CN115807070A - Marker for screening memory sample NK (Natural killer) cells of mycobacterium tuberculosis and application of marker - Google Patents
Marker for screening memory sample NK (Natural killer) cells of mycobacterium tuberculosis and application of marker Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a marker for screening memory sample NK (natural killer) cells of mycobacterium tuberculosis and application thereof. A marker for screening memory-like NK (natural killer) cells of Mycobacterium tuberculosis is a CRTAM receptor. Furthermore, the CRTAM gene is highly expressed after being stimulated by the tuberculosis specific antigen again. The invention has the advantages that: the invention adopts the flow cytometry technology to sort IFN-gamma-NK cells and IFN-gamma + NK cell groups after being stimulated by Mtb lytic antigen, adopts the single cell transcriptome technology to analyze the expression difference of two groups of cells, and screens out the activation receptor marker CRTAM molecules with high expression of tuberculosis specific memory sample NK cells. The CRTAM provided by the invention can be used as a marker of memory-like NK cells activated by tuberculosis specificity and a target for screening and researching antitubercular drugs, and has wide application prospects in the aspects of prevention and treatment of tuberculosis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mycobacterium tuberculosis memory sample NK cell screening marker and application thereof.
Background
Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), and currently, tuberculosis still faces one of major public health problems worldwide.
The innate immune system plays a dominant role in protecting the host from early infection with Mtb. NK cells are important immune cells to participate in the innate immune process against Mtb infection, and play an essential role in early clearance of Mtb infection. In recent years, some NK cells show the characteristics of adaptive immunity, and specific memory-like NK cells can rapidly respond to pathogen reinfection so as to carry out early elimination, and the memory-like NK cells have great potential application values in immunotherapy of infectious diseases and enhancement of vaccine protection effect.
The mechanism research related to Mtb infection resistance also discovers the existence of memory-like NK cells, and the research related to the memory-like NK cells in the immune response caused in the tuberculosis infection resistance process is based on BCG immunization animal liver or pleural effusion of tuberculosis patients, and peripheral blood binding specificity of the memory-like NK cells is not found.
Our studies show that NK cells releasing a large amount of IFN-gamma after being stimulated by tuberculosis antigen exist in peripheral blood of patients with active tuberculosis and patients with latent tuberculosis infection, and the expression of various genes (such as IL-12 and IL-15 receptors and the like) related to memory-like NK cells in the cells is up-regulated, which indicates that the cells are possibly memory-like NK cells in the peripheral blood of the patients with tuberculosis.
Memory-like NK cells have important value in the host innate immune system for rapid response to Mtb infection, but there are currently no biomarkers in memory-like NK cells for screening memory-like NK cells with tuberculosis specificity.
Disclosure of Invention
The invention aims to identify the memory-like NK cell with the specificity of the Mtb antigen and screen a surface activation receptor CRTAM (CD 355) of the memory-like NK cell with the specificity of the Mtb antigen, and the receptor CRTAM can be used for anti-tuberculosis treatment and drug development of people. And provides a marker for screening mycobacterium tuberculosis memory sample NK cells and application thereof;
in order to realize the purpose, the invention adopts the technical scheme that:
a marker for screening memory-like NK (natural killer) cells of Mycobacterium tuberculosis is a CRTAM receptor.
Furthermore, the CRTAM gene is highly expressed after being stimulated by the tuberculosis specific antigen again.
The invention also provides a substance for screening the immunity of the organism, wherein the substance is memory-like NK cells in peripheral blood of the organism, further, the organism refers to a human body or an animal body with infectious disease risk, and further, the organism refers to a group needing to judge whether to inject a booster vaccine or not. The infectious disease is preferably tuberculosis. The substance includes an isolation reagent for PBMCs in peripheral blood.
The application of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis is to screen the memory-like NK cells of the tuberculosis specificity.
The invention also provides application of another mycobacterium tuberculosis memory-like NK cell screening marker.
The application of the mycobacterium tuberculosis memory-like NK cell screening marker is used for research and development of anti-tuberculosis related medicines and/or research on curative effect.
The invention also provides application of the third mycobacterium tuberculosis memory-like NK cell screening marker.
