CN111579779A - Marker for evaluating immune cell function of hepatitis B patient and application thereof - Google Patents
Marker for evaluating immune cell function of hepatitis B patient and application thereof Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/7051—T-cell receptor (TcR)-CD3 complex
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- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70514—CD4
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70517—CD8
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Abstract
The invention relates to a marker for evaluating the immune cell function of a hepatitis B patient and application thereof, belonging to the technical field of cells. The marker includes: CD4+, CD8+, Tfh, and Th cells. The marker can accurately reflect the immune function state of HBV infected patients, and finally, if the analysis shows that the immune function is low, the marker prompts that the immune intervention is possibly needed, such as the improvement of the immune function of an organism by a medicament, functional food or biological treatment method, thereby achieving the purpose of improving the treatment efficiency of the HBV infected patients.
Description
Technical Field
The invention relates to the technical field of cells, in particular to a marker for evaluating the function of immune cells of a hepatitis B patient and application thereof.
Background
Hepatocellular carcinoma (HCC) is the most common liver malignancy, accounting for up to about 90%, and is a serious health threat to humans. Hepatitis B Virus (HBV) infection and non-alcoholic fatty liver disease (NAFLD) are high risk factors for HCC. In China, HBV is one of the main factors causing HCC.
At present, researches at home and abroad prove that HBV infection is closely related to virus, liver microenvironment and host antiviral immune response. The immune mechanism comprises natural immunity and acquired immunity, and is extremely important in the process. The acquired immune system can be divided into cellular immunity and humoral immunity which are mediated by T lymphocytes and B lymphocytes respectively, the T lymphocytes are mainly divided into CD4+ T cells and CD8+ T cells, and the CD4+ T cells can generate cytokines under antigen stimulation to play an important immunoregulation role. Effector CD8+ T cells can clear viruses by killing virus-infected hepatocytes, or a non-lethal mechanism that releases cytokines.
In summary, the HBV infection process is closely related to the function of the immune system of the patient. However, based on the complexity of the immune system and the diversity of HBV disease spectrum, the immunological research on HBV infection is not clear, and in clinical practice, HBV infected patients have five main examination items, such as hepatitis B, and then have more common assay indexes, such as hematology, electrolytes, liver function, kidney function, blood coagulation function, etc., detected, and the virus quantitative detection is used to determine whether there is significant virus replication in peripheral blood. How to more systematically and precisely evaluate the immune function of HBV-infected patients in the whole disease process of HBV infection is a key problem to be solved urgently.
The methods reported in the literature to detect immune function include: 1. immune function is analyzed through the diversity of immune cell receptors (TCR), the method belongs to an indirect method, is complex, needs special instruments and equipment, has extremely high requirements on analysts, has high data analysis difficulty and high cost, and is not beneficial to popularization and application; 2. the method has the advantages of high cost, high operation difficulty and high requirement on the technical level of personnel, and is not favorable for clinical popularization and application.
To date, there is no technology or product on the market that can fully analyze and detect the immune cell function of HBV infected patients, and there is no detailed, energy-based, rapid and low-cost immune function detection and evaluation scheme in clinic, that is, there is no immune cell function evaluation method for HBV infected patients, and there are no more reference parameters.
Disclosure of Invention
Therefore, there is a need to provide an immune cell function analysis system and an application thereof, which are used for analyzing a liver disease patient and can provide reliable basis for adjusting a later treatment scheme of a HBV infected patient in clinic.
A marker for assessing immune cell function in a hepatitis b patient, the marker comprising: CD4+, CD8+, Tfh, and Th cells.
Since HBV infection is closely related to T cell dysfunction and the like, the invention mainly aims at the function detection of T cells. The invention comparatively analyzes the peripheral immune function of a healthy human sample (a healthy control group) and a liver disease sample (a patient group, which can be subdivided into an HBV + HCC group, an HCC + HBV group and an HBV + HCC group), mainly analyzes various functional indexes of immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells and the like in peripheral blood, and finds that 9 immune function indexes have high correlation with the disease. For example, CD4+ T cells were significantly elevated in HBV-infected patients, whereas CD8+ T cells were significantly reduced, both of which are highly correlated with the developmental changes in immune function during the course of disease in HBV-infected patients.
