CN105664155A - DC (dendritic cell) based breast cancer therapeutic vaccine and preparation method thereof - Google Patents
DC (dendritic cell) based breast cancer therapeutic vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a DC (dendritic cell) based breast cancer therapeutic vaccine and a preparation method thereof. The preparation method of the DC based breast cancer therapeutic vaccine comprises steps as follows: immature DCs are acquired; a breast cancer antigen compound and the immature DCs are subjected to co-culture in a DC culture medium for 5-9 days; a tumor necrosis factor-alpha is added for continuous culture until the DCs are mature and are loaded with the breast cancer antigen compound, wherein the breast cancer antigen compound is prepared from inactivated breast cancer cells and a MUC1 monoclonal antibody compound through co-culture. The DC based breast cancer therapeutic vaccine has higher immunogenicity and can be used for regulating the body immune mechanism for breast cancer very well.
Description
Technical field
The present invention relates to biological engineering and biomedicine field, particularly to a kind of breast cancer treatment vaccine based on DC and preparation method thereof.
Background technology
Breast carcinoma is the common cancer that women ranks the first, and sickness rate is in ascendant trend year by year. In existing breast cancer treatment process to the infringement of patients immune system greatly, causing that tumor therapeutic procedure runs into the bottleneck that cannot break through, kill tumor ability, tumor cannot be cured completely. Present stage cytokine induced kill cell is the immunologically competent cell with cytotoxicity; Dendritic cell is the antigen presenting cell of most potentiality, has important function in primality immunity and acquired immunity; Dendritic cell is that known body endoantigen offers the cell that the ability of passing is the strongest, and it can catch antigen, and passes information to T, bone-marrow-derived lymphocyte, thus causing a series of specific immune response to react. Therefore, if tumor antigen is injected dendritic cell, the specific anti tumor immune response of body can be caused.
Summary of the invention
The technical problem to be solved is, has for breast cancer treatment vaccine in prior art that immunoreation is weak, kill the defects such as tumor ability, it is provided that breast cancer treatment vaccine based on DC of a kind of immunogenicity and high specificity and preparation method thereof.
The technical solution adopted for the present invention to solve the technical problems is: the preparation method providing a kind of breast cancer treatment vaccine based on DC, and described preparation method comprises the following steps:
Obtain immature DC;
Carry out co-culturing 5～9 days in DC culture medium by breast cancer antigen complex and immature DC;
Add tumor necrosis factor-alpha to continue to cultivate, ripe to DC and be loaded with breast cancer antigen complex;
Wherein, described breast cancer antigen complex is to be co-cultured acquisition by the breast cancer cell after inactivateing and MUC1 monoclonal antibody complex.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC, the volume ratio of described immature DC and described breast cancer antigen complex is (3～7): 1.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC, the concentration of described MUC1 monoclonal antibody complex is (0.5～1.5) μ g/ (1 × 105) individual breast cancer cell.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC,
The process that breast cancer cell after described inactivation and MUC1 monoclonal antibody complex co-culture, comprises the following steps:
Take the breast cancer cell after inactivation, add 0.5～2% bovine serum albumin, add MUC1 monoclonal antibody complex and co-culture 0.5～1.5 hour.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC,
Breast cancer cell acquisition process after described inactivation, comprises the following steps:
Take the logarithm the breast cancer cell of trophophase, washing after centrifugal, make (0.5～2) × 107The breast cancer cell suspension of individual/ml, puts in liquid nitrogen, and melts completely in water-bath, is placed again in liquid nitrogen, repeated multiple times is placed in liquid nitrogen and water-bath, then filters, and collects filtrate, i.e. breast cancer cell after inactivation.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC, the concentration of described immature DC is (2～5) × 105Individual/ml.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC,
The acquisition process of described immature DC comprises the following steps:
Take peripheral blood, filter out CD14 by magnetic bead+Mononuclear cell, adds external evoked culture medium and carries out inducing culture 2～6 days, to CD14+Mononuclear cell induces differentiation into immature DC.