The application of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis is disclosed, and the marker is used for a CRTAM detection kit.
The invention also provides an application method of the mycobacterium tuberculosis memory-like NK cell screening marker.
An application method of a marker for screening memory-like NK (natural killer) cells of mycobacterium tuberculosis, which comprises the following steps: activated tuberculosis-specific memory-like NK cells are screened by detecting the mRNA transcription level and/or protein expression level of NK cells CRTAM under the condition that the peripheral blood PBMCs of the TB patients are stimulated again by Mtb antigens.
The invention also provides a use method of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis.
A method of using a marker for mycobacterium tuberculosis memory-like NK cell screening, the method comprising:
(1) Taking 10ml of peripheral blood, placing the peripheral blood into a blood collection tube containing an EDTA anticoagulant, and separating PBMCs by using TBD (tert-butyl-D) human whole blood mononuclear cell separation liquid;
(2) The resulting PBMCs were plated into 12-well cell culture plates, and 2X 10 cells were added to each well 6 (ii) individual cells;
inoculating and culturing an Mtb standard strain H37Rv 3-4 weeks in advance, collecting Mtb in a logarithmic phase of growth, centrifuging, washing for 2 times by PBS, adding PBS for resuspension, centrifuging after ultrasonic lysis, collecting supernatant, and filtering;
the stimulation time was set at 24h, the protein concentration of Mtb lysate was 10. Mu.g/ml, and the IFN-. Gamma. + NK cell number was analyzed by flow-based analysis after the stimulation was completed.
The invention also provides application of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis.
A mycobacterium tuberculosis memory-like NK cell screening marker is used for immunotherapy research of specific targeting CRTAM genes.
The invention utilizes the tuberculosis specific memory-like NK cells in peripheral blood, screens out the activation receptor marker CRTAM molecules with high expression of the tuberculosis specific memory-like NK cells through transcriptome analysis, and provides a new thought and a specific target point for antitubercular treatment and drug research and development.
The invention adopts the flow cytometry technology to sort IFN-gamma-NK cells and IFN-gamma + NK cell groups after being stimulated by Mtb lytic antigen, adopts the single cell transcriptome technology to analyze the expression difference of two groups of cells, and screens out the activation receptor marker CRTAM molecules with high expression of tuberculosis specific memory sample NK cells.
The CRTAM provided by the invention can be used as a marker of memory-like NK cells activated by tuberculosis specificity and a target for screening and researching antitubercular drugs, and has wide application prospects in the aspects of prevention and treatment of tuberculosis.
Compared with the prior art, the mycobacterium tuberculosis memory-like NK cell screening marker and the application thereof have the advantages that:
(1) Specific biomarkers of mycobacterium tuberculosis memory-like NK cells are provided.
(2) Provides a new thought and a specific target point for anti-tuberculosis treatment and drug research and development.
(3) Has wide application prospect in the aspects of preventing and treating tuberculosis.
Drawings
FIG. 1 is a flow cytometer of example 1 of the present invention to analyze peripheral blood PBMCs for 24h IFN-. Gamma. + NK cells stimulated with Mtb lysate for HC (n = 5), LTBI (n = 5), TB (n = 5);
FIG. 2 shows the number of IFN-. Gamma. + NK cells counted in example 1 of the present invention. * P <0.05, P < 0.01, P < 0.001 (one-way ANOVA).
FIG. 3 is a diagram of the tuberculosis specific memory-like NK cell differential gene Venn in example 2 of the present invention.
FIG. 4 is the tuberculosis specific memory like NK cell differential gene KEGG enriched forest Lin Tu in example 2 of this invention.
FIG. 5 is the IFN-. Gamma. + NK cells IL-12RB2, IL-15RA and IL-21R gene mRNA expression level analysis (n = 4) of example 3 of the present invention. * P <0.05, P < 0.01, P < 0.001 (Paired T-test).
