CN110646619A - Method for detecting cell factor secreted by specific T cell in lung cancer or intestinal cancer - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Abstract
The invention belongs to the technical field of medical detection application, and discloses a method for detecting cytokines secreted by specific T cells in lung cancer or intestinal cancer. The sample in the detection system is mononuclear cells separated from peripheral blood of a patient with lung cancer or intestinal cancer, and then the cell factors secreted by specific T cells are detected after polypeptide multi-round stimulation. The invention achieves the purpose of shortening the detection time of the whole system under the condition of not influencing the detection sensitivity by inducing the mononuclear cells into DC cells, then co-culturing the DC cells and the T cells, adding the polypeptide to stimulate the DC cells for three times and then detecting the cell factors secreted by the specific T cells. The detection system provided by the invention is simple and convenient to use, rapid and efficient; the detection time can be shortened, and the detection efficiency is improved. The detection system provided by the invention has wide application range and huge market value.
Description
Technical Field
The invention belongs to the technical field of medical detection application, and particularly relates to a method for detecting a cytokine secreted by a specific T cell in lung cancer or intestinal cancer.
Background
Currently, the closest prior art:
cytokines (CK) are small molecules proteins with a wide range of biological activities that are synthesized and secreted by immune cells (e.g., monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. Has multiple functions of regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, damaged tissue repair and the like. Cytokines can be classified as interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, and the like. Many cytokines play roles in vivo through paracrine, autocrine or endocrine and other modes, have multiple physiological characteristics such as multiple effects, overlapping, antagonistic, cooperativity and the like, form a very complex cytokine regulation network and participate in multiple important physiological functions of human bodies.
When the body is infected with a disease, a series of related cytokines participate in the immune response process of the body by coordination. Taking tuberculosis immunity as an example, mycobacterium tuberculosis induces IV-type hypersensitivity of the body through T lymphocyte mediation, the body mainly plays the role of T cell immunity, and various cytokines are synergistically involved in immune response, which is mainly mediated by CD4+ T cells, namely T helper 1 cells (Th1) and CD8+ T cells, namely T helper 2 cells (Th 2). Relevant cytokines include Tumor Necrosis Factor (TNF), interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), and the like.
The state of body infection disease can be revealed by detecting the extent of response of memory T cells to stimulation by specific antigen, usually expressed as secretion of various cytokines, which is a reliable detection method, such as gamma interferon release assay, which is a technique for detecting the ability of T cells to produce and release gamma-interferon after stimulation by binding specific antigen in vitro, thereby determining cell-mediated immune response. Stimulating peripheral blood mononuclear cells of a patient to enable T lymphocytes to produce a large amount of gamma-interferon, and then detecting the concentration of the gamma-interferon by an enzyme-linked immunosorbent assay.
The in vitro diagnosis of pathogenic microorganism infection by using the cellular immune reaction of antigen-specific T cells is a new detection method developed in the present year. Peripheral blood mononuclear cells in fresh whole blood are separated and cultured in a stimulation mode, and then the number of cells capable of secreting IFN-gamma is detected by using ELISPOT. The method is currently mainly applied to the diagnosis of mycobacterium tuberculosis infection. The current diagnosis of tuberculosis generally adopted in clinic mainly depends on clinical symptoms, influential diagnosis and etiological diagnosis, and is insensitive to the diagnosis of latent infection of mycobacterium tuberculosis. Meanwhile, in the tuberculosis screening process, the sensitivity and specificity for directly detecting pathogens or detecting mycobacterium tuberculosis antibodies are not ideal.
In summary, the problems of the prior art are as follows:
(1) the current diagnosis of tuberculosis generally adopted in clinic mainly depends on clinical symptoms, influential diagnosis and etiological diagnosis, and is insensitive to the diagnosis of latent infection of mycobacterium tuberculosis.
(2) The sensitivity and specificity of direct detection of pathogens or detection of antibodies to mycobacterium tuberculosis are also not ideal in the tuberculosis screening process.
