CN109374901B

CN109374901B - Myocardial infarction prognosis risk index detection device and establishment method of myocardial infarction prognosis early warning model

Info

Publication number: CN109374901B
Application number: CN201811161440.XA
Authority: CN
Inventors: 郝盼盼; 刘燕萍; 李贵双
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2018-09-30
Filing date: 2018-09-30
Publication date: 2021-08-27
Anticipated expiration: 2038-09-30
Also published as: CN109374901A

Abstract

The disclosure relates to a myocardial infarction prognosis risk index detection device and a method for establishing a myocardial infarction prognosis early warning model. The method analyzes the correlation between 48 protein factor levels such as Ang- (1-7) and the like in baseline plasma and delta EDV, delta LVEF and delta E '/A' by utilizing two correlation test methods of Pearson and Spearman, and determines a regression equation and independent early warning factors of left ventricular reconstruction and functional indexes such as delta EDV, delta LVEF and delta E '/A' by utilizing a multiple stepwise regression analysis method.

Description

Myocardial infarction prognosis risk index detection device and establishment method of myocardial infarction prognosis early warning model

Technical Field

The invention relates to a myocardial infarction prognosis risk index detection device and a method for establishing a myocardial infarction prognosis early warning model, and belongs to the technical field of biomedicine.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

With the continuous Progress of Coronary Intervention (PCI) technology, the mortality and heart failure incidence rate in the acute phase of myocardial infarction are decreasing year by year, while the incidence and mortality rate in the distant phase of heart failure due to chronic myocardial remodeling are increasing year by year. Myocardial remodeling generally refers to left ventricular remodeling, which varies widely in speed and severity from patient to patient. There are many factors that affect left ventricular remodeling, including the location and severity of myocardial infarction, the time of reperfusion, the patient's age, sex, and complications. The ability to make a scientific prediction of the severity of the long-term left ventricular remodeling directly impacts the assessment of the prognosis of patients with acute myocardial infarction and the development of individualized therapy. Therefore, the early warning technology of acute myocardial infarction left ventricular remodeling becomes a research hotspot in the cardiovascular field at present.

The early stage of heart failure progression is myocardial remodeling, which manifests as changes in heart size, shape, and function, a common response of the heart to injury or inflammation. The mechanism by which myocardial infarction induces myocardial remodeling and dysfunction is not known, but it has been confirmed that there is a common mechanism by which myocardial remodeling due to various causes is overactivated by the renin-angiotensin system (RAS). Myocardial ischemia can activate RAS, and promote Angiotensin Converting Enzyme (ACE) to catalyze the conversion of angiotensin I (angiotensin I, Ang-I) to angiotensin II (angiotensin II, Ang-II). Ang-II acts by binding to angiotensin II type 1receptor (AT 1R), inducing myocardial remodeling and fibrosis, leading to the development and progression of heart failure. ACE inhibitors and AT1R antagonists can prolong the life of heart failure patients, reduce or even reverse the deterioration of certain parameters of myocardial remodeling, and are the cornerstone of treatment for myocardial remodeling. Other mechanisms such as microvascular lesions, vegetative nerve functional disorders, metabolic disorders and interstitial fibrosis may also be involved in the development of heart failure.

Angiotensin (1-7) [ angiotensin- (1-7), Ang- (1-7) ] which is a new RAS member in cardiac muscle is produced by catalytic decomposition of Ang-I and Ang-II by several endopeptidases and carboxypeptidases including angiotensin-converting enzyme 2 (ACE 2). Recently, both the applicant and other researchers have reported that ACE2 and Ang- (1-7) exert cardioprotective effects both in vivo and in vitro. In addition, the applicant also found that plasma Ang- (1-7) levels are closely related to improvement of myocardial injury and recovery of left ventricular function after reperfusion therapy in patients with acute myocardial infarction. However, the relationship between plasma Ang- (1-7) levels and myocardial remodeling and function in patients with acute myocardial infarction is not clear.