FIG. 6 shows that the expression level of mRNA of the IFN-gamma + NK cell surface activation receptor CRTAM is high, and the expression levels of mRNA of NKG2D, NKG2E, NKp, NKp30, CD244, CD27 and NCR2 genes are not obviously different between IFN-gamma-NK cells and IFN-gamma + NK cells (n = 4) in example 4 of the present invention. P <0.05 (Paired T-test).
FIG. 7 is a flow cytometry analysis of the ratio of peripheral blood tuberculosis specific memory-like CRTAM + IFN- γ + NK cells in healthy people and TB patients.
FIG. 8 is a statistical analysis of the ratio of peripheral blood tuberculosis specific memory sample CRTAM + IFN-. Gamma. + NK cells in healthy people and TB patients.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention but not to limit the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
A marker for screening memory-like NK (natural killer) cells of Mycobacterium tuberculosis is a CRTAM receptor.
Furthermore, the CRTAM gene is highly expressed after being stimulated by the tuberculosis specific antigen again.
The invention also provides application of the mycobacterium tuberculosis memory sample NK cell screening marker.
An application of a mycobacterium tuberculosis memory-like NK cell screening marker, wherein the marker is used for screening tuberculosis specific memory-like NK cells.
The invention also provides application of another mycobacterium tuberculosis memory-like NK cell screening marker.
The application of the mycobacterium tuberculosis memory-like NK cell screening marker is used for research and development of anti-tuberculosis related medicines and/or research on curative effect.
The invention also provides application of the third mycobacterium tuberculosis memory-like NK cell screening marker.
The application of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis is disclosed, and the marker is used for a CRTAM detection kit.
The invention also provides an application method of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis.
An application method of a marker for screening memory-like NK (natural killer) cells of mycobacterium tuberculosis, which comprises the following steps: activated tuberculosis-specific memory-like NK cells are screened by detecting the mRNA transcription level and/or the protein expression level of NK cells CRTAM under the condition that peripheral blood PBMCs of a TB patient are stimulated again by Mtb antigen.
The invention also provides a use method of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis.
A method of using a marker for mycobacterium tuberculosis memory-like NK cell screening, the method comprising:
(1) Taking 10ml of peripheral blood, placing the peripheral blood into a blood collection tube containing an EDTA anticoagulant, and separating PBMCs by using TBD (tert-butyl-D) human whole blood mononuclear cell separation liquid;
(2) The resulting PBMCs were plated into 12-well cell culture plates, and 2X 10 cells were added to each well 6 (ii) individual cells;
inoculating and culturing an Mtb standard strain H37Rv 3-4 weeks in advance, collecting Mtb in a logarithmic phase of growth, centrifuging, washing for 2 times by PBS, adding PBS for heavy suspension, centrifuging after ultrasonic lysis, collecting supernatant, and filtering;
the stimulation time was set at 24h, the protein concentration of Mtb lysate was 10. Mu.g/ml, and the IFN-. Gamma. + NK cell number was analyzed by flow-based analysis after the stimulation was completed.
The invention also provides application of the marker for screening the memory-like NK cells of the mycobacterium tuberculosis.
A mycobacterium tuberculosis memory-like NK cell screening marker is used for immunotherapy research of specific targeting CRTAM genes.
The invention utilizes the tuberculosis specific memory-like NK cells in peripheral blood, screens out the activation receptor marker CRTAM molecules with high expression of the tuberculosis specific memory-like NK cells through transcriptome analysis, and provides a new thought and a specific target point for antitubercular treatment and drug research and development.
The invention adopts the flow cytometry technology to sort IFN-gamma-NK cells and IFN-gamma + NK cell groups after being stimulated by Mtb lytic antigen, adopts the single cell transcriptome technology to analyze the expression difference of two groups of cells, and screens out the activation receptor marker CRTAM molecules with high expression of tuberculosis specific memory sample NK cells.
The CRTAM provided by the invention can be used as a marker of memory-like NK cells activated by tuberculosis specificity and a target for screening and researching antitubercular drugs, and has wide application prospects in the aspects of prevention and treatment of tuberculosis.
