CN115074430A - Immune aging detection system for immune-related disease patients and application thereof - Google Patents
Immune aging detection system for immune-related disease patients and application thereof Download PDFInfo
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Abstract
The invention provides a set of immune aging detection system for immune-related disease patients and application thereof, belonging to the technical field of biomedicine. The immune aging detection system for the immune-related disease patient comprises a detection module and an analysis module; the detection module comprises a first detection module, a second detection module and a third detection module; the first detection module is used for detecting the content of the immunosenescence cell marker in the sample; the second detection module is used for detecting the content of the immunosenescence inflammatory factor in the sample; the third detection module is used for detecting the content of the bone immune decline marker in the sample; the analysis module is used for receiving and analyzing the correlation between the data obtained by the detection module. The invention monitors the aging signals of immune-related disease patients more comprehensively and finely from three layers of cell markers, inflammatory factors and bone immune decline markers, and plays an important role in research and development of prevention, diagnosis and treatment drugs for the pathogenesis of immune-related diseases.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an immune aging detection system for immune-related disease patients and application thereof.
Background
Immune diseases are a large group of chronic diseases caused by immune cell dysfunction and are important public health problems. The immune diseases include inflammatory diseases such as ankylosing spondylitis and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Although the pathogenesis of most immune diseases is unknown, in recent years, the problem of immunosenescence has been long-lived in the study of various immune diseases. The immune system is a defense system for an organism to execute immune response and immune function, consists of 3 parts of immune organs, immune cells, immune molecules and the like, can recognize heterosis and eliminate non-self to maintain stable environment in the organism. The immunosenescence can cause the reduction of the immune function of the body, and the main manifestations are the following 3 aspects: one is impaired immune response and lack of reactivity to foreign pathogens; secondly, the immune protection function is weakened, and cancer cells in the body cannot be eliminated; third, it affects the established immunological memory response. Therefore, immunosenescence is likely to lead to infection and tumorigenesis. Recent studies have found that aging immunity may also lead to chronic inflammation, increasing the risk of immune disease development and the risk of osteoporosis. In addition, epidemiological studies have found that patients with immune diseases are often accompanied by infection and tumorigenesis, and besides being associated with immunosuppressive therapy, there may be some correlation with the body's immunosenescence.
From a mechanistic perspective, immunosenescence involves a change in multi-cellular, multi-molecular, multi-pathway function. The most obvious manifestations of immunosenescence are a reduction in the number and function of immune cells, such as a reduction in T cell production accompanied by a loss of TCR diversity, an increase in memory B cells, an increase in NK cell number but a reduction in killing capacity. Senescent cells secrete a number of soluble factors that promote the formation of the inflammatory microenvironment, known as senescent secretory phenotype factors, including IL1 β, IL6, TNFa, IFN γ, and the like. Furthermore, senescent immune cells are dysregulated in response to unfolded proteins, resulting in a loss of intracellular protein homeostasis. In addition, aging immune cells may also be characterized by disturbances in cell cycle and cell metabolism, DNA and telomere damage, and the like. From the bone immunology perspective, the aging immune cells produce a series of cytokines, inhibit the osteogenesis process, promote the osteoclastic process, easily cause bone loss, and present the bone aging phenotype. Epidemiological statistics indicate that osteoporosis has an increased incidence among immune disease populations. In addition, aging immune cells may cause chondrocyte aging, promote local articular cartilage degradation, aggravate osteoarthritis, and the like.
At present, there are 3 international immunosenescence evaluation indexes, which are T cell dynamic balance, T cell receptor polymorphism and thymus activity, and the specific implementation method comprises the following steps: flow cytometry is used for detecting the proportion of T cells and NK cells of peripheral blood, and the immune repertoire technology is used for evaluating TCR diversity and thymus T cell proportion. These methods are either rough, fail to accurately reflect the aging state, or are difficult to operate and analyze, expensive, and inconvenient to popularize.
