CN114107202A - Immune cell capable of preventing tumorigenesis and preparation method and application thereof - Google Patents
Immune cell capable of preventing tumorigenesis and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an immune cell capable of preventing tumorigenesis. The invention discloses a preparation method of the immune cell, which comprises the following steps: putting peripheral blood mononuclear cells and a culture medium into a culture bottle coated with Retronectin, and adding a cytokine IFN-gamma 500-2000 mu g/ml for culture; culturing to day 2, adding cytokine CD3McAb 0.5-5 μ g/mL, CD28McAb 0.5-2 μ g/mL, IL-2500-; culturing to 7 days, adding cytokine CD 40L 0.1-0.5 μ g/mL and GM-CSF0.1-0.5 μ g/mL, and continuing culturing; the culture process is supplemented with culture medium every 3-4 days, and with cytokine IL-2500-.
Description
Technical Field
The invention relates to the technical field of immune cells, in particular to an immune cell capable of preventing tumorigenesis and a preparation method and application thereof.
Background
Tumors are common and frequently encountered diseases seriously threatening human health. More than 160 million malignant tumor patients in China per year, the cancer exceeding cardiovascular diseases becomes the first cause of death, the biological treatment of tumors is the fourth major treatment method after surgery, radiotherapy and chemotherapy, and as the development of the traditional surgery and radiotherapy and chemotherapy enters the plateau stage, people put more and more eyes on the prevention of tumor prevention.
The avoidance of expensive treatment protocols and intensive therapeutic drugs is beneficial to cancer patients and healthcare facilities worldwide if the ever increasing incidence of cancer can be stopped, reduced, or even prevented, and thus the concept of cancer chemoprevention is essential both from the cancer patient's perspective and to healthcare facilities that are under great economic stress.
Immune cells are the core components in tumor immunity, and the immune cells mainly comprise lymphocytes, antigen presenting cells, hematopoietic stem cells, eosinophils, neutrophils, basophils and the like. In international tumor immunotherapy, a killer cell (LAK cell) activated by a lymphokine, a killer cell induced by an anti-CD 3 monoclonal antibody and a tumor infiltrating lymphocyte (TIL cell) are sequentially subjected to, and as some cellular immunotherapies have difficulty in obtaining effector cells (such as TIL) or have large side effects (such as LAK) in treatment, research and reports are less and less at present.
CN 102321577B discloses a preparation method of anti-tumor adoptive immune cells and prepared immune cells, the method comprises the steps of collecting and separating peripheral blood mononuclear cells, adding cytokines to induce and culture to obtain killer cells CIK, the cytokines comprise CD28McAb, the prepared immune cells can enhance the expansion multiple of effector cells and enhance the cytotoxic activity of the killer cells, although the obtained DC-CIK becomes tumor specific cell killer cells and improves the overall tumor killing effect of the CIK population, the method does not disclose the effect of preventing tumor occurrence.
The inventor finds that the effect is not obvious by applying the immune cell in preventing tumorigenesis, and the immune cell has excellent research prospect if being prepared to achieve the effect of preventing tumorigenesis at present.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides an immune cell capable of preventing tumorigenesis, and a preparation method and application thereof.
A method for preparing immune cells capable of preventing tumorigenesis comprises the following steps:
s1, placing the peripheral blood mononuclear cells and the culture medium into a culture bottle coated with Retrocin, and adding the cell factor IFN-gamma 500-;
s2, culturing until the 2 nd day, adding the cytokines CD3McAb 0.5-5 μ g/mL, CD28McAb 0.5-2 μ g/mL, IL-2500-;
s3, culturing to 7 th day, adding 0.1-0.5 mug/mL of cell factor CD-40L and 0.1-0.5 mug/mL of GM-CSF, and continuing culturing;
s3, supplementing the culture medium every 3-4 days in the culture process, supplementing the cytokine IL-2500-.
GM-CSF is granulocyte-macrophage colony stimulating factor, and has effects of exciting hematopoietic function of bone marrow, stimulating proliferation of granulocyte, monocyte, and T cell, promoting maturation of monocyte and granulocyte, enhancing monocyte, granulocyte, eosinophil, and macrophage functions, and improving anti-tumor and anti-infection immunity.
