Kit for predicting atrial fibrillation disease course progress and ablation postoperative recurrence

Abstract

The invention relates to the technical field of biological monitoring, in particular to a kit for predicting atrial fibrillation disease progress and ablation postoperative recurrence. The invention provides application of a biomarker to preparation of a product for predicting atrial fibrillation progress or preparation of a product for predicting atrial fibrillation postoperative recurrence. The biomarker comprises aged CD8 + T cells. The present application discloses, for the first time, senescent CD8 + T cells as biomarkers to predict the progression of the course of disease or the risk of postoperative recurrence in atrial fibrillation patients. The kit for predicting atrial fibrillation progress or postoperative recurrence is developed, by means of the kit, high-risk patients with atrial fibrillation recurrence can be rapidly and accurately predicted before an operation, and guidance is provided for operation selection and early clinical intervention. The recurrence risk can be predicted only by using 1mL of blood sample, and the method is convenient, small in trauma and easy to accept by patients.

Description

Technical Field

[0001] The present application relates to the field of biological monitoring technology, and in particular to a kit for predicting the progression of atrial fibrillation and recurrence after ablation. Background Art

[0002] With the increasing aging of the population and the increasing incidence of other cardiovascular diseases, the incidence of atrial fibrillation (AF) and its related complications has been increasing year by year. Because AF can cause hemodynamic changes and lead to cardiac structural remodeling, the risk of thrombosis, stroke and sudden cardiac death in patients with AF is significantly higher than that of the general population. Compared with non-surgical methods that use drugs to control heart rhythm, reduce AF attacks and prevent thrombosis, ablation surgery based on pulmonary vein isolation can significantly improve the prognosis of patients with AF and improve their quality of life. Although there have been great advances in the improvement of surgical procedures and devices such as cryoablation and radiofrequency ablation, surgical treatment of AF still has a high recurrence rate, which seriously affects the long-term prognosis of patients.

[0003] Atrial fibrillation is one of the cardiovascular diseases most closely related to aging, and aging is also one of the most important risk factors for the occurrence of atrial fibrillation. In addition to the fact that the incidence of atrial fibrillation increases significantly with age, the risk of related adverse events and the progression of atrial fibrillation in patients with atrial fibrillation also increase significantly with age. Studies have shown that the aging of immune cells can promote the aging of other tissues and organs, while transplanting young immune cells can delay individual aging. This suggests the important role of immune aging in individual aging and aging-related diseases. Currently, the immune cells that have been proven to undergo significant changes during aging include CD4 + T cells, CD8 + T cells and monocytes. In addition to common signs of cellular aging, such as impaired proliferation, mitochondrial dysfunction, and telomere shortening, aging T cells are often accompanied by changes in surface CD molecules, such as downregulation of the cell surface co-stimulatory molecule CD28 and upregulation of CD57. Studies have reported the potential relationship between aging immune cells and some cardiovascular diseases: CD28 in peripheral blood is upregulated. - CD4 + Increased numbers of T cells are associated with the risk of death from cardiovascular disease; high CD28 - CD4 + T subpopulations can serve as independent predictors and are associated with worsening of heart failure in patients; senescent CD28 in unstable plaques - CD4 + The number of T cells increased significantly, which was positively correlated with the risk of plaque rupture. However, there are relatively few studies on the relationship between aging immune cells and atrial fibrillation. Whether aging immune cells are related to the recurrence of atrial fibrillation, and whether changes in immune cells can be used to predict the recurrence of atrial fibrillation, there are currently no relevant studies. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, in order to solve the technical problem that the relationship between aging immune cells and recurrence of atrial fibrillation in the prior art is unknown, the purpose of this application is to utilize aging CD8 + T cells as biomarkers to predict the risk of atrial fibrillation progression or recurrence after surgery.

[0005] To achieve the above-mentioned and other related purposes, the present application provides, in a first aspect, the use of a biomarker in the preparation of a product for predicting the progression of atrial fibrillation or a product for predicting the recurrence of atrial fibrillation after surgery, wherein the biomarker comprises aging CD8 + T cells.

[0006] The second aspect of the present application provides a system for predicting the progression or postoperative recurrence of atrial fibrillation, the system comprising a data analysis module, the data analysis module being used to analyze the test value of a sample biomarker, the biomarker comprising aging CD8 + T cells.

[0007] The third aspect of the present application provides a kit for predicting the progression or postoperative recurrence of atrial fibrillation, wherein the kit comprises a detection reagent for the biomarker in the aforementioned use.

[0008] The fourth aspect of the present application provides a method for predicting the progression or postoperative recurrence of atrial fibrillation for non-diagnostic treatment purposes, comprising analyzing the test value of a sample biomarker, wherein the biomarker includes aging CD8 + T cells.

[0009] A fifth aspect of the present application provides a computer-readable storage medium having a computer program stored thereon, which implements the steps of the aforementioned prediction method when executed by a processor.

[0010] A sixth aspect of the present application provides a terminal, comprising a processor and the aforementioned computer-readable storage medium, wherein the processor executes a computer program on the computer-readable storage medium to implement the steps of the aforementioned prediction method.

[0011] Compared with the prior art, the beneficial effects of this application are:

[0012] 1. This application discloses for the first time the aging CD8 + T cells serve as biomarkers to predict the risk of disease progression or recurrence after surgery in patients with atrial fibrillation.

[0013] 2. This application has developed a kit for predicting the progression of atrial fibrillation or postoperative recurrence. This kit can be used to quickly and accurately predict patients at high risk of atrial fibrillation recurrence before surgery, providing guidance for surgical selection and early clinical intervention.

[0014] 3. The test kit of the present application can predict the risk of recurrence using only 1 mL of blood sample, which is convenient and less invasive, and is easy for patients to accept. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Flow chart for streaming data analysis.

[0016] Figure 2 For aging CD8 + All CD8 T cells + The proportion of T cells.

