CN111228471A - Preparation method of tumor cell vaccine based on low-temperature plasma - Google Patents

Preparation method of tumor cell vaccine based on low-temperature plasma Download PDF

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CN111228471A
CN111228471A CN201911324973.XA CN201911324973A CN111228471A CN 111228471 A CN111228471 A CN 111228471A CN 201911324973 A CN201911324973 A CN 201911324973A CN 111228471 A CN111228471 A CN 111228471A
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temperature plasma
cells
tumor
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韩伟
马洁
陈国栋
程诚
聂莉莉
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Hefei Institutes of Physical Science of CAS
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/812—Breast
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]

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Abstract

The invention discloses a preparation method of a tumor cell vaccine based on low-temperature plasma, and relates to the field of biological medicine. The invention discovers that the low-temperature plasma can obviously kill tumor cells, such as melanoma cells and the like; the tumor cells inactivated by injecting the low-temperature plasma can effectively inhibit the growth of in vivo tumors, or the occurrence and development of metastatic tumors can be effectively inhibited by directly irradiating the in-situ tumors by utilizing the low-temperature plasma, so that a new idea is opened up for the application of the subsequent low-temperature plasma in the field of cancer treatment.

Description

Preparation method of tumor cell vaccine based on low-temperature plasma
Technical Field
The invention relates to the technical field of biomedicine, in particular to a preparation method of a tumor cell vaccine based on low-temperature plasma.
Background
The malignant tumor, i.e. cancer, is one of major diseases facing the world, and the existing cancer treatment means, such as radiotherapy, surgery, targeted therapy and the like, have certain curative effect but also have great limitation. Tumor vaccines are one of the hot spots in tumor treatment research in recent years, and the tumor vaccines are introduced into patients, so that the immunogenicity is enhanced, the immune system is activated, and the cellular immunity and humoral immunity response of organisms are induced, thereby controlling the tumor growth and metastasis or eliminating the tumor. Tumor vaccines are mainly comprised of four types: cell vaccine, polypeptide vaccine, genetic engineering vaccine, and antibody tumor vaccine. At present, the means for obtaining the tumor cell vaccine mainly comprises treating autologous or allogeneic tumor cells by physical, chemical or biological methods (ultraviolet irradiation, heating and the like), and then obtaining the cell vaccine which retains immunogenicity and has no tumorigenicity.
Low-temperature Plasma (NTP), which is the fourth state of matter, mainly contains various free radicals such as electrons, ions and neutral particles, is widely used in the biomedical field, such as sterilization, whitening, and wound healing. In recent years, studies have demonstrated that low temperature plasma can significantly kill a variety of cancer cells in an in vitro cell model. Meanwhile, the low-temperature plasma is found to be capable of inhibiting the growth rate of tumor bodies in a tumor-bearing mouse model. As a new technology, low-temperature plasma has the advantages of strong controllability, precise action range, approach to the temperature of human body, and the like, and is being developed into a new cancer treatment means. Therefore, the preparation of tumor cell vaccines based on the low-temperature plasma technology and the development of novel low-temperature plasma cancer treatment means have important significance.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a preparation method of a tumor cell vaccine based on low-temperature plasma.
The inventor is devoted to researches on killing tumor cells by low-temperature plasma for a long time. Researches show that in vitro cell experiments, the low-temperature plasma can effectively kill various tumor cells; in a tumor-bearing mouse model, low-temperature plasma can inhibit the growth of tumor bodies in vivo. On the basis of the former stage, the application mainly develops related tumor cell vaccines, inhibits the formation rate and the growth speed of tumor bodies by injecting low-temperature plasma inactivated tumor cells, and expands the application range of low-temperature plasma in tumor immunotherapy.
In a first aspect, the present invention provides a method for preparing a low temperature plasma tumor cell vaccine, said method comprising the steps of:
1) taking tumor cells as objects, culturing and adjusting the cell state to a growth log phase;
2) collecting the tumor cells in the logarithmic growth phase in the step 1) and washing the cells, performing centrifugal collection after protease enzymolysis of the cells, and adjusting the cell concentration to 3 x 105～5*105Cells/ml;
3) directly or indirectly treating the tumor cells treated in the step 2) for 30 seconds to 30 minutes by using low-temperature plasma;
4) continuously culturing the tumor cells obtained by the low-temperature plasma treatment in the step 3) for 4-48 hours, and centrifugally collecting inactivated cells after protease enzymolysis, thereby obtaining the tumor cell vaccine.
Preferably, the tumor cells in step 1) are at 37 ℃ and 5% CO2Under the condition, culturing in a DMEM high-sugar medium containing 10% fetal calf serum and 1% penicillin-streptomycin, and carrying out subculture when the cell fusion degree reaches 80-90%.
Preferably, step 2) washing the cells with pre-warmed PBS, protease preferably trypsin, adjusting the cell concentration to 3 x 10 using pre-warmed PBS or DMDM high sugar medium5～5*105Cells/ml.
Preferably, the low-temperature plasma in step 3) may be selected from one or more of jet low-temperature plasma, dielectric barrier discharge low-temperature plasma, and air multi-needle low-temperature plasma.
In a specific embodiment, the low-temperature plasma is a dielectric barrier discharge low-temperature plasma.
Preferably, the working gas of the low-temperature plasma in the step 3) is air and/or inert gas.
The inert gas comprises one or more of helium, argon and nitrogen, and preferably the inert gas is helium.
Preferably, the power of the low-temperature plasma in the step 3) is 0.5W/cm2～5W/cm2(ii) a The working time of the low-temperature plasma is 30 seconds to 30 minutes.
In one embodiment, the low temperature plasma has a power of 0.9W/cm2(ii) a The working time of the low-temperature plasma is 120 seconds.
Preferably, the tumor cells obtained in step 3) are cultured for a further 12 hours, the protease preferably being trypsin.
Preferably, the tumor cell is a melanoma cell, a head and neck cancer cell, a cervical cancer cell, a breast cancer cell, preferably a melanoma cell.
In a second aspect, the present invention provides the use of a tumor cell vaccine prepared according to the first aspect.
The invention has the beneficial effects that:
