CN110596399B - Application of substance for detecting content of angiopoietin-like protein8 - Google Patents
Application of substance for detecting content of angiopoietin-like protein8 Download PDFInfo
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- CN110596399B CN110596399B CN201910807777.1A CN201910807777A CN110596399B CN 110596399 B CN110596399 B CN 110596399B CN 201910807777 A CN201910807777 A CN 201910807777A CN 110596399 B CN110596399 B CN 110596399B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The application provides an application of a substance for detecting the content of angiopoietin-like protein 8. The application comprises the application of the substance for detecting the content of the angiopoietin-like protein8 in preparing a product for predicting or assisting in predicting the recurrence of adverse cardiovascular events after percutaneous coronary intervention. The application of the substance for detecting the content of the angiopoietin-like protein8 can apply the substance for detecting the content of the angiopoietin-like protein8 to the prediction of adverse cardiovascular events of patients with coronary heart disease after PCI surgery, has high accuracy of the risk probability of predicting the adverse cardiovascular events of patients with coronary heart disease after PCI surgery, can make early decision-making time of active clinical intervention before the adverse cardiovascular events occur, makes up the blank of medical industry research and clinical application, and has huge potential and value of clinical and market application.
Description
Technical Field
The application relates to the technical field of biomedicine, in particular to application of a substance for detecting the content of angiopoietin-like protein8 and application of angiopoietin-like protein8 as a serum marker in predicting a recurrent adverse cardiovascular event product after percutaneous coronary intervention.
Background
Coronary heart disease (CAD) is a disease caused by myocardial ischemia and hypoxia necrosis due to stenosis or obstruction of arterial lumens caused by Coronary atherosclerosis. The experiment data published in 2013 shows that the death factor of coronary heart disease in China is increased from 7 th in 1990 to the second in 2010, and the number of deaths of coronary heart disease in 2010 reaches 94.5 thousands, thereby seriously threatening the public health of China.
At present, Coronary Angiography (CAG) and Percutaneous Coronary Intervention (PCI) have become important means for diagnosing and treating coronary heart disease. Among them, PCI is a therapeutic method for improving myocardial perfusion by opening a narrow or even an occluded coronary artery lumen through a cardiac catheter technique. Compared with the traditional surgical operation, the PCI has the advantages of small wound, quick recovery, low cost, high safety and the like, and particularly, the appearance and wide use of the drug eluting stent greatly improve the long-term prognosis of a patient.
However, there are still some patients who have Major Adverse Cardiovascular Events (MACE) after receiving PCI treatment, resulting in poor prognosis and increased mortality. Therefore, the control of the persistent risk factors after PCI surgery of patients with coronary heart disease is generally concerned and valued, and simple and effective observation indexes are needed to evaluate the risk of MACE recurrence after PCI surgery in clinical practice so as to take active intervention measures to improve the short-term and long-term curative effects after PCI surgery.
Disclosure of Invention
In view of the above, the invention provides an application of a substance for detecting the content of angiopoietin-like protein8 and an application of angiopoietin-like protein8 as a serum marker in a product for predicting adverse cardiovascular events after percutaneous coronary intervention, so as to solve the technical problems in the prior art.
One aspect of the invention provides an application of a substance for detecting the content of angiopoietin-like protein8 in preparing a product for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention.
Furthermore, the substance for detecting the content of the angiopoietin-like protein8 is angiopoietin-like protein8 binding protein or a kit containing the angiopoietin-like protein8 binding protein.
Further, the kit containing the angiopoietin-like protein8 binding protein is selected from an enzyme linked immunosorbent kit containing the angiopoietin-like protein8 binding protein, a chemiluminescence kit containing the angiopoietin-like protein8 binding protein, a time-resolved fluoroimmunoassay kit containing the angiopoietin-like protein8 binding protein, a flow fluorescence detection kit containing the angiopoietin-like protein8 binding protein, and any combination thereof.
Further, the angiopoietin-like protein8 binding protein is an angiopoietin-like protein8 antibody or an angiopoietin-like protein8 fusion protein.
Further, the antibody of the angiopoietin-like protein8 is an angiopoietin-like protein8 monoclonal antibody, and the fusion protein of the angiopoietin-like protein8 comprises an angiopoietin-like protein8 antibody variable region and a crystallizable fragment.
Further, the substance for detecting the content of the angiopoietin-like protein8 and the substance for detecting the content of serum low-density lipoprotein cholesterol are used in combination to predict or assist in predicting adverse cardiovascular events after percutaneous coronary intervention.
Further, the substance for detecting the content of the angiopoietin-like protein8 is used in combination with the substance for detecting the content of serum low-density lipoprotein cholesterol and the substance for detecting other indexes to predict or assist in predicting adverse cardiovascular events after percutaneous coronary intervention.