Example 1
Peripheral blood nodule specific memory sample NK cell detection
1. Sample collection
(1) Population inclusion criteria:
grouping the crowds: healthy people (HC), latent infected people (LTBI), tuberculosis patients (TB)
HC group: no clinical symptoms of TB, negative INF-gamma release test (IGRA), no other serious medical history;
LTBI group: no clinical symptoms of TB; positive IGRA, negative bacteriological examination, no abnormality in imaging examination and no history of tuberculosis treatment.
TB group: has clinical symptoms of TB, positive sputum smear/sputum culture, positive pulmonary lesion found by chest X-ray or CT, positive IGRA test, and tuberculosis pathological change of extrapulmonary tissues or pulmonary tissues.
Exclusion criteria: HIV co-infection and co-infection with other disease individuals, as well as anyone who exhibits uncertain IGRA outcomes.
(2) Sample processing
According to inclusion and exclusion criteria, 15 samples were included: 5 healthy, 5 latently infected, 5 TB patients, 5 in each of 3 groups of people, volunteers signed informed consent.
10ml of peripheral blood of each subject in three groups of people is respectively taken and placed in a blood collection tube containing EDTA anticoagulant, and separation of mononuclear cells (PBMCs) is carried out by using TBD human whole blood mononuclear cell separation liquid.
Mtb cleavage protein stimulation of PBMCs
The mononuclear cells obtained in the above (2) were plated in 12-well cell culture plates, and 2X 10 cells were added to each well 6 And (4) cells. Inoculating and culturing an Mtb standard strain H37Rv 3-4 weeks in advance, collecting Mtb in a logarithmic phase of growth, centrifuging, washing for 2 times by PBS, adding PBS for resuspension, centrifuging after ultrasonic lysis, collecting supernatant, and filtering. The stimulation time was set at 24h, the protein concentration of Mtb lysate was 10. Mu.g/ml, and the number of IFN-. Gamma. + NK cells was analyzed by flow analysis after the stimulation was completed.
3. Pretreatment of cell flow analysis
(1) Preparation of solution required for flow analysis (IFN-. Gamma.Secretion Assay, no. 130-090-762):
a. buffer solution: phosphate buffered saline (PBS, PH = 7.2) containing 0.5% bovine serum albumin and 2mM EDTA (ethylenediaminetetraacetic acid), i.e. 4 ml of 0.5EDTA stock solution per 1 l of PBS buffer.
b.0.5EDTA stock solution: 56g of sodium hydroxide are dissolved in 900ml of ddH 2 To O, 146.2g of EDTA was added, the pH was adjusted to 7.5, and the solution was made up to 1 liter with ddH 2O.
c. Culture medium: RPMI 1640 medium containing 5% human serum.
(2) The pretreatment process comprises the following steps:
(1) collecting cells stimulated by Mtb lysate protein for 24h in a 1.5ml EP tube, adding 1-2 ml of precooling buffer solution, centrifuging at 300g and 4 ℃ for 10min, and removing supernatant;
(2) add 90. Mu.l of pre-cooled PBS to resuspend the cells.
(3) Add 10. Mu.l IFN-. Gamma.capture reagent, mix and incubate for 5min on ice.
(4) Adding 10ml of the preheated medium at 37 ℃, transferring into a 50ml centrifuge tube, placing at 37 ℃, uniformly mixing in a test tube mixer at a low speed, and incubating for 45min.
(5) After completion, the tube was placed on ice. 10ml of pre-cooled buffer was added, 300g was centrifuged at 4 ℃ for 10min, and the supernatant was removed.
(6) Add 90. Mu.l of pre-cooled buffer to resuspend the cells.
(7) Add 10. Mu.l IFN-. Gamma. -APC detection antibody. Additional 10. Mu.l of dye reagent (20. Mu.l of CD3-FITC, 20. Mu.l of CD16-PE, 20. Mu.l of CD56-PE, 5. Mu.l of CD19-PE-Cy7, 5. Mu.l of CD14-PE-Cy 7) were added.
(8) Finally, 2ml of pre-cooled PBS was added, 300g was centrifuged at 4 ℃ for 5min, and the supernatant was removed. Add 300. Mu.l PBS and test on the machine.
