CN116650629A - Preparation of mouse dendritic cell exosome mediated liver cancer vaccine - Google Patents
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Abstract
The invention belongs to the field of biotechnology and medicine, and in particular relates to a preparation method of mouse dendritic cell exosomes as liver cancer vaccines. The purpose of the invention is realized in the following way: the mouse dendritic cell exosome vaccine is obtained by extracting an exosome from a mouse dendritic cell, loading a mouse-derived liver cancer specific antigen dominant epitope peptide and an immune adjuvant.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and in particular relates to a preparation method of mouse dendritic cell exosomes as liver cancer vaccines.
Background
Liver cancer has become the second leading cause of cancer death worldwide due to its high malignancy and rapid progression, and is the cause of death of the 4 th common malignant tumor and the 2 nd tumor in our country. Liver malignancy remains a global health threat, with an estimated incidence of more than one million in 2025. The clinical treatment effect of the liver cancer is not ideal due to the multiple drug-resistant mechanism of the liver cancer, complex physiological barriers, immune microenvironment and the like. The occurrence, development, metastasis and recurrence of liver cancer are closely related to the immune system of the body. Immunotherapy has become a hotspot in clinical research of tumors by enhancing the immune function of the organism to activate an anti-tumor immune response, thereby inhibiting tumor progression, recurrence and metastasis. The liver cancer vaccine can activate anti-tumor immune response through antigen, effectively inhibit tumor recurrence and metastasis, including cell vaccine, peptide vaccine, DC vaccine, DNA vaccine, etc., generally, the liver cancer vaccine is in an inhibiting state due to long-term chronic liver disease background of liver cell cancer patients, and the immune function of the patients is insufficient to cause effective immune response. However, clinical treatments have not achieved the desired results.
Dendritic Cells (DCs) are used to load tumor antigens due to their powerful antigen presentation, and the preparation of cellular vaccines has been demonstrated to be effective in inducing antigen-specific immune responses, such as the earliest approved DC vaccines against prostate cancer. However, the in vitro cultured and proliferated DC cells have the disadvantages of rejection, complicated operation, time consumption and high cost, and the Tumor Associated Antigen (TAA) -specific T cells have low affinity for tumor cells and limited anti-tumor immune response capability.
This series of problems severely limits the development and use of DC clinical vaccines. For this, exosome vaccines have been developed which co-deliver adjuvants and polyepitope antigens to a certain extent into lymphoid organs and antigen presenting cells, through which intracellular release of the vaccine and cross-expression of the antigen can be fine-tuned. Exosomes secreted by dendritic cells (DEX) have been shown to carry a substantial portion of DC cell surface molecules, particularly the histocompatibility complex associated with immune activation (MHC-I and MHC-II) and co-stimulatory factors CD80, CD86, CD54, etc., and due to these characteristics, DEX is considered a non-cellular biological nanovaccine that can be used to activate immune responses in place of dendritic cells. DEX vaccines loaded with specific antigenic peptides are one of the emerging immunotherapeutic strategies. The DEX vaccine is used as a non-cell vaccine, has definite composition, stable structure and high safety, can be frozen and stored for a long time, can be produced in batches according to GMP, overcomes a plurality of defects of DC cell vaccines, and has more superiority in tumor immunotherapy. However, the side effects and complexity of the preparation process make it alarming in terms of preparation and application.
Disclosure of Invention
Based on this, the present invention aims to overcome the above-mentioned shortcomings of the prior art and provide a dendritic cell exosome as a liver cancer vaccine.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following aspects:
the purpose of the invention is realized in the following way: a dendritic cell exosome vaccine is prepared from the exosome from mouse dendritic cells, the peptide loaded with liver cancer specific antigen dominant epitope and immunoadjuvant.
For this purpose, the invention provides a dendritic cell exosome vaccine comprising an exosome derived from a mouse dendritic cell, a liver cancer specific antigen and an immunoadjuvant.
Wherein the liver cancer specific antigen is selected from the group consisting of: the AFP 212 short peptide has the amino acid sequence: GSMLNEHVM, SEQ ID NO.1; GPC3 127-136 short peptide with amino acid sequence: AMFKNNYPSL, SEQ ID NO.2.