Further, the content of the angiopoietin-like protein8 is the content of the angiopoietin-like protein8 in the serum of the subject.
Another aspect of the invention provides the use of angiopoietin-like protein8 as a serum marker in a product for predicting recurrent adverse cardiovascular events following percutaneous coronary intervention.
Further wherein the angiopoietin-like protein8 is combined with low density lipoprotein cholesterol as a serum marker.
The application of the substance for detecting the content of the angiopoietin-like protein8 can apply the substance for detecting the content of the angiopoietin-like protein8 to the prediction of adverse cardiovascular events after PCI (peripheral component interconnect) operation of patients with coronary heart disease, has high accuracy in predicting the risk probability of adverse cardiovascular events after PCI operation of patients with coronary heart disease, can make early decision-making time of clinical active intervention before the adverse cardiovascular events occur, makes up the blank of medical industry research and clinical application, and has great potential and value of clinical and market application.
Drawings
FIG. 1 is a schematic flow chart of an assay method according to an embodiment of the present application;
FIG. 2 is a graphical representation of the distribution of angiopoietin-like protein8 levels in a patient according to an embodiment of the present application;
FIG. 3 is a bar graph showing the relationship between the level of angiopoietin-like protein8 and the presence or absence of adverse cardiovascular events in a patient according to an embodiment of the present application;
FIG. 4 is a schematic K-M curve based on the levels of angiopoietin-like protein8 in a patient according to an embodiment of the present application;
FIG. 5 is a forest plot showing the ratio of major adverse events at the end of the follow-up period as described in an embodiment of the present application;
figure 6 is a forest chart showing the risk ratio of major adverse events at the end of the follow-up period as described in an embodiment of the present application.
Detailed Description
The following description of specific embodiments of the present application refers to the accompanying drawings.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the reagents, materials and procedures used herein are those widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
Angiopoietin-like protein (Angiopoietin-like protein, ANGPTL): is a family of secreted protein factors which are not only related to angiogenesis, but also closely related to lipid metabolism, glucose metabolism, energy metabolism, insulin sensitivity and the like. The ANGPTLs family contains 8 members and is a group of secreted proteins. Except Angiopoietin-like protein 5(Angiopoietin-like protein5, ANGPTL5) which is expressed only in humans, other members of ANGPTL are expressed in both humans and mice.
Angiopoietin-like protein8 (angiopietin-like protein8, ANGPTL 8): is one of the members of the ANGPTLs family. ANGPTL8, also known as GM6484, RIFL, Lipasin, and Betatrophin, consists of 198 amino acids, has a molecular weight of 22kDa, and is located on chromosome 19p13.2 a. Physiologically, ANGPTL8 is secreted by liver-specific expression in humans, while ANGPTL8 is secreted primarily by the liver and adipose tissue in mice.
Angiopoietin-like protein8 may be involved in plasma lipid metabolism. The concentration of circulating angiopoietin-like protein8 is positively correlated with serum Triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) level, and negatively correlated with high density lipoprotein cholesterol (HDL-C) level of diabetic patients. The recombinant protein of the angiopoietin-like protein8 stimulates HepG2 cells and 3T3-L1 cells for 48 hours, regulates lipid homeostasis by inhibiting the expression of adipose tissue triglyceride hydrolase (ATGL), and promotes the accumulation of TG in the cells; angiopoietin-like protein8 knockout mice, with reduced weight gain, reduced plasma TG levels, reduced Very Low Density Lipoprotein (VLDL) secretion but enhanced low density lipoprotein (LPL) activity; in contrast, overexpression of angiopoietin-like protein8 results in increased TG levels in mice and decreased plasma LPL activity; injection of angiopoietin-like protein8 monoclonal antibody into mice revealed increased LPL activity but decreased mouse serum TG levels. Angiopoietin-like protein8 may also be involved in the development of coronary heart disease.
Binding protein: is formed by combining simple protein and other compounds, including chromoprotein, ovalbumin, lipoprotein, metalloprotein, glycoprotein, nucleoprotein, etc.
Fusion protein (fusion protein): is the expression product of two recombined genes obtained by DNA recombination technology or a group of proteins mediating the fusion of two cell plasma membranes.
Antibody: it refers to a protein with protective action produced by the body due to the stimulation of antigen.
Low Density Lipoprotein cholesterol (Low Density Lipoprotein cholesterol, LDL-C): is the major lipoprotein in fasting plasma, approximately 2/3 of plasma lipoproteins, and is the major vehicle for the transport of cholesterol to extrahepatic tissues. LDL-C is converted in plasma from very low density lipoprotein cholesterol (VLDL-C), the site of its synthesis being mainly intravascular and the site of degradation in the liver.