Provided by the invention based on, in the preparation method of the breast cancer treatment vaccine of DC, described DC culture medium being added with the basal medium of 750～850U/mL grain-macrophage and 900～1100U/mL interleukin-4.
Provided by the invention based in the preparation method of the breast cancer treatment vaccine of DC, described tumor necrosis factor-alpha final concentration of 800～1200U/ml in the medium.
The present invention also provides for a kind of breast cancer treatment vaccine based on DC, the above-mentioned breast cancer treatment vaccine preparation method based on DC obtain.
Implement breast cancer treatment vaccine based on DC provided by the invention and preparation method thereof, following beneficial effect can be reached: adopt and stimulated DC by the breast cancer cell inactivated and MUC1 monoclonal antibody complex altogether collectively as the supplier of tumor antigen information, promote the angtigen presentation of DC, improve the immunoreation of body, the breast cancer treatment vaccine not only character based on DC obtained by the present invention is more stable, and immunogenicity and specificity are stronger, kill tumor ability stronger, it is possible to well regulate the immunity of organism mechanism of breast carcinoma.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated. Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
A kind of breast cancer treatment vaccine based on DC provided by the invention, its preparation method comprises the following steps:
S1, acquisition immature DC;
S2, breast cancer antigen complex and immature DC are co-cultured 5～9 days in DC culture medium; Wherein, breast cancer antigen complex is to be co-cultured obtained by the breast cancer cell after putting out a fire and MUC1 monoclonal antibody complex;
S3, then add tumor necrosis factor-alpha and continue to cultivate, ripe to DC and be loaded with breast cancer antigen complex.
In step S1, immature DC can derive from blood of human body, but only account for the 0.5-1.0% that mononuclear cell is total in blood due to immature DC, and quantity is little; Therefore, in the preferred embodiment of the present invention, take the mode of induced monocyte to obtain immature DC, concretely comprise the following steps:
S11, from peripheral blood, isolate mononuclear cell;
S12, add external evoked culture medium culturing 2～6 days, the mononuclear cell obtained is carried out inducing culture become immature DC to monocyte transformation in step S11.
In step s 11, mononuclear cell can be CD34+And/or CD14+Mononuclear cell, wherein, CD34+Mononuclear cell can separate from bone marrow, Cord blood or spleen etc. and obtain; CD14+ mononuclear cell obtains from human peripheral separation. Owing to being purified into CD34 from Cord blood+Mononuclear cell somewhat expensive; Bone marrow collection not easily, and easily pollutes, and patient is more painful, expands somewhat expensive; And spleen source is limited etc. makes CD34+Mononuclear cell obtains more difficult; Therefore, in the present invention, it is preferred to adopt and separate CD14 from the fresh blood of people periphery+Mononuclear cell, obtains from fresh blood, compares the CD14 after freezer storage or subculture+Mononuclear cell, the CD14 extracted from fresh blood+The monocytes ex vivo time is not long, its cytoactive and state weaken or morphologic change degree is all less, be more favorable for obtaining DC. Preferably, in the present invention, immunomagnetic beads method is adopted to isolate CD14 from peripheral blood+Mononuclear cell.
External evoked culture medium in step S12 is preferably the basal medium containing 5%～15% hyclone, 750～850U/mL grain-macrophage and 900～1100U/mL interleukin-4 being suitable for DC growth. It is significant to note that, owing to DC compares non-adherent cell, it can have adherent property, but its adherent performance is not strong, but is partial to half adherent type, therefore the incubation of mononuclearcell in step s 13 needs to adopt half amount to change liquid, suspend to avoid blowing out purpose DC and run off.
Of course, it should be understood that the approach that the present invention obtains immature DC is also not limited thereto, the immature DC obtained by other abductive approach induced monocyte is equally applicable to the present invention.
In step s 2, it is preferable that adopt and immature DC and breast cancer antigen complex are co-cultured 5～9 days by the DC culture medium containing 750～850U/mL grain-macrophage cytokines and 900～1100U/mL interleukin-4; Wherein, immature DC is (4～6) with the volume ratio of breast cancer antigen complex: 1.