Other plasma protein factors are known to the Applicant, such as renin, Ang-II, Bradykinin (BK), aldosterone, cortisol, Cathelicidin (CST), creatine kinase-isozyme (CK-MB), cardiac troponin I (CTNI), N-terminal pro-B-natriuretic peptide (N-terminal pro-brain peptide, NT-proBNP), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), hepatocyte growth factor (hepatocyte growth factor, HGF), endothelin 1(endothelin-1, ET-1), Osteoprotegerin (OPG), Periostin (PN), syndecan-1 (SDC-1), metalloproteinases (MMP-1, metalloproteinases (MMP-1, etc.) MMP-2, MMP-3, MMP-8, MMP-9, matrix metalloproteinase tissue inhibitor-1 (tissue inhibitor of metalloproteinases-1, TIMP-1), TIMP-2, TIMP-4, and Peptidin, Von Willebrand Factor (VWF), tissue plasminogen activator (t-PA), fibrin-2 (FCN 2), mannose-agglutinin (MBL), mannose-agglutinin/fibrin-associated Protein 1 (MBL/fibrin-associated Protein-1, MAP-1), C-reactive Protein (C-reactive Protein, CRP), Interleukin-1 beta (Interleukin-1 beta, IL-1 beta), IL-6, IL-21, tumor necrosis factor alpha (alpha factor alpha), TNF- α), soluble tumor necrosis factor type 1receptor (sTNFR 1) and type 2 receptor (sttnfr 2), macrophage Migration Inhibitory Factor (MIF), CC chemokine ligand 4 (chemikine C-C motif ligand 4, CCL4), CXC chemokine ligand 8(CXC chemokine ligand 8, CXCL8), CXCL10, CXCL16, Myeloperoxidase (MPO), adiponectin, oxidized low density lipoprotein (oxidized LDL, OX-LDL), granzyme B, Tenascin C (Tenascin-C, Tn-C) and type I collagen carboxy-terminal peptide (carbon-terminal peptide of collagen type I, ict p) may also be associated with acute ventricular infarction after myocardial infarction.

However, in the existing acute myocardial infarction left ventricular reconstruction prognosis early warning technology, a plurality of related protein factors are not adopted for research and analysis, and the accuracy and reliability of a prognosis result are influenced.

The essence of the microbead chip is that two mature technologies of flow cytometry and enzyme labeling detection are organically combined, and the reaction of microbeads in a solution is utilized, so that the influence of surface tension, space effect and the like on reaction kinetics when a film chip detects macromolecules is overcome, and the accuracy and the repeatability of sample detection are greatly improved.

In conclusion, a microbead suspension chip which is used for integrally detecting batch protein factors is not available at present, and reports of early warning of left ventricular remodeling after acute myocardial infarction by adopting various related protein factors are not available.

Disclosure of Invention

Aiming at the background technology, the invention aims to establish a microbead chip integrated detection system for quantitatively and multiply detecting the left ventricular remodeling related protein factor and search a rapid, accurate and simple left ventricular remodeling related factor detection method compared with the prior art. Meanwhile, the correlation between the detection result of the bead chip and the left ventricular remodeling severity of the acute myocardial infarction patient is evaluated, and an early warning model is constructed by combining clinical factors. The novel early warning technology established by taking the early warning technology as the foundation stone can assist clinicians to carry out early risk stratification and early individualized treatment on acute myocardial infarction patients, and finally reduces or delays the occurrence of heart failure.

In a first aspect of the present disclosure, a bead chip capable of detecting a protein factor associated with a myocardial infarction prognosis early warning is provided, the bead chip comprising a bead solid substrate, i.e., chloromethylated polystyrene resin, the bead solid substrate being coupled with a protein factor antibody associated with a myocardial infarction prognosis early warning through an amide bond.

In a second aspect of the present disclosure, a bead suspension chip product capable of detecting a protein factor associated with a pre-warning of myocardial infarction is provided, which is characterized in that: each 100 μ L of the extract contains 1 × 10⁶～2×10⁶The bead comprises the bead chip and an activated bead solid matrix which is not coupled, wherein the proportion (number) of the bead chip is 70-80%.