Major adverse cardiovascular events (major adverse cardiovascular events, MACE): short for adverse cardiovascular events, including primary endpoint events and secondary endpoint events. Wherein the primary endpoint events include cardiac death, cardiac arrest, myocardial infarction, stroke, heart failure, acute ischemic heart disease (including stable and unstable angina), Transient Ischemic Attack (TIA), and secondary endpoint events include all-cause death, peripheral vascular disease, atrial fibrillation/flutter, valvular disease, new onset diabetes.
The kit comprises: the box is used for containing chemical reagents for detecting chemical components, drug residues, virus types and the like. The kit comprises but is not limited to a nucleic acid extraction and purification kit, a protein detection kit and other kits, wherein the protein detection kit comprises but is not limited to an enzyme linked immunosorbent assay kit, a co-immunoprecipitation kit, a chemiluminescence kit, an immunohistochemical kit, a radioimmunoassay kit and an immunofluorescence kit.
Body Mass Index (Body Mass Index, BMI): is an important standard which is commonly used internationally to measure the obesity degree and the health of the human body.
Clinical baseline characteristics: including various clinical characteristics and prognostic factors of patients, such as sex, age, body mass index, general data of basic diseases and the like, and etiological diagnosis and functional diagnosis of diseases.
Proportional hazards regression model (proportional hazards model): the Cox model is a semi-parameter regression model, takes survival outcome and survival time as dependent variables, can simultaneously analyze the influence of a plurality of factors on the survival period, can analyze data with ending survival time, and does not require to estimate the survival distribution type of the data. Cox regression analysis is used in this application to determine prognostic predictors of predefined adverse events and to evaluate risk models after correcting covariates.
Receiver Operating Characteristics (ROC): the balance between sensitivity and specificity is reflected, the area under the ROC curve is an important test accuracy index, and the larger the area under the ROC curve is, the higher the diagnostic value of the test is. ROC is used herein to evaluate the discrimination of ANGPTL8 for adverse events.
Skewed distribution (skewness distribution): the peak of the frequency distribution is located at one side, and the tail part extends to the other side. It is divided into a positive bias and a negative bias.
Product-limit method (product-limit estimate): the Kaplan-Meier method, K-M for short, is the most common survival analysis method, and is mainly suitable for estimating the survival rate of patients and drawing a survival curve aiming at the ungrouped survival data. The Kaplan-Meier curve is a continuous stepped curve drawn by taking the survival time r as a horizontal axis and the survival rate S (tk) as a vertical axis, and is used for explaining the relationship between the survival time and the survival rate.
Forest diagrams (Forest plots): drawing a graph by using a numerical operation result based on the statistical index and a statistical analysis method. In a rectangular plane coordinate system, a vertical invalid line (the scale of the abscissa is 1 or 0) is taken as the center, a plurality of line segments parallel to the horizontal axis describe each effect quantity and Confidence Interval (CI) included in the research, and a prism (or other graphs) describes the combined effect quantity and confidence interval of a plurality of researches.
Odds Ratio (OR): the ratio of the number of exposed to the number of unexposed persons in a case group divided by the ratio of the number of exposed to the number of unexposed persons in a control group is also called odds ratio, which is a common index in case-control studies in epidemiological studies.
Relative Risk (RR): also called risk ratio, refers to the cumulative morbidity (or mortality) of the exposure group/the cumulative morbidity (or mortality) of the control group. The relative risk indicates how many times the morbidity or mortality of the exposed group is compared to the control group. Indicating the risk of morbidity or mortality in the exposed group was a multiple of the non-exposed group. The greater the RR value, indicating a greater effect of exposure, the greater the intensity of the association of exposure with outcome.
Confidence intervals (confident intervals, CI): refers to the estimation interval of the overall parameter constructed from the sample statistics. In statistics, the confidence interval for a probability sample is an interval estimate for some overall parameter of the sample. The confidence interval, which represents the extent to which the true value of the parameter has a certain probability of falling around the measurement result, gives the confidence level of the measured value of the measured parameter, i.e. the "one probability" required above.
95% confidence interval (95% CI): meaning that if the confidence interval is calculated by selecting the sample in the same way, then 100 such independent processes have a 95% probability that the calculated interval will contain the true parameter value, i.e. there will be approximately 95 confidence intervals containing true values.
The application provides the following application of the substance for detecting the content of the angiopoietin-like protein 8.
The application of the substance for detecting the content of the angiopoietin-like protein8 in preparing a product for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention is disclosed.
Wherein, the protein sequence of the angiopoietin-like protein8 is shown as a sequence 1 in a sequence table, and the DNA sequence is shown as a sequence 2 in the sequence table.