Grain-macrophage cytokines and interleukin-4 not only contribute to the growing multiplication of DC, and are conducive to DC antigen uptaking information. Owing to DC has the custom that tufted grows, concentration is too high, assemble agglomerating, the DC being centrally located is not easily ripe, affects the load of breast cancer antigen, thus affecting the immune effect of vaccine, therefore, DC concentration is not easily too high, in this step, treats that immature DC concentration in DC culture medium is for (2～5) × 105Individual/ml or by step S1 obtain immature DC concentration be adjusted to (2～5) × 105Individual/ml time, add breast cancer antigen complex carry out preload.
Owing to immature DC has extremely strong antigen phagocytic activity, in antigen uptaking (include external processing and carry out endocytosis, process antigen in interior body), and offer antigen to MHC I and MHC II, thus starting Peptide-specific CTL reaction; And immature DC expresses some membrane receptors such as Fc receptor that can mediate DC antigen uptaking, thus compare ripe DC and have higher antigen uptake, processing and disposal ability, therefore, step S2 utilize immature DC and breast cancer antigen complex co-culture the preload being capable of breast cancer antigen, promote and improve the load capacity of breast cancer antigen, meanwhile, immature DC can to ripe through antigen sensibilization.
Further, the acquisition process of breast cancer antigen complex, comprise the following steps:
S21, obtain exponential phase breast cancer cell;
S22, the breast cancer cell of exponential phase obtained in step S21 is carried out inactivation treatment;
S23, breast cancer cell and the MUC1 monoclonal antibody complex after the inactivation obtained in step S22 is co-cultured 0.5～2 hour.
Wherein, in step 21, breast cancer cell can derive from autologous, it is possible to derives from allosome; The breast cancer cell adopted in the present invention comes from the MCF-7 breast carcinoma cell strain that hematopathy hospital of the Chinese Academy of Medical Sciences provides.
In step S22, the process of breast cancer cell inactivation, comprise the following steps:
Take the exponential phase breast cancer cell obtained in step S21, centrifugal after washing, make concentration for (0.5～2) × 107The breast cancer cell suspension of individual/ml, add in normal saline, put into liquid nitrogen, within 2～8 minutes, put into rapidly in 30～45 DEG C of water-baths, after breast cancer cell melts completely, it is placed again in liquid nitrogen, repeated multiple times it is placed in liquid nitrogen and water-bath, filtering with microporous membrane, collect filtrate, namely the breast cancer cell after results inactivation, saves backup in 4 DEG C.
It is understood that these are only a kind of method that breast cancer cell provided by the invention inactivates, the breast cancer cell after the inactivation of additive method acquisition is adopted to be equally applicable to the present invention.
In step S23, the process that breast cancer cell after inactivation and MUC1 monoclonal antibody complex co-culture is: in the breast cancer cell after inactivation, add 0.5～2% bovine serum albumin, add MUC1 monoclonal antibody complex and co-culture 0.5～1.5 hour, namely obtain breast cancer antigen complex; Wherein, the concentration of MUC1 monoclonal antibody complex is (0.5～1.5) μ g/ (1 × 105) individual cell.
Wherein, expressed by MUC1 (people's mucin core peptide) gene almost has in all of epithelial cell adenocarcinoma, and it limits two ways induced activation CTL by MHC restriction and non-MHC, to expressing tumor cell positive for MUC1, there is lethal effect, thus MUC1 becomes the desirable target antigen of active specific immunotherapy cancer. Breast cancer cell and MUC1 monoclonal antibody complex are stimulated DC cell by the present invention altogether, it is possible to the MUC1 immunne response of inducing producing specificity, it is possible to block or postpone the development of spontaneous mammary cancer, and except activation CD8+T cell, it is also possible to activation CD4+T cell; And the DC merged can move to local drainage lymph node and interact with T cell, promote T cell to breed, produce MUC1 specific C D8+CTL and secretion IL-2, IFN-γ, IL-10, IL-4, CD4+T cell, thus producing multiple Graft Versus Tumor substrate, it is achieved kill tumor comprehensively, improves the purpose killing ratio of outflow.