(3) The in vitro detection of T cells has the defect of overlong detection time.
The difficulty of solving the technical problems is as follows:
(1) firstly, how to use the minimum sample amount to achieve the detection purpose is related to the sample collection amount.
(2) Secondly, how to shorten the detection time of the whole system.
(3) And finally how to achieve the detection sensitivity in the minimum sample size and the minimum time.
The significance of solving the technical problems is as follows: the significance for solving the technical problems is that the detection effect can be achieved by using the minimum amount of samples in a shorter time, so that the detection efficiency is improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for detecting a cytokine secreted by a specific T cell in lung cancer or intestinal cancer.
The invention is realized in such a way that the method for detecting the cell factors secreted by the specific T cells in the lung cancer or the intestinal cancer comprises the following steps:
firstly, collecting a blood sample into a heparin anticoagulant blood collection tube, and uniformly mixing;
secondly, transferring the blood sample into a new centrifugal tube, and adding physiological saline with the same volume for dilution;
thirdly, adding Ficoll-Histopaque separating medium into a new centrifugal tube, inclining the centrifugal tube to 30 degrees, and then adding the blood sample diluted in the second step into the separating medium along the centrifugal tube;
fourthly, centrifuging the sample prepared in the third step for 30min at 2500r/min in a density gradient manner, dividing the sample into three layers after centrifugation, and sucking out the middle white layer to a new centrifugal tube; adding physiological saline for dilution, centrifuging at 1500r/min for 10min, discarding supernatant, adding physiological saline for dilution, and counting cells;
fifthly, centrifuging the cells in the fourth step at low speed for 10min at 1200r/min, and discarding the supernatant, wherein the lower layer is PBMC cells;
sixthly, resuspending the obtained PBMC cells by using an AIM-V serum-free culture medium, carrying out adherent culture on the PBMC cells for 2 hours by using a 6-pore plate, sucking out the non-adherent cells on the upper layer, adding the non-adherent cells into a new pore, and continuously culturing for 1 hour; adding a DC culture medium and 5% serum into the original hole for culturing;
seventhly, after culturing for 1 hour, sucking the upper layer cells out, adding the upper layer cells into a new centrifugal tube, centrifuging the upper layer cells at 1200r/min for 10min, counting the cells and freezing the cells; adding DC culture medium and 5% serum into the new hole to continue culturing;
eighthly, changing the culture medium for half of the cultured cells by the third day, and adding culture factors with the same quantity;
ninth, by the fifth day, performing resuscitation culture on the cryopreserved non-adherent walls; plating according to the quantity of the polypeptide to be detected, wherein the number of cells per well is 1x105Culturing for 24 hours;
step ten, inducing the DC cells to become DC cells by the sixth day, adding the DC cells into the recovered nonadherent cells in the step ninth step according to the cell number of 10:1, and adding corresponding polypeptide into each hole to stimulate the culture;
step eleven, performing half-amount liquid change every three days, and adding culture factors and polypeptides in corresponding proportions;
in the twelfth step, after three rounds of stimulation, cytokine content was measured using an Elispot assay kit.
The invention also aims to provide a system for detecting the cytokine secreted by the specific T cell in the lung cancer or the intestinal cancer, which is used for the method for detecting the cytokine secreted by the specific T cell in the lung cancer or the intestinal cancer, wherein the system for detecting the specific T cell consists of a detection sample, a target, a stimulation antigen and/or a positive control antigen of the detection sample and a conventional reagent of the detection sample;
the detection sample is heparin anticoagulated blood; the sample size is 5mL, 10mL, 15mL, 20mL, 30mL, or 40 mL;
the stimulation antigen is a specific antigen for stimulating memory T cells;
the positive control antigen is a non-specific stimulant for stimulating immune cells;
the conventional reagent refers to a conventional detection reagent for detecting the cytokine.
Further, the target of the detection system is a substance secreted by the cell after stimulation.