Preferably, the cell density is controlled to 1-5X 10 during the culture of S17/mL。
Preferably, the cell density is controlled to 1-5X 10 during the culture of S27/mL。
Preferably, the culture temperature is 37 +/-0.5 ℃ and the carbon dioxide concentration is 5 +/-0.2% in the culture process of S2.
Preferably, the peripheral blood mononuclear cells are prepared by the following steps: aseptic extraction peripheral blood adds the centrifuging tube that has put heparin saline, and the gentle shake is fully anti-freezing, presses 1 with normal saline: 1, adding the diluted solution into a lymphocyte separating medium, centrifuging the solution at room temperature for 10 to 15min, and sucking out peripheral blood mononuclear cells.
Preferably, the centrifugation speed is 1500-.
Preferably, the peripheral blood mononuclear cells are aspirated and then transferred into a new centrifuge tube, the physiological saline is added, the mixture is lightly blown by a pipette and centrifuged for 10-15min, the supernatant is discarded, and the culture medium is added for culture.
Preferably, the culture medium is GIBCO AIM-V serum-free culture medium, and the cell density is adjusted to 1-5 × 10 by adding the culture medium7The culture temperature is 37 +/-0.5 ℃ and the carbon dioxide concentration is 5 +/-0.2 percent.
An immune cell capable of preventing tumorigenesis is prepared by the preparation method of the immune cell capable of preventing tumorigenesis.
The immune cell capable of preventing tumorigenesis is applied to the preparation of medicaments for preventing the growth or metastasis of breast cancer cells.
The technical effects of the invention are as follows:
(1) in the CIK induction system, the cytokine CD28McAb is introduced, so that although the induction effect can be improved, the cell proliferation multiple is improved, and the CIK induction system has a good tumor killing effect, the prevention effect is poor; the CD-40L and GM-CSF stimulating factors are added in the seventh day, so that the T cell proliferation can be effectively stimulated, the anti-tumor immune response can be effectively enhanced, the tumor regression can be induced, and the prevention effect on tumors is excellent;
(2) through a large number of experiments, the inventor finds that the co-stimulation of the CD28McAb and the CD 40L, GM-CSF stimulating factor can induce the capacity of preventing the tumor, which is not possessed by the CD28McAb stimulating factor added alone, after the immune cells prepared by the invention are injected into ten mice, the survival rate of 180d rats reaches 90%, but the immune cells only adopting the CD28McAb do not generate the tumor and die within 60 days.
Drawings
FIG. 1 is a graph showing the change of tumor volumes with time between the rats in the experimental group and the rats in the blank group in example 3.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example 1
SD rats purchased from the Experimental animal center of Wenzhou medical university were bred in an animal room SPF environment, and were allowed to obtain feed and water ad libitum, acclimatized for 1 week. All principles and procedures of animal experiments are in accordance with relevant provisions of the regulations on the management of laboratory animals.
Numbering the rats, then extracting peripheral blood of the rats in sequence under the aseptic condition, adding a centrifugal tube containing heparin saline, shaking gently, fully anticoagulating, and pressing 1: diluting 1, adding into lymphocyte separation solution, centrifuging at 2000r/min for 15min, sucking out PBMC, transferring into new centrifuge tube, adding physiological saline, mixing with pipette, centrifuging at 2000r/min for 15min, discarding supernatant, adding AIM-V (GIBCO) serum-free culture medium to adjust cell density to 1-5 × 107one/mL, 5% CO at 37 ℃2Culturing for 3h under the condition.
Example 2
1 tube of 2.5mg Retronectin was dissolved in 250mL of physiological saline in advance to prepare a coating solution at a final concentration of 10. mu.g/mL. The coating solution is put into a culture flask and is kept stand overnight. The next day, the coating solution was aspirated and the flask was washed with physiological saline for 2 times to obtain a flask coated with Retronectin.