In a third aspect of the present disclosure, a method for preparing the bead suspension chip product is provided, which includes the following steps:

(1) activation of microbeads:

washing with a microbead washing solution, separating, adding a microbead activation buffer solution, oscillating, and performing ultrasonic treatment to completely suspend microbeads;

adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy thiosuccinimide (Sulfo-NHS) into suspended microbeads to react, then adding a PBS buffer solution, vibrating and then performing ultrasonic treatment to obtain a carboxylated microbead solution;

(2) coupling antibody:

adding a protein factor antibody related to the pre-warning of myocardial infarction into the carboxylated microbead solution, and carrying out vibration incubation in a dark place; after incubation, washing, separating, and adding a confining liquid to block the microbead antibody; and washing, and adding a storage solution to resuspend the microbeads to prepare the microbead suspension chip product.

In a fourth aspect of the present disclosure, a kit for detecting a protein factor associated with a pre-warning of myocardial infarction is provided, the kit at least includes the bead suspension chip product, and further includes: antigen (namely protein factors related to the myocardial infarction prognosis early warning) standards with different concentrations, PBS buffer solution, micro-bead activation buffer solution, micro-bead washing solution, confining liquid and the like.

In a fifth aspect of the present disclosure, there is provided a method for detecting a protein factor associated with pre-warning of myocardial infarction, the method comprising the step of performing detection using the bead suspension chip or bead suspension chip product or kit.

In a sixth aspect of the present disclosure, there is provided a myocardial infarction prognosis risk index detection device, comprising at least:

a protein factor detection system capable of detecting at least one of the following three groups of values in plasma: the content values of Ang- (1-7) and TNF-alpha in the sample, the content values of renin activity and CTNI in the sample, and the content values of Ang-II and NT-proBNP in the sample;

a prognostic data processor for receiving values from at least one of the following three groups: the content values of Ang- (1-7) and TNF-alpha in the sample, the content values of renin activity and CTNI in the sample, and the content values of Ang-II and NT-proBNP in the sample; further, at least one of Δ EDV,. DELTA.LVEF,. DELTA.E '/A', wherein. DELTA.edv is 137 to 0.412 × Ang- (1 to 7) +0.334 × TNF-. alpha., Δ LVEF is 85 to 0.358 × renin activity-0.315 × Log-CTNI, and. DELTA.E '/A' is 32 to 0.415 × Ang-II to 0.304 × Log-NT-proBNP, is calculated;

wherein Δ EDV represents the left ventricular end diastolic volume change value; Δ LVEF represents the left ventricular ejection fraction change value; Δ E '/A' represents the variation of the ratio of the peak diastolic early velocity (E ') to the peak diastolic late velocity (A') of the mitral valve annulus.

In a seventh aspect of the present disclosure, there is provided a method for establishing an early warning model for myocardial infarction prognosis, the method comprising:

(1) at least one group of values in at least three groups before treatment of a plurality of myocardial infarction patients is respectively obtained: the content values of Ang- (1-7) and TNF-alpha in the sample, the content values of renin activity and CTNI in the sample, and the content values of Ang-II and NT-proBNP in the sample;

acquiring LVEDV, LVEF and/or E '/A' of a plurality of patients before and after treatment respectively, and calculating delta EDV, delta LVEF and/or delta E '/A' of the patients;

(2) and (3) processing data by adopting SPSS software:

the detection result is expressed by the average value +/-standard error or median [ interquartile distance ], if the detection result obeys normal distribution, the comparison among groups is carried out by adopting variance analysis, and if the difference among groups has statistical significance, the comparison between every two groups is further carried out by adopting a Bonferroni method; if not, performing comparison among groups by using Kruskal-Wallis rank sum test, and if the difference among the groups has statistical significance, further performing multiple comparison by using a Dwass-Steel-Critchlow-Fligner method; differences are considered to be statistically significant when P on both sides is less than 0.05;

(3) selecting a Pearson or Spearman correlation test method according to the result of the normality test to analyze the correlation of the level of each protein factor in the plasma with the delta EDV, the delta LVEF and the delta E '/A'; selecting Pearson correlation analysis if the sample accords with normal distribution; if the samples do not conform to normal distribution, Spearman rank correlation analysis is selected; differences are considered to be statistically significant when P on both sides is less than 0.05;

(4) determining a regression equation of the reconstruction and the functional index of the left ventricle of the delta EDV, the delta LVEF and/or the delta E '/A' by using a multivariate stepwise regression analysis method, determining independent early warning factors of the delta EDV, the delta LVEF and/or the delta E '/A', and establishing an early warning model; differences were considered statistically significant when P was < 0.05 on both sides.