[0017] Figure 3 To compare the recurrence-free survival of patients with atrial fibrillation in different groups.

[0018] Figure 4 Based on senescent CD8 + T cell model for predicting recurrence of atrial fibrillation.

[0019] Figure 5 Evaluation of a prediction model for recurrence of atrial fibrillation.

[0020] Figure 6 CD57 detected by flow cytometry in Example 3 + CD8 + T cell subsets.

[0021] Figure 7 This is the result of external validation of the atrial fibrillation recurrence prediction model in Example 3. DETAILED DESCRIPTION

[0022] In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

[0023] After extensive exploration and research, the inventor of the present application discovered a kit for predicting the progression of atrial fibrillation and recurrence after ablation, and completed the present application on this basis.

[0024] The first aspect of the present application provides the use of biomarkers in preparing a product for predicting the progression of atrial fibrillation or a product for predicting the recurrence of atrial fibrillation after surgery, wherein the biomarkers include aging CD8 + This application discloses for the first time the aging of CD8 + T cells serve as biomarkers to predict the risk of disease progression or recurrence after surgery in patients with atrial fibrillation.

[0025] In the use provided in this application, aging CD8 + T cells and normal CD8 + Compared with T cells, WT cells showed cell senescence characteristics and / or changes in surface CD molecules. Cell senescence characteristics include impaired proliferation ability, mitochondrial dysfunction, and telomere shortening. Changes in surface CD molecules include downregulation of cell surface co-stimulatory molecules CD28 and / or upregulation of CD57.

[0026] In the use provided in the present application, according to the aged CD8 + All CD8 T cells + The risk of postoperative recurrence is assessed by measuring the proportion of T cells in the sample. The sample is selected from blood, and further, the sample is selected from peripheral blood and / or left atrial blood. + All CD8 T cells + When the proportion of T cells is greater than a threshold, it is predicted that atrial fibrillation is more likely to recur after surgery; preferably, the threshold is 50.4%.

[0027] The second aspect of the present application provides a system for predicting the progression or postoperative recurrence of atrial fibrillation, the system comprising a data analysis module, the data analysis module is used to analyze the test value of the sample biomarker, the biomarker includes aging CD8 + T cells.

[0028] In the system provided by the present application, the sample is selected from blood, and further, the sample is selected from peripheral blood and / or left atrial blood. The test value of the biomarker includes the aging CD8 + All CD8 T cells + The data analysis module uses aging CD8 + All CD8 T cells + The proportion of T cells in the senescent cells is used to assess the risk of recurrence of atrial fibrillation after surgery. + All CD8 T cells + When the proportion of T cells is greater than 50.4%, it predicts that atrial fibrillation is more likely to recur after surgery.

[0029] In the system provided by the present application, the data analyzed by the data analysis module also includes: a combination of one or more of age, gender, body mass index, smoking, drinking, diabetes, coronary heart disease, heart failure, stroke or transient ischemic attack, atrial fibrillation type and left atrial diameter.

[0030] In the system provided in the present application, the data analysis module calculates the 10-month recurrence risk of the subject to be tested in the following manner: obtaining the individual score of each individual data item, and converting it to obtain the 10-month recurrence risk of the subject to be tested.

[0031] In the system provided by this application, the data analysis module obtains a calculation formula through Cox regression modeling, and the calculation formula is:

[0032]

[0033] Wherein, h0(t) is the baseline risk function, Age represents the age index, which is the age of the subject to be tested, and its unit is years;

[0034] Male represents the gender index. When the subject to be tested is female, the gender index is 0, and when the subject to be tested is male, the gender index is 1;

[0035] BMI stands for body mass index, BMI = weight (kg) / height (m) 2 ;

[0036] Smoke represents the smoking index; when the subject has no smoking history, Smoke is 0, and when the subject has a smoking history, Smoke is 1;

[0037] Drink represents the drinking index. When the subject has no drinking history, Drink is 0; when the subject has a drinking history, Drink is 1;

[0038] DM represents the diabetes index. When the subject has no history of diabetes, DM is 0, and when the subject has a history of diabetes, DM is 1.

[0039] HBp represents the hypertension index. When the subject has no history of hypertension, HBp is 0, and when the subject has a history of hypertension, HBp is 1.

[0040] CHD represents the coronary heart disease index. When the subject has no history of coronary heart disease, CHD is 0, and when the subject has a history of coronary heart disease, CHD is 1;

[0041] HF represents the heart failure index. When the subject has no history of heart failure, HF is 0; when the subject has a history of heart failure, HF is 1.

[0042] Stroke (TIA) represents the index of stroke and transient ischemic attack. When the subject has no history of stroke or transient ischemic attack, Stroke (TIA) is 0; when the subject has a history of stroke or transient ischemic attack, Stroke (TIA) is 1.

[0043] AF type indicates the atrial fibrillation type index. When the subject has paroxysmal atrial fibrillation, AF type is 1; when the subject has persistent atrial fibrillation, AF type is 2.

[0044] LAD stands for left atrial diameter index, which is the left atrial diameter of the subject to be measured, and its unit is mm;

[0045] Senescent CD8+T>50.4% indicates senescent CD8 + T cell ratio index, when the subject is aging CD8 + All CD8 T cells + When the proportion of T cells is greater than 50.4%, Senescent CD8+T>50.4% is 1. When the senescent CD8 + All CD8 T cells + When the proportion of T cells is ≤50.4%, Senescent CD8+T>50.4% is 0;

[0046] The data analysis module substitutes each individual data into the calculation formula to obtain h(t), which is the recurrence risk of the subject to be tested. Figure 4 As shown, the formula can be used to evaluate the various biological indicators of the subjects to be tested in detail and obtain an accurate recurrence risk. By using the nomogram function of the "rms" R package to visualize the formula and obtain a nomogram, the 10-month recurrence-free rate can be intuitively seen. It can quickly and accurately predict patients at high risk of atrial fibrillation recurrence before surgery, providing guidance for surgical selection and early clinical intervention.