4. Analysis of results
Stimulating Peripheral Blood Mononuclear Cells (PBMCs) of HC, LTBI and TB by using a virulent strain Mtb lysate, analyzing and releasing NK cells of IFN-gamma by using flow cytometry after acting for 24 hours, wherein the result is shown in figure 1, and the experimental result shows that under the condition of stimulating the virulent strain Mtb lysate, more peripheral blood IFN-gamma + NK cells are generated in HC, LTBI and TB groups compared with HC, LTBI and TB; compared with LTBI, TB patients generate more peripheral blood IFN-gamma + NK cells, and the quantity of the IFN-gamma + NK cells of the peripheral blood PBMCs of HC populations is not obviously changed under the stimulation of virulent strain Mtb lysate. As the LTBI and TB population were exposed to BCG or Mtb, there was a primary stimulus, which when restimulated, suggested a memory-like response. The results are shown in FIG. 2, which illustrates that both LTBI and TB present tuberculosis specific memory-like NK cells, and TB produced more memory-like NK cells upon renewed exposure to Mtb stimulation.
Example 2
Single cell sequencing gene enrichment results
Peripheral blood of 4 TB patients was collected, IFN-. Gamma. -NK cells and IFN-. Gamma. + NK cells were collected by flow sorting before and after stimulation with lysate of virulent strain Mtb according to the procedure of example 1, and differences in gene expression between these two groups of cells were analyzed by single cell transcriptome, and the results are shown in FIG. 3.
Single cell sequencing results showed that there were 669 differentially expressed genes shared by IFN-. Gamma. -NK cells and IFN-. Gamma. + NK cells among 4 patients. Further channel enrichment analysis shows that the genes are mainly enriched in immune-related signal channels such as cytokines and cytokine receptors, and the like, which indicates that IFN-gamma + NK cells may release more cytokines to further activate the related immune signal channels, and the results are shown in FIG. 4.
Example 3
Analysis of memory-like NK-related genes
The existing research results show that IL-12, IL-15 and the like are closely related to the formation and maintenance of memory-like NK cells, IFN-gamma-NK cells and IFN-gamma + NK cells are collected and sorted according to the operation steps of example 1, and the expression condition of genes related to the memory-like NK cells in the sequencing result of the transcriptome is further analyzed. The results are shown in figure 5, and the analysis result shows that IL-12RB2, IL-15RA and IL-12RB2 are highly expressed in IFN-gamma + NK cells, which indicates that the peripheral blood IFN-gamma + NK cells have the characteristics of memory-like NK.
Example 4
Screening IFN-gamma + NK cell surface activation receptor, identifying CRTAM activation receptor high expression
In order to further screen for activated receptors on the surface of IFN-gamma + NK cells, the expression of activated receptors on the surface of NK cells in the sequencing result of the transcriptome was further analyzed. The result is shown in figure 6, and the result shows that the differential up-regulation gene is screened for the NK cell surface activation receptor to find that IFN-gamma + NK cell activation receptor CRTAM is highly expressed, and the expression levels of NKG2D, NKG2E, NKp, NKp30, CD244, CD27 and NCR2 gene mRNA have no obvious difference in IFN-gamma-NK cells and IFN-gamma + NK cells, so that the result indicates that the peripheral blood nodule-specific memory-like NK cells highly express the CRTAM receptor.
Example 5
Detection of peripheral blood nodule specific memory CRTAM + IFN-gamma + NK cell
1. Sample collection
(1) Population inclusion criteria:
grouping the crowds: healthy people (HC), latent infected people (LTBI), tuberculosis patients (TB)
Group HC: no clinical symptoms of TB, negative INF-gamma release test (IGRA), no other serious medical history;
TB group: has clinical symptoms of TB, positive sputum smear/sputum culture, positive pulmonary lesion found by chest X-ray or CT, positive IGRA test, and tuberculosis pathological change of extrapulmonary tissues or pulmonary tissues.
Exclusion criteria: HIV co-infection and co-infection with other disease individuals, as well as anyone who exhibits uncertain IGRA outcomes.
(2) Sample processing
According to inclusion and exclusion criteria, 17 samples were included: 8 healthy, 9 TB patients, volunteers signed informed consent.