Further, the substance secreted by the cell after stimulation is any one or the combination of at least two of cytokines, chemokines or proteins;
the specific antigen for stimulating the memory T cells is any one or the combination of at least two of natural protein, polypeptide, nucleic acid, polysaccharide, small molecule compound, virus specific antigen or bacteria specific antigen.
Furthermore, the cell factor is secreted by immune cells and/or non-immune cells after being stimulated by a non-specific stimulant;
the cytokine is specifically any one or combination of at least two of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-alpha, IFN-beta, IFN-gamma, TNF-beta, SCF, G-CSF, M-CSF, GM-CSF, neuroleukin, IgA, IgG, perforin, granulin, IP10 or TGF-beta;
the natural protein, polypeptide, nucleic acid, polysaccharide and small molecule compound are allergens capable of stimulating cellular reactions.
Further, the immune cell may be selected from, but not limited to, a T cell, a B cell, an NK cell, or a macrophage;
the nonspecific stimulator for stimulating immune cells is selected from one or more of concanavalin A, phytohemagglutinin, human phytolaccin, peanut agglutinin, soybean agglutinin, thallus of staphylococcal protein A, lipopolysaccharide, and tumor stimulator.
The invention also aims to provide the application of the method for detecting the specific T cell secreted cytokine in the lung cancer or the intestinal cancer in detecting the memory immune cells.
The invention also aims to provide the application of the method for detecting the specific T cell secreted cytokine in the lung cancer or the intestinal cancer in preparing a kit for detecting and/or diagnosing diseases.
The invention also aims to provide the application of the method for detecting the cytokines secreted by the specific T cells in the lung cancer or the intestinal cancer in detecting the secretory substances of the memory immune cells.
The invention also aims to provide the application of the method for detecting the specific T cell secreted cytokine in the lung cancer or the intestinal cancer in the diseases of cytokine detection.
In summary, the advantages and positive effects of the invention are:
(1) the invention detects specific T cells by separating peripheral blood mononuclear cells, inducing the cells into DC cells, co-culturing the DC cells and the T cells, adding polypeptide for stimulation.
(2) When the sample is reduced to 10mL under the condition that the final concentration of the stimulating antigen is consistent, compared with the conventional sample volume of more than 20mL, the detection amount of the sample is reduced, and the detection of the final specific T cells is not influenced.
(3) The detection system provided by the invention is simple and convenient to use, rapid and efficient; the detection time can be shortened, and the detection efficiency is improved.
(4) The detection system provided by the invention has wide application range and huge market value.
Drawings
FIG. 1 is a flowchart of a method for detecting cytokines secreted from specific T cells in lung cancer or intestinal cancer according to an embodiment of the present invention.
Fig. 2 is a diagram of ELISPOT detection results provided by the embodiment of the present invention.
FIG. 3 is a diagram showing the amount of PBMC obtained from different sample amounts according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In view of the problems of the prior art, the present invention provides a method for detecting cytokines secreted by specific T cells in lung cancer or intestinal cancer, which is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for detecting cytokines secreted by specific T cells in lung cancer or intestinal cancer according to the embodiment of the present invention includes the following steps:
s101: mononuclear cells are extracted after separating peripheral blood of a patient with lung cancer.
S102: monocytes are induced into DC cells.
S103: the DC cells were co-cultured with T cells.
S104: the cytokines secreted by specific T cells were detected after three stimulations with polypeptide.
The technical effects of the present invention will be described in detail with reference to specific embodiments.
The method for detecting the cell factors secreted by the specific T cells in the lung cancer or the intestinal cancer, provided by the embodiment of the invention, comprises the following steps:
(1) detecting cytokine IFN-gamma generated by specific T cells stimulated by peripheral blood lymphocyte polypeptide antigen of lung cancer patients, performing anticoagulation on peripheral whole blood of lung cancer patients, performing density gradient centrifugation (room temperature, 400g and 30 minutes) on lymphocyte separating medium (Ficoll-Histopaque1.077), taking intermediate leucocyte layer cells, putting the intermediate leucocyte layer cells into a 50ml centrifuge tube, and washing the cells for 3 times by PBS (phosphate buffer solution) to obtain Peripheral Blood Mononuclear Cells (PBMC). The obtained mononuclear cells were suspended in PBMC with complete medium at 37 ℃ in 5% CO2After 2 hours of incubation, the plates were gently shaken, the non-adherent cells were aspirated off and frozen for future use.
