6th day the antigen mixture for disliking black cell M3 (after irradiation), BCG vaccine and bar-shaped corpusculum persistently to cultivate 24h；
3rd group is that black cell M3+ BCG vaccine+DC groups (DNP-M3+ BCG vaccines+DC) are disliked in DNP modifications, is cultivated the 6th day in DC
The black cell M3 (after irradiation) of evil containing DNP modifications and the antigen mixture of BCG vaccine is added persistently to cultivate 24h；
4th group, to dislike black cell M3+ BCG vaccine+DC groups (M3+ BCG vaccines+DC), is cultivated to add for the 6th day and dislikes black cell in DC
The antigen mixture of M3 (after irradiation) and BCG vaccine persistently cultivates 24h；
5th group is that black bar-shaped corpusculum+DC groups of cell M3+ (the bar-shaped corpusculum+DC of DNP-M3+) are disliked in DNP modifications, in DC cultures the
The black cell M3 (after irradiation) of evil containing DNP modifications and the antigen mixture of bar-shaped corpusculum is added within 6 days persistently to cultivate 24h；
6th group, to dislike the black bar-shaped corpusculum+DC groups of cell M3+ (the bar-shaped corpusculum+DC of M3+), is cultivated in DC and adds within the 6th day evil black
The antigen mixture of cell M3 (after irradiation) and bar-shaped corpusculum persistently cultivates 24h；
7th group is simple DC groups, and 7d is cultivated in DC.
Each group cell is collected with standby after flow cytometry cell phenotype.
4th, excitation experiments of each group DC to T cell
Using MACS magnetic bead sortings instrument and the positive CD3 cells for sorting BALB/c mouse peripheral blood of CD3 monoclonal antibodies, (total T is thin
Born of the same parents), the CD that previous step is obtained3+RPMI1640 of the cell containing IL-2 (100U/mL) and 10% hyclone be diluted to 3 ×
106/ mL, is separately added into 24 well culture plates (1mL/ holes) and 96 well culture plates (100L/ holes), is separately added into by the 1/10 of T cell
1-7 groups, in 5%CO2, cultivate 72h under the conditions of 37 DEG C.The cell in 24 orifice plates is collected, is washed with RPMI1640 2 times, it is standby
As effector cell.Added in 96 orifice plates3H-TdR (37kBq/ holes) continues to cultivate 12h, determines radionuclide sudden strain of a muscle per minute
Bright numeration (CPM) value, SI (SI) is calculated according to formula (I).
The negative group CPM (I) of SI=experimental groups CPM/
5th, T cell is tested to the specific killing of malignant melanoma cell
Respectively with the different groups of T cells of DC activation as effector cell, with3The M3 cells of the exponential phase of H-TdR marks
It is target cell, non-MHC matching cells B16 is control group, is 50 by effect target ratio：1st, incubation time is 24h to effect target altogether, carries out cell
Poison experiment, while setting maximum release group (target cell+1%TritonX100) and spontaneous release group (target cell+nutrient solution), determines
Every group of supernatant CPM value.Before T cell is added, the B16 cells of non-marked are first added with 10 times of amounts of target cell, exclude nature
Kill the activity of (NK) cell.T cell specific killing activity is calculated according to formula (II).
6th, statistical procedures data represent with ± S, are checked using variance analysis and t, with SPSS11.0 software statistics, P<
0.05 has conspicuousness for difference.
7th, interpretation of result
7.1st, the result of T cell propagation
(1) antigen+DC of various concentrations stimulates the proliferation function to T cell
Volume ratio with antigen and DC is 1：1 antigen+the DC for investigating various concentrations respectively stimulates and the propagation of T cell is made
With wherein malignant melanoma antigen is that DNP modifies malignant mela noma attenuated cell.Using3H-TdR methods determine various concentrations
Antigen+DC stimulate to the proliferation function of T cell, the results are shown in Table 1.
As it can be seen from table 1 93% concentration malignant melanoma antigen, 5% concentration BCG vaccine and 2% concentration is bar-shaped small
Relatively strong (the P of stimulate proliferation effect of the body to T cell<0.05), the results are shown in Table 1.