Compared with the related technology known by the inventor, one technical scheme of the present disclosure has the following beneficial effects:

the microsphere suspension chip prepared by the method overcomes the influence of surface tension, space effect and the like on reaction kinetics when a film chip is used for detecting macromolecules due to the reaction of microspheres in a solution, and greatly improves the accuracy and repeatability of sample detection by using a laser detection technology, so that the microsphere suspension chip has the characteristic of high flux of the film chip, and has the characteristics of simplicity and convenience in operation, good repeatability, high sensitivity, wide dynamic range and the like which are superior to the film chip. Compared with ELISA, the micro-bead suspension chip has higher detection sensitivity and has the advantages of high efficiency, rapidness, sensitivity, specificity, low cost and the like. Through the optimization and detection of the microbead material, we find that: chloromethyl polystyrene resin is selected as the microbead of the microbead solid matrix, and the antibody coating efficiency of the microbead solid matrix is 1.64 times that of the traditional polystyrene resin. Therefore, chloromethyl polystyrene resin is selected as the bead material in the application. The method is superior to the prior preparation technology.

The bead chip integrated detection system for synchronously detecting 48 left ventricular remodeling related protein factors such as Ang- (1-7) of blood plasma is developed, the correlation between the levels of the 48 protein factors such as Ang- (1-7) and the like and delta EDV, delta LVEF and delta E '/A' in baseline blood plasma is analyzed by utilizing two correlation detection methods of Pearson and Spearman, and a regression equation and independent early warning factors of left ventricular remodeling and functional indexes such as delta EDV, delta LVEF and delta E '/A' are determined by utilizing a multiple stepwise regression analysis method.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.

FIG. 1: a process flow for preparing the micro-bead suspension chip.

FIG. 2: Δ EDV correlates with Ang- (1-7) levels.

FIG. 3: Δ EDV correlates with TNF- α levels.

FIG. 4: Δ LVEF correlates with renin levels.

FIG. 5: Δ LVEF vs Log-CTNI levels.

FIG. 6: Δ E '/A' is related to Ang-II levels.

FIG. 7: Δ E '/A' correlates with Log-NT-proBNP levels.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

Interpretation of terms:

prognosis refers to predicting the likely course and outcome of a disease.

Left ventricular remodeling refers to the change in overall left ventricular morphology and size following an acute myocardial infarction.

As introduced in the background art, a microbead suspension chip for simultaneously and integrally detecting a batch of protein factors is not available at present, and no report of early warning of left ventricular remodeling after acute myocardial infarction by adopting various related protein factors is available, and in order to solve the above technical problems, in a first typical embodiment of the disclosure, a microbead chip capable of detecting a protein factor related to the early warning of the prognosis of myocardial infarction is provided, wherein the microbead chip comprises a microbead solid substrate, namely chloromethylated polystyrene, and the microbead solid substrate is coupled with a protein factor antibody related to the early warning of the prognosis of myocardial infarction through an amido bond. The amino group of the side chain of the antibody amino acid and the carboxylated microsphere form amido bond coupling.