Adherent cells were cultured in DC serum-free medium containing human recombinant rhGM-CSF (500U/ml) and human recombinant rhIL-4(10ng/ml) at 37 deg.C and 5% CO2Culturing in an incubator. Culturing till the seventh day. Simultaneously recovering cryopreserved nonadherent cells (rich in lymphocytes), suspending in 10% fetal bovine serum RPMI1640 culture medium, respectively extracting into the above autologous DCs, and treating with 5% CO at 37 deg.C2The cells were co-cultured under the same conditions, and IL-2(100U/ml), 10% FBS and the polypeptide were added to stimulate the culture. This is the first round of stimulation. IL-2(100U/ml) was added every 3 days during the culture, and the fresh medium was replaced half a day for 2-3 days. Three rounds of polypeptide stimulated culture were performed.
As shown in FIG. 2, the cell concentration of the cells after three cycles of co-culture stimulation was adjusted to 1X10 with the medium5And/m, directly transferring the cell suspension into an ELISPOT detection plate coated with anti-IFN-gamma antibody, wherein the volume of the detection plate is 200 mu l/hole, and adding corresponding polypeptide. IFN-gamma secreting T cell colonies were detected by methods described in the IFN-gamma ELISPOT assay kit instructions.
(2) Selection of sample size
Since the peripheral blood sample of the patient is extremely precious, the subsequent experiment for detecting the secretion after the specific T cell re-stimulation is achieved by using the minimum sample size. The sample size may be 5mL, 10mL, 15mL, 20mL, 30mL or 40 mL. Through experimental verification, when the detection sample amount is 10ml, the number of the subsequently separated mononuclear cells (PBMC) can be ensured to reach 1x107Thereby ensuring that a suitable number of atopic T cells can be reached in the subsequent system.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for detecting cytokines secreted by specific T cells in lung cancer or intestinal cancer, which is characterized in that the method for detecting the cytokines secreted by the specific T cells in the lung cancer or the intestinal cancer comprises the following steps:
firstly, collecting a blood sample into a heparin anticoagulant blood collection tube, and uniformly mixing;
secondly, transferring the blood sample into a new centrifugal tube, and adding physiological saline with the same volume for dilution;
thirdly, adding Ficoll-Histopaque separating medium into a new centrifugal tube, inclining the centrifugal tube to 30 degrees, and then adding the blood sample diluted in the second step into the separating medium along the centrifugal tube;
fourthly, centrifuging the sample prepared in the third step for 30min at 2500r/min in a density gradient manner, dividing the sample into three layers after centrifugation, and sucking out the middle white layer to a new centrifugal tube; adding physiological saline for dilution, centrifuging at 1500r/min for 10min, discarding supernatant, adding physiological saline for dilution, and counting cells;
fifthly, centrifuging the cells in the fourth step at low speed for 10min at 1200r/min, and discarding the supernatant, wherein the lower layer is PBMC cells;
sixthly, resuspending the obtained PBMC cells by using an AIM-V serum-free culture medium, carrying out adherent culture on the PBMC cells for 2 hours by using a 6-pore plate, sucking out the non-adherent cells on the upper layer, adding the non-adherent cells into a new pore, and continuously culturing for 1 hour; adding a DC culture medium and 5% serum into the original hole for culturing;
seventhly, after culturing for 1 hour, sucking the upper layer cells out, adding the upper layer cells into a new centrifugal tube, centrifuging the upper layer cells at 1200r/min for 10min, counting the cells and freezing the cells; adding DC culture medium and 5% serum into the new hole to continue culturing;
eighthly, changing the culture medium for half of the cultured cells by the third day, and adding culture factors with the same quantity;
ninth, by the fifth day, performing resuscitation culture on the cryopreserved non-adherent walls; plating according to the quantity of the polypeptide to be detected, wherein the number of cells per well is 1x105Culturing for 24 hours;
step ten, inducing the DC cells to become DC cells by the sixth day, adding the DC cells into the recovered nonadherent cells in the step ninth step according to the cell number of 10:1, and adding corresponding polypeptide into each hole to stimulate the culture;
step eleven, performing half-amount liquid change every three days, and adding culture factors and polypeptides in corresponding proportions;
in the twelfth step, after three rounds of stimulation, cytokine content was measured using an Elispot assay kit.