Antigen+the DC of the various concentrations of table 1 stimulates the proliferation function to T cell
(2) each group DC stimulates the proliferation function to T cell
Volume ratio with antigen and DC is 1：1 investigates proliferation function of each group DC stimulations to T cell, wherein DNP-M3 respectively
In+BCG vaccine+bar-shaped corpusculum+DC groups and M3+ BCG vaccines+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+ BCG vaccines+DC
In group and M3+ BCG vaccines+DC, the content of malignant melanoma antigen is 80% (volume), and the content of tuberculosis antigen is 20%
(volume)；In the bar-shaped corpusculum+DC groups of DNP-M3+ and the bar-shaped corpusculum+DC groups of M3+, the content of malignant melanoma antigen is 80%
(volume), the content of bar-shaped corpusculum is 20% (volume).
Using3H-TdR methods determine each group DC stimulations to the proliferation function of T cell, as a result see Fig. 1.It will be seen from figure 1 that
The proliferation function of combination T cell of DNP, M3, BCG vaccine, bar-shaped corpusculum and DC is added simultaneously compared with without DNP modifications group, single half
Antigen BCG vaccine or bar-shaped corpusculum and simple DC groups are strong, i.e., M3 cells and haptens BCG vaccine, bar-shaped corpusculum after being modified through DNP
Combination activation DC, the T cell multiplication capacity that it induces illustrated containing tuberculosis antigen, pernicious black apparently higher than other groups
The DC of the antigen mixture activation of pigment tumor antigen and bar-shaped corpusculum can induce the black specific T-cells effect of stronger evil.
(3) synantigen-DC does not compare the influence of T cell propagation
The shadow that not synantigen-DC compares T cell propagation is investigated as antigen using DNP-M3+ BCG vaccines+bar-shaped corpusculum+DC groups
Ring, use3H-TdR methods determine the antigen+DC stimulations of different proportion to the proliferation function of T cell, the results are shown in Table 2
The different proportion antigen mixture of table 2 compares the proliferation function of T cell with DC culture volumes
| Antigen mixture：DC(v/v) | SI SI values |
| 1:1 | 63 |
| 1.5:1 | 50 |
| 2:1 | 41 |
From Table 2, it can be seen that when antigen composition (DNP-M3, BCG vaccine, bar-shaped corpusculum) is cultivated with BMDC
The volume ratio of thing is 1：When 1, it is most strong to the proliferation function that T cell is produced.
7.2nd, killing activity of the T cell to tumour cell
In the presence of radionuclide method measure DNP-M3+ BCG vaccines+bar-shaped corpusculum+DC groups, M3-DC groups and DC groups
T cell is compareed to the lethal effect of tumour cell (M3 cells) with physiological saline group, the results are shown in Table 3.
From table 3 it can be seen that the T cell of DNP-M3+ BCG vaccines+bar-shaped corpusculum+DC groups activation is 50 in effect target ratio：When 1,
M3 cells can be substantially killed, but it is faint to the lethal effect of B16 cells；The T cell of M3-DC groups activation is activated with simple DC
T cell is very low to 2 kinds of lethal effects of tumour cell.
Table 3T cells cytotoxicity ()
Embodiment 2：
1st, the preparation of malignant melanoma antigen
(1) preparation of malignant mela noma attenuated cell
Take the logarithm the malignant melanoma cell system B16 (H-2 of growthb)(1xl07/ mL) add 6 orifice plates in, Ran Houjing6030Gy is standby for Co irradiations.
(2) DNP modifies the preparation of malignant mela noma attenuated cell
Take the logarithm the malignant melanoma cell system B16 (H-2 of growthb)(1xl07/ mL) add 6 orifice plates in, then add DNP it is molten
The μ L of liquid 100 to final concentration of 0.2% (5mmol/L, pH7.2), 37 DEG C are incubated 10min, Ran Houjing6030Gy is standby for Co irradiations, agent
Dose rate 1.88Gy/min.
The preparation of the antigen mixture the 2nd, containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum
Malignant melanoma antigen B16 attenuated cells are measured according to 80%-98% (volume) respectively, according to 2%-10%
(volume) measures tuberculosis antigen BCG vaccine, according to 2-10% (volume) measure bar-shaped corpusculum it is well mixed after it is standby.