the invention discovers that the low-temperature plasma can obviously kill tumor cells, such as mouse melanoma cells; the tumor cells treated by the low-temperature plasma are used as tumor cell vaccines, so that the formation rate and the growth speed of tumors can be effectively inhibited, and a new idea is provided for the application of the subsequent low-temperature plasma in the aspect of tumor immunotherapy.
Drawings
FIG. 1 shows the change of cell viability of mouse melanoma cells B16-F10-OVA after low temperature plasma treatment, Ctrl represents the control group.
FIG. 2 shows the effect of tumor cell vaccine prepared by low temperature plasma on the tumor formation rate and tumor growth rate of B16-F10-OVA cells. a & b, schematic representation of tumor-bearing mice and tumor bodies; and c, a tumor volume change schematic diagram.
Detailed Description
The following description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the technical principle of the present invention, and these modifications and improvements should also be regarded as the protection scope of the present invention.
Example 1 Cell Counting Kit-8(CCK-8 Kit) for detecting the killing effect of low-temperature plasma on cancer cells
A dielectric barrier discharge low-temperature plasma device is adopted, and the device mainly comprises 2 parts: high voltage power supply, plasma reaction chamber. Wherein, the plasma reaction chamber is composed of 4 pairs of electrodes, and helium is selected as the working gas in all the embodiments of the application.
The frozen mouse melanoma cells B16-F10-OVA were recovered from mouse melanoma cells B16-F10-OVA as a test subject (purchased from the cell bank of the Committee for culture Collection of Chinese academy of sciences), and then treated at 37 ℃ with 5% CO2Under the condition, culturing in a DMEM high-sugar medium containing 10% fetal calf serum and 1% penicillin-streptomycin, and carrying out subculture when the cell fusion degree reaches 80-90%.
Cells grown in log phase were taken, washed with pre-warmed PBS, added with 1.0mL of trypsin, centrifuged at 2000rpm for 5 minutes, and collected. Resuspend cells using DMEM, count, 3X 10 per dish5Cells were seeded in 35mm diameter cell culture dishes at 1.5mL DMEM medium per dish, followed by low temperature plasma treatment. Helium was introduced for 5 minutes before the treatment to exhaust the air from the plasma reaction chamber at a helium flow rate of 120L/h. The parameters of the low-temperature plasma device are set as follows: the experimental power supply is 12kV, 24kHz, and the discharge power is 0.9W/cm2The distance between the electrode and the liquid surface of the culture medium is 5 mm. Placing the cell culture dish inoculated in the step 1 in a reaction chamber of a dielectric barrier discharge low-temperature plasma device, introducing helium for 5 minutes, and adopting different low-temperature plasma treatment doses of 0, 60, 90 and 120 seconds respectively; after low-temperature plasma treatment, each group of cells was placed at 37 ℃ in 5% CO2Under the condition, the culture is continued for 24 hours, the culture medium is discarded, CCK-8 working solution (800-1000 mu L) is added into each dish, the culture is continued for 0.5-1 hour, 100 mu L of supernatant is respectively taken and placed into a 96-well plate, and the absorbance value (OD value) at 450nm is detected by an enzyme-labeling instrument. Medium only and CCK-8 solution, cell-free wells were used as blanks; wells not subjected to low temperature plasma treatment were used as a control group. Cell viability was calculated according to the following formula:
cell viability ═ 100% x [ (OD treated-OD blank)/(OD control-OD blank) ].
As shown in FIG. 1, the low temperature plasma treatment can effectively kill mouse melanoma cells B16-F10-OVA, and the efficiency and the dosage show positive correlation. After 120 seconds of low temperature plasma treatment, the cell viability was less than 10%, which is considered a suitable dose for making tumor cell vaccines in the present application.
Example 2 in vivo experiments to verify the effectiveness of tumor cell vaccines
The low-temperature plasma apparatus, the test materials, and the low-temperature plasma treatment method were the same as in example 1. After low-temperature plasma treatment for 120 seconds, the mouse melanoma cells B16-F10-OVA are cultured for 12 hours, trypsinized, centrifuged at 2000rpm for 5 minutes, and the cells are collected. Precooled PBS was used to wash the cells, resuspended and counted, and the concentration adjusted to 107Cells per ml. 20C 57BL/6 mice aged 6-8 weeks were divided into 2 groups of 10 mice each, control and experimental groups, respectively. On day 1, PBS (100ul) was intraperitoneally injected into control mice, and low-temperature plasma-inactivated tumor cells (100ul, about 1-3X 10) were intraperitoneally injected into experimental mice6A cell). On day 3, the experimental mice were anesthetized, shaved, and right-side dorsal subcutaneous tumors were injected with untreated B16-F10-OVA cells at 5X 105(100 ul). The mice are kept for about 30 days, and the tumor formation rate of the back transplanted tumor and the change of the tumor volume are continuously monitored. Measuring the length and the width of the tumor body by using a vernier caliper according to a formula: v0.52 length width2And calculating the volume of the tumor body. When the tumor volume reaches 50mm3Thereafter, tumor volume was measured 3 times per week and tumor growth rate was plotted.
As shown in FIG. 2, the tumor vaccine prepared by injecting low temperature plasma can effectively inhibit the growth rate of melanoma tumor body (FIG. 2a, c). After the experiment, tumor bodies were dissected out, and the tumor body volume of the experimental group injected with the tumor vaccine was found to be significantly smaller than that of the control group without injection (fig. 2 b).
In conclusion, the low-temperature plasma treatment can remarkably kill the melanoma cells of the mice; the tumor cell vaccine killed by low-temperature plasma is injected into the abdominal cavity, so that the tumor formation rate and the tumor body growth speed of melanoma can be inhibited. The tumor cells are inactivated by adopting a low-temperature plasma technology to further obtain the tumor cell vaccine, and a new idea and method are provided for the future clinical application of the low-temperature plasma.
The invention takes a melanoma tumor model of a mouse as an example, and proves that the tumor cell vaccine inactivated by low-temperature plasma is injected into an abdominal cavity, so that the tumor formation rate of the subcutaneous transplanted melanoma tumor and the growth speed of a tumor body can be effectively inhibited. However, it should be understood by those skilled in the art that the equipment of low temperature plasma is diversified, and the injection mode of the vaccine is multi-site, and all the various injection modes of low temperature plasma for preparing tumor cell vaccine are within the protection scope of the present invention.

