CN110124021A - A kind of preparation method of novel tumor vaccine - Google Patents
A kind of preparation method of novel tumor vaccine Download PDFInfo
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- CN110124021A CN110124021A CN201910403408.6A CN201910403408A CN110124021A CN 110124021 A CN110124021 A CN 110124021A CN 201910403408 A CN201910403408 A CN 201910403408A CN 110124021 A CN110124021 A CN 110124021A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- A61K2039/5152—Tumor cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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Abstract
The present invention relates to antitumor technical field, especially a kind of preparation method of novel tumor vaccine is mixed by hypochlorous acid treated tumor cell lysis liquid and immunoactivator.This method step are as follows: 1) carry out primary tumor cell culture after obtaining fresh tumor tissue, and obtain primary tumor cell；2) primary tumor cell and culture solution obtained the step 1) carries out hypochlorous acid processing, collects the mixed liquor obtained after hypochlorous acid processing cell, the tumor cell debris mixture of lysate and apoptosis needed for obtaining；3) mixed liquor that the step 2) obtains is centrifuged, and is concentrated and purifies repeatedly to get the concentrate containing cell pyrolysis liquid is arrived；4) cell pyrolysis liquid after purifying concentration obtained in the step 3) is subjected to radiotherapy inactivation.It is low that the present invention can be used for solving Common tumors antigen vaccine immunogenicity, inaccurately, the low problem of immune response activation efficiency.
Description
Technical field
The present invention relates to antitumor technical field more particularly to a kind of preparation methods of novel tumor vaccine.
Background technique
With cancer genomics and immunologic fast development, immunotherapy is had become after radiotherapy, chemotherapy, operation
The 4th kind for the treatment of means after treating.The targeting of chemicotherapy is poor, is easy to damage normal cell, generates adverse reaction.And it is immunized
The antigen on the tumor cell surface of specificity is capable of in treatment, and the hair of side reaction is reduced while target killing tumor cell
It is raw.In immunization therapy, tumor vaccine is one of hot spot of Recent study.Tumor vaccine by expression specificity, have
The tumour antigen of immunogenicity, such as tumor cell lysis liquid, tumor correlated albumen or antigen polypeptide and expression tumour antigen
Related gene etc. activates body immune system to generate specificity antineoplastic under the auxiliary of the adjuvants such as cell factor, chemotactic factor (CF)
Cell immunoreceptor enhances the anti-cancer ability of body, prevents the growth, diffusion and recurrence of tumour, swollen to reach removing or control
The purpose of tumor.
Clinically granted tumor vaccine includes dendron shape vaccine (DC vaccine), vaccine for cervical cancer and individuation at present
Sipuleucel-T vaccine, but these vaccine activation efficiencies are low, antigen is few, clinical efficacy is not good enough and expensive, certain
Its large-scale use clinically is limited in degree.Hypochlorous acid (HClO) is a kind of effective oxidant, in acute inflammation
Phase is by neutrophil leucocyte myeloperoxidase (MPO) catalyzing hydrogen peroxide (H2O2) and chloride ion (cl) generation.In addition to powerful kills
Wound effect is outer, has studies have shown that HClO that can dramatically increase the immunogenicity of proteantigen.Albumen after HClO is aoxidized more holds
It is easily swallowed by antigen presenting cell (APC) and offers the activation and proliferation that promote T cell to T cell, enhanced the immune of body and kill
Wound effect.Therefore, the powerful immune activation effect after how efficiently using HClO oxidation tumour cell, enhances the antitumor of body
Ability, it is still necessary to further go to explore.Immunologic adjuvant is mainly used to helper antigen induction body and generates immune response, it can be
The activation of T cell provides second signal, is a part of vaccine development.The artificial synthesized widow containing non A-G hepatitis
Polynucleotide chain (CpG-ODN) is a kind of aqueous adjuvants, itself non-immunogenicity can improve the immunogenicity of proteantigen, directly
It connects activated mononuclear cell, macrophage and Dendritic Cells and stimulates the cells such as these cells secretion IL-12, TNF-α, IFN-γ
The factor induces Th1 type immune response.It goes back synergistic activation T cell simultaneously.Therefore, CpG ODN can be used as the immune of a variety of antigens
Adjuvant and play unique immunological enhancement.Therefore, develop a kind of immune activation ability is strong, spectrotype extensively, preparation process
Simple vaccine, becomes urgent problem to be solved.