[0047] A third aspect of the present application provides a kit for predicting the progression or postoperative recurrence of atrial fibrillation, the kit comprising a detection reagent for the biomarker used in the aforementioned purpose.

[0048] The kit provided in the present application also includes a lymphocyte separation reagent and / or a flow staining reagent. In a specific embodiment of the present application, the lymphocyte separation reagent includes conventional reagents such as an anticoagulant blood vessel containing ethylenediaminetetraacetic acid (EDTA), a human lymphocyte separation tube, Dulbecco's phosphate buffered saline (DPBS), and RPMI 1640 culture medium. The flow staining reagent includes conventional reagents such as a flow cytometer tube, a flow staining buffer, a cell death dye, and an Fc receptor blocker.

[0049] In the kit provided in the present application, the detection reagent includes an antibody, and further, the antibody includes an anti-CD3 antibody, an anti-CD8 antibody, and an anti-CD57 antibody. Among them, CD3 cells are a common surface marker of T cells, and CD57 cells are a marker for identifying senescent T cells. Anti-CD3 antibodies, anti-CD8 antibodies, and anti-CD57 antibodies are all fluorescent anti-human monoclonal flow cytometry antibodies, and the fluorescent dyes labeled with anti-human monoclonal flow cytometry antibodies include: FITC, APC, Cy5.5 and other conventional dyes.

[0050] The kit provided in the present application also includes an atrial fibrillation recurrence prediction tool card. The tool card includes the individual scoring criteria for each data and its conversion relationship with the 10-month recurrence risk, and the biomarkers include aging CD8 + All CD8 T cells + In some embodiments, the data also includes a combination of one or more of age, gender, body mass index, smoking, drinking, diabetes, coronary heart disease, heart failure, stroke or transient ischemic attack, atrial fibrillation type and left atrial diameter. For the conversion relationship of specific information, see Figure 4 ,according to Figure 4 The single score of each indicator is obtained by the indication, and then the total score is accumulated. According to the corresponding relationship between the total score and the 10-month recurrence-free rate in the tool card, the 10-month recurrence-free rate of the subject to be tested is obtained. The scale marked on the line segment represents the predicted value of the 10-month recurrence-free rate. The higher the value of the 10-month recurrence-free rate, the greater the possibility that the patient will not have a recurrence of atrial fibrillation. In clinical terms, it is more suitable for evaluating the patient's suitability for surgery, so that patients with high risk of atrial fibrillation recurrence can be quickly and accurately predicted before surgery, providing guidance for surgical selection and early clinical intervention.

[0051] In a specific embodiment of the present application, the kit includes the following substances:

[0052]

[0053]

[0054] The fourth aspect of the present application provides a method for predicting the progression or postoperative recurrence of atrial fibrillation for non-diagnostic and therapeutic purposes, comprising analyzing the test value of a sample biomarker, wherein the biomarker includes aging CD8 + T cells. Non-diagnostic therapeutic purposes can be, for example, statistical analysis purposes.

[0055] In the prediction method provided in the present application, the sample is selected from blood, and further, the sample is selected from peripheral blood and / or left atrial blood. The test value of the biomarker includes the aging CD8 + All CD8 T cells + The percentage of T cells in the sample. The test values ​​of biomarkers analyzed include: + All CD8 T cells + The proportion of senescent CD8 T cells in the AF tissue was used to assess the risk of recurrence after AF surgery. + All CD8 T cells + When the proportion of T cells is greater than 50.4%, it predicts that atrial fibrillation is more likely to recur after surgery.

[0056] In the prediction method provided in the present application, the analyzed data also includes: a combination of one or more of age, gender, body mass index, smoking, drinking, diabetes, coronary heart disease, heart failure, stroke or transient ischemia, atrial fibrillation type and left atrial diameter.

[0057] In the prediction method provided by the present application, analyzing the test value of the sample includes calculating the 10-month recurrence risk of the subject to be tested based on the test value of each data. In a specific embodiment of the present application, the 10-month recurrence risk is calculated in the following manner: obtaining the individual score of each individual data, and converting it to obtain the 10-month recurrence risk of the subject to be tested.

[0058] In the prediction method provided in the present application, the test value of the analyzed sample includes a calculation formula obtained by Cox regression modeling, and the calculation formula is:

[0059]

[0060] Where h0(t) is the baseline risk function, Age represents the age index, which is the age of the subject to be tested, and its unit is years;

[0061] Male represents the gender index. When the subject to be tested is female, the gender index is 0, and when the subject to be tested is male, the gender index is 1;

[0062] BMI stands for body mass index, BMI = weight (kg) / height (m) 2 ;

[0063] Smoke represents the smoking index; when the subject has no smoking history, Smoke is 0, and when the subject has a smoking history, Smoke is 1;

[0064] Drink represents the drinking index. When the subject has no drinking history, Drink is 0; when the subject has a drinking history, Drink is 1;

[0065] DM represents the diabetes index. When the subject has no history of diabetes, DM is 0, and when the subject has a history of diabetes, DM is 1.

[0066] HBp represents the hypertension index. When the subject has no history of hypertension, HBp is 0, and when the subject has a history of hypertension, HBp is 1.

[0067] CHD represents the coronary heart disease index. When the subject has no history of coronary heart disease, CHD is 0, and when the subject has a history of coronary heart disease, CHD is 1;

[0068] HF represents the heart failure index. When the subject has no history of heart failure, HF is 0; when the subject has a history of heart failure, HF is 1.

[0069] Stroke (TIA) represents the index of stroke and transient ischemic attack. When the subject has no history of stroke or transient ischemic attack, Stroke (TIA) is 0; when the subject has a history of stroke or transient ischemic attack, Stroke (TIA) is 1.