The substance for detecting the content of the angiopoietin-like protein8 can comprise a reagent and/or a kit and/or an instrument for detecting the content of the angiopoietin-like protein 8. For example, the substance for detecting the content of angiopoietin-like protein8 may include angiopoietin-like protein8, angiopoietin-like protein8 antibody and other reagents and instruments required for enzyme-linked immunosorbent assay (ELISA), and of course, the substance for detecting the content of angiopoietin-like protein8 may consist of angiopoietin-like protein8 and angiopoietin-like protein8 antibody alone, angiopoietin-like protein8 alone, or angiopoietin-like protein8 antibody alone. The angiopoietin-like protein8, the angiopoietin-like protein8 antibody and other reagents required for performing enzyme-linked immunosorbent assay can be packaged independently.
The product for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention can be various reagents or kits for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention.
Furthermore, the substance for detecting the content of the angiopoietin-like protein8 is angiopoietin-like protein8 binding protein or a kit containing the angiopoietin-like protein8 binding protein.
Further, the kit containing the angiopoietin-like protein8 binding protein is selected from an enzyme linked immunosorbent kit containing the angiopoietin-like protein8 binding protein, a chemiluminescence kit containing the angiopoietin-like protein8 binding protein, a time-resolved fluoroimmunoassay kit containing the angiopoietin-like protein8 binding protein, a flow fluorescence detection kit containing the angiopoietin-like protein8 binding protein, and any combination thereof.
Further, the angiopoietin-like protein8 binding protein is an angiopoietin-like protein8 antibody or an angiopoietin-like protein8 fusion protein.
Furthermore, the angiopoietin-like protein8 antibody is an angiopoietin-like protein8 monoclonal antibody, and the angiopoietin-like protein8 fusion protein comprises an angiopoietin-like protein8 antibody variable region and a crystallizable fragment.
Among them, monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone and directed against only a specific epitope. The variable regions are regions of immunoglobulin light and heavy chains that vary widely in amino acid sequence near the N-terminus. The fragment (Fc fragment) which can be crystallized is also called Fc fragment, one of 3 fragments formed by hydrolyzing disulfide bond near N end of IgG hinge region by papain is equivalent to CH of IgG2And CH3The functional region can form crystals, has no antigen binding activity, and has the functions of fixing complement, binding Fc receptor and the like.
Further, the substance for detecting the content of the angiopoietin-like protein8 and the substance for detecting the content of serum low-density lipoprotein cholesterol are used together to predict or assist in predicting adverse cardiovascular events after percutaneous coronary intervention.
The substance for detecting the content of the angiopoietin-like protein8 can comprise a reagent and/or a kit and/or an apparatus for detecting the content of the angiopoietin-like protein8, and the substance for detecting the content of serum low-density lipoprotein cholesterol can comprise a reagent and/or a kit and/or an apparatus for detecting the content of serum low-density lipoprotein cholesterol.
Further, the substance for detecting the content of the angiopoietin-like protein8 is combined with the substance for detecting the content of serum low-density lipoprotein cholesterol and the substance for detecting other indexes to be used for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention.
The substance for detecting the content of the angiopoietin-like protein8 can comprise a reagent and/or a kit and/or an instrument for detecting the content of the angiopoietin-like protein8, the substance for detecting the content of serum low-density lipoprotein cholesterol can comprise a reagent and/or a kit and/or an instrument for detecting the content of serum low-density lipoprotein cholesterol, and the substance for detecting other indexes can be a reagent and/or a kit and/or an instrument for detecting other indexes.
Further, the content of the angiopoietin-like protein8 is the content of the angiopoietin-like protein8 in the serum of the subject.
Specifically, the subject is a coronary heart disease patient who receives the percutaneous coronary intervention for the first time, and the content of the angiopoietin-like protein8 in the serum of the subject can be the concentration of the angiopoietin-like protein8 in the serum of the subject.
Application of angiopoietin-like protein8 as a serum marker in a product for predicting adverse cardiovascular events after percutaneous coronary intervention.
Further, the angiopoietin-like protein8 is combined with serum low density lipoprotein cholesterol as a serum marker.
The following statistical tests were performed using SPSS 21.0(IBM, Chicago, IL, USA) and version R3.4.0 (R Core Team, Vienna, Austria). Where all statistical tests are two-tailed, a P-value less than 0.05 is considered statistically significantly different, i.e., the difference is statistically significant.
First, the correlation between the level of angiopoietin-like protein8 in serum and the occurrence of adverse events after PCI surgery in patients with coronary heart disease
As shown in fig. 1, fig. 1 shows a schematic flow chart of the testing method in the present embodiment.
Selecting the study subjects: 467 patients who are diagnosed in cardiology department of a certain hospital and who are subjected to PCI surgery for the first time are selected.
Screening of the study subjects: the 328 patients with congestive heart failure, past stroke history, valvular disease or arrhythmia, cardiovascular disease, cancer, acute infectious disease, hepatic insufficiency, renal dysfunction, pregnancy, and clinical baseline data insufficiency were excluded from the study and were eligible for the standard.
The following relevant measurements and statistics were performed on 328 patients who met the criteria.