Nonadherent cells obtained after 3h adherent culture in example 1 were resuspended in 10ml of LAIM-V medium, transferred to a Retronectin-coated flask, and the cytokine IFN-. gamma.1000. mu.g/ml was added at 37 ℃ with 5% CO2The cell density is controlled to be 1-5 multiplied by 10 during the culture process7/mL。
On day 2, the cytokines CD3McAb 2. mu.g/mL, CD28McAb 1. mu.g/mL, IL-21000. mu.g/mL were added to the flask at 37 ℃ with 5% CO2The cell density is controlled to be 1-5 multiplied by 10 during the culture process7/mL；
On day 7, the culture flask was further charged with 0.1-0.5. mu.g/mL of cytokine CD 40L and 0.1-0.5. mu.g/mL of GM-CSF at 37 ℃ with 5% CO2Continuously culturing in the culture environment;
the culture process is supplemented with culture medium every 3-4 days, and with cytokine IL-21000 μ g/mL every 1-2 days, continuously culturing for 14 days, maturing cells, and collecting mature cells.
Comparative example
1 tube of 2.5mg Retronectin was dissolved in 250mL of physiological saline in advance to prepare a coating solution at a final concentration of 10. mu.g/mL. The coating solution is put into a culture flask and is kept stand overnight. The next day, the coating solution was aspirated and the flask was washed with physiological saline for 2 times to obtain a flask coated with Retronectin.
Nonadherent cells obtained after 3h adherent culture in example 1 were resuspended in 10ml of LAIM-V medium, transferred to a Retronectin-coated flask, and the cytokine IFN-. gamma.1000. mu.g/ml was added at 37 ℃ with 5% CO2The cell density is controlled to be 1-5 multiplied by 10 during the culture process7/mL。
On day 2, the cytokines CD3McAb 2. mu.g/mL, CD28McAb 1. mu.g/mL, IL-21000. mu.g/mL were added to the flask at 37 ℃ with 5% CO2The cell density is controlled to be 1-5 multiplied by 10 during the culture process7/mL；
The culture process is supplemented with culture medium every 3-4 days, and with cytokine IL-21000 μ g/mL every 1-2 days, continuously culturing for 14 days, maturing cells, and collecting mature cells.
Test examples
The rats were randomly assigned to 3 groups of 3 replicates each, 7 rats per replicate, according to the rat numbering in example 1.
The test group was re-dispersed by adding the mature cells obtained in example 2 to RPMI-1640 medium and adjusting the cell concentration to 5X 106one/mL, 0.1mL by tail vein injection in rats from each source.
Experimental control group mature cells obtained in comparative example were added to RPMI-1640 medium to re-disperse and adjust the cell concentration to 5X 106one/mL, 0.1mL by tail vein injection in rats from each source.
The rats in the blank group were injected with 0.1mL of physiological saline by tail vein injection.
After 1 day, Walker 256 rat mammary carcinoma cells purchased from Shanghai Zea leaf Biotech Co., Ltd were re-dispersed with RPMI-1640 medium and the cell concentration was adjusted to 2X 106one/mL, 0.35mL was injected subcutaneously in the back of the neck of all rats, respectively.
Body weight and tumor volume of rats were measured 1 time every 2 days and based on relative tumor volume (V)t/V0) The change of the time is used for judging the prevention/inhibition effect on the tumorThe application is as follows. On day 14, all rats were sacrificed, tumors and lungs were harvested, and after washing with pre-cooled PBS, tumor weight was measured and Tumor Inhibition Rate (TIR) was calculated.
TIR＝(1-WTest of/WBlank space)×100％
WTest ofIs the mean weight of the tumors in the test group, WBlank spaceIs the average weight of the tumors in the blank group.
The results are shown in FIG. 1, and it can be seen from FIG. 1 that: although the tumor volumes of the rats in the three groups are increased, the change of the tumor volumes of the test group is obviously smaller than that of the blank group and the test control group, which shows that the invention has better tumor prevention/inhibition effect in vivo.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A method for preparing immune cells capable of preventing tumorigenesis, which comprises the following steps:
s1, placing the peripheral blood mononuclear cells and the culture medium into a culture bottle coated with Retrocin, and adding the cell factor IFN-gamma 500-;
s2, culturing until the 2 nd day, adding the cytokines CD3McAb 0.5-5 μ g/mL, CD28McAb 0.5-2 μ g/mL, IL-2500-;
s3, culturing to 7 th day, adding 0.1-0.5 mug/mL of cell factor CD-40L and 0.1-0.5 mug/mL of GM-CSF, and continuing culturing;
s4, supplementing the culture medium every 3-4 days in the culture process, supplementing the cytokine IL-2500-.