[0070] AF type indicates the atrial fibrillation type index. When the subject has paroxysmal atrial fibrillation, AF type is 1; when the subject has persistent atrial fibrillation, AF type is 2.

[0071] LAD stands for left atrial diameter index, which is the left atrial diameter of the subject to be measured, and its unit is mm;

[0072] Senescent CD8+T>50.4% indicates senescent CD8 + T cell ratio index, when the subject is aging CD8 + All CD8 T cells + When the proportion of T cells is greater than 50.4%, Senescent CD8+T>50.4% is 1. When the senescent CD8 + All CD8 T cells + When the proportion of T cells is ≤50.4%, Senescent CD8+T>50.4% is 0;

[0073] Analyzing the test value of the sample data includes substituting each individual data into a calculation formula to obtain h(t), that is, the recurrence risk of the subject to be tested.

[0074] In the prediction method provided in this application, aging CD8 + The T cell test value is obtained by taking fresh anticoagulated whole blood from the patient, separating lymphocytes, incubating with anti-CD3 antibody, anti-CD8 antibody and anti-CD57 antibody, and then detecting by flow cytometry.

[0075] In a specific embodiment of the present application, the prediction method specifically includes the following steps:

[0076] 1. Isolation of lymphocytes

[0077] Collect fresh anticoagulated whole blood using an anticoagulant blood collection tube containing EDTA. Use a human lymphocyte separation tube to extract lymphocytes. Add 1 mL of whole blood to each tube and centrifuge (20°C, 800g, 30 minutes). Aspirate the lymphocytes below the plasma layer and wash 1-2 times with RPMI1640 medium (20°C, 350g, 10 minutes). Use DPBS to resuspend the lymphocytes for later use. This application only uses 1 mL of blood sample to predict the risk of recurrence, which is convenient and less invasive, and easy for patients to accept.

[0078] 2. Flow Cytometry Staining

[0079] Resuspend lymphocytes in 1mL DPBS into flow cytometer tubes, add 1μL cell death and viability dye to each tube, incubate at room temperature in the dark for 5 minutes, and centrifuge (20℃, 350g, 5 minutes). Wash once with 1mL pre-cooled flow staining buffer, and then resuspend with 100μL pre-cooled flow staining buffer. Add 1μL Fc receptor blocker to each tube, mix well, and incubate at room temperature in the dark for 10 minutes. Add the flow staining antibodies to be tested to each tube: APC Mouse Anti-Human CD3, PerCP-Cy TM 5.5 5 μL of Mouse Anti-Human CD8 and FITC Mouse anti-human CD57 were added and incubated at room temperature in the dark for 20 minutes. Centrifuged (20°C, 350 g, 5 minutes), then resuspended with 300 μL of flow staining buffer and detected on a flow cytometer.

[0080] 3 Streaming Data Analysis

[0081] Flow cytometry data analysis software was used for data analysis. FSC-A and live / dead cells were used to circle live cells, FSC-A and FSC-H were used to circle single cells, and FSC-A and SSC-A were used to circle lymphocytes. T cells, CD8 + T cells and senescent CD8 + T cells (CD57 + CD8 + T) to obtain the proportion of each subpopulation of cells.

[0082] 4 Predicting recurrence of atrial fibrillation

[0083] Acquisition of senescent CD8 + After the T cell ratio is calculated, the atrial fibrillation recurrence prediction tool card (such as Figure 4 ) predict the recurrence of atrial fibrillation. The patient's age, gender, body mass index, smoking, drinking and other personal information, diabetes, hypertension, coronary heart disease, heart failure, stroke / transient ischemic stroke, atrial fibrillation type, left atrial diameter, and aging CD8 + By mapping information such as the percentage of T cells to the number axis at the top one by one, we can get the scores for each indicator. After adding up all the individual scores, we can get the total score, which can be used to predict the patient's median recurrence-free survival time.

[0084] The prediction method of the present application can quickly and accurately predict patients at high risk of atrial fibrillation recurrence before surgery, providing guidance for surgical selection and early clinical intervention.

[0085] A fifth aspect of the present application provides a computer-readable storage medium having a computer program stored thereon, which implements the steps of the aforementioned prediction method when executed by a processor.

[0086] The embodiments of the present invention may be implemented by hardware, software or a combination of software and hardware. The software portion may be implemented using the logic of the aforementioned prediction method; the software portion may be stored in a memory and executed by an appropriate instruction execution system, such as a microprocessor or dedicated hardware. A person of ordinary skill in the art will appreciate that the above-mentioned apparatus and method may be implemented using computer executable instructions and / or contained in a processor control code, such as a carrier medium such as a disk, CD or DVD-ROM, a memory or a data carrier such as an optical or electronic signal carrier.

[0087] It is understood that the memory can be a volatile memory or a non-volatile memory, and can also include both volatile and non-volatile memories. Among them, the non-volatile memory can be a read-only memory (ROM), a programmable read-only memory (PROM), which is used as an external cache. By way of exemplary but not limiting explanation, many forms of RAM are available, such as static random access memory (SRAM), synchronous static random access memory (SSRAM). The memory described in the embodiment of the present invention is intended to include but is not limited to these and any other suitable categories of memory. The memory of the present invention is used to store various categories of data to support the operation of the controller. Examples of these data include: any executable program for operating on the controller, such as an operating system and an application; the operating system includes various system programs, such as a framework layer, a core library layer, a driver layer, etc., for implementing various basic services and processing hardware-based tasks. The application can include various applications, such as a media player (MediaPlayer), a browser (Browser), etc., for implementing various application services.

[0088] A sixth aspect of the present application provides a terminal, comprising a processor and the aforementioned computer-readable storage medium, wherein the processor executes a computer program on the computer-readable storage medium to implement the steps of the aforementioned prediction method.