1. Demographic indices and statistics of cardiovascular related risk factors:
a unified form filling guide is formulated.
The medical staff who is trained on the current day of the doctor checks the doctor in a face-to-face mode item by item to ensure the accuracy.
The investigation contents comprise personal living habits, hypertension history, diabetes history, stroke history, myocardial infarction history, dyslipidemia history, family history, smoking history, drinking habits, physical exercise, sleeping time and quality, educational conditions, general conditions of occupation, family income, working environment and the like.
All patients were followed every 3 months. Subsequent data are obtained through medical records, telephone interviews, regular visit of patients to medical staff in outpatient clinics and the like, and follow-up visit lasts for 1 year.
2. Blood sample collection and index measurement:
after the patient takes an empty stomach for 10-12 hours, 3 venous blood tubes are collected, 10ml of each venous blood tube is respectively an anticoagulation tube, an Ethylene Diamine Tetraacetic Acid (EDTA) anticoagulation tube and a heparin anticoagulation tube.
The anticoagulation tube is centrifuged on site to separate serum. And centrifuging an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube and a heparin anticoagulation tube, and subpackaging blood cells and a plasma sample. All blood samples were stored at-80 ℃.
The biochemical indicators were measured using a fully automatic biochemical analyzer (Hitachi-7600; Hitachi, Tokyo, Japan): blood sugar, blood fat, blood uric acid, hypersensitive C-reactive protein and the like, and the used detection reagents are all purchased from Zhongsheng Bei Zhi Biotech GmbH.
3. Statistical analysis:
sequencing according to the measured level of the angiopoietin-like protein8 in the serum of the patients, dividing the patients into three groups according to the number trisection of the patients, and obtaining the clinical baseline characteristics of the patients with coronary heart disease through statistics, as shown in tables 1-1 and 1-2.
TABLE 1-1
Tables 1 to 2
Table 1-1 shows the clinical baseline profile of all 328 patients, and Table 1-2 shows the clinical baseline profile of each of three groups of patients with different levels of angiopoietin-like protein8 in serum. As shown in tables 1-2, after sorting and grouping according to the level of the angiopoietin-like protein8 in the serum of patients, 110 patients are counted in T1 group, the content of the angiopoietin-like protein8 in the serum is less than or equal to 538.381pg/ml, 109 patients are counted in T2 group, the content of the angiopoietin-like protein8 in the serum is 538.381-734.285pg/ml, 109 patients are counted in T3 group, and the content of the angiopoietin-like protein8 in the serum is greater than or equal to 734.285 pg/ml.
At a median follow-up time of 17(14-19) months, a total of 40 patients experienced an adverse cardiovascular event and the remaining 288 patients did not experience an adverse cardiovascular event, and the clinical baseline characteristics of patients who experienced an adverse cardiovascular event and patients who did not experience an adverse cardiovascular event were measured and counted separately, with the results shown in table 2, figure 2, and figure 3.
TABLE 2
Wherein, represents P < 0.05.
In this example, a high sensitivity sandwich immunoassay (ELISA) was used to determine the amount of serum circulating angiopoietin-like protein 8. The high-sensitivity sandwich immunoassay method comprises the steps of coating an angiopoietin-like protein8 antibody in a 96-hole micropore plate to prepare a solid phase carrier, respectively adding a standard substance or a specimen into each micropore, combining the angiopoietin-like protein8 with the antibody connected on the solid phase carrier, then adding a biotinylated angiopoietin-like protein8 antibody, washing the unbound biotinylated antibody, adding horseradish peroxidase (HRP) labeled avidin, thoroughly washing again, and adding a 3,3',5,5' -Tetramethylbenzidine (TMB) substrate for color development. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The shade of the color was positively correlated with angiopoietin-like protein8 in the sample. The concentration of angiopoietin-like protein8 was calculated by measuring the absorbance (o.d. value) at a wavelength of 450nm with a microplate reader.
As shown in fig. 2, fig. 2 is a graph showing the distribution of the angiopoietin-like protein8 levels of patients, wherein the horizontal axis represents the content of angiopoietin-like protein8 in the serum of the patients and the vertical axis represents the frequency with which different angiopoietin-like protein8 levels appear in the patients, i.e. the number of patients with the same level of angiopoietin-like protein 8. The serum level distribution of angiopoietin-like protein8 of 328 patients was statistically biased, with a median of 616.37 pg/ml.
As shown in table 2 and fig. 3, the bar corresponding to "NO" in fig. 3 indicates the content of angiopoietin-like protein8 in the serum of patients without adverse cardiovascular events, the value corresponding to the top of the bar is the average value of the content of angiopoietin-like protein8 in the serum of patients without adverse cardiovascular events, and the value corresponding to the horizontal line above the top of the bar is the standard deviation of the content of angiopoietin-like protein8 in the serum of patients without adverse cardiovascular events. The bar corresponding to "YES" indicates the content of angiopoietin-like protein8 in the serum of a patient suffering from an adverse cardiovascular event, the value corresponding to the top of the bar is the average value of the content of angiopoietin-like protein8 in the serum of a patient suffering from an adverse cardiovascular event, and the value corresponding to the horizontal line above the top of the bar is the standard deviation of the content of angiopoietin-like protein8 in the serum of a patient suffering from an adverse cardiovascular event.