Disclosure of Invention
In view of the above problems, the present invention provides a set of immune aging detection system for immune-related disease patients and the application thereof, which combines bone immunology from the perspective of immune aging to monitor aging signals of immune-related disease patients more comprehensively and finely from three directions of cells, inflammatory factors and bone immune decline markers.
In order to achieve the purpose, the invention adopts the technical scheme that: a set of immune aging detection system for immune-related disease patients comprises a detection module and an analysis module; the detection module comprises a first detection module, a second detection module and a third detection module; the first detection module is used for detecting the content of the immunosenescence cell marker in the sample; the second detection module is used for detecting the content of the immunosenescence inflammatory factor in the sample; the third detection module is used for detecting the content of the bone immune decline marker in the sample; the analysis module is used for receiving and analyzing the correlation between the data obtained by the detection module.
Research shows that the most obvious expression of immunosenescence is the reduction of the number and the function of immune cells, and the aged immune cells are disordered along with the reaction of unfolded proteins, so that the steady state of intracellular proteins is lost; the aging immune cells produce a series of cytokines, inhibit the osteogenesis process, promote the osteoclastic process, easily cause bone loss and show a bone aging phenotype. The immune aging detection system for immune-related disease patients detects the content of cell markers, the content of inflammatory factors and the content of bone immune decline markers in immune-related disease patient samples, monitors aging signals of immune-related and inflammatory disease patients more comprehensively and finely from three layers of the cell markers, the inflammatory factors and the bone immune decline markers, and reflects the degree of immune aging of the immune-related disease patients more completely through differential analysis.
As a preferred embodiment of the immunosenescence detection system for patients with immune-related diseases, the immunosenescence cell marker comprises one or more of CD3, CD45, CD4, CD8, CD16, CD56, CDKN1A, TNF, PSMB10, PSMB9, UBE2N, UBE2F, VPS33A and ATF 4. Through a large amount of researches, the inventor screens the immunosenescence cell marker, and detects the immunosenescence cell marker as an antibody panel, so that the condition of cellular immunosenescence of patients with immune-related diseases is reflected more comprehensively.
As a preferred embodiment of the immunosenescence detection system for patients with immune-related diseases, the immunosenescence inflammatory factors include one or more of IL1 β, IL2, IL4, IL5, IL6, IL8, IL10, IL12p70, IL17, IFN γ, IFN α and TNC factors. Immune related diseases cause inflammation of the body, and inflammatory T cells secrete inflammatory factors. The inventor screens the immune aging inflammatory factor as a signal factor of immune aging through a great deal of research, and reflects the inflammatory state of the organism and the immune aging process through detecting the content of the immune aging inflammatory factor.
As a preferred embodiment of the immunosenescence test system for patients with immune-related diseases, the bone immune decline marker comprises one or more of a special sequence of beta-collagen, amino-terminal extension peptide of total type I collagen, N-MID osteocalcin, parathyroxine, 25 hydroxyvitamin D, RAD51, BMP1, BMP6, OPN3, OSM and SPOCK 2. Through a large amount of researches, the inventor adds the bone immune decline marker into an immunosenescence detection system for the first time, and the immunosenescence is comprehensively evaluated by combining with an aging secretion phenotype, and the risk of a patient suffering from immune related diseases and complicating bone metabolism and bone immune imbalance diseases is well predicted.
As a preferred embodiment of the immune aging detection system for patients with immune-related diseases, the method for detecting the sample by the detection module comprises flow cytometry, gene chip, qPCR method, ELISA method, immunomagnetic bead method and electrochemical luminescence method.
In a preferred embodiment of the immunosenescence detection system for patients with immune-related diseases, the samples include, but are not limited to, peripheral blood, synovial fluid, skin, bone marrow, spleen, intestinal tract, lymph node, thymus, bone tissue and other parenchymal organs. The immune aging detection system for the immune-related disease patient can comprehensively analyze and detect the immune aging condition of the immune-related disease patient by any one of the samples.