In step s3, tumor necrosis factor-alpha by vivo immunization cell (such as T lymphocyte, T cell hybridoma, T lymphoid cell line is with NK cell etc.) a kind of monokine of secreting, tumor necrosis factor-alpha is stronger to the stimulus effects effect of immature DC, the load factor of tumor antigen can be improved, and can stablize DC angtigen presentation simultaneously facilitate DC maturation, the MHC-II quasi-molecule that ripe DC expresses, costimulatory molecules, the more non-ripe DC of level of adhesion molecule is high, therefore the relatively non-ripe DC of its HLA-II antigen is stronger, organism immune response can be improved, preferably, tumor necrosis factor-alpha final concentration of 800～1200U/ml in the medium.
Detailed description of the invention is:
Embodiment 1
Based on the breast cancer treatment vaccine of DC, its preparation method comprises the following steps:
S1, breast cancer antigen complex acquisition:
S11, trophophase of taking the logarithm MCF-7 breast cancer cell, purchased from hematopathy hospital of the Chinese Academy of Medical Sciences;
Breast cancer cell in step S11 is carried out inactivation treatment by S12, employing freeze-thaw method;
Take the logarithm the breast cancer cell of trophophase, brine twice, 1800 revs/min centrifugal after, PBS solution re-suspended cell liquid, it is thus achieved that 1 × 107The breast cancer cell suspension of individual/ml, take 20 μ L cell suspension and add in 100 μ L normal saline, it is then placed in liquid nitrogen, put into rapidly in 37 DEG C of water-baths after 5 minutes, after breast cancer cell melts completely, be placed again in liquid nitrogen, 3 times repeatedly, then filtering with microporous membrane, collects filtrate, and 4 DEG C save backup.
S13, breast cancer cell and the MUC1 monoclonal antibody complex after inactivation is co-cultured;
Take the bovine serum albumin closing cell surface heterogenetic antigen epi-position that the breast cancer cell after 20 μ L inactivations adds 1%, stand 30 minutes at 4 DEG C, be subsequently adding the 1 μ g/ (1 × 10 of 2 μ L5) after the MUC1 monoclonal antibody complex (purchased from Ai Bokang (Shanghai) trade Co., Ltd) of individual cell co-cultures 1 hour in 37 DEG C, PBS washs 2 times, collect inactivation breast cancer cell-MUC1 monoclonal antibody complex and adopt cellular immunofluorescence indirect method to identify, be i.e. results breast cancer antigen complex.
S2, acquisition immature DC;
S21, separation obtain CD14+Mononuclear cell;
Taking 20ml human peripheral, use Germany Mei Tian Ni company MonocyteIsolationKitIIOrderno.130-091-153, by specification isolates CD14+Mononuclear cell.
S22, by step S21 obtain CD14+Mononuclear cell induces differentiation into immature DC;
By CD14+Mononuclear cell is inoculated in the RPMI-l640 culture medium containing 10% hyclone, 800U/mL grain-macrophage cytokines and 1000U/mL interleukin-4 and at 37 DEG C, CO2Concentration be 5% incubator in inducing culture 4 days, half amount changes liquid every other day, supplements grain-macrophage cytokines and interleukin-4, maintains grain-macrophage cytokines and interleukin-4 concentration is constant, it is thus achieved that immature DC.
S3, immature DC and breast cancer antigen complex are co-cultured
Immature DC being mixed by the volume ratio of 5:1 with breast cancer antigen complex, wherein, immature DC concentration is 3 × 105Individual/ml; Then, add in the DC culture medium containing the 800U/mL granular leukocyte macrophage factor and the IL-4 of 1000U/mL and co-culture 7 days;
Then, add that the tumor necrosis factor-alpha inducing culture two days of 1000U/ml is ripe to DC and breast cancer antigen complex in load.