It is clear that the levels of angiopoietin-like protein8 are significantly higher in patients with an adverse cardiovascular event than in patients without an adverse cardiovascular event, as are the levels of total serum cholesterol (TC), low density lipoprotein cholesterol (LDL-C) in patients with an adverse cardiovascular event.
As shown in FIG. 4, the relevance of baseline levels of angiopoietin-like protein8 to adverse events over the follow-up time was assessed visually using the Kaplan-Meier method in this application, and probabilistic estimates were made for risk stratification. Based on baseline ANGPTL8 levels for three groups of patients as shown in tables 1-2, a Kaplan-Meier curve is plotted, with the horizontal axis representing survival time and the vertical axis representing cumulative survival function, T1 representing a K-M curve for patients with serum angiopoietin-like protein8 levels of less than or equal to 538.381pg/ml, T2 representing a K-M curve for patients with serum angiopoietin-like protein8 levels between 538.381pg/ml and 734.285pg/ml, and T3 representing a K-M curve for patients with serum angiopoietin-like protein8 levels greater than 734.285 pg/ml.
As can be seen in FIG. 4, patients with serum levels of angiopoietin-like protein8 greater than 734.285pg/ml had the lowest survival rate, i.e., the highest incidence of adverse cardiovascular events, and patients with serum levels of angiopoietin-like protein8 less than or equal to 538.381pg/ml had the highest survival rate, i.e., the lowest incidence of adverse cardiovascular events.
As shown in fig. 5, fig. 5 shows the ratio of adverse cardiovascular events at the end of the follow-up period, the OR values of adverse cardiovascular events of three groups of patients with different levels of angiopoietin-like protein8 in the serum shown in tables 1-2 were counted, and the analysis results were obtained by adjusting the relevant variables such as age, sex, BMI, smoking history, systolic blood pressure, diastolic blood pressure, triglyceride, total serum cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, fasting blood glucose, glutamic-pyruvic transaminase, and serum creatinine using a multifactorial Logistic regression model, and then counting the OR values of adverse cardiovascular events of three groups of patients with different levels of angiopoietin-like protein8 in the serum shown in tables 1-2 again.
Where 5-95% confidence intervals are represented by the length of the bar and the OR value (odds ratio) is represented by the diamonds. If the OR value is greater than 1, this indicates that the incidence of an adverse cardiovascular event increases with increasing levels of angiopoietin-like protein8, a positive correlation is made between the levels of angiopoietin-like protein8 and the incidence of an adverse cardiovascular event; if the OR value is less than 1, this indicates that the incidence of an adverse cardiovascular event is decreased by an increase in the level of angiopoietin-like protein8, and there is a negative correlation between the level of angiopoietin-like protein8 and the incidence of an adverse cardiovascular event.
In the case of not excluding the influence of the related variables, the OR values of the T1 group are taken as reference, the OR values of the T2 group and the T3 group are respectively 4.830 and 10.071, in the case of excluding the influence of the related variables, the OR values of the T1 group are taken as reference, and the OR values of the T2 group and the T3 group are respectively 5.854 and 11.895, which shows that the occurrence rate of adverse cardiovascular events is increased along with the increase of the level of the angiopoietin-like protein8, and the exclusion OR absence of the related variables does not influence the OR values of each group, so that the angiopoietin-like protein8 is independently related to the occurrence of the adverse cardiovascular events after PCI surgery of the coronary heart disease patient, and the angiopoietin-like protein8 is an independent predictor of the occurrence of the adverse cardiovascular events after PCI surgery of the coronary heart disease patient.
As shown in fig. 6, fig. 6 shows the risk ratio of adverse cardiovascular events at the end of the follow-up period, RR values of adverse cardiovascular events of three groups of patients with different levels of angiopoietin-like protein8 in serum shown in tables 1-2 were counted, and after adjusting relevant variables such as age, sex, BMI, smoking history, systolic pressure, diastolic pressure, triglyceride, serum total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, fasting blood glucose, glutamic-pyruvic transaminase, serum creatinine and the like using a multivariate Cox model, RR values of adverse cardiovascular events of three groups of patients with different levels of angiopoietin-like protein8 in serum shown in tables 1-2 were counted again to obtain analysis results.