The invention also provides a detection method of the immunosenescence detection system for the patient with the immune-related disease, which comprises the following steps:
(1) setting an immune-related disease group and a healthy control group, and obtaining samples of the immune-related disease group and the healthy control group;
(2) detecting the contents of the immunosenescence cell markers, the immunosenescence inflammatory factors and the bone immunological decline markers of the two groups of samples by using a detection module to obtain immunosenescence cell marker detection data, immunosenescence inflammatory factor detection data and bone immunological decline marker detection data;
(3) inputting the immunosenescence cell marker detection data, the immunosenescence inflammatory factor detection data and the bone immunological decline marker detection data obtained in the step (2) into an analysis module for analysis; the analysis comprises the steps of carrying out comparative analysis on the detection data of the same type of markers of the immune related disease group and the healthy control group, and carrying out correlation analysis on the detection data of different types of markers of the immune related disease group.
The invention also provides an immunosenescence detection kit for patients with immune-related diseases, which comprises a first detection reagent, a second detection reagent and a third detection reagent; the first detection reagent comprises a reagent for detecting one or more of the immunosenescence cell markers; the second detection reagent comprises a reagent for detecting one or more of the immunosenescence cell markers; the third detection reagent comprises a reagent for detecting one or more of the bone immune decline markers.
The invention also provides an application of the immune aging detection system for the immune related disease patients or the immune aging detection kit for the immune related disease patients in any one of the following items:
(1) preparing a product for predicting the bone metabolism and bone immune imbalance of the patient suffering from the immune-related disease;
(2) study of pathogenesis of immune-related diseases;
(3) prevention, diagnosis or treatment of immune-related diseases;
(4) preparing a product for immunosenescence evaluation.
As a preferred embodiment of the immunosenescence detection system or the application of the immune-related disease patient, the immune-related disease comprises but is not limited to ankylosing spondylitis, psoriasis, systemic lupus erythematosus, rheumatoid arthritis, vasculitis, mixed connective tissue disease, scleroderma, immune premature ovarian failure, recurrent abortion, Behcet's disease, Reiter syndrome, inflammatory bowel disease, polymyositis and dermatomyositis, infection and tumor immune-related disease.
The invention has the beneficial effects that: the invention provides a set of immune aging detection system and a kit for immune-related disease patients, the immune aging detection system for immune-related disease patients detects the content of specific cell markers, the content of inflammatory factors and the content of bone immune decline markers in immune-related disease patient samples, aging signals of immune-related disease patients are monitored more comprehensively and finely from three layers of the cell markers, the inflammatory factors and the bone immune decline markers, the degree of immunosenescence of patients with the immune-related diseases is reflected more completely and integrally through differential analysis, scientific support is provided for the evaluation of immunosenescence of the patients with the immune-related diseases and the risk prediction effect of diseases related to bone immunity decline (osteogenesis attenuation and osteoclast activation), and the method plays an important role in the research and development of medicaments for treating the immune-related diseases; the invention screens out specific immunosenescence cell markers as antibody panel for detection, and more comprehensively reflects the condition of cellular immunosenescence; the invention screens out specific immune aging inflammatory factors and captures aging signals more comprehensively; the invention adopts the bone immune decline marker for the immune aging evaluation for the first time, and has good prediction effect on the risk of the immune-related disease patients with the concurrent bone metabolism and bone immune imbalance diseases; the immune aging detection system for immune-related disease patients has the advantages of simple and efficient detection and analysis process, low cost and convenient popularization.