Wherein the immunoadjuvant is selected from: n1ND, its amino acid sequence is:
MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQT, SEQ ID NO.3.
Another object of the present invention is to provide a preparation method of a dendritic cell exosome liver cancer vaccine, comprising the steps of:
(1) Mouse bone marrow dendritic cell exosomes (DEX) supernatants were collected: firstly, extracting mouse bone marrow dendritic cell precursor mononuclear cells, culturing in a 6-hole plate, adding 1640 complete culture medium, inducing the mouse bone marrow dendritic cell precursor mononuclear cells to differentiate and mature into dendritic cells, and collecting supernatant of the dendritic cells;
(2) Extraction of mouse Dendritic Cell (DC) exosomes: centrifuging the collected supernatant of dendritic cells for 500g and 5min to remove large cell clusters, and reserving the supernatant; subsequently, the supernatant was centrifuged in sequence. Centrifuging for 2000g and 10min, taking out supernatant, centrifuging for 30min and removing large vesicles and fragments; finally transferring the supernatant into an overspeed centrifuge tube, carrying out centrifugation for 70min at 110000g to obtain exosome sediment. Then adding a large amount of PBS, again 110000g, centrifuging for 70min, discarding the supernatant, and re-suspending the exosomes with 200ul of PBS;
(3) Preparation of mouse dendritic cell exosome vaccine: and (3) incubating the exosomes obtained in the previous step with the mouse liver cancer antigen peptide AFP 212, GPC 3-136 and immunoadjuvant N1ND for 12-48 hours in a refrigerator.
Most preferably, the preparation of the dendritic cell exosome liver cancer vaccine comprises the following steps:
(1) Mouse bone marrow dendritic cell exosomes (DEX) supernatants were collected: firstly, extracting mouse bone marrow dendritic cell precursor mononuclear cells, culturing in a 6-hole plate, adding 10%1640 complete culture medium into each hole by 2ml, and collecting dendritic cell supernatant after the differentiation and maturation of the dendritic cells into the dendritic cells are induced;
(2) Extraction of mouse Dendritic Cell (DC) exosomes: centrifuging the collected supernatant of dendritic cells for 500g and 5min to remove large cell clusters, and reserving the supernatant; subsequently, the supernatant was centrifuged in sequence. Centrifuging for 2000g and 10min, taking out supernatant, centrifuging for 30min and removing large vesicles and fragments; finally transferring the supernatant into an overspeed centrifuge tube, carrying out centrifugation for 70min at 110000g to obtain exosome sediment. Then a large amount of PBS was added, 110000g again, centrifuged for 70min, the supernatant was discarded, and the exosomes were resuspended with 200ul PBS. All steps were performed at 4 ℃;
(3) Preparation of mouse dendritic cell exosome vaccine: the exosomes were resuspended in 200ul pbs, and after repeated pipetting transferred to 1.5ml ep tubes, BCA quantified for exosomes concentration of 50ug/ml. The polypeptides were formulated according to the manufacturer's instructions. The concentration was 1mg/ml. Adding a murine liver cancer antigen peptide AFP 212: GSMLNEHVM, GPC3 AMFKNNYPSL and immunoadjuvant N1ND 10ul each, and incubated in a refrigerator at 4deg.C for 24h.
In step (1), 10%1640 complete medium: 10%1640 complete medium refers to complete medium prepared by adding 10% Fetal Bovine Serum (FBS) and 90%1640 incomplete medium.
In step (2), 2000g,10min centrifugation: 2000g,10min centrifugation refers to a centrifugal force of 2000g, centrifugation for 10 minutes.
Wherein, PBS: phosphate buffer is a buffer solution, and can be used as washing solution
BCA quantitative exosomes in step (3): refers to the detection of exosome protein concentration by BCA protein quantitative method, which is a method for exosome quantitative detection
Wherein the polypeptide refers to antigen peptide AFP 212, GPC3 127-136, and immunoadjuvant N1ND.
It is another object of the present invention to provide the use of a mouse dendritic cell exosome vaccine for the preparation of a medicament for the treatment of cancer.