Embodiment 2
It is different in that with embodiment 1 provided by the invention, in the present embodiment,
The concentration of immature DC is 2 × 105Individual/ml;
The concentration of breast cancer cell suspension is 0.5 × 107Individual/ml;
The concentration of MUC1 monoclonal antibody complex is 0.5 μ g/ (1 × 105) individual cell;
The concentration of tumor necrosis factor-alpha is 800U/ml;
The volume ratio of immature DC and breast cancer antigen complex is 4:1.
Embodiment 3
It is different in that with embodiment 1 provided by the invention, in the present embodiment,
The concentration of immature DC is 5 × 105Individual/ml;
The concentration of breast cancer cell suspension is 2 × 107Individual/ml;
The concentration of MUC1 monoclonal antibody complex is 1.5 μ g/ (1 × 105) individual cell;
The concentration of tumor necrosis factor-alpha is 1200U/ml;
The volume ratio of immature DC and breast cancer antigen complex is 6:1.
For verifying the remarkable result of the breast cancer treatment vaccine based on DC provided by the invention (lower abbreviation " DC vaccine ") further, following experiment is set and carries out detection checking.
Test experience one, flow cytomery cell surface molecule expression rate
Detection object
Detection group: the DC vaccine that the embodiment of the present invention 1 provides;
Matched group: DC-tumor cell fusion bacterin in prior art;
After cultivating one week for the DC being loaded with tumor antigen information obtained in embodiment 1, it is 50.06 ± 1.92% through Flow cytometry DC surface molecular CD80 expression rate, surface molecular CD83 expression rate 60.07 ± 2.09%, surface molecular CD86 expression rate 52.43 ± 1.67%, surface molecular HLA-DR expression rate 58.80 ± 3.37%, and DC-tumor cell fusion bacterin is 38.06 ± 2.02% through Flow cytometry DC surface molecular CD80 expression rate, surface molecular CD83 expression rate 40.07 ± 1.06%, surface molecular CD86 expression rate 30.03 ± 1.04%, surface molecular HLA-DR expression rate 35.190 ± 2.29%, as can be seen here, DC vaccine provided by the invention is stronger relative to the immunoreation caused by DC-tumor cell fusion bacterin.
Test experience two,
Detection object:
Detection group 1～3 DC vaccine that the corresponding embodiment of the present invention 1～3 obtains respectively;
The vaccine being jointly made up of DC and tumor cell in matched group prior art;
Detection process:
Target cell (breast cancer cell) is inoculated in 96 orifice plates (three wells), respectively experimental group 1～3 and matched group is performed following operation:
Every hole adds vaccine by effect target than for 5:1,25:1,50:1 and 100:1 respectively, every kind of effect target ratio sets three multiple holes, sets maximum release group (with the concentration hydrochloric acid substitution effect cell as 2mol/L) and minimum release group (the RPMI1640 culture medium substitution effect cell with containing the hyclone that mass percentage concentration is 10%) simultaneously; After centrifugal 30 seconds of 96 orifice plate 500r/min, temperature 37 DEG C hatches 4 hours, centrifugal 10 minutes of 1000r/min, each hole takes supernatant 100 μ L, radioactivity per minute (cpm value) is measured with gamma counter, killing rate is calculated: killing rate (%)=[(experimental group cpm average-minimum release group cpm average)/(maximum release group cpm average-minimum release group cpm average)] × 100% by following formula, calculating killing rate, result of calculation is shown in following table.
Table 1
| Effect target compares E/T | 5:1 | 25:1 | 50:1 | 100:1 |
| Detection group 1 | 19.80±2.11 | 41.29±2.58 | 59.37±1.50 | 48.20±2.17 |
| Detection group 2 | 17.42±1.64 | 39.85±2.65 | 54.42±2.85 | 47.63±2.41 |
| Detection group 3 | 25.22±1.52 | 47.49±2.57 | 64.83±1.61 | 52.73±1.82 |
| Matched group | 9.37±1.21 | 16.67±2.31 | 21.59±140 | 17.39±1.71 |
Experimental result: as shown in table 1 below, detection group 1～3 all has specific killing action at each effect target comparison breast cancer cell, and along with effect target ratio increases, specific killing action strengthens gradually, when E/T is 50: 1, killing rate reaches maximum, and most High Fragmentation rate is 64.83%, and breast cancer cell killing rate is 21.59% by matched group; It is indicated above that the breast cancer treatment vaccine based on DC provided by the invention effectively can produce the specificity kill and wounding effect to breast cancer cell by activated t cell.