Wherein the protein factors related to the prognosis warning of myocardial infarction are angiotensin (1-7) (Ang- (1-7)), renin, Ang-II, Bradykinin (BK), aldosterone, cortisol, catechol (cathestin, CST), creatine kinase isoenzyme (creatine kinase-MB, CK-MB), cardiac troponin I (cardiac troponin-I, CTNI), N-terminal pro-B-type natriuretic peptide (N-terminal pro-polypeptide, NT-BNP), Vascular Endothelial Growth Factor (VEGF), hepatocyte growth factor (hepatocyte growth factor, HGF), endothelin 1 (other-1, ET-1), osteoprotegerin (osteoprotegerin, BMP), periostin (synopsystin, SDC-1-ligand (SDC-1, SDC-1), and VEGF), Matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, matrix metalloproteinase tissue inhibitor-1 (tissue inhibitor of metalloproteinases-1, TIMP-1), TIMP-2, TIMP-4, and peptin, Von Willebrand Factor (VWF), tissue plasminogen activator (t-PA), fibrin-2 (FCN 2), mannose-agglutinin (mannose-binding lectin, MBL), mannose-agglutinin/fibrin-related Protein 1 (MBL/fibrin-associated Protein-1, MAP-1), C-reactive Protein (C-reactive Protein, CRP), Interleukin-1 beta (Interleukin-1, IL-1, and so on, IL-6, IL-21, tumor necrosis factor alpha (TNF-alpha), soluble tumor necrosis factor type 1receptor (soluble tumor necrosis factor receptor 1, sTNFR1) and type 2 receptor (sTNFR2), macrophage Migration Inhibitory Factor (MIF), CC chemokine ligand 4(chemokine C-C ligand 4, CCL4), CXC chemokine ligand 8(CXC chemokine ligand 8, CXCL8), CXCL10, CXCL16, Myeloperoxidase (MPO), adiponectin, oxidized low density lipoprotein (oxidized, OX-tendon), granzyme B, Tenascin C (Tenascin-C, Tn-C) or I-carboxyl type collagen (collagen type I ). The corresponding antibodies in this disclosure are derived from Abcam, UK and Cell Signaling Technology, Inc., USA.

The present disclosure firstly selects the traditional polystyrene resin microbeads with color code codes for testing, and although the sample to be tested can be rapidly detected by using a Bio-Plex microbead chip system, the inventor further studies and finds that the antibody coating efficiency is low. Therefore, in one or some specific embodiments of the present disclosure, based on the protein factor, the material of the microbead solid substrate is chloromethyl polystyrene resin, the particle size of the microbead is 3-8 μm, CAS: 55844-94-5, the antibody coating efficiency is 1.64 times that of the traditional polystyrene resin with color code. Therefore, the chloromethyl polystyrene resin is selected as the bead material in the present disclosure. The antibody coating efficiency of the method is superior to that of the prior preparation technology, and a fluorescence spectrometer is adopted for detecting the concentration of the protein factor.

In a second exemplary embodiment of the present disclosure, a bead suspension chip product capable of detecting a protein factor associated with a pre-warning of myocardial infarction is provided, which is characterized in that: the product contains 1 × 10 per 100 μ L⁶～2×10⁶The bead comprises the bead suspension chip and an activated bead solid matrix which is not coupled, wherein the bead chip accounts for 70-80%.

Further, the product contains 1.25X 10 per 100 μ L⁶And (4) micro beads.

In a third exemplary embodiment of the present disclosure, a method for preparing the above bead suspension chip product is provided, which includes the steps of:

(1) activation of microbeads:

(2) coupling antibody:

By the preparation method, high antibody coating efficiency can be ensured, and accurate detection of protein factors is facilitated.

In a fourth exemplary embodiment of the present disclosure, there is provided a kit for detecting a protein factor associated with pre-warning of myocardial infarction, the kit comprising the bead suspension chip product, and further comprising: antigen standard substances with different concentrations, a microbead solid matrix, a PBS buffer solution, a microbead activation buffer solution, a microbead washing solution, a confining liquid and the like.

In a fifth exemplary embodiment of the present disclosure, there is provided a method for detecting a protein factor associated with pre-warning of myocardial infarction, the method comprising the step of performing detection using the bead suspension chip or the bead suspension chip product or the kit.

Further, a double antibody sandwich method is adopted for detection.