The cancer is liver cancer.
In summary, the beneficial effects of the invention are as follows:
the invention provides a liver cancer epidemic which is carried on mouse dendritic cell exosome by combining a mouse liver cancer specific antigen AFP, GPC3 and an immunological adjuvant N1NDSeedling. On the one hand, it can enhance DC immunogenicity, induce immune response, activate antigen-specific CD8 + T cells; on the other hand, the polypeptide is directly combined with the MHC of the exosome to directly activate the T cells, thereby promoting the anti-tumor immune response. The key point of the technology is also to construct a mouse liver cancer specific antigen short peptide AFP 212: GSMLNEHVM and GPC3
127-136:AMFKNNYPSL。
The dendritic cell exosome vaccine of the invention is loaded with the mouse liver cancer specific polypeptide, is a non-cell liver cancer specific vaccine, and has simple and convenient preparation method. Meanwhile, the DEX vaccine combines the advantages of the DC vaccine and the unique advantages of exosomes, has very remarkable effect as a tumor vaccine, and can be used for tumor immunotherapy.
The results of the DEX vaccine T stimulation experiment and the LDH experiment show that: the expression level of IFN-gamma released by the stimulated T cells of the DEXAFP-GPC3-N1ND group is 2 times that of the control group, and the killing efficiency (toxicity) of liver cancer cells of the DEXAFP-GPC3-N1ND group is 2-3 times that of the control group (see the attached drawing for details). Thus, the DEX modified by the antigen can activate immune response, excite specific CD8+T cell cytotoxicity effect and achieve the anti-tumor effect.
Interpretation of nouns and terms:
dendritic Cells (DC): the stem cells are derived from bone marrow multipotent hematopoietic stem cells, which are the cells with the strongest antigen presenting ability in human body, and can efficiently ingest and process antigens.
DC vaccine: DC vaccine is prepared by culturing mature DC in vitro, and infusing the DC stimulated by antigen back into human body to exert antitumor effect, i.e. DC through different forms of antigen loaded to induce human body to generate T lymphocyte with specific killing effect to control tumor
Exosomes secreted by dendritic cells (DEX): firstly, the exosomes are nanovesicles secreted by cells, and the exosomes secreted by dendritic cells are exosomes derived from dendritic cells (DEX) obtained by collecting a culture solution (supernatant) of cells after the dendritic cells are mature and then centrifuging the culture solution
DEX vaccine: the DEX vaccine is a tumor vaccine prepared from exosomes derived from dendritic cells. Since almost all antigen presenting molecules such as co-stimulatory molecules CD86 and MHC class molecules are involved, it is possible to induce or amplify the acquired immunity and activate the innate immunity by natural killer cells. The novel tumor vaccine is constructed by combining the novel tumor vaccine with a mouse liver cancer specific antigen and an immune adjuvant and loading on DEX.
Liver cancer specific antigen: specific antigen which exists on the surface of liver cancer cells but does not exist in normal cells is often used as a marker of liver cancer.
Immune adjuvants: refers to some non-specific immunopotentiators. Refers to auxiliary substances which are injected into the body together with the antigen or in advance and can enhance the immune response capability of the body to the antigen or change the immune response type.
1640 complete medium: is a complete culture medium prepared by adding 1% of chain and penicillin into the fetal bovine serum and 1640 incomplete culture medium according to the proportion of 1:9.
PE-AFP, FITC-GPC3: PE and FITC are fluorescent dyes commonly used in flow cytometry, PE fluoresces orange and FITC fluoresces yellow-green, both of which are used to label antibodies. PE-AFP and FITC-GPC3 refer to labeling both streaming antibodies of AFP and GPC3 with PE and FITC.
Drawings
FIG. 1 shows the binding efficiency of polypeptides AFP, GPC3, N1ND and DEX
AFP and DEX binding efficiency, GPC3 and DEX binding efficiency, and N1ND and DEX binding efficiency
FIG. 2 is a mixed leukocyte reaction experiment
A, amnis quantitative imaging flow cytometry for detecting CD4 + T cells, CD8 + T cell and IFN-gamma fluorescence schematic
B under different stimulation conditions, CD4 + IFN-gamma release from T cells
CD8 under different stimulation conditions + IFN-gamma release from T cells
FIG. 3 is an experiment of LDH lactate dehydrogenase release
Detailed Description
The following detailed description is made with specific examples in order to make the present invention clear to those skilled in the art. The methods used in the respective examples are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified.