In contrast, previous studies have shown that hepatitis B virus clearance is dependent on T cell activity, and one of the causes of poor T cell responses, is due to the inhibition of CD4/CD8T cells by the large number of regulatory T cells (Tregs) that are generated by inhibiting Notch signaling activation. The result of the targeted index change matches the result of the analysis, and thus the marker can be used as an indicator of the immune function of HBV-infected patients.
Based on the design and accuracy of the index combination, the inventor screens the 9 immune function indexes, finally selects the 4 markers as detection indexes, and can be used for the adjuvant therapy and prognosis monitoring of HBV infected patients.
In one embodiment, the T cells of CD4+ are immune cells of CD3+ and CD4+, the T cells of CD8+ are immune cells of CD3+ and CD8+, the Tfh cells are immune cells of CD3+, CD4+ and CD185+, and the Th cells are immune cells of CD3+, CD4+ and CD 185-.
In one embodiment, the marker is used for evaluating the immune cell function of a hepatitis B patient, the ratio of the T cells of the CD4+ in the T cells is 40-67%, the ratio of the T cells of the CD8+ in the T cells is 31-58%, the ratio of the Tfh cells in the T cells of the CD4+ is 23-41.2%, and the ratio of the Th cells in the T cells of the CD4+ is … …
The invention also discloses the application of the marker in diagnosing, treating and/or prognosticating hepatitis B.
The invention also discloses application of the reagent for detecting the marker in the biological sample in preparing an immune cell function evaluation reagent for hepatitis B patients.
The invention also discloses a kit for evaluating the immune cell function of a hepatitis B patient, which comprises a reagent for detecting the marker in a biological sample.
In one embodiment, anti-human CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody and anti-human CD185 antibody are included.
In one embodiment, the kit is used for flow cytometry, and the anti-human CD3 antibody, the anti-human CD4 antibody, the anti-human CD8 antibody and the anti-human CD185 antibody are all fluorescent anti-human monoclonal flow antibodies, and the fluorescent dye labeled by the anti-human monoclonal flow antibodies comprises: FITC, VioGreen, PE/Cy7, PerCP/Cy5.5.
In one embodiment, the biological sample is human peripheral blood.
The invention also discloses a hepatitis B patient immune cell function evaluation method for scientific research, which comprises the following steps:
and (3) taking peripheral blood, extracting and separating to obtain peripheral blood mononuclear cells, incubating with an anti-human CD3 antibody, an anti-human CD4 antibody, an anti-human CD8 antibody and an anti-human CD185 antibody, detecting by using a flow cytometer to obtain the content of the marker, and evaluating the function of the immune cells.
Compared with the prior art, the invention has the following beneficial effects:
the marker for evaluating the immune cell function of the hepatitis B patient can accurately reflect the immune function state of the HBV infected patient, and if the immune function is analyzed and judged to be low, the marker indicates that immune intervention is possibly needed, for example, the immune function of an organism is improved by a medicament, functional food or biological treatment method, so that the aim of improving the treatment efficiency of the HBV infected patient is fulfilled.
The marker for evaluating the immune cell function of the hepatitis B patient is suitable for all HBV infected patients, so that whether the current immune function state of the HBV infected patient is suitable for a certain clinical treatment scheme or not is accurately evaluated in terms of the immune cell function state, and a certain reliable basis is provided for selecting a more suitable treatment scheme.
The marker is used for evaluating the immune cell function of a hepatitis B patient, has the advantages of good specificity, strong specificity and high accuracy, and the determination accuracy of the immune cell subset can reach more than 90%.
Therefore, the marker and the method provided by the invention can simply, accurately and efficiently evaluate the state of the immune cell function of the HBV infected patient. Has important clinical significance for improving the treatment effective rate (namely auxiliary treatment) of HBV infected patients and making a later treatment scheme.