[0089] The processor of the present application may be an integrated circuit chip with signal processing capabilities. In the implementation process, each step of the above prediction method can be completed by an integrated logic circuit of hardware in the processor or instructions in the form of software. The above processor may be a general-purpose processor, a digital signal processor (DSP, Digital Signal Processor), or other programmable logic devices, discrete gates or transistor logic devices, discrete hardware components, etc. The processor can implement or execute the various methods, steps and logic block diagrams disclosed in the embodiments of the present invention. The general-purpose processor may be a microprocessor or any conventional processor, etc. In combination with the steps of the accessory optimization method provided in the embodiment of the present invention, it can be directly embodied as a hardware decoding processor to execute, or it can be executed by a combination of hardware and software modules in the decoding processor.

[0090] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0091] Example 1

[0092] method

[0093] 1. Research subjects

[0094] Patients with atrial fibrillation who underwent radiofrequency ablation in the Department of Cardiology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from March 2021 to August 2021 were included. Inclusion criteria: (1) patients with a clear clinical diagnosis of atrial fibrillation; (2) ≥18 years old; (3) signed written informed consent. Exclusion criteria: (1) severe heart failure (left ventricular ejection fraction ≤35%); (2) left atrial diameter ≥55mm as shown by cardiac ultrasound; (3) pregnant women; (4) with a family history of arrhythmia; (5) with acute infection or high-sensitivity C-reactive protein ≥10mg / L; (6) with allergic or autoimmune diseases; (7) with tumors or receiving radiotherapy / chemotherapy; (8) taking or using growth hormone, dehydroepiandrosterone, spermidine, NAD + Enhancer or lithium preparation. The diagnostic criteria for atrial fibrillation refer to the latest clinical guidelines. All patients signed informed consent before surgery and stopped taking antiarrhythmic drugs for at least 5 half-lives.

[0095] 2. Electrophysiological examination and ablation

[0096] All patients received standard anticoagulation therapy. After transesophageal echocardiography showed no atrial thrombus, each patient underwent electrophysiological studies in the awake state. After transesophageal echocardiography showed no atrial thrombus, electrophysiological studies were performed in the awake state. TM RMT ThermoCool TM; BiosenseWebster Inc.) 3D mapping system (CARTO TM ,Biosense Webster or EnSite TM , St. Jude Medical) and a robotic magnetic navigation system (Stereotaxis Inc, St. Louis, MO) were used for three-dimensional mapping and ablation. Additional fractionated potential ablation or linear ablation was performed when necessary.

[0097] 3. Follow-up

[0098] All enrolled patients were scheduled for regular outpatient follow-up. The main method for diagnosing AF recurrence was the 24-hour dynamic electrocardiogram results. In addition, if patients felt symptoms that might indicate AF recurrence, they were advised to go to the nearest hospital for an electrocardiogram in a timely manner. AF recurrence was defined as: (1) recurrence of AF lasting (>30 seconds); (2) atrial flutter or atrial tachycardia; (3) use of class I or class III antiarrhythmic drugs within 3 months after surgery; (4) re-ablation. A recurrence during follow-up after the blank period (3 months after surgery) was defined as AF recurrence.

[0099] 4. Flow Cytometry

[0100] 4.1 Isolation of lymphocytes

[0101] Collect fresh anticoagulated whole blood from the patient's periphery and left atrium. Avoid freezing and long-term refrigeration during collection. Add 1 mL of whole blood to each tube and centrifuge (20°C, 800g, 30 minutes, set the acceleration and deceleration to 3 gears, centrifuge EppendorfCentrifuge 5810R). Pipette the lymphocytes below the plasma layer and wash them 1-2 times with RPMI 1640 medium (20°C, 350g, 10 minutes). Resuspend the lymphocytes with DPBS for later use.

[0102] 4.2 Flow staining

[0103] Resuspend lymphocytes in flow cytometry tubes with 1 mL of DPBS, add 1 μL of cell death dye (DAPI staining solution) to each tube, incubate at room temperature in the dark for 5 minutes, and centrifuge (20°C, 350g, 5 minutes). Wash once with 1 mL of pre-cooled flow staining buffer (PBS solution containing 3% fetal bovine serum), and then resuspend with 100 μL of pre-cooled flow staining buffer. Add 1 μL of Fc receptor blocker to each tube, mix well, and incubate at room temperature in the dark for 10 minutes. Add the flow staining antibodies to be tested to each tube: APC Mouse Anti-Human CD3, PerCP-Cy TM5.5 5 μL of Mouse Anti-Human CD8 and FITC Mouse anti-human CD57 were added and incubated at room temperature in the dark for 20 minutes. Centrifuged (20°C, 350 g, 5 minutes), and then resuspended with 300 μL of flow staining buffer and placed in LSRFortessa TM The cells were detected using X-20 flow cytometer (BD Biosciences).

[0104] 4.3 Streaming Data Analysis

[0105] Data analysis was performed using FlowJo (BD Biosciences). Live cells were circled using FSC-A and live / dead cells, single cells were circled using FSC-A and FSC-H, and lymphocytes were circled using FSC-A and SSC-A. T cells, CD8 + T cells and senescent CD8 + T cells (CD57 + CD8 + T), and obtain the percentage of each subpopulation of cells. Figure 1 .

[0106] 5. Statistical Analysis

[0107] Statistical analysis and visualization were performed using R language. Quantitative data were expressed as mean ± standard deviation, and count data were expressed as number and percentage. Continuous variables were analyzed using t-test or rank sum test, and categorical variables were analyzed using χ2 test. 2 Test or Fisher's exact probability method. Kaplan-Meier curve was used to evaluate the recurrence-free survival time of patients, and Log-rank test was used to compare the recurrence-free survival rate of each group. The consistency index (index of concordance, C-index) was used to evaluate the prediction accuracy of the model, and the calibration curve was constructed by Bootstrap method (1000 samplings) to evaluate the calibration of the model. Two-tailed p < 0.05 was considered statistically significant.