Drawings
FIG. 1 is a graph of CD4+ T cell trace analysis in peripheral blood mononuclear cells of ankylosing spondylitis patients and healthy persons;
FIG. 2 is an enrichment analysis diagram of differential gene GO of CD4+ T cells in different differentiation states of ankylosing spondylitis patients;
FIG. 3 is a graph of mRNA differential expression violins from peripheral blood inflammatory factors IFNG, TNC and TNF from ankylosing spondylitis patients and healthy persons;
FIG. 4 is a graph showing the correlation between inflammatory factors such as peripheral blood IFNG, TNC and TNF and immunosenescence cell markers;
FIG. 5 is a graph showing the correlation between the ratio of inflammatory factors such as IFNG, TNC and TNF to immune cells in peripheral blood;
FIG. 6 is a diagram of differentially expressed violins from mRNA of peripheral blood bone metabolism genes BMP1, BMP6, OPN3, OSM and SPOCK2 of ankylosing spondylitis patients and healthy persons;
FIG. 7 is a diagram of the correlation analysis of peripheral blood bone metabolism genes BMP1, BMP6, OPN3, OSM and SPOCK2 and immunosenescence cell markers.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1
In this embodiment, the method for researching the immunosenescence mechanism of ankylosing spondylitis by using the immunosenescence detection system for patients with immune-related diseases of the present invention specifically comprises the following steps:
1. the method for screening the ankylosing spondylitis immunosenescence cell marker by single cell sequencing specifically comprises the following steps:
s1: selecting 10 ankylosing spondylitis patients and 10 healthy controls as a disease group and a control group, and respectively drawing 10ml of venous blood into an anticoagulation tube by each person;
s2: adding equal volume of 1xPBS into peripheral blood of S1 for equal ratio dilution, blowing and uniformly mixing by using a Pasteur tube, slowly adding the mixture into a centrifugal tube filled with equal volume of lymphocyte separation liquid, and taking care not to damage a liquid level boundary; and then centrifuging at 20min × 3000rpm by using a fast rising and slow falling method, sucking the middle peripheral blood mononuclear cell layer into another centrifuge tube, washing for 2 times by using a 1xPBS solution, centrifuging for 5min at 300g, discarding supernatant, re-suspending cells by using 1ml of HBSS, filtering cell clusters by using a cell screen, and counting by trypan blue to obtain the peripheral blood mononuclear cells.
S3: taking a proper amount of peripheral blood mononuclear cells, constructing a cDNA library of the mononuclear cells according to the instruction of a 10xGenomics single cell sequencing kit, sequencing by using an Illumina platform, and performing a series of conventional single cell analysis according to pipeline disclosed by 10 xGenomics.
2. The method for detecting the inflammatory factors related to immunosenescence of ankylosing spondylitis specifically comprises the following steps:
s1: selecting 10 ankylosing spondylitis patients and 10 healthy controls as a disease group and a control group, and respectively drawing 10ml of venous blood into an anticoagulation tube by each person;
s2: adding equal volume of 1xPBS into peripheral blood of S1 for equal ratio dilution, blowing and uniformly mixing by using a Pasteur tube, slowly adding the mixture into a centrifugal tube filled with equal volume of lymphocyte separation liquid, and taking care not to damage a liquid level boundary; centrifuging at 20min × 3000rpm by using a fast rising and slow falling method, sucking the middle peripheral blood mononuclear cell layer into another centrifuge tube, washing for 2 times by using 1xPBS solution, centrifuging for 5min at 300g, discarding supernatant, re-suspending cells by using 1ml of HBSS, filtering cell clusters by using a cell screen, and counting by trypan blue to obtain peripheral blood mononuclear cells;
s3: extracting RNA of peripheral blood mononuclear cells by adopting a traditional Trizol method, and quantifying the RNA;
s4: performing RNAseq library construction sequencing by utilizing Illumina 6000;
s5: quality control, splicing, genome comparison, expression matrix construction and differential gene analysis.
S6, comparing the inflammatory pathway related gene characteristics of the MsigDB database, selecting immune aging related inflammatory factors, and carrying out related analysis on the immune aging related inflammatory factors, the immune cell ratio and the found immune aging cell markers.