Where the 5-95% confidence interval is represented by the length of the crossline and the RR values (hazard ratio) are represented by diamonds. Under the condition that the influence of related variables is not eliminated, the RR values of a T1 group are taken as reference values, the RR values of a T2 group and a T3 group are respectively 4.575 and 8.928, under the condition that the influence of the related variables is eliminated, the RR values of a T1 group are taken as reference values, and the RR values of a T2 group and a T3 group are respectively 4.959 and 10.002, so that whether the related variables are eliminated or not does not influence the RR values of the groups, the angiopoietin-like protein8 is independently related to the occurrence of adverse cardiovascular events of the coronary heart disease patient after PCI surgery, and the angiopoietin-like protein8 is an independent predictor of the occurrence of the adverse cardiovascular events of the coronary heart disease patient after PCI surgery.
II, comparing the correlation between the level of angiopoietin-like protein8 in serum, the level of low-density lipoprotein cholesterol and other related variables and the occurrence of adverse events after PCI surgery of patients with coronary heart disease
Among the risk factors of coronary heart disease, dyslipidemia plays an important pathogenic role, and elevated ldl cholesterol levels are considered to be a central factor in the development and progression of atherosclerosis. The low-density lipoprotein cholesterol can enter the intima of a blood vessel wall through damaged vascular endothelium and is further modified into oxidized low-density lipoprotein cholesterol (ox LDL-C), the oxidized low-density lipoprotein cholesterol is phagocytosed by macrophages to form macrophage-derived foam cells, and the macrophage-derived foam cells and smooth muscle cells migrate from a middle membrane to the intima and swallow the lipid to form muscle-derived foam cells, so that the lipid core of the atheromatous plaque is formed together.
Therefore, the low-density lipoprotein cholesterol is an independent prediction factor for adverse cardiovascular events after percutaneous coronary intervention treatment of coronary heart disease patients, and the incidence rate of adverse cardiovascular events of heart and cerebral vessels can be obviously reduced by reducing the level of serum low-density lipoprotein cholesterol. The substance for detecting the content of the low-density lipoprotein can be applied to the preparation of products for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention. Similarly, the low-density lipoprotein cholesterol can be used as a serum marker in products for predicting recurrent adverse cardiovascular events after percutaneous coronary intervention.
The results of the analysis of low-density lipoprotein cholesterol for predicting the risk of adverse cardiovascular events in patients with coronary heart disease, the analysis of angiopoietin-like protein8 for predicting the risk of adverse cardiovascular events in patients with coronary heart disease, the analysis of low-density lipoprotein cholesterol in combination with angiopoietin-like protein8 for predicting the risk of adverse cardiovascular events, and the analysis of low-density lipoprotein cholesterol, angiopoietin-like protein8 in combination with related variables for predicting the risk of adverse cardiovascular events are shown in table 3, respectively, using a risk assessment model (Cox model).
TABLE 3
Wherein, represents P < 0.05, and represents P < 0.001.
The C-statistic (C-statistic) may reflect how well the model's predicted outcome is consistent with the actual event occurrence. As can be seen from the above table, the C-statistic of the analysis of low density lipoprotein cholesterol for predicting adverse cardiovascular events is 0.653, so that the low density lipoprotein cholesterol is an independent predicting factor for adverse cardiovascular events after percutaneous coronary intervention of coronary heart disease patients, and can predict the risk probability of adverse cardiovascular events after percutaneous coronary intervention of coronary heart disease patients.
The C-statistic of the angiopoietin-like protein8 for the analysis for predicting the risk of the occurrence of the adverse cardiovascular event is 0.738, and is larger than the C-statistic of the low-density lipoprotein cholesterol for the analysis for predicting the risk of the adverse cardiovascular event, so that the angiopoietin-like protein8 can predict the risk probability of the occurrence of the adverse event after the percutaneous coronary intervention of the coronary heart disease patient, and the prediction capability of the angiopoietin-like protein8 for the adverse cardiovascular event is larger than the prediction capability of the low-density lipoprotein cholesterol for the adverse cardiovascular event.
The C-statistic of the combination of the angiopoietin-like protein8 and the low-density lipoprotein cholesterol relative to the analysis for predicting the adverse cardiovascular event is 0.794, which is larger than the C-statistic of the two items, and the combination of the angiopoietin-like protein8 and the low-density lipoprotein cholesterol can remarkably improve the risk prediction capability of the low-density lipoprotein cholesterol for the adverse cardiovascular event after the percutaneous coronary intervention treatment of the coronary heart disease patient.
The combination of angiopoietin-like protein8, low density lipoprotein cholesterol and related variables has the highest C-statistic value of 0.819 for the analysis of predicting adverse cardiovascular events, which shows that the combination of angiopoietin-like protein8, low density lipoprotein cholesterol and related variables can further improve the risk prediction capability of low density lipoprotein cholesterol on adverse cardiovascular events after percutaneous coronary intervention treatment of patients with coronary heart disease.