The kit for evaluating the immune cell function of the hepatitis B patient detects 4 immune function indexes (biomar) by a flow cytometer, and has the advantages of obvious advantages, simple operation, relatively low requirement on personnel and low detection cost, and is favorable for popularization and application although special instruments and equipment are also needed, for example, the detected data directly reflects the functions of peripheral immune cells.
Drawings
FIG. 1 is a schematic diagram showing the comparison of B cells analysis among different groups of Lymphocytes;
FIG. 2 is a schematic diagram showing the comparison of CD3+ T cells analysis among different groups of None B Lymphocytes;
FIG. 3 is a schematic diagram showing the comparison of CD4+ T cells analysis among different groups of CD3+ T cells;
FIG. 4 is a schematic diagram showing the comparison of CD8+ T cells analysis among different groups of CD3+ T cells;
FIG. 5 is a schematic diagram showing the comparison of DNT cells in different groups of CD3+ T cells;
FIG. 6 is a diagram showing comparison of analysis of NK cells in different groups of None B Lymphocytes;
FIG. 7 is a graph showing the comparison of γ T cells analysis among different groups of Lymphocytes;
FIG. 8 is a comparison of Tfh analyses in CD4+ T cells of different groups;
FIG. 9 is a comparison of analysis of Tfh cells for CD183+ CD196-cells of different groups;
FIG. 10 is a diagram showing the comparison of analysis of CD183-CD196-cells in Tfh cells of different groups;
FIG. 11 is a diagram showing a comparison of Th analysis in different groups of CD4+ T cells;
FIG. 12 is a schematic diagram showing the comparison of analysis of CD183+ CD196-cells in different groups of Th cells;
FIG. 13 is a schematic diagram showing the comparison of analysis of CD183-CD196-cells in different groups of Th cells;
FIG. 14 is a comparison of CD185+ cells analysis among different groups of CD4+ T cells;
FIG. 15 is a comparison of CD185-cells analysis in different groups of CD4+ T cells;
FIG. 16 is a comparison of CD183-cells analysis among different groups of CD4-CD 185-;
FIG. 17 is a graph showing a comparison of Memory T cells analysis among different groups of CD3+ T cells;
FIG. 18 is a schematic diagram showing a comparison of analysis of Naive T cells in different groups of CD3+ T cells;
FIG. 19 is a graph showing a comparison of the analysis of Activated T cells in different groups of CD3+ T cells;
FIG. 20 is a schematic comparison of Tfh cells in high and low copy HBV virus groups;
FIG. 21 is a schematic diagram showing the comparison of the analysis of Th cells in the high copy group and the low copy group of HBV virus;
FIG. 22 is a schematic diagram showing the comparison of the analysis of CD4+ T cells in the high copy group and the low copy group of HBV virus;
FIG. 23 is a schematic diagram showing the comparison of the analysis of CD8+ T cells in the high copy group and the low copy group of HBV virus;
FIG. 24 is a schematic view of flow cytometry detection.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present description, "+" indicates positive and "-" indicates negative.
Example 1
Analyzing immune function indexes of healthy people and liver patients.
Firstly, a sample source.
Peripheral blood samples from liver disease patients or healthy volunteers from organ transplantations at the first hospital affiliated to Zhongshan university were used as the study subjects.
The above samples are grouped in the following way:
healthy control group: healthy human sample
Liver disease patient group: HBV + HCC-sample set, HCC + HBV-sample set, HBV + HCC + sample set
The HBV + HCC-sample group refers to hepatitis B virus negative hepatocellular carcinoma patient group, and the HBV + HCC + HBV-sample group refers to hepatitis B virus positive hepatocellular carcinoma patient group
And II, a method.
The peripheral immune function of the patients of different subgroups is analyzed by using conventional reagents and methods, and mainly functional indexes of immune cells such as CD4+ T cells, CD8+ T cells, B cells, NK cells and the like in peripheral blood are analyzed.