Above embodiments of the invention are described; but the invention is not limited in above-mentioned detailed description of the invention; above-mentioned detailed description of the invention is merely schematic; rather than it is restrictive; those of ordinary skill in the art is under the enlightenment of the present invention; without departing under present inventive concept and scope of the claimed protection situation, it may also be made that a lot of form, these belong within the protection of the present invention.
Claims (10)
1. the preparation method based on the breast cancer treatment vaccine of DC, it is characterised in that: described preparation method comprises the following steps:
Obtain immature DC;
Breast cancer antigen complex and immature DC are co-cultured 5～9 days in DC culture medium;
Add tumor necrosis factor-alpha to continue to cultivate, ripe to DC and be loaded with described breast cancer antigen complex;
Wherein, described breast cancer antigen complex is to be co-cultured acquisition by the breast cancer cell after inactivateing and MUC1 monoclonal antibody complex.
2. the preparation method of the breast cancer treatment vaccine based on DC according to claim 1, it is characterised in that the volume ratio of described immature DC and described breast cancer antigen complex is (3～7): 1.
3. the preparation method of the breast cancer treatment vaccine based on DC according to claim 1, it is characterised in that the concentration of described MUC1 monoclonal antibody complex is (0.5～1.5) μ g/ (1 × 105) individual breast cancer cell.
4. the preparation method of the breast cancer treatment vaccine based on DC according to claim 1 or 3, it is characterised in that the process that the breast cancer cell after described inactivation and MUC1 monoclonal antibody complex co-culture, comprises the following steps:
Take the breast cancer cell after inactivation, add 0.5～2% bovine serum albumin, add MUC1 monoclonal antibody complex and co-culture 0.5～1.5 hour.
5. the preparation method of the breast cancer treatment vaccine based on DC according to claim 4, it is characterised in that the breast cancer cell acquisition process after described inactivation, comprises the following steps:
Take the logarithm the breast cancer cell of trophophase, washing after centrifugal, make (0.5～2) × 107The breast cancer cell suspension of individual/ml, puts in liquid nitrogen, and melts completely in water-bath, is placed again in liquid nitrogen, repeated multiple times is placed in liquid nitrogen and water-bath, then filters, and collects filtrate, i.e. breast cancer cell after inactivation.
6. the preparation method of the breast cancer treatment vaccine based on DC according to claim 1, it is characterised in that the concentration of described immature DC is (2～5) × 105Individual/ml.
7. the preparation method of the breast cancer treatment vaccine based on DC according to claim 6, it is characterised in that the acquisition process of described immature DC comprises the following steps:
Take peripheral blood, filter out CD14 by magnetic bead+Mononuclear cell, adds external evoked culture medium and carries out inducing culture 2～6 days, to CD14+Mononuclear cell induces differentiation into immature DC.
8. the preparation method of the breast cancer treatment vaccine based on DC according to claim 1, it is characterised in that be added with the basal medium of 750～850U/mL grain-macrophage and 900～1100U/mL interleukin-4 in described DC culture medium.
9. the preparation method of the breast cancer treatment vaccine based on DC according to claim 1, it is characterised in that described tumor necrosis factor-alpha final concentration of 800～1200U/ml in the medium.
10. the breast cancer treatment vaccine based on DC, it is characterised in that the breast cancer treatment vaccine preparation method based on DC described in any one of claim 1-9 obtains.
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| CN109575118A (en) * | 2018-12-17 | 2019-04-05 | 英普乐孚生物技术(上海)有限公司 | It is used to prepare the polypeptide fragment and DC vaccine of DC vaccine |
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