Example 1: liver cancer exosome vaccine
The preparation method provided by the invention comprises the following steps:
(1) Extracting mouse bone marrow dendritic cell precursor mononuclear cells, culturing in a 6-well plate, adding 10%1640 complete culture medium, 2ml per well, inducing differentiation and maturation into dendritic cells, and collecting DC supernatant by changing liquid every other day;
(2) Centrifuging the obtained DC supernatant at 500g for 5min, removing large cell mass, and retaining the supernatant; then, the supernatant was centrifuged in order. Centrifuging 2000g for 10min, centrifuging 10000g of supernatant for 30min, and removing large vesicles and fragments; finally transferring the supernatant into an overspeed centrifuge tube, carrying out centrifugation for 70min at 110000g to obtain exosome sediment. Then a large amount of PBS was added, 110000g again, centrifuged for 70min, the supernatant was discarded, and the exosomes were resuspended with 200ul PBS. All steps were performed at 4 ℃;
(3) Preparation of mouse dendritic cell exosome vaccine: the exosomes were resuspended in 200ul pbs, and after repeated pipetting transferred to 1.5ml ep tubes, BCA quantified for exosomes concentration of 50ug/ml. The polypeptides were formulated according to the manufacturer's instructions at concentrations of 1mg/ml. Then adding the murine liver cancer antigen peptide AFP 212: GSMLNEHVM, GPC3 AMFKNNYPSL and immunoadjuvant N1ND 10ul each, and incubating at 4deg.C for 24 hr.
Example 2: detection of polypeptide binding efficiency to exosomes
The Amnis quantitative imaging flow cytometer detects the binding efficiency of polypeptide and exosome, and the specific embodiments are as follows:
1) The exosomes 10ug and polypeptides obtained in example 1 were placed in 10ul of each of a 1.5ml EP tube at 4 ℃
Co-incubating for 24 hours in a refrigerator;
2) Adding 2.5ul PE-labeled anti-mouse AFP antibody and FITC-labeled anti-mouse GPC3 antibody to the exo-polypeptide obtained in 1), respectively; placing on ice, and incubating for 30min in dark;
3) Transferring the exosome-polypeptide of 2) to an ultracentrifuge tube, adding a large amount of PBS, centrifuging at 110000g for 70min, and washing away unbound antibody;
4) Discarding the supernatant, adding 200ul PBS for resuspension, fully blowing and uniformly mixing, and then sucking and placing into a 1.5ml EP tube for detection;
5) Amnis quantitative imaging flow meters detect the binding efficiency of AFP, GPC3, N1ND to exosomes. N1ND can be directly detected without adding flow type antibody for dyeing because of FAM green fluorescence,
results display (fig. 1): the AFP binding efficiency to DEX was 57.21%, the GPC3 binding efficiency to DEX was 62.19%, and the N1ND binding efficiency to DEX was 57.61%.