10ml of peripheral blood of each subject in two groups of people is respectively taken and placed in a blood collection tube containing EDTA anticoagulant, and separation of mononuclear cells (PBMCs) is carried out by using TBD human whole blood mononuclear cell separation liquid.
Mtb cleavage protein stimulation of PBMCs
The mononuclear cells obtained in the above (2) were plated in 12-well cell culture plates, and 2X 10 cells were added to each well 6 And (4) cells. Inoculating and culturing an Mtb standard strain H37Rv 3-4 weeks in advance, collecting Mtb in a logarithmic phase of growth, centrifuging, washing for 2 times by PBS, adding PBS for resuspension, centrifuging after ultrasonic lysis, collecting supernatant, and filtering. The stimulation time is set to 24h, the protein concentration of Mtb lysate is set to 10 mu g/ml, protein transport inhibitors, namely 0.7 mu l of Brefeldin A and 1 mu l of Monensin are added when the stimulation is carried out for 20h, and the number of CRTAM + IFN-gamma + NK cells is analyzed by flow type after the stimulation is finished.
3. Pretreatment of cell flow analysis
(1) Preparation of a solution required for flow analysis:
a. buffer solution: phosphate buffered saline (PBS, PH = 7.2) containing 0.5% bovine serum albumin and 2mM EDTA (ethylenediaminetetraacetic acid), i.e. 4 ml of 0.5EDTA stock solution per 1 l of PBS buffer.
b.0.5EDTA stock solution: 56g of sodium hydroxide was dissolved in 900ml of ddH2O, 146.2g of EDTA was added, the pH was adjusted to 7.5, and the solution was made up to 1 liter with ddH 2O.
c. Culture medium: RPMI 1640 medium containing 5% human serum.
(2) The pretreatment process comprises the following steps:
(1) collecting the cells stimulated by Mtb lysate protein for 24h in a flow analysis tube, centrifuging for 5min at 500g, and discarding the supernatant;
(2) add 2ml buffer to resuspend the cells, 500g, centrifuge for 5min, discard the supernatant.
(3) 1ml of the dead-end dye Zombie NIR was added TM Fixable Viabilitykit working solution is mixed, incubated at room temperature for 10min, added with 2ml of buffer solution to resuspend cells, centrifuged for 5min, and the supernatant is discarded.
(4) Add 5. Mu.l of blocking agent Human TruStain FcX, mix well, incubate 10min at room temperature in the dark.
(5) Adding surface antibody mixture (CD 3-FITC, CD16-BV480, CD56-PE, CD 355-APC), mixing, incubating at room temperature in dark for 25min, adding 2ml buffer solution to resuspend cells, 500g, centrifuging for 5min, and discarding supernatant.
(6) Adding 2ml membrane-breaking lotion, mixing, incubating at room temperature in dark for 10min,500g, centrifuging for 5min, and discarding the supernatant.
(7) Adding intracellular antibody IFN-gamma-BV 421, mixing, and incubating at room temperature in dark for 30min.
(8) Adding 2ml buffer solution to resuspend the cells, 500g, centrifuging for 5min, and discarding the supernatant; the cells were resuspended in 2ml buffer, 500g, centrifuged for 5min and the supernatant discarded.
(9) Adding 300 mul buffer solution, and detecting on a machine.
4. Analysis of results
Peripheral Blood Mononuclear Cells (PBMCs) of HC and TB are stimulated by using a virulent strain Mtb lysate, and the ratio of CRTAM + IFN-gamma + NK cells is analyzed by flow cytometry after 24 hours of action. The results are shown in figure 7, and the experimental results show that TB produces more peripheral blood CRTAM + IFN-gamma + NK cells in two groups of HC and TB under the stimulation of lysate of virulent strain Mtb; and the number of CRTAM + IFN-gamma + NK cells of the peripheral blood PBMCs of HC population is not obviously changed under the stimulation of a lysate of a virulent strain Mtb. Since the TB population was exposed to Mtb, there was an initial stimulus, which when restimulated, suggested a memory-like response. The results are shown in FIG. 8, which illustrates that TB presents tuberculosis specific memory-like CRTAM + IFN-. Gamma. + NK cells, and that TB produces more CRTAM + IFN-. Gamma. + NK cells upon further exposure to Mtb stimulation.