Claims (10)

1. A preparation method of a tumor cell vaccine based on low-temperature plasma is characterized by comprising the following steps: the method comprises the following steps:
1) taking tumor cells as objects, culturing and adjusting the cell state to a growth log phase;
2) collecting the tumor cells in the logarithmic growth phase in the step 1) and washing the cells, performing centrifugal collection after protease enzymolysis of the cells, and adjusting the cell concentration to 3 x 105～5*105Cells/ml;
3) directly or indirectly treating the tumor cells treated in the step 2) for 30 seconds to 30 minutes by using low-temperature plasma;
4) continuously culturing the tumor cells obtained by the low-temperature plasma treatment in the step 3) for 4-48 hours, and centrifugally collecting inactivated cells after protease enzymolysis, thereby obtaining the tumor cell vaccine.
2. The method of claim 1, wherein: step 1) tumor cells were incubated at 37 ℃ with 5% CO2Under the condition, culturing in a DMEM high-sugar medium containing 10% fetal calf serum and 1% penicillin-streptomycin, and carrying out subculture when the cell fusion degree reaches 80-90%.
3. The method of claim 1, wherein: step 2) washing the cells with pre-warmed PBS, the protease is preferably trypsin, and the cells are conditioned using pre-warmed PBS or DMDM high sugar mediumCell concentration to 3 x 105～5*105Cells/ml.
4. The method of claim 1, wherein: the low-temperature plasma in the step 3) can be selected from one or more of jet low-temperature plasma, dielectric barrier discharge low-temperature plasma and air multi-needle low-temperature plasma; preferably, the low-temperature plasma is dielectric barrier discharge low-temperature plasma.
5. The method of claim 1, wherein: the working gas of the low-temperature plasma in the step 3) is air and/or inert gas, and the power of the low-temperature plasma is 0.5W/cm2～5W/cm2。
6. The method of claim 5, wherein: the inert gas comprises one or more of helium, argon and nitrogen, and preferably the inert gas is helium.
7. The method of claim 1 or 5, wherein: the power of the low-temperature plasma is 0.9W/cm2The processing time of the low-temperature plasma is 120 seconds.
8. The method of claim 1, wherein: the tumor cells in step 4) are cultured for 12 hours, and the protease is preferably trypsin.
9. The method of claim 1, wherein: the tumor cells are melanoma cells, head and neck cancer cells, cervical cancer cells and breast cancer cells, and are preferably melanoma cells.
10. Use of a tumor cell vaccine obtained by the method of any one of claims 1 to 9.
CN201911324973.XA 2019-12-20 2019-12-20 Preparation method of tumor cell vaccine based on low-temperature plasma Pending CN111228471A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113244261A (en) * 2021-03-26 2021-08-13 西安交通大学 Application of CAP activated physiological saline in cell metabolism regulation and tumor treatment and medicine

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