In one or some embodiments of the present disclosure, the method for detecting protein factors related to pre-warning of myocardial infarction specifically comprises the following steps:

adding the micro-bead suspension chip product into a 96-hole filter plate, and performing vacuum drying; adding a microbead washing solution, washing and then vacuum-pumping; adding a blood plasma sample to be detected and antigen standard products with different concentrations respectively, and attaching a sealing plate film; incubating at room temperature in a dark place, and vacuum filtering; adding a microbead washing solution, washing and then vacuum-pumping; adding biotin-labeled secondary antibody (such as biotin-labeled goat anti-mouse antibody, biotin-labeled goat anti-rabbit antibody, etc.), incubating at room temperature in dark place, and vacuum filtering; adding a microbead washing solution, washing and then vacuum-pumping; adding streptavidin-phycoerythrin; incubating at room temperature in a dark place, and vacuum-pumping; adding a microbead washing solution, washing and then vacuum-pumping; adding confining liquid heavy suspension microbeads, detecting the fluorescence intensity (wavelength 565nm) of the confining liquid heavy suspension microbeads by using a fluorescence spectrometer to obtain the fluorescence intensity change curves of the standard substances with different antigen concentrations, making a linear relation curve and obtaining the fluorescence intensity of the plasma sample to be detected, and then obtaining the content of the protein factors in the plasma sample to be detected according to the linear relation curve.

In a sixth exemplary embodiment of the present disclosure, there is provided a myocardial infarction prognosis risk index detecting device, including at least:

a protein factor detection system capable of detecting at least one of the following three groups of values in plasma: the content values of Ang- (1-7) and TNF-alpha, the content values of renin activity and CTNI, and the content values of Ang-II and NT-proBNP;

a prognostic data processor for receiving values from at least one of the following three groups: the content values of Ang- (1-7) and TNF-alpha, the content values of renin activity and CTNI, and the content values of Ang-II and NT-proBNP; further, at least one of Δ EDV,. DELTA.LVEF,. DELTA.E '/A', wherein. DELTA.edv is 137 to 0.412 × Ang- (1 to 7) +0.334 × TNF-. alpha., Δ LVEF is 85 to 0.358 × renin activity-0.315 × Log-CTNI, and. DELTA.E '/A' is 32 to 0.415 × Ang-II to 0.304 × Log-NT-proBNP, is calculated;

wherein Δ EDV represents the left ventricular end diastolic volume change value; Δ LVEF represents the left ventricular ejection fraction change value; Δ E '/A' represents the variation of the ratio of the peak diastolic early velocity (E ') to the peak diastolic late velocity (A') of the mitral valve annulus; ang- (1-7), renin activity, Ang-II, CTNI, TNF-alpha and NT-proBNP represent the amount or activity, respectively, of the corresponding protein factor in the plasma sample.

In one or some specific embodiments of the present disclosure, the protein factor detection system comprises the microbead suspension chip or microbead suspension chip product or kit.

Further, the detection device also comprises a prognosis result display for displaying the values of delta EDV, delta LVEF and delta E '/A'.

In a seventh exemplary embodiment of the present disclosure, there is provided a method for establishing an early warning model for myocardial infarction prognosis, the method comprising:

(2) and (3) processing data by adopting SPSS software:

By the establishing method, the myocardial reconstruction early warning model is obtained as follows:

ΔEDV＝137-0.412×Ang-(1-7)+0.334×TNF-α；

the left ventricular contraction function change early warning model is as follows:

Δ LVEF ═ 85-0.358 × renin activity-0.315 × Log-CTNI;

wherein the renin activity unit is ng multiplied by mL/hr, and the CTNI unit is ng/mL.

The left ventricular diastolic function change early warning model is as follows:

ΔE'/A'＝32-0.415×Ang-II-0.304×Log-NT-proBNP；

wherein the Ang-II unit is pg/mL, and the NT-proBNP unit is pg/mL.

Δ EDV represents the left ventricular end diastolic volume change value; Δ LVEF represents the left ventricular ejection fraction change value; Δ E '/A' represents the variation of the ratio of the peak diastolic early velocity (E ') to the peak diastolic late velocity (A') of the mitral valve annulus.