Example 3 Mixed leukocyte reaction experiment (MLR experiment)
S1: spleens of C57 mice were taken, placed in a 70uM screen, placed on a 50ml centrifuge tube, and gently milled with a 5ml syringe handle. Rinsing with 1640 medium while milling;
s2: a new 15ml centrifuge tube is taken and added with lymphocyte separation liquid (the lymphocyte separation liquid is equal to the spleen cell suspension obtained by S1 in volume); sucking the spleen cell suspension in the S1 by using a suction pipe, and slowly adding the spleen cell suspension to the separating liquid;
s3:900g,30min, centrifuge up 3 down 1 centrifuge;
s4: obvious layering in the centrifuge tube can be seen, the middle white membrane layer is gently sucked into another new centrifuge tube with 15ml, 10ml of washing liquid (or PBS) is added, 250g is centrifuged for 10min, and the mixture is centrifugally washed twice to obtain T cell sediment
S5: uniformly spreading T cells obtained in S4 into 6-well plates according to density, wherein each well is 10 6 cells；
S6: t cells in S5 above were subjected to different intervention treatments, divided into 3 groups: NC group (200 ul PBS was added to T cells), DEX group (200 ul of exosome heavy suspension of 50ug was added to T cells), DEXAFP-GPC3-N1ND group (200 ul exosome pre-treated with polypeptide was added to T cells). Placing the prepared marks in a 37 ℃ incubator for incubation for 12 hours;
s7: adding 3ul of Golgi transporter blocker to the T cells in the step S6, and continuing to incubate for 5-6h;
s8: t cells in S7 were collected in 15ml centrifuge tubes, and centrifuged at 500g for 5min. The supernatant was discarded, added with 5ml PBS,500g,5min, and washed twice by centrifugation, and transferred to a 1.5ml EP tube;
s9: to each EP tube obtained in S8, 2.5ul of anti-mouse CD3, CD4, CD8 antibodies were added, respectively.
Incubating for 30min on ice in dark place;
s10: to each EP tube was added 1ml PBS,500g,5min, and washed by centrifugation;
s11: adding 200ul of membrane breaker, and standing for 30min at 4 ℃ in a refrigerator; after 30min, adding 1ml PBS for centrifugal washing;
s12: then adding 2.5ul of anti-mouse IFN-gamma streaming antibody, and continuing to incubate for 30min in a dark place;
s13: after the incubation, the cells were washed once again with PBS and examined for release of IFN- γ by cd4+ T cells and cd8+ T cells using an amais quantitative imaging flow cytometer.
Results show (see fig. 2): first: among T cells, CD8+ T cells release more IFN-gamma than CD4+ T cells. It is explained that the cytotoxic effect is exerted mainly by CD8+ T cells. Second,: compared with the control group, the amount of IFN-gamma released by DEXAFP-GPC3-N1ND group was 2 times that of the control group. In conclusion, the exosome vaccine provided by the invention can excite antigen-specific CD8+T cells, further promote anti-tumor immune response and make up for the defect of limited immune response capability in the prior art.
Example 4: LDH lactate dehydrogenase Release assay
S1: t cells under different stimulation conditions in MLR experiments and mouse liver cancer cells hepa1-6 were plated in 96-well plates (wherein T cells 10) at a cell number of 20:1 5 cells, hepa1-6 cells 5000 cells); and a background blank well, a sample control well, a sample maximum enzyme activity control well, and an experimental group were set. Placing the prepared marks in a 37 ℃ incubator for incubation for 48 hours;
s2: 1h before the preset detection time, taking out the cell culture plate S1 from the incubator, adding an LDH release reagent with 10 percent of the original culture solution volume (namely 20 ul) into a 'sample maximum enzyme activity control hole', repeatedly blowing and uniformly mixing, and then continuously placing the cell culture plate into the incubator for incubation;
s3: after 1h, the cell culture plate obtained in S2 was removed, and centrifuged for 5min with 400g in a multi-well plate centrifuge. Taking 120ul of supernatant of each well respectively, and adding the supernatant into a corresponding well of another new 96-well plate;
s4: preparing an LDH working solution: according to the instruction, preparing the LDH working solution according to the number of samples to be tested.
S5: adding 60ul LDH working solution to each well in the 96-well plate in the step S3; mixing, and incubating at room temperature for 60min in dark place;
s6: absorbance was measured at 490 nm. Calculation (measured absorbance of each group should be subtracted from background blank control well absorbance)
S7: cytotoxicity or mortality (%) = (absorbance of treated sample-absorbance of sample control wells)/(absorbance of maximum enzyme activity of cells-absorbance of sample control wells) ×100%
Results show (see fig. 3): compared with the control group, the DEXAFP-GPC3-N1ND group exerts a cytotoxic effect or cancer cell death rate 2-3 times that of the control group. The vaccine of the invention has obvious effect of killing tumor cells, has good anti-tumor effect, and solves the shortages of immunosuppression in the prior art.