1. And (4) screening candidate markers.
The difference in functional index between the healthy control group and the group of patients with liver disease was analyzed first, and then the difference in functional index between the healthy control group and the group of HBV-positive hepatocellular carcinoma and the group of HBV-negative hepatocellular carcinoma was analyzed.
The results are shown in the following tables and FIGS. 1-18.
TABLE 1 analysis of various functional indicators of immune cells
In the figure, P is not more than 0.05, P is not more than 0.01, P is not more than 0.001, and ns is not less than 0.001.
From the results, it can be seen that 9 immune function indexes such as B cells in Lymphocytes, CD4+ T cells in CD3+ Tcells, CD8+ T cells in CD3+ Tcells, Tfh in CD4+ T cells, Th in CD4+ T cells, CD183-CD196-cells in Th cells, CD183-CD 185-cell in CD4-CD185-, Naive T cells in CD3+ T cells, and Activated T cells in CD3+ T cells have high correlation with liver diseases (including HBV infection and hepatocellular carcinoma).
2. And (4) screening markers.
On the basis, the inventor screens indexes based on the design and accuracy of index combination, and if the indexes are different from the indexes of healthy people and patients, the markers shown in the table below are obtained.
TABLE 2 preferred markers
| Sign (immune function index) | Marker molecules | Percentage of | Reference range |
| CD4+ T cells | CD3+CD4+ | ％of |
40～67％ |
| CD8+ T cells | CD3+CD8+ | ％of T cells | 31％～58％ |
| Tfh cell | CD3+CD4+CD185+ | ％of CD4+T cells | 23％～41.2％ |
| Th cells | CD3+CD4+CD185- | ％of CD4+T cells | 22.6％～39.6％ |
Among the above markers, the function of CD4+ T cells plays a key role in adaptive immunity, and signals need to be transmitted from the cells, such as antibody production, phagocytic function of macrophages, and killing effect of killer cells. CD8+ T cells function to specifically kill target cells directly. The main function of Tfh cells is to assist B cells in proliferation and antibody production, and to participate in humoral immunity. It is important for the establishment of the immune system and the improvement of the function, and in particular plays a decisive role in the regulation of humoral immunity against infectious diseases. Th cells function as helper B cell responses.
Namely, CD4+ T cells, CD8+ T cells, Tfh cells and Th cells can be used as markers (biomuraker) for evaluating the immune cell function of hepatitis B patients.
The above reference ranges are confirmed based on the healthy human immune parameters of the healthy control group.
Example 2
Peripheral blood samples from liver disease patients in organ transplantable department of the first hospital affiliated to the university of Zhongshan are classified into a high copy group (high copies) and a low copy group (low copies) according to the copy amount of HBV, and the content of CD4+ T cells, CD8+ T cells, Tfh cells and Th cells in the samples are respectively detected.
The results are shown in fig. 20-23, and it can be seen that the percentage of Tfh cells in CD4T cells is significantly different between the high copy group and the low copy group of HBV virus, i.e. the biomar obtained in example 1 above is correlated with the hepatitis b DNA copy number, and can reflect the immune function status of HBV infected patients.
Example 3
A kit for evaluating the function of immune cells of hepatitis B patients comprises 4 fluorescent monoclonal flow antibodies of anti-human CD3, CD4, CD8 and CD185 (i.e. CXCR 5).
The kit can be used for flow cytometry detection, wherein the fluorescent dye for antibody labeling comprises: FITC, VioGreen, PE/Cy7, PerCP/Cy5.5, and the like.
The antibody reagent is packaged in a standard antibody reagent tube in a dark place. And (4) storing at 4 ℃. Each fluorescent monoclonal antibody is a liquid preparation, does not need to be dissolved, and can be directly used. The number of uses of each antibody reagent may vary from 50 to 500, but the number of uses of each antibody reagent is consistent. The requirement for the antibody reagent tube is a light-tight brown vial, of size 0.5ml to 5 ml.