The delta EDV is the most important index for reflecting the reconstruction of the left ventricle, the larger the numerical value is, the heavier the reconstruction degree of the left ventricle is, and the negative value is, the reversal of the reconstruction of the left ventricle is; Δ LVEF reflects the change of the contraction function of the left ventricle, the larger the value is, the better the contraction function of the left ventricle is, the negative value is, the decline of the contraction function of the left ventricle is, and the positive value is, the improvement of the contraction function of the left ventricle is; Δ E '/A' reflects the change in left ventricular diastolic function, with larger values indicating better left ventricular diastolic function, negative values indicating decreased left ventricular diastolic function, and positive values indicating improved left ventricular diastolic function.

Step (1) further comprises detecting the content of other protein factors, such as aldosterone, cortisol, catechol inhibin (CST), etc., as listed above.

The present disclosure is to predict the degree of myocardial remodeling and cardiac function in the future by blood index detection when a patient has just suffered a myocardial infarction. One of the indexes can be adopted for the left ventricular remodeling prognosis of myocardial infarction, but for more comprehensive early warning, the three indexes delta EDV, delta LVEF and delta E '/A' can be combined to evaluate the risk.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.

The reagents employed in the present disclosure were as follows:

preparing a PBS buffer solution: 8g of NaCl, 0.2g of KCl, KH₂PO₄ 0.2g，Na₂HPO₄·12H₂O2.88 g, and adding triple distilled water to 1000 mL. The pH value is 7.4, the mixture is sterilized by conventional high pressure and stored at 4 ℃.

Preparing a microbead activation buffer: weighing NaH₂PO₄3g, 200mL of double distilled water, and fully stirring and dissolving; the pH value is adjusted to 6 by using 5mol/L NaOH, and the volume is adjusted to 250 mL. Sterilizing by conventional autoclaving, and storing at 4 deg.C.

Preparing a microbead washing solution: na (Na)₂HPO₄ 1.44g，NaCI 8.073g，KH₂PO₄0.24g, KCI 0.2015g and Tween 200.5 mL, and triple distilled water is added to 1000 mL. pH 7.4, filtering with 0.22um filter membrane for sterilization, and storing at 4 deg.C.

Preparing a sealing liquid: na (Na)₂HPO₄ 1.44g，NaCI 8.073g，KH₂PO₄0.24g, KCI 0.2015g, BSA 10g, Thimerosal 200mg, and triple distilled water to 1000 mL. pH 7.4, filtering with 0.22um filter membrane for sterilization, and storing at 4 deg.C.

Preparing a storage solution: na (Na)₂HPO₄ 1.44g，NaCI 8.073g，KH₂PO₄0.24g, KCI 0.2015g, BSA 1g, Thimerosal 200mg, Tween 200.5 mL, and triple distilled water to 1000 mL. pH 7.4, filtering with 0.22um filter membrane for sterilization, and storing at 4 deg.C.

Preparing an EDC solution: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was purchased from Merck, Germany and 50mg EDC was added to 1mL of triple distilled water and used immediately after mixing at a concentration of 50 mg/mL.

Preparing a Sulfo-NHS solution: N-Hydroxythiosuccinimide (Sulfo-NHS) was purchased from Merck, Germany, and 50mg of Sulfo-NHS was added to 1mL of triple distilled water and used immediately after mixing at a concentration of 50 mg/mL.

Example 1

A microbead chip capable of detecting protein factors related to myocardial infarction prognosis early warning comprises a microbead solid substrate, wherein the microbead solid substrate is coupled with protein factor antibodies related to myocardial infarction prognosis early warning through amido bonds.

The amino group of the side chain of the antibody amino acid and the carboxylated microsphere form amido bond coupling.

The material of the microbead solid matrix is chloromethyl polystyrene resin, and the antibody coating efficiency of the microbead solid matrix is 1.64 times that of the conventional polystyrene resin. Therefore, the chloromethyl polystyrene resin is selected as the bead material in the present disclosure. The method is superior to the prior preparation technology.