3. The detection of the ankylosing spondylitis bone immunity decline marker specifically comprises the following steps:
s1: selecting 10 ankylosing spondylitis patients and 10 healthy controls as a disease group and a control group, and respectively drawing 10ml of venous blood into an anticoagulation tube by each person;
s2: adding equal volume of 1xPBS into peripheral blood of S1 for equal ratio dilution, blowing and uniformly mixing by using a Pasteur tube, slowly adding the mixture into a centrifugal tube filled with equal volume of lymphocyte separation liquid, and taking care not to damage a liquid level boundary; centrifuging at 20min × 3000rpm by using a fast rising and slow falling method, sucking the middle PBMC layer into another centrifuge tube, washing with 1xPBS solution for 2 times, centrifuging at 300g for 5min, discarding supernatant, re-suspending cells with 1ml of HBSS, filtering cell clusters with a cell screen, and counting with trypan blue to obtain peripheral blood mononuclear cells;
s3: extracting RNA of peripheral blood mononuclear cells by adopting a traditional Trizol method, and quantifying the RNA;
s4: performing RNAseq library construction sequencing by utilizing Illumina 6000;
s5: quality control, splicing, genome comparison, expression matrix construction and differential gene analysis;
s6, comparing according to the bone metabolism gene characterization reported by PubMed, selecting a bone immune decline marker, and carrying out correlation analysis on the marker and the found immune aging cell marker.
The experimental results are shown in FIGS. 1 to 7.
The detection results of the immunosenescence cell marker of ankylosing spondylitis are shown in FIGS. 1 to 2. As can be seen from fig. 1, the single cell sequencing result shows that CD4+ T cells of the ankylosing spondylitis patients are more differentiated into the cell fate1 of the senescent branch compared with healthy people; differential expression shows that the cells of the group highly express an immunosenescence marker gene CDKN1A and an senescence-associated secretory phenotype gene TNF; low expression protein steady state related genes PSMB10, PSMB9, UBE2N, UBE2F and VPS 33A; all highly express endoplasmic reticulum stress related transcription factor ATF 4. As can be seen from fig. 2, differential pathway enrichment analysis shows that the protein processing signal pathway in endoplasmic reticulum, the MAPK signal pathway related to mitochondrial dysfunction are mainly up-regulated, and the proteasome signal pathway and the Fc γ receptor-mediated phagocytosis signal pathway are down-regulated.
The detection results of inflammatory factors related to immunosenescence of ankylosing spondylitis are shown in fig. 3 to 5. As can be seen from fig. 3, compared with healthy people, serum mRNA levels of inflammatory factors such as TNC and TNF in ankylosing spondylitis patients are significantly up-regulated, while IFNG mRNA levels have an up-regulation tendency but are not significant; as can be seen in fig. 4, TNC and IFNG were found to be associated with the presence of one or more of the above immunosenescence cell markers, respectively, by correlation analysis, while TNF has been identified as a novel senescence marker; as can be seen from fig. 5, correlation analysis revealed that the immunosenescence-related inflammatory factor was correlated with the ratio of several peripheral blood immature cells and effector cells, and thus, the immunosenescence-related inflammatory factor assay could reflect the immunosenescence and senescence secretion phenotype to some extent.
The detection results of the bone immune decline marker of ankylosing spondylitis are shown in fig. 6 to 7. As can be seen from fig. 6, compared with healthy people, the serum mRNA levels of bone immune decline markers BMP6 and OPN3 of ankylosing spondylitis patients are up-regulated, and the serum mRNA levels of BMP1, OPN3 and SPOCK2 are down-regulated; as can be seen from fig. 7, the correlation analysis revealed that the immunosenescence-related bone metabolism markers were correlated with one or more of the immunosenescence cell markers, and thus the bone immunosenescence phenotype was reflected to some extent by the bone immunosenescence marker detection.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A set of immune aging detection system for immune-related disease patients is characterized by comprising a detection module and an analysis module; the detection module comprises a first detection module, a second detection module and a third detection module; the first detection module is used for detecting the content of the immunosenescence cell marker in the sample; the second detection module is used for detecting the content of the immunosenescence inflammatory factor in the sample; the third detection module is used for detecting the content of the bone immune decline marker in the sample; the analysis module is used for receiving and analyzing the correlation between the data obtained by the detection module.