2. The method for producing an immune cell according to claim 1, wherein the cultured product of S1 is S1In the process, the cell density is controlled to be 1-5X 107/mL。
3. The method for preparing immunocyte for preventing tumorigenesis according to claim 1, wherein the cell density is controlled to 1-5X 10 during the culture of S27/mL。
4. The method for preparing immunocyte for preventing tumorigenesis according to claim 1, wherein the culture temperature is 37 ± 0.5 ℃ and the carbon dioxide concentration is 5 ± 0.2% in the culture process of S2.
5. The method of claim 1, wherein the mononuclear cells from peripheral blood are prepared by the steps of: aseptic extraction peripheral blood adds the centrifuging tube that has put heparin saline, and the gentle shake is fully anti-freezing, presses 1 with normal saline: 1, adding the diluted solution into a lymphocyte separating medium, centrifuging the solution at room temperature for 10 to 15min, and sucking out peripheral blood mononuclear cells.
6. The method of claim 5, wherein the centrifugation speed is 1500-3000 r/min.
7. The method of claim 5, wherein the mononuclear cells of peripheral blood are aspirated and transferred to a new centrifuge tube, and the mononuclear cells are mixed with normal saline by gentle blowing with a pipette, centrifuged for 10-15min, the supernatant is discarded, and the culture medium is added for culturing.
8. The method for preparing immunocyte for preventing tumorigenesis according to claim 7, wherein the culture medium is GIBCO AIM-V serum-free medium, and the cell density is adjusted to 1-5X 10 by adding the culture medium7The culture temperature is 37 +/-0.5 ℃ and the carbon dioxide concentration is 5 +/-0.2 percent.
9. An immunocyte for preventing tumorigenesis, which is produced by the method for producing an immunocyte for preventing tumorigenesis according to any one of claims 1 to 8.
10. Use of the immune cell of claim 9 for preventing tumorigenesis in the preparation of a medicament for preventing breast cancer cell growth.
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| JPH06121672A (en) * | 1992-10-13 | 1994-05-06 | Cellcor Inc | Production of immunoreactive cell |
| CN102321577A (en) * | 2011-01-26 | 2012-01-18 | 深圳市中美康士生物科技有限公司 | Preparation method of antitumor adoptive immune cells and prepared immune cells |
| US20140065096A1 (en) * | 2012-09-05 | 2014-03-06 | Regen BioPharma, Inc. | Cancer therapy by ex vivo activated autologous immune cells |
| CN103937742A (en) * | 2014-04-29 | 2014-07-23 | 湖北华赛生物医药技术有限公司 | Immune cell culture method capable of simultaneously activating CD4<+> and CD8<+>T cells |
| CN105349489A (en) * | 2015-12-07 | 2016-02-24 | 广州赛莱拉干细胞科技股份有限公司 | Culture method of CIK cell |
| CN113046313A (en) * | 2021-03-18 | 2021-06-29 | 重庆福美干细胞生物科技发展有限公司 | Composition and kit for efficiently inducing and amplifying human peripheral blood killer immune cells and culture method of immune cells |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06121672A (en) * | 1992-10-13 | 1994-05-06 | Cellcor Inc | Production of immunoreactive cell |
| CN102321577A (en) * | 2011-01-26 | 2012-01-18 | 深圳市中美康士生物科技有限公司 | Preparation method of antitumor adoptive immune cells and prepared immune cells |
| US20140065096A1 (en) * | 2012-09-05 | 2014-03-06 | Regen BioPharma, Inc. | Cancer therapy by ex vivo activated autologous immune cells |
| CN103937742A (en) * | 2014-04-29 | 2014-07-23 | 湖北华赛生物医药技术有限公司 | Immune cell culture method capable of simultaneously activating CD4<+> and CD8<+>T cells |
| CN105349489A (en) * | 2015-12-07 | 2016-02-24 | 广州赛莱拉干细胞科技股份有限公司 | Culture method of CIK cell |
| CN113046313A (en) * | 2021-03-18 | 2021-06-29 | 重庆福美干细胞生物科技发展有限公司 | Composition and kit for efficiently inducing and amplifying human peripheral blood killer immune cells and culture method of immune cells |
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