Claims (10)
1. A mouse dendritic cell exosome vaccine is characterized by comprising an exosome from a mouse dendritic cell source, a mouse-derived liver cancer specific antigen and an immune adjuvant,
wherein the murine liver cancer specific antigen is selected from the group consisting of: AFP 212 short peptide, amino acid sequence is: SEQ ID NO.1;
GPC3 127-136 short peptide, amino acid sequence is: SEQ ID NO. 2;
the immunological adjuvant is selected from: n1ND, amino acid sequence: SEQ ID NO.3.
2. A preparation method of a mouse dendritic cell exosome liver cancer vaccine is characterized in that,
(1) Collecting mouse bone marrow dendritic cell exosome supernatant: firstly, extracting mouse bone marrow dendritic cell precursor mononuclear cells, culturing in a 6-hole plate, adding 1640 complete culture medium, inducing the mouse bone marrow dendritic cell precursor mononuclear cells to differentiate and mature into dendritic cells, and collecting supernatant of the dendritic cells;
(2) Extraction of mouse dendritic cell exosomes: centrifuging the collected supernatant of the dendritic cells, removing large cell clusters, discarding the supernatant, and adding PBS to resuspend exosomes;
(3) Preparation of mouse dendritic cell exosome vaccine: and (3) incubating the exosomes obtained in the previous step with the mouse liver cancer antigen peptide AFP 212, GPC 3-136 and immunoadjuvant N1ND for 12-48 hours in a refrigerator.
3. The method of claim 2, wherein step (1) comprises adding 5-20% 1640 complete medium.
4. The method according to claim 2, wherein the step (2) is to centrifuge 500g of the collected supernatant of dendritic cells for 5min to remove large cell mass and to retain the supernatant; centrifuging 2000g for 10min, taking out the supernatant, centrifuging 10000g for 30min, and removing large vesicles and fragments; finally transferring the supernatant into an overspeed centrifuge tube, carrying out centrifugation for 70min at 110000g to obtain exosome sediment. A large amount of PBS was added, again 110000g, centrifuged for 70min, the supernatant was discarded, and then 200ul PBS was added to resuspend the exosomes.
5. The process according to claim 2, wherein all steps of step (2) are carried out at 4 ℃.
6. The method of claim 2, wherein the exosome concentration in step (3) is 50ug/ml.
7. The method according to claim 2, wherein the polypeptide concentration in step (3) is 1mg/ml and the amount added is 10ul.
8. The preparation method according to claim 2, characterized by comprising the steps of:
(1) Mouse bone marrow dendritic cell exosomes (DEX) supernatants were collected: firstly, extracting mouse bone marrow dendritic cell precursor mononuclear cells, culturing in a 6-hole plate, adding 10%1640 complete culture medium into each hole by 2ml, and collecting dendritic cell supernatant after the differentiation and maturation of the dendritic cells into the dendritic cells are induced;
(2) Extraction of mouse Dendritic Cell (DC) exosomes: centrifuging the collected supernatant of dendritic cells for 500g and 5min to remove large cell clusters, and reserving the supernatant; centrifuging 2000g for 10min, taking out the supernatant, centrifuging 10000g for 30min, and removing large vesicles and fragments; finally transferring the supernatant into an overspeed centrifuge tube, carrying out centrifugation for 70min at 110000g to obtain exosome sediment. Adding a large amount of PBS, again 110000g, centrifuging for 70min, discarding the supernatant, then adding 200ul PBS to resuspend the exosomes, all at 4 ℃;
(3) Preparation of mouse dendritic cell exosome vaccine: the exosomes were resuspended in 200ul pbs, and after repeated pipetting transferred to 1.5ml ep tubes, BCA quantified for exosomes concentration of 50ug/ml. The polypeptides were formulated according to the manufacturer's instructions at concentrations of 1mg/ml. Adding a murine liver cancer antigen peptide AFP 212: GSMLNEHVM, GPC3 AMFKNNYPSL and immunoadjuvant N1ND 10ul each, and incubated in a refrigerator at 4deg.C for 24h.
9. Use of the mouse dendritic cell exosome vaccine of claim 1 for the preparation of a medicament for treating cancer.
10. The use according to claim 9, wherein the cancer is liver cancer.
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