[0108] Example 2

[0109] result

[0110] 1. Patient baseline information

[0111] A total of 71 patients with atrial fibrillation were included. The average age was 65.28±9.60 years, of whom 49.3% were male, 69.0% had paroxysmal atrial fibrillation, and 31.0% had persistent atrial fibrillation. Baseline data such as personal history, past medical history, and left atrial diameter are shown in Table 1.

[0112] Table 1 Baseline data of patients with atrial fibrillation

[0113]

[0114] TIA = transient ischemic attack.

[0115] 2. Comparison of immune cell subsets

[0116] Tables 2 and 3 show the differences in peripheral blood and left atrial blood of patients with recurrent and non-recurrent atrial fibrillation after radiofrequency ablation using flow cytometry for each immune cell subset. + The levels of T cells in both peripheral blood and left atrial blood were significantly higher than those in non-relapse patients. Figure 2 Demonstrated the detection of senescent CD8 + Representative results and statistical analysis results of the proportion of T cells in peripheral blood and left atrial blood of patients with recurrent and non-recurrent atrial fibrillation.

[0117] Table 2 Comparison of peripheral blood immune cell subsets in patients with recurrent and non-recurrent atrial fibrillation

[0118]

[0119] Table 3 Comparison of immune cell subsets in left atrial blood in patients with recurrent and non-recurrent atrial fibrillation

[0120]

[0121] 3. Aging CD8 + T cells predict recurrence of atrial fibrillation

[0122] In order to clarify the aging CD8 + Can T cells predict the recurrence of patients with atrial fibrillation after radiofrequency ablation? This application uses the Kaplan-Meier curve to compare the recurrence of patients in different groups. Figure 3 It can be seen that patients with persistent atrial fibrillation are more likely to relapse than patients with paroxysmal atrial fibrillation (p = 0.24); the recurrence rate of patients with left atrial diameter > 44 mm is higher than that of patients with atrial fibrillation with left atrial diameter ≤ 44 mm (p = 0.032), and the recurrence rate of patients with aging CD8 + T>50.4% of patients were compared with aging CD8 + The relapse rate of patients with T≤50.4% was significantly increased (p<0.0001), indicating that senescent CD8 + T cells can better distinguish patients with AF who are prone to recurrence than AF type and left atrial diameter.

[0123] Furthermore, the present application constructs a novel senescent CD8 + The atrial fibrillation recurrence model based on the proportion of T cells and the atrial fibrillation recurrence prediction model were calculated by Cox regression modeling. The calculation formula is:

[0124]

[0125] Where Age represents the age index, which is the age of the subject to be tested, and its unit is years;

[0126] Male represents the gender index. When the subject to be tested is female, the gender index is 0, and when the subject to be tested is male, the gender index is 1;

[0127] BMI stands for body mass index, BMI = weight (kg) / height (m) 2 ;

[0128] Smoke represents the smoking index; when the subject has no smoking history, Smoke is 0, and when the subject has a smoking history, Smoke is 1;

[0129] Drink represents the drinking index. When the subject has no drinking history, Drink is 0; when the subject has a drinking history, Drink is 1;

[0130] DM represents the diabetes index. When the subject has no history of diabetes, DM is 0, and when the subject has a history of diabetes, DM is 1.

[0131] HBp represents the hypertension index. When the subject has no history of hypertension, HBp is 0, and when the subject has a history of hypertension, HBp is 1.

[0132] CHD represents the coronary heart disease index. When the subject has no history of coronary heart disease, CHD is 0, and when the subject has a history of coronary heart disease, CHD is 1;

[0133] HF represents the heart failure index. When the subject has no history of heart failure, HF is 0; when the subject has a history of heart failure, HF is 1.

[0134] Stroke (TIA) represents the index of stroke and transient ischemic attack. When the subject has no history of stroke or transient ischemic attack, Stroke (TIA) is 0; when the subject has a history of stroke or transient ischemic attack, Stroke (TIA) is 1.

[0135] AF type indicates the atrial fibrillation type index. When the subject has paroxysmal atrial fibrillation, AF type is 1; when the subject has persistent atrial fibrillation, AF type is 2.

[0136] LAD stands for left atrial diameter index, which is the left atrial diameter of the subject to be measured, and its unit is mm;

[0137] Senescent CD8+T>50.4% indicates senescent CD8 + T cell ratio index, when the subject is aging CD8+ All CD8 T cells + When the proportion of T cells is greater than 50.4%, Senescent CD8+T>50.4% is 1. When the senescent CD8 + All CD8 T cells + When the proportion of T cells is ≤50.4%, Senescent CD8+T>50.4% is 0;

[0138] The atrial fibrillation recurrence prediction model obtains h(t), i.e., the recurrence risk of the subject to be tested, by substituting each individual data into the calculation formula. The nomogram function of the "rms" R package is used to obtain Figure 4 The visualization tool card shown in the figure combines the patient's age, gender, body mass index, personal information such as smoking and drinking, history of diabetes, hypertension, coronary heart disease, heart failure, stroke / transient ischemic stroke, atrial fibrillation type, left atrial diameter, and aging CD8 + By mapping information such as the percentage of T cells to the number axis at the top one by one, you can get the scores for each indicator. After adding up all the individual scores to get the total score, you can intuitively see the 10-month recurrence-free rate and predict the patient's 10-month recurrence risk.

[0139] Figure 5 This is the result of the evaluation of the atrial fibrillation recurrence prediction model. The C-index score is 0.877, which shows that the model has good accuracy. The Bootstrap method is used to randomly sample the samples 1000 times, and the calibration curve obtained is very close to the diagonal line. It can be seen that the model has good calibration.