Example 4
A hepatitis B patient immune cell function assessment method for scientific research application comprises the following steps:
when the kit of example 3 is used, the 4 fluorescent anti-human monoclonal antibodies are taken out under clean environment, and if the antibodies are powder antibodies, a proper amount of Phosphate Buffer Solution (PBS) is added for dissolving according to the requirements of users to obtain the required concentration. If the preparation is a liquid preparation, the preparation can be used directly without dissolution.
Taking 1-5 microliter (mu.l) from each antibody reagent tube, mixing all the antibody reagent tubes with Peripheral Blood Mononuclear Cells (PBMC) extracted from human peripheral blood (2-10ml), and incubating for 10-30 minutes at 4 ℃ in the dark, thus carrying out subsequent detection and analysis.
The prepared cell sample was examined by a flow cytometer to obtain a flow cytogram as shown in fig. 24, and 4 immune function-related indicators (biomarker) shown in example 1 in the human peripheral blood-derived immune cells were analyzed.
The assessment method can meet the requirements on specificity, accuracy, precision (including repeatability, daytime difference, personnel operation error) and the like in the experimental process, and has important significance for improving the treatment success rate of HBV infected patients.
And (3) inspecting whether the contents of the obtained CD4+ T cells, CD8+ T cells, Tfh cells and Th cells meet the reference range in the table 2, if the contents are analyzed and judged to be low in immune function, indicating that immune intervention is possibly needed, such as improving the immune function of an organism by a medicament, functional food or biological treatment method, and further achieving the purpose of improving the treatment efficiency of the HBV infected patient.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A marker for assessing immune cell function in a hepatitis b patient, said marker comprising: CD4+, CD8+, Tfh, and Th cells.
2. The marker according to claim 1, wherein the T cells of CD4+ are immune cells of CD3+ and CD4+, the T cells of CD8+ are immune cells of CD3+ and CD8+, the Tfh cells are immune cells of CD3+, CD4+ and CD185+, and the Th cells are immune cells of CD3+, CD4+ and CD 185-.
3. The marker according to claim 1, wherein the marker has a reference value of 40-67% for the ratio of T cells of CD4+ to T cells, 31-58% for the ratio of T cells of CD8+ to T cells, 23-41.2% for the ratio of Tfh cells to T cells of CD4+ and 22.6-39.6% for the ratio of Th cells to T cells of CD4+ in the evaluation of the function of immune cells of a hepatitis b patient.
4. Use of a marker according to any of claims 1-3 for the diagnostic, therapeutic and/or prognostic assessment of hepatitis b.
5. Use of a reagent for detecting the marker of any one of claims 1 to 3 in a biological sample for the preparation of an agent for evaluating the function of immune cells of a hepatitis b patient.
6. A kit for evaluating immune cell function of a hepatitis B patient, comprising a reagent for detecting the marker of any one of claims 1 to 3 in a biological sample.
7. The kit according to claim 6, comprising an anti-human CD3 antibody, an anti-human CD4 antibody, an anti-human CD8 antibody and an anti-human CD185 antibody.
8. The kit of claim 7, wherein the kit is used for flow cytometry, and the anti-human CD3 antibody, the anti-human CD4 antibody, the anti-human CD8 antibody and the anti-human CD185 antibody are all fluorescent anti-human monoclonal flow antibodies, and the fluorescent dye labeled by the anti-human monoclonal flow antibodies comprises: FITC, VioGreen, PE/Cy7, PerCP/Cy5.5.
9. The kit of claim 5, wherein the biological sample is human peripheral blood.
10. A hepatitis B patient immune cell function assessment method for scientific research is characterized by comprising the following steps:
peripheral blood is taken, extracted and separated to obtain peripheral blood mononuclear cells, and the peripheral blood mononuclear cells are incubated with an anti-human CD3 antibody, an anti-human CD4 antibody, an anti-human CD8 antibody and an anti-human CD185 antibody, and then the content of the marker in any one of claims 1 to 3 is detected by a flow cytometer, so that the function of the immune cells is evaluated.
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