2. The immunosenescence detection system of the immune-related disease patient according to claim 1, wherein the immunosenescence cell marker comprises one or more of CD3, CD45, CD4, CD8, CD16, CD56, CDKN1A, TNF, PSMB10, PSMB9, UBE2N, UBE2F, VPS33A and ATF 4.
3. The immunosenescence test system for a patient with an immune-related disease according to claim 1, wherein the immunosenescence inflammatory factors include one or more of factors IL1 β, IL2, IL4, IL5, IL6, IL8, IL10, IL12p70, IL17, IFN γ, IFN α and TNC.
4. The immunosenescence test system of a patient with an immune-related disease according to claim 1, wherein the bone immune-degeneration marker comprises one or more of a specific sequence of beta-collagen, amino-terminal extension peptide of total type I collagen, N-MID osteocalcin, parathyroxine, 25 hydroxyvitamin D, RAD51, BMP1, BMP6, OPN3, OSM and SPOCK 2.
5. The immunosenescence detection system for patients with immune-related diseases according to claim 1, wherein the detection module is used for detecting samples by methods including flow cytometry, gene chips, qPCR (quantitative polymerase chain reaction), ELISA (enzyme-linked immunosorbent assay), microfluidic technology, immunomagnetic bead method and electrochemiluminescence method.
6. The immunosenescence test system of a patient with an immune-related disease according to claim 1, wherein the sample includes, but is not limited to, peripheral blood, joint fluid, skin, bone marrow, spleen, intestine, lymph node, thymus, bone tissue and other parenchymal organs.
7. The method for detecting the immunosenescence detection system of the patient with the immune-related disease according to any one of claims 1 to 6, which comprises the following steps:
(1) setting an immune-related disease group and a healthy control group, and obtaining samples of the immune-related disease group and the healthy control group;
(2) detecting the contents of the immunosenescence cell markers, the immunosenescence inflammatory factors and the bone immunological decline markers of the two groups of samples by using a detection module to obtain immunosenescence cell marker detection data, immunosenescence inflammatory factor detection data and bone immunological decline marker detection data;
(3) inputting the immunosenescence cell marker detection data, the immunosenescence inflammatory factor detection data and the bone immunological decline marker detection data obtained in the step (2) into an analysis module for analysis; the analysis comprises the steps of carrying out comparative analysis on the detection data of the same type of markers of the immune related disease group and the healthy control group, and carrying out correlation analysis on the detection data of different types of markers of the immune related disease group.
8. An immunosenescence detection kit for a patient with an immune-related disease, which is characterized by comprising a first detection reagent, a second detection reagent and a third detection reagent; the first detection reagent comprises a reagent for detecting one or more of the immunosenescence cell markers of claim 2; the second detection reagent comprises a reagent for detecting one or more of the immunosenescence cell markers of claim 3; the third detection reagent comprises a reagent for detecting one or more of the bone immune decline markers of claim 4.
9. The immunosenescence detection system for patients with immune-related diseases according to any one of claims 1 to 6 or the immunosenescence detection kit for patients with immune-related diseases according to claim 8, wherein the immunosenescence detection system comprises:
(1) preparing a product for predicting the bone metabolism and bone immune imbalance of the patient suffering from the immune-related disease;
(2) study of pathogenesis of immune-related diseases;
(3) prevention, diagnosis or treatment of immune-related diseases;
(4) preparing a product for immunosenescence evaluation.
10. The immunosenescence detection system for patients with immune-related diseases according to any one of claims 1 to 6 or the use according to claim 9, wherein the immune-related diseases include but are not limited to ankylosing spondylitis, psoriasis, systemic lupus erythematosus, rheumatoid arthritis, vasculitis, mixed connective tissue disease, scleroderma, immune premature ovarian failure, recurrent abortion, behcet disease, Reiter syndrome, inflammatory bowel disease, polymyositis and dermatomyositis, infections, tumor immune-related diseases.
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