Summary of the invention
The purpose of the present invention is to solve disadvantages existing in the prior art, and a kind of novel tumor vaccine proposed
Preparation method.
To achieve the goals above, present invention employs following technical solutions:
The preparation method for designing a kind of novel tumor vaccine, by hypochlorous acid (HClO) treated tumor cell lysis liquid and
Immunoactivator mixes.
Preferably, the immunoactivator is specially CpG.
Preferably, the preparation method of the hypochlorous acid (HClO) treated tumor cell lysis liquid, specific steps are such as
Under:
1): carrying out primary tumor cell culture after obtaining fresh tumor tissue, and obtain primary tumor cell；
2): handling primary tumor cell with hypochlorous acid (HClO), obtained after collection hypochlorous acid (HClO) processing cell upper
Clear liquid, the tumor cell debris mixture of lysate and apoptosis needed for obtaining；
3): the lysate that the step 2) obtains being centrifuged, and is concentrated and purifies repeatedly to get thin to tumour is contained
The concentrate of cellular lysate liquid；
4): the tumor cell lysis liquid after purifying concentration obtained in the step 3) being subjected to radiotherapy inactivation to get arriving
The tumor cell lysis liquid.
Preferably, the tumor tissues in step 1) are self tumor tissue in vitro.
Preferably, it before the hypochlorous acid in step 2) (HClO) processing, needs to clean tumour with HBSS balanced salt solution thin
Born of the same parents 2-3 times, concentration when hypochlorous acid (HClO) is handled is 60-80 μM, and the supernatant collection time is at hypochlorous acid (HClO)
2 hours after reason.
Preferably, when being centrifuged in step 3) to lysate, used centrifuge speed is 1000r, and is centrifuged
Time is 5min, and the cell fragment of centrifugation bottom of the tube is discarded after centrifugation.
Preferably, when carrying out purifying concentration to lysate in step 4), the supernatant after centrifugation is transferred to super filter tube
Inner tube, using angle rotor centrifuge is determined, revolving speed 6000*g, centrifugation time 20min are gently blown after centrifugation with 200 μ l pipette tips
Concentrate is bought, 3-5ml ultrapure water is added to concentration tube inner tube, is centrifuged 6000*g again, and the time is 20min, it is finally obtained
The volume of the concentrated liquid is about 300ul, as the concentrate containing tumor cell lysis liquid.
Preferably, it also needs to carry out radiotherapy inactivation to the tumor cell lysis liquid after purifying concentration using radiotherapy technology, and puts
Treatment dosage is 20Gy, obtains the tumor cell lysis liquid after radiotherapy inactivation.
Preferably, tumor cell lysis liquid and CpG aqueous solution are mixed, the final concentration of 1-2mg/ml of CpG aqueous solution,
The solvent of CpG aqueous solution is deionized water.
Preferably, the concentration of the tumor cell lysis liquid is about 20-80 μ g/ml.
A kind of preparation method of novel tumor vaccine proposed by the present invention, beneficial effect are:
1, medicament made from the microparticle of autologous has preferable biological safety and biocompatibility.
2, the present invention is inactivated using radiotherapy technology, in the case where guaranteeing tumour cell immunogenicity, loses tumour cell
It is devitalized, avoid the generation that tumour is induced in use process.
3, preparation process is simple, is convenient for large-scale production.
4, it is low to can be used for solving Common tumors antigen vaccine immunogenicity by the present invention, inaccurately, is immunoreacted activation efficiency
Low problem.
Detailed description of the invention
Fig. 1 is the schematic diagram to the activation capability assessment result of DC cell.
Fig. 2 is the schematic diagram of prophylactic treatment anti-tumor capacity assessment.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Embodiment 1
The present invention provides a kind of preparation method of novel tumor vaccine, by hypochlorous acid (HClO) treated tumour cell
Lysate and immunoactivator mix, and the immunoactivator is specially CpG.