Example 6
Application of CRTAM activated receptor high expression in anti-tuberculosis infection and drug research and development
In the immune process of Mtb infection, NK cells are used as important immune cells to participate in the natural immune process of resisting Mtb infection, and play an essential role in early clearing of Mtb infection. And the specific memory-like NK cells can quickly respond to the re-infection of pathogens so as to carry out early clearing, and the memory-like NK cells have great potential application values for immunotherapy of infectious diseases and enhancement of vaccine protection effects.
According to the invention, tuberculosis specific memory-like NK cells are confirmed to exist in primary NK cells of tuberculosis patients and latent infected persons, proinflammatory cytokine IFN-gamma can be rapidly released after exogenous antigen stimulation, the tuberculosis specific memory-like NK cells in the patients are separated by adopting a flow cell sorting technology, the expression condition of a surface receptor participating in the formation of the tuberculosis specific memory-like NK cells is identified by virtue of a single cell sequencing technology, and the surface receptor CRTAM is confirmed to be an activation marker of the tuberculosis specific memory-like NK cells, so that an important theoretical support is provided for developing a pathogen-oriented immunological treatment method based on the NK cells.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various changes may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features and steps described in the foregoing embodiments may be combined in any suitable manner without contradiction, and various combinations that are possible in the present invention are not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.
Claims (10)
1. A marker for screening Mycobacterium tuberculosis memory-like NK cells, which is characterized in that: the marker is CRTAM receptor.
2. The marker for screening mycobacterium tuberculosis memory-like NK cells as claimed in claim 1, wherein: the CRTAM gene is highly expressed after being stimulated by the tuberculosis specific antigen again.
3. The application of the marker for screening the memory-like NK (natural killer) cells of the mycobacterium tuberculosis is characterized in that: the marker is used for screening the tuberculosis specific memory-like NK cells.
4. The application of the marker for screening the memory-like NK (natural killer) cells of the mycobacterium tuberculosis is characterized in that: the marker is used for research and development of anti-tuberculosis related drugs and/or research on curative effect.
5. The application of the marker for screening the memory-like NK (natural killer) cells of the mycobacterium tuberculosis is characterized in that: the marker is used for a CRTAM detection kit.
6. An application method of a marker for screening memory-like NK (natural killer) cells of mycobacterium tuberculosis is characterized by comprising the following steps: the method is to screen the activated tuberculosis specific memory-like NK cells by detecting the mRNA transcription level and/or the protein expression level of NK cells CRTAM of PBMCs of peripheral blood of a TB patient under the condition of Mtb antigen re-stimulation.
7. A method for using a marker for screening memory-like NK (natural killer) cells of Mycobacterium tuberculosis, which comprises the following steps:
(1) Taking 10ml of peripheral blood, placing the peripheral blood into a blood collection tube containing an EDTA anticoagulant, and separating PBMCs by using TBD (tert-butyl-D) human whole blood mononuclear cell separating medium;
(2) The resulting PBMCs were plated into 12-well cell culture plates, and 2X 10 cells were added to each well 6 (ii) individual cells;
inoculating and culturing an Mtb standard strain H37Rv 3-4 weeks in advance, collecting Mtb in a logarithmic phase of growth, centrifuging, washing for 2 times by PBS, adding PBS for heavy suspension, centrifuging after ultrasonic lysis, collecting supernatant, and filtering;
the stimulation time was set at 24h, the protein concentration of Mtb lysate was 10. Mu.g/ml, and the number of IFN-. Gamma. + NK cells was analyzed by flow analysis after the stimulation was completed.
8. A marker for screening memory NK cells of mycobacterium tuberculosis is used for immunotherapy research of specific targeting CRTAM genes.
9. Application of memory-like NK cells in peripheral blood in preparation of substances for screening organism immunity.
10. Use according to claim 9, wherein the infectious disease is tuberculosis and the substance comprises an isolated reagent of PBMCs in peripheral blood.
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