In conclusion, the substance for detecting the content of the angiopoietin-like protein8 can be applied to the preparation of products for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention treatment so as to predict or assist in predicting the risk probability of adverse cardiovascular events after PCI of patients with coronary heart disease. In other words, angiopoietin-like protein8 can also be used as a serum marker in products for predicting adverse cardiovascular events after percutaneous coronary intervention to predict or assist in predicting the risk probability of adverse cardiovascular events after PCI of patients with coronary heart disease.
Secondly, substances that detect the content of angiopoietin-like protein8 may also increase the prediction rate of low-density lipoprotein cholesterol for predicting the risk probability of recurrent adverse cardiovascular events after percutaneous coronary intervention. In other words, the angiopoietin-like protein8 can be combined with serum low-density lipoprotein cholesterol to be used as a serum marker in products for predicting adverse cardiovascular events after percutaneous coronary intervention treatment, so as to improve the prediction rate of the serum low-density lipoprotein cholesterol on the risk probability of adverse cardiovascular events after PCI operation of coronary heart disease patients.
Furthermore, the substance for detecting the content of the angiopoietin-like protein8 can be combined with the substance for detecting the content of serum low-density lipoprotein cholesterol and the substances for detecting other indexes to improve the prediction rate of adverse cardiovascular events after percutaneous coronary intervention.
The application of the substance for detecting the content of the angiopoietin-like protein8 can apply the substance for detecting the content of the angiopoietin-like protein8 to the prediction of adverse cardiovascular events of patients with coronary heart disease after PCI surgery, and can apply the angiopoietin-like protein8 as a serum marker to the prediction of adverse cardiovascular events of patients with coronary heart disease after PCI surgery.
The substance for detecting the content of the angiopoietin-like protein8 can be used together with a substance for detecting the content of low-density lipoprotein cholesterol and a substance for detecting other indexes to be applied to the prediction of adverse cardiovascular events of patients with coronary heart disease after PCI surgery, and the angiopoietin-like protein8 can also be used together with the low-density lipoprotein cholesterol as a serum marker to be applied to the prediction of the adverse cardiovascular events of patients with coronary heart disease after PCI surgery, so that the sensitivity and the accuracy of predicting the risk probability of the adverse cardiovascular events of patients with coronary heart disease after surgery can be improved, and more 'high-risk' patients can be identified.
In this document, "upper", "lower", "front", "rear", "left", "right", and the like are used only to indicate relative positional relationships between relevant portions, and do not limit absolute positions of the relevant portions.
In this document, "first", "second", and the like are used only for distinguishing one from another, and do not indicate the degree and order of importance, and the premise that each other exists, and the like.
In this context, "equal," "same," and the like are not strictly mathematical and/or geometric limitations, but also encompass errors that may be understood by one skilled in the art and that may be allowed for manufacturing or use, etc.
Unless otherwise indicated, numerical ranges herein include not only the entire range within its two endpoints, but also several sub-ranges subsumed therein.
The preferred embodiments and examples of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the embodiments and examples described above, and various changes can be made within the knowledge of those skilled in the art without departing from the concept of the present application.
Claims (9)
1. The application of the antibody for detecting the content of the angiopoietin-like protein8 in preparing a reagent or a kit for predicting or assisting in predicting adverse cardiovascular events after percutaneous coronary intervention.
2. The use according to claim 1, wherein the antibody that detects the amount of angiopoietin-like protein8 is angiopoietin-like protein8 binding protein.
3. The use according to claim 2, wherein the kit is selected from the group consisting of an enzyme linked immunosorbent kit containing angiopoietin-like protein8 binding protein, a chemiluminescent kit containing angiopoietin-like protein8 binding protein, a time-resolved fluoroimmunoassay kit containing angiopoietin-like protein8 binding protein, a flow-based fluorescent detection kit containing angiopoietin-like protein8 binding protein, and any combination thereof.
4. The use of claim 3 wherein the angiopoietin-like protein8 binding protein is angiopoietin-like protein8 fusion protein.
5. The use according to claim 3 wherein the antibody that detects the amount of angiopoietin-like protein8 is angiopoietin-like protein8 monoclonal antibody.
6. The use of claim 4, wherein the fusion protein of angiopoietin-like protein8 comprises the variable region and crystallizable fragment of angiopoietin-like protein8 antibody.
7. The use according to any one of claims 1 to 6, wherein the antibody for detecting the amount of angiopoietin-like protein8 is used in combination with a substance for detecting the amount of serum low density lipoprotein cholesterol to predict or assist in predicting the recurrence of adverse cardiovascular events following percutaneous coronary intervention.
8. The use according to claim 1 wherein the amount of angiopoietin-like protein8 is the amount of angiopoietin-like protein8 in the serum of the subject.
9. Application of angiopoietin-like protein8 as a serum marker in preparing a product for predicting adverse cardiovascular events after percutaneous coronary intervention.
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