The preparation method of the hypochlorous acid (HClO) treated tumor cell lysis liquid, the specific steps are as follows:
1): carrying out primary tumor cell culture after obtaining fresh tumor tissue, and obtain primary tumor cell, and tumor group
It is woven to self tumor tissue in vitro；
2): handling primary tumor cell with hypochlorous acid (HClO), before hypochlorous acid (HClO) processing, need to be balanced with HBSS
Washed with saline solution tumour cell 2-3 times, concentration when hypochlorous acid (HClO) is handled are 60-80 μM, the supernatant collection time
It is 2 hours after hypochlorous acid (HClO) processing, collects the supernatant obtained after hypochlorous acid (HClO) processing cell, cracking needed for obtaining
The tumor cell debris mixture of liquid and apoptosis；
3): the lysate that the step 2) obtains is centrifuged, when being centrifuged to lysate, used centrifugation
Machine revolving speed be 1000r, and centrifugation time be 5min, discarded after centrifugation centrifugation bottom of the tube cell fragment, and repeatedly concentration and it is pure
Change to get the concentrate containing tumor cell lysis liquid is arrived；
4): the tumor cell lysis liquid after purifying concentration obtained in the step 3) being subjected to radiotherapy inactivation, in counterincision
When solution liquid carries out purifying concentration, the supernatant after centrifugation is transferred to super filter tube inner tube, using determining angle rotor centrifuge, revolving speed is
6000*g, centrifugation time 20min gently blow and beat concentrate with 200 μ l pipette tips after centrifugation, and 3-5ml is added to concentration tube inner tube
Ultrapure water is centrifuged 6000*g again, and the time is 20min, and the finally obtained volume of the concentrated liquid is about 300ul, as containing swollen
The concentrate of oncocyte lysate.
It also needs to carry out radiotherapy inactivation to the tumor cell lysis liquid after purifying concentration using radiotherapy technology, and Radiotherapy dosimetry is
20Gy obtains the tumor cell lysis liquid after radiotherapy inactivation.
Tumor cell lysis liquid and CpG aqueous solution are mixed, the final concentration of 1-2mg/ml of CpG aqueous solution, CpG aqueous solution
Solvent be deionized water.
The concentration of the tumor cell lysis liquid is about 20-80 μ g/ml.
Embodiment 2:
Referring to Fig.1-2, activation of the assessment novel tumor to DC cell:
Stem cell is obtained from 6-8 week old male C57 mouse femur and shin bone, with 1640 culture mediums containing M-CSF
It cultivates 5 days and is planted in 24 orifice plates after obtaining immature DC cell, be divided into PBS group, Cell lysate group (multigelation method
Obtain), HClO-lysate group, each group takes the corresponding solution of 40 μ l to be incubated for altogether with immature DC cell, stimulate DC cell at
It is ripe, Flow cytometry CD11c, CD80, CD86 cell-stimulating situation (as shown in Figure 1), experimental result are utilized after 24 hours
Show the maturation that can promote DC cell with novel tumor vaccine.
Embodiment 3: novel tumor vaccine prophylaxis therapy antitumous effect research
C57 mouse is randomly divided into 3 groups, first group of mouse tail root+foot pad inject 50 μ l PBS, second group of root of the tail portion+
Foot pad injects 50 μ l Cell lysate, third group root of the tail portion+foot pad injects 50 μ l HClO-lysate, is recorded as the 1st at this time
It, is administered once every other day, is administered 3 times altogether, the 7th day subcutaneous implantation malignant melanoma cell B16 (2*105/ 100 μ l/ are only), it is fixed
Phase monitors survival condition, experiment display, and novel tumor vaccine can significantly inhibit tumour growth.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of preparation method of novel tumor vaccine, which is characterized in that by hypochlorous acid (HCl) treated tumor cell lysis
Liquid and immunoactivator mix.
2. a kind of preparation method of novel tumor vaccine according to claim 1, which is characterized in that the immunoactivator
Specially CpG.
3. the preparation method of novel tumor vaccine according to claim 2, which is characterized in that the hypochlorous acid (HClO)
The preparation method of treated tumor cell lysis liquid, the specific steps are as follows:
1): carrying out primary tumor cell culture after obtaining fresh tumor tissue, and obtain primary tumor cell；
2): primary tumor cell is handled with hypochlorous acid (HClO), collects the supernatant obtained after hypochlorous acid (HClO) processing cell,
The tumor cell debris mixture of lysate and apoptosis needed for obtaining；
3): the lysate that the step 2) obtains being centrifuged, and is concentrated and purifies repeatedly and split to get to containing tumour cell
Solve the concentrate of liquid；
4): it is described to get arriving that the tumor cell lysis liquid after purifying concentration obtained in the step 3) being subjected to radiotherapy inactivation
Tumor cell lysis liquid.
4. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that swollen in step 1)
Tumor tissue is self tumor tissue in vitro.
5. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that time in step 2)
Before chloric acid (HClO) processing, need to be cleaned tumour cell 2-3 times with HBSS balanced salt solution, it is dense when hypochlorous acid (HClO) is handled
Degree is 60-80 μM, and the supernatant collection time is 2 hours after hypochlorous acid (HClO) processing.
6. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that the counterincision in step 3)
When solution liquid is centrifuged, used centrifuge speed is 1000r, and centrifugation time is 5min, and centrifuge tube bottom is discarded after centrifugation
The cell fragment in portion.
7. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that the counterincision in step 4)
When solution liquid carries out purifying concentration, the supernatant after centrifugation is transferred to super filter tube inner tube, using determining angle rotor centrifuge, revolving speed is
6000*g, centrifugation time 20min gently blow and beat concentrate with 200 μ l pipette tips after centrifugation, and 3-5ml is added to concentration tube inner tube
Ultrapure water is centrifuged 6000*g again, and the time is 20min, and the finally obtained volume of the concentrated liquid is about 300ul, as containing swollen
The concentrate of oncocyte lysate.
8. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that also need using radiotherapy skill
Art carries out radiotherapy inactivation to the tumor cell lysis liquid after purifying concentration, and Radiotherapy dosimetry is 20Gy, is obtained after radiotherapy inactivation
The tumor cell lysis liquid.
9. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that by tumor cell lysis
Liquid and the mixing of CpG aqueous solution, the final concentration of 1-2mg/ml of CpG aqueous solution, the solvent of CpG aqueous solution are deionized water.
10. a kind of preparation method of novel tumor vaccine according to claim 3, which is characterized in that the tumour is thin
The concentration of cellular lysate liquid is about 20-80 μ g/ml.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228473A (en) * | 2020-02-14 | 2020-06-05 | 杭州憶盛医疗科技有限公司 | Anti-tumor novel composite vaccine for treating fibrosarcoma |
| CN111603554A (en) * | 2020-07-07 | 2020-09-01 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of anti-tumor vaccine antigen raw material |
| CN112870340A (en) * | 2021-01-27 | 2021-06-01 | 四川大学 | Tumor vaccine based on breast cancer extracellular vesicles and preparation method thereof |
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| CN109096386A (en) * | 2018-08-31 | 2018-12-28 | 上海莱馥生命科学技术有限公司 | A kind of preparation method and application of breast carcinoma stem cell antigen |
| CN109568570A (en) * | 2019-01-14 | 2019-04-05 | 华中科技大学同济医学院附属协和医院 | A kind of anti-tumor vaccine compound and preparation method, injection and application |
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2019
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| US20030228300A1 (en) * | 2002-02-22 | 2003-12-11 | Haspel Martin V. | Sterile immunogenic non-tumorigenic tumor cell compositions and methods |
| CN109096386A (en) * | 2018-08-31 | 2018-12-28 | 上海莱馥生命科学技术有限公司 | A kind of preparation method and application of breast carcinoma stem cell antigen |
| CN109568570A (en) * | 2019-01-14 | 2019-04-05 | 华中科技大学同济医学院附属协和医院 | A kind of anti-tumor vaccine compound and preparation method, injection and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111228473A (en) * | 2020-02-14 | 2020-06-05 | 杭州憶盛医疗科技有限公司 | Anti-tumor novel composite vaccine for treating fibrosarcoma |
| CN111603554A (en) * | 2020-07-07 | 2020-09-01 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of anti-tumor vaccine antigen raw material |
| CN112870340A (en) * | 2021-01-27 | 2021-06-01 | 四川大学 | Tumor vaccine based on breast cancer extracellular vesicles and preparation method thereof |
| CN112870340B (en) * | 2021-01-27 | 2022-09-06 | 四川大学 | Tumor vaccine based on breast cancer extracellular vesicles and preparation method thereof |
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