[0140] Example 3

[0141] The method is the same as in Example 2

[0142] result

[0143] 1. Patient baseline information

[0144] A total of 77 patients with atrial fibrillation who underwent radiofrequency ablation in the Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from September 2021 to February 2022 were included as an external validation cohort. The average age was 63.64±10.92 years, 57.1% were male, 54.5% had paroxysmal atrial fibrillation, and 45.5% had persistent atrial fibrillation. Baseline data are shown in Table 4.

[0145] Table 4 Baseline data of patients with atrial fibrillation

[0146]

[0147] TIA = transient ischemic attack.

[0148] 2. Comparison of immune cell subsets

[0149] Figure 6 CD57 detected by flow cytometry + CD8 + Comparison of T cell subsets in peripheral blood between patients with recurrent and non-recurrent atrial fibrillation after radiofrequency ablation. + T cells were significantly higher than those in non-relapsed patients (p<0.0001).

[0150] 3. Aging CD8 + T cells predict recurrence of atrial fibrillation

[0151] Figure 7 The results show that the constructed atrial fibrillation recurrence prediction model can accurately predict the recurrence of atrial fibrillation in patients 8 months after radiofrequency ablation in the validation cohort. The area under the ROC curve reaches 0.832, which indicates the stability of the model.

[0152] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims

1. Use of a biomarker in the preparation of a product for predicting the progression of atrial fibrillation or a product for predicting the recurrence of atrial fibrillation after surgery, characterized in that: The biomarkers include senescent CD8 + T cells.

2. The use according to claim 1, characterized in that The surgical method in the product for predicting postoperative recurrence of atrial fibrillation is selected from radiofrequency ablation; and / or, the aged CD8 + T cells and normal CD8 + Compared with T cells, they showed signs of cellular senescence and / or changes in surface CD molecules; Furthermore, the cell aging characteristics include one or more combined features of impaired proliferation capacity, mitochondrial dysfunction, and telomere shortening; the changes in surface CD molecules include downregulation of cell surface co-stimulatory molecules CD28 and / or upregulation of CD57.

3. The use according to claim 1, characterized in that According to the aging CD8 + All CD8 T cells + The proportion of T cells in the AF cells is used to assess the risk of recurrence of AF after surgery. Further, the sample is selected from blood; further, the sample is selected from peripheral blood and / or left atrial blood; and / or, when senescent CD8 + All CD8 T cells + When the proportion of T cells is greater than a threshold, it is predicted that atrial fibrillation is more likely to recur after surgery; preferably, the threshold is 50.4%.

4. A system for predicting progression or postoperative recurrence of atrial fibrillation, characterized in that: The system includes a data analysis module, which is used to analyze the test value of the sample biomarker, wherein the biomarker includes aging CD8 + T cells.

5. The system according to claim 4, characterized in that The surgical method for postoperative recurrence is selected from radiofrequency ablation; And / or, the test value of the biomarker includes aging CD8 + All CD8 T cells + The proportion of T cells; And / or, the sample is selected from a blood sample; further, the sample is selected from peripheral blood and / or left atrial blood; And / or, the data analyzed by the data analysis module also includes: a combination of one or more of age, gender, body mass index, smoking, drinking, diabetes, coronary heart disease, heart failure, stroke or transient ischemic attack, atrial fibrillation type and left atrial diameter.

6. The system according to claim 5, characterized in that The data analysis module uses senescent CD8 + All CD8 T cells + The proportion of senescent CD8 T cells in the AF tissue was used to assess the risk of recurrence after surgery. + All CD8 T cells + When the proportion of T cells is greater than a threshold, it is predicted that atrial fibrillation is more likely to recur after surgery; preferably, the threshold is 50.4%; And / or, the data analysis module calculates the 10-month recurrence risk of the subject to be tested by the following method: obtaining a single score of each single data item, and converting it to obtain the 10-month recurrence risk of the subject to be tested.

7. The system according to claim 6, characterized in that The data analysis module obtains a calculation formula through Cox regression modeling. Preferably, the calculation formula is: Where h0(t) is the baseline risk function, Age represents the age index, which is the age of the subject to be tested, and its unit is years; Male represents the gender index. When the subject to be tested is female, the gender index is 0, and when the subject to be tested is male, the gender index is 1; BMI stands for body mass index, BMI = weight (kg) / height (m) 2 ; Smoke represents the smoking index; when the subject has no smoking history, Smoke is 0, and when the subject has a smoking history, Smoke is 1; Drink represents the drinking index. When the subject has no drinking history, Drink is 0; when the subject has a drinking history, Drink is 1; DM represents the diabetes index. When the subject has no history of diabetes, DM is 0, and when the subject has a history of diabetes, DM is 1. HBp represents the hypertension index. When the subject has no history of hypertension, HBp is 0, and when the subject has a history of hypertension, HBp is 1. CHD represents the coronary heart disease index. When the subject has no history of coronary heart disease, CHD is 0, and when the subject has a history of coronary heart disease, CHD is 1; HF represents the heart failure index. When the subject has no history of heart failure, HF is 0; when the subject has a history of heart failure, HF is 1. Stroke (TIA) represents the index of stroke and transient ischemic attack. When the subject has no history of stroke or transient ischemic attack, Stroke (TIA) is 0; when the subject has a history of stroke or transient ischemic attack, Stroke (TIA) is 1. AF type indicates the atrial fibrillation type index. When the subject has paroxysmal atrial fibrillation, AF type is 1; when the subject has persistent atrial fibrillation, AF type is 2. LAD stands for left atrial diameter index, which is the left atrial diameter of the subject to be measured, and its unit is mm; Senescent CD8+T>50.4% indicates senescent CD8 + T cell ratio index, when the subject is aging CD8 + All CD8 T cells + When the proportion of T cells in the test object is greater than the threshold, Senescent CD8+T>50.4% is 1, and when the senescent CD8 + All CD8 T cells + When the proportion in T cells is ≤ the threshold, Senescent CD8+T>50.4% is 0; preferably, the threshold is 50.4%; The data analysis module substitutes each data into a calculation formula to obtain h(t), ie, the recurrence risk of the subject to be tested.