3rd, the preparation of DC and urgency are living
(1) preparation of DC
Take C57BL/6 mouse (H-2b) peripheral blood and bone marrow cell, separated with Ficoll and obtain mononuclearcell, use
RPMI1640 is washed 2 times, in 5%CO in the RPMI1640 containing 10% hyclone2, cultivate 2 hours under the conditions of 37 DEG C, gently
Rinse out suspension cell standby.
(2) activation of DC
Contain GM-CSF (800U/mL), IL-4 (500U/mL) and 5% hyclone to being added in the suspension cell for being washed out
RPMI1640, in 5%CO2, cultivate under the conditions of 37 DEG C, per 2d, half measures and changes liquid 1 time.Basis of microscopic observation cellular morphology.6th day
Above-mentioned cell is divided into 7 groups：
1st group is that black cell B16+ BCG vaccines+bar-shaped corpusculum+DC groups (DNP-B16+ BCG vaccines+bar-shaped small are disliked in DNP modifications
Body+DC), cultivated in DC and add within the 6th day the anti-of the black cell M3 (after irradiation) of evil containing DNP modifications, BCG vaccine and bar-shaped corpusculum
Original mixture persistently cultivates 24h；
2nd group is the black cell B16+ BCG vaccines+bar-shaped corpusculum+DC groups (B16+ BCG vaccines+bar-shaped corpusculum+DC) of evil, in DC
Cultivate and add within the 6th day the antigen mixture for disliking black cell B16 (after irradiation), BCG vaccine and bar-shaped corpusculum persistently to cultivate 24h；
3rd group is that black cell B16+ BCG vaccine+DC groups (DNP-B16+ BCG vaccines+DC) are disliked in DNP modifications, and the 6th is cultivated in DC
It adds the black cell B16 (after irradiation) of evil containing DNP modifications and the antigen mixture of BCG vaccine persistently to cultivate 24h；
4th group, to dislike black cell B16+ BCG vaccine+DC groups (B16+ BCG vaccines+DC), is cultivated in DC and adds within the 6th day evil black thin
The antigen mixture of born of the same parents B16 (after irradiation) and BCG vaccine persistently cultivates 24h；
5th group is that the black bar-shaped corpusculum+DC groups of cell B16 (the bar-shaped corpusculum+DC of DNP-B16+) are disliked in DNP modifications, in DC cultures
The black cell B16 (after irradiation) of evil containing DNP modifications and the antigen mixture of bar-shaped corpusculum is added within 6th day persistently to cultivate 24h；
6th group, to dislike the black bar-shaped corpusculum+DC groups of cell B16+ (the bar-shaped corpusculum+DC of B16+), is cultivated to add for the 6th day and disliked in DC
The antigen mixture of black cell B16 (after irradiation) and bar-shaped corpusculum persistently cultivates 24h；
7th group is simple DC groups, and 7d is cultivated in DC.
Each group cell is collected with standby after flow cytometry cell phenotype.
4th, excitation experiments of each group DC to T cell
Using MACS magnetic bead sortings instrument and the positive CD3 cells for sorting C57BL/6 mouse peripheral bloods of CD3 monoclonal antibodies, (total T is thin
Born of the same parents), the CD that previous step is obtained3+RPMI1640 of the cell containing IL-2 (100U/mL) and 10% hyclone be diluted to 3 ×
106/ mL, is separately added into 24 well culture plates (1mL/ holes) and 96 well culture plates (100L/ holes), is separately added into by the 1/10 of T cell
1-7 groups, in 5%CO2, cultivate 72h under the conditions of 37 DEG C.The cell in 24 orifice plates is collected, is washed with RPMI1640 2 times, it is standby
As effector cell.Added in 96 orifice plates3H-TdR (37kBq/ holes) continues to cultivate 12h, determines radionuclide sudden strain of a muscle per minute
Bright numeration (CPM) value, SI (SI) is calculated according to formula (I).