2. The system for detecting the cytokines secreted by the specific T cells in the lung cancer or the intestinal cancer, which is the method for detecting the cytokines secreted by the specific T cells in the lung cancer or the intestinal cancer according to claim 1, is characterized in that the system for detecting the specific T cells consists of a detection sample, a target, a stimulation antigen and/or a positive control antigen of the detection sample, and a conventional reagent of the detection sample;
the detection sample is heparin anticoagulated blood; the sample size is 5mL, 10mL, 15mL, 20mL, 30mL, or 40 mL;
the stimulation antigen is a specific antigen for stimulating memory T cells;
the positive control antigen is a non-specific stimulant for stimulating immune cells;
the conventional reagent refers to a conventional detection reagent for detecting the cytokine.
3. The system according to claim 2, wherein the target of the detection system is a substance secreted by a cell after stimulation.
4. The system according to claim 3, wherein the substance secreted by the cells after stimulation is any one or a combination of at least two of cytokines, chemokines or proteins;
the specific antigen for stimulating the memory T cells is any one or the combination of at least two of natural protein, polypeptide, nucleic acid, polysaccharide, small molecule compound, virus specific antigen or bacteria specific antigen.
5. The system for detecting cytokines secreted from specific T cells in lung cancer or intestinal cancer according to claim 4, wherein the cytokines are cytokines secreted from immune cells and/or non-immune cells after stimulation with a non-specific stimulant;
the cytokine is specifically any one or combination of at least two of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-alpha, IFN-beta, IFN-gamma, TNF-beta, SCF, G-CSF, M-CSF, GM-CSF, neuroleukin, IgA, IgG, perforin, granulin, IP10 or TGF-beta;
the natural protein, polypeptide, nucleic acid, polysaccharide and small molecule compound are allergens capable of stimulating cellular reactions.
6. The system for detecting cytokines secreted by specific T cells in lung or intestinal cancer according to claim 5, wherein the immune cells include T cells, B cells, NK cells, or macrophages;
the nonspecific stimulator for stimulating immune cells is selected from one or more of concanavalin A, phytohemagglutinin, human phytolaccin, peanut agglutinin, soybean agglutinin, thallus of staphylococcal protein A, lipopolysaccharide, and tumor stimulator.
7. Use of the method of claim 1 for detecting cytokines secreted by specific T cells in lung or intestinal cancer for detecting memory immune cells.
8. Use of a method according to claim 1 for the detection of cytokines secreted by specific T cells in lung or intestinal cancer for the preparation of a kit for the detection and/or diagnosis of a disease.
9. Use of the method of claim 1 for detecting cytokines secreted from specific T cells in lung or intestinal cancer for detecting secreted substances from memory immune cells.
10. Use of the method of claim 1 for detecting cytokines secreted by specific T cells in lung or intestinal cancer in diseases detected by cytokines.
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| CN112300988A (en) * | 2020-05-11 | 2021-02-02 | 浙江格源致善生物医药科技有限公司 | T cell sorting preparation technology based on tumor neoantigen and application thereof |
| CN112666358A (en) * | 2021-01-14 | 2021-04-16 | 深圳裕康医学检验实验室 | Method for detecting antigen immunogenicity by using peripheral blood |
| CN114317435A (en) * | 2021-12-30 | 2022-04-12 | 杭州芯递力生物科技有限公司 | Method for obtaining antigen specific T cells |
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