8. A kit for predicting the progression or postoperative recurrence of atrial fibrillation, comprising a detection reagent for the biomarker for use according to any one of claims 1 to 3.

9. The kit according to claim 8, characterized in that The surgical method for postoperative recurrence is selected from radiofrequency ablation; And / or, the kit further comprises a lymphocyte separation reagent and / or a flow staining reagent; preferably, the lymphocyte separation reagent comprises a combination of one or more of an ethylenediaminetetraacetic acid anticoagulation blood vessel, a human lymphocyte separation tube, Dulbecco's phosphate buffer, and an RPMI1640 culture medium; the flow staining reagent comprises a combination of one or more of a flow cell tube, a flow staining buffer, a cell death and viability dye, and an Fc receptor blocker; And / or, the detection reagent includes an antibody; further, the antibody includes a monoclonal antibody; further, the monoclonal antibody includes an anti-CD3 antibody, an anti-CD8 antibody and an anti-CD57 antibody.

10. The kit according to claim 8, characterized in that The kit also includes an atrial fibrillation recurrence prediction tool card, which includes a single scoring standard for each data and its conversion relationship with the 10-month recurrence risk. The biomarkers include aging CD8 + All CD8 T cells + The data also include a combination of one or more of age, gender, body mass index, smoking, drinking, diabetes, coronary heart disease, heart failure, stroke or transient ischemic attack, atrial fibrillation type and left atrial diameter.

11. A method for predicting progression or postoperative recurrence of atrial fibrillation for non-diagnostic and therapeutic purposes, characterized in that: The test value of the sample biomarker is analyzed, and the biomarker includes senescent CD8 + T cells.

12. The prediction method according to claim 11, characterized in that: The surgical method for postoperative recurrence is selected from radiofrequency ablation; And / or, the test value of the biomarker includes aging CD8 + All CD8 T cells + The proportion of T cells; And / or, the sample is selected from a blood sample; further, the sample is selected from peripheral blood and / or left atrial blood; And / or, the analyzed data further includes: a combination of one or more of age, gender, body mass index, smoking, drinking, diabetes, coronary heart disease, heart failure, stroke or transient ischemic attack, atrial fibrillation type and left atrial diameter.

13. The prediction method according to claim 12, characterized in that: The test values ​​of the biomarkers of the analyzed samples include: + All CD8 T cells + The proportion of senescent CD8 T cells in the AF tissue was used to assess the risk of recurrence after surgery. + All CD8 T cells + When the proportion of T cells is greater than a threshold, it is predicted that atrial fibrillation is more likely to recur after surgery; preferably, the threshold is 50.4%; And / or, the test value of the analyzed sample includes calculating the 10-month recurrence risk of the subject to be tested based on the test value of each data; further, the 10-month recurrence risk is calculated in the following manner: obtaining the single score of each single data, and converting it to obtain the 10-month recurrence risk of the subject to be tested.

14. The prediction method according to claim 13, characterized in that: The test value of the analyzed sample includes a calculation formula obtained by Cox regression modeling, and the calculation formula is: Where h0(t) is the baseline risk function, Age represents the age index, which is the age of the subject to be tested, and its unit is years; Male represents the gender index. When the subject to be tested is female, the gender index is 0, and when the subject to be tested is male, the gender index is 1; BMI stands for body mass index, BMI = weight (kg) / height (m) 2 ; Smoke represents the smoking index; when the subject has no smoking history, Smoke is 0, and when the subject has a smoking history, Smoke is 1; Drink represents the drinking index. When the subject has no drinking history, Drink is 0; when the subject has a drinking history, Drink is 1; DM represents the diabetes index. When the subject has no history of diabetes, DM is 0, and when the subject has a history of diabetes, DM is 1. HBp represents the hypertension index. When the subject has no history of hypertension, HBp is 0, and when the subject has a history of hypertension, HBp is 1. CHD represents the coronary heart disease index. When the subject has no history of coronary heart disease, CHD is 0, and when the subject has a history of coronary heart disease, CHD is 1; HF represents the heart failure index. When the subject has no history of heart failure, HF is 0; when the subject has a history of heart failure, HF is 1. Stroke (TIA) represents the index of stroke and transient ischemic attack. When the subject has no history of stroke or transient ischemic attack, Stroke (TIA) is 0; when the subject has a history of stroke or transient ischemic attack, Stroke (TIA) is 1. AF type indicates the atrial fibrillation type index. When the subject has paroxysmal atrial fibrillation, AF type is 1; when the subject has persistent atrial fibrillation, AF type is 2. LAD stands for left atrial diameter index, which is the left atrial diameter of the subject to be measured, and its unit is mm; Senescent CD8+T>50.4% indicates senescent CD8 + T cell ratio index, when the subject is aging CD8 + All CD8 T cells + When the proportion of T cells in the test object is greater than the threshold, Senescent CD8+T>50.4% is 1, and when the senescent CD8 + All CD8 T cells + When the proportion in T cells is ≤ the threshold, Senescent CD8+T>50.4% is 0; preferably, the threshold is 50.4%; The test value of the analyzed sample includes obtaining h(t), ie, the recurrence risk of the subject to be tested, by substituting each individual data into a calculation formula.

15. The prediction method according to claim 11, characterized in that: The senescent CD8 + The T cell test value is obtained by taking fresh anticoagulated whole blood from the patient, separating lymphocytes, incubating with anti-CD3 antibody, anti-CD8 antibody and anti-CD57 antibody, and then detecting by flow cytometry.

16. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the program is executed by a processor, the steps of the prediction method described in any one of claims 11 to 15 are implemented.

17. A terminal comprising a processor and the computer-readable storage medium according to claim 16, characterized in that: The processor executes the computer program on the computer-readable storage medium to implement the steps of the prediction method according to any one of claims 11 to 15.

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