The negative group CPM (I) of SI=experimental groups CPM/
5th, T cell is tested to the specific killing of malignant melanoma cell
Respectively with the different groups of T cells of activation as effector cell, with3The B16 cells of exponential phase of H-TdR marks are
Target cell, non-MHC matching cells M3 is control group, is 50 by effect target ratio：1st, incubation time is 24h to effect target altogether, carries out cell toxicant
Experiment, while setting maximum release group (target cell+1%TritonX100) and spontaneous release group (target cell+nutrient solution), determines every
Group supernatant CPM values.Before T cell is added, the M3 cells of non-marked are first added with 10 times of amounts of target cell, exclude NKT
(NK) activity of cell.T cell specific killing activity is calculated according to formula (II).
6th, the immunization therapy that mouse dislikes black model and hapten transformation is set up
C57BL/ mouse (H-2b) totally 30 are taken, black cell line B16 (H-2 will be dislikedb)1×107Individual cell seeding is carried on the back in mouse
Portion, accessible subcutaneous tumors tubercle after 10d.Tumor-bearing mice is randomly divided into 9 groups, every group 6, respectively in the left dorsal injection of mouse not
With the composition and physiological saline (physiological saline group) of group, all animal periodic evaluation tumor sizes.Every 3d slide measures
Measurement knurl body major diameter (a), minor axis (b), calculate the volume (V) of knurl body：V=ab2/2；After inoculation 28d, peel off each group tumour and claim
Weight, tumour inhibiting rate is calculated after 4 weeks according to formula (III)：
Tumour inhibiting rate=each experimental group tumor weight-physiological saline group tumor weight/physiological saline group tumor weight × 100% (III)
7th, statistical procedures data represent with ± S, are checked using variance analysis and t, with SPSS11.0 software statistics, P<
0.05 has conspicuousness for difference.
8th, interpretation of result
(1) each group DC stimulates the proliferation function to T cell
Volume ratio with antigen and DC is 1：1 investigates proliferation function of each group DC stimulations to T cell, wherein DNP- respectively
In B16+ BCG vaccines+bar-shaped corpusculum+DC groups and B16+ BCG vaccines+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+ cards are situated between
Seedling+DC is organized with B16+ BCG vaccines+DC, and the content of malignant melanoma antigen is 80% (volume), the content of tuberculosis antigen
It is 20% (volume)；In DNP-B16+ bar-shaped corpusculum+DC groups and the bar-shaped corpusculum+DC groups of B16+, malignant melanoma antigen contains
It is 80% (volume) to measure, and the content of bar-shaped corpusculum is 20% (volume).
Using3H-TdR methods determine each group DC stimulations to the proliferation function of T cell, as a result see Fig. 2.Figure it is seen that
The proliferation function of DNP, B16, BCG vaccine, the combination T cell of bar-shaped corpusculum and DC is added simultaneously compared with without DNP modifications group, single
Haptens BCG vaccine or bar-shaped corpusculum and simple DC groups are strong, i.e., the B16 cells after being modified through DNP and haptens BCG vaccine, bar-shaped
The DC of the combination activation of corpusculum, the T cell multiplication capacity that it induces is illustrated containing tuberculosis antigen, disliked apparently higher than other groups
Property melanoma-associated antigen and the DC of antigen mixture activation of bar-shaped corpusculum the black specific T-cells of stronger evil can be induced to imitate
Should.
(2) killing activity of the T cell to tumour cell
Determined under the effect of DNP-B16+ BCG vaccines+bar-shaped corpusculum+DC groups, B16-DC groups and DC groups using radionuclide method
T cell compares group to the lethal effect of tumour cell (B16 cells) with physiological saline group, the results are shown in Table 4.
T cell is determined to the lethal effect of B16 cells using isotope method, is as a result shown, DNP-B16+ BCG vaccines+bar-shaped
The T cell of corpusculum+DC group activation is 50 in effect target ratio：When 1, B16 cells can be substantially killed, but to M3 cells, (H-2d dislikes black
Cell line) lethal effect is faint；The T cell that the T cell of B16-DC groups activation is activated with simple DC is killed to two kinds of tumour cells
Wound effect is very low, is shown in Table 4.
Table 4T cells cytotoxicity ()
(4) immunization therapy of the anti-malignant mela noma vaccine combination to tumor-bearing mice
After B16 cell implantation in vivo, the accessible subcutaneous tumors tubercles of 8~12d.After the 1 mouse medication of hapten transformation group
20d dies unexpectedly.Physiological saline group and DC group tumor volumes gradually increase, and B16-DC groups contract since the tumor volume after the 17th day
Small, tumour inhibiting rate is 50.11% after 4 weeks；There is analog result, tumour inhibiting rate 57.79 in DNP+ BCG vaccines+bar-shaped corpusculum group；DNP-B16
+ BCG vaccine+bar-shaped corpusculum+- DC groups from the 10th~12 day knurl body be to be obviously reduced, hence it is evident that earlier than B16-DC groups, and during 28d press down
Ratio of outflow is up to 98.96%, hence it is evident that higher than other each groups (P<0.01), tumour is almost completely inhibited, this is in the prior art very
It is inaccessible, it is shown in Table 5.
Influence of the anti-malignant mela noma vaccine combination of table 5 to 28d tumor-bearing mices
From above-described embodiment as can be seen that the malignant melanoma cell modified with simple DNP and BMDC
(DC) combine and compare, will be thin containing tuberculosis antigen, malignant melanoma antigen and bar-shaped corpusculum antigen mixture and dendron shape
The culture of born of the same parents (DC) anti-malignant mela noma vaccine combination obtained after being mixed 24 hours, it is formed with DC
Compound produces synergy from each other, can preferably excite DC, promotes the propagation of specific T-cells, and then produce extremely strong
SC lymphocyte immunity reaction.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (10)
1. a kind of anti-malignant mela noma vaccine combination, it is mixed system by antigen mixture and BMDC culture
Into wherein the antigen mixture includes malignant melanoma antigen and tuberculosis
Antigen and/or bar-shaped corpusculum；
The antigen mixture is (2-1) with the volume ratio of BMDC culture：1；
In the antigen mixture, the content of the malignant melanoma antigen is 80%-93% volumes, tuberculosis antigen
Content is 2%-10% volumes, and the content of bar-shaped corpusculum is 2%-10% volumes.
2. vaccine combination according to claim 1, it is characterised in that the antigen mixture is cultivated with BMDC
The volume ratio of thing is 1：1.
3. vaccine combination according to claim 1, it is characterised in that in the vaccine combination, the tuberculosis
The content of antigen is 5% volume, and the content of bar-shaped corpusculum is 2% volume.
4. vaccine combination according to claim 1, it is characterised in that the malignant melanoma antigen includes pernicious black
Melanoma attenuated cell and/or its malignant mela noma attenuated cell modified body.
5. vaccine combination according to claim 4, it is characterised in that the malignant mela noma attenuated cell modified body
It is to be modified through 2,4- dinitro benzenes by malignant melanoma cell and obtained after inactivation or break process；The maligna element
Knurl attenuated cell is obtained by malignant melanoma cell after inactivation or break process.
6. vaccine combination according to claim 5, it is characterised in that the malignant melanoma cell includes that B16 is thin
Born of the same parents or M3 cells.
7. the vaccine combination according to any one in claim 1-6, it is characterised in that the BMDC culture
Thing by BMDC in the RPMI1640 containing GM-CSF, IL-4 and hyclone, in 5%CO2, cultivate under the conditions of 37 DEG C
Obtain within 6 days.
8. the vaccine combination according to any one in claim 1-6, it is characterised in that the culture of the mixed culture
Condition is in the RPMI1640 containing GM-CSF, IL-4 and hyclone, in 5%CO2, cultivate 1 day under the conditions of 37 DEG C.
9. the vaccine combination according to any one in claim 1-6, it is characterised in that the tuberculosis antigen is card
Jie's seedling.
10. the vaccine combination in a kind of 1-9 according to claim described in any one is preparing prevention and treatment maligna
Application in the medicine of plain knurl.
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| 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;Masaru Udagawa等;《Clinical Cancer Research》;20061215;第12卷(第24期);7465 * |
| Preventive antitumor activity against hepatocellular carcinoma (HCC) induced by immunization with fusions of dendritic cells and HCC cells in mice;Sadamu Homma;《Journal of Gastroenterol》;20011231;第36卷;764-771 * |
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