The protein factors related to the prognosis warning of myocardial infarction are angiotensin (1-7) (Ang- (1-7)), renin, Ang-II, Bradykinin (BK), aldosterone, cortisol, catechol (cat, CST), creatine kinase isozyme (CK-MB), cardiac troponin I (CTNI), N-terminal pro-B-type natriuretic peptide (N-terminal pro-protein, NT-proBNP), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), endothelin 1 (endothien-1, ET-1), osteoprotegerin (osteoprotegerin, G), periostin (periostin, PN), multiple protein-substrate protein (SDC-1 ), metalloproteinases (metalloproteinases, SDC-1), MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, matrix metalloproteinase tissue inhibitor-1 (tissue inhibitor of metalloproteinases-1, TIMP-1), TIMP-2, TIMP-4, and Peptidin, Von Willebrand Factor (VWF), tissue plasminogen activator (t-PA), fibrin-2 (FCN 2), mannose-binding lectin (MBL), mannose-lectin/fibrin-related Protein 1 (MBL/fibrin-associated Protein-1, MAP-1), C-reactive Protein (C-reactive Protein, CRP), Interleukin-1 beta (Interleukin-1 beta, IL-1 beta), IL-6, IL-21, IL-1 beta, and so on, Tumor necrosis factor alpha (TNF-alpha), soluble tumor necrosis factor type 1receptor (soluble tumor necrosis factor receptor 1, sTNFR1), type 2 receptor (sttnfr 2), macrophage Migration Inhibitory Factor (MIF), CC chemokine ligand 4(chemokine C-C chemokine ligand 4, CCL4), CXC chemokine ligand 8(CXC chemokine ligand 8, CXCL8), CXCL10, CXCL16, Myeloperoxidase (MPO), adiponectin, oxidized low density lipoprotein (oxidized LDL, OX-LDL collagen), granzyme B, Tenascin C (Tenascin-C, Tn-C), or carboxy-terminal peptide (carbon-terminal peptide of ict p).

Example 2

A micro-bead suspension chip product capable of detecting protein factors related to myocardial infarction prognosis early warning is characterized in that: the product contains 1.25 × 10 per 100 μ L⁶A microbead comprising the microbead suspension chip of example 1 and an uncoupled activated microbead solid matrix, the microbead suspension chip comprising about 75% of the microbead solid matrix.

The preparation method of the micro-bead suspension chip product comprises the following steps:

(1) activation of microbeads:

the beads were first placed in water and shaken on an oscillator for 1 minute (2000 rpm), and sonicated in a water bath for 10 seconds/time, 3 times. 4mL of the bead solution was added to a light-shielded EP tube in 48 parts total. Centrifuge at 20000 rpm for 5 minutes and discard the supernatant. 4mL of bead washing solution was added, followed by shaking at 2000 rpm for 1 minute, and water bath sonication for 10 seconds/time and 3 times, respectively. Centrifuge at 20000 rpm for 5 minutes and discard the supernatant. Respectively adding 4mL of microbead activation buffer solution, oscillating for 1 minute at 2000 rpm, and performing water bath ultrasonic treatment for 10 seconds/time and 3 times to completely suspend microbeads.

Each EP tube was charged with 500ul each of 50mg/mL EDC and 50mg/mL Sulfo-NHS prepared extemporaneously, respectively, for 30 minutes at 2000 rpm at room temperature. 4mL of PBS buffer was added, shaking at 2000 rpm for 1 min, and sonicated in a water bath for 10 sec/3 times. Centrifuge at 20000 rpm for 5 minutes and discard the supernatant. 4mL of PBS buffer was added repeatedly, shaking at 2000 rpm for 1 min, and sonicating in a water bath for 10 sec/3 times.

(2) Coupling antibody:

40ul of antibody of 48 protein factors is respectively added into the carboxylated microbead solution, the solution is diluted to 25mL by PBS buffer solution, and the mixture is incubated overnight at 4 ℃ in a dark place with shaking. Centrifuge at 20000 rpm for 5 minutes and discard the supernatant. The cells were washed 3 times with shaking by adding 25mL of PBS buffer. Centrifugation was repeated at 20000 rpm for 5 minutes and the supernatant was discarded. Adding 25mL of blocking solution, blocking the microbead antibody at 1000 rpm, and oscillating for 2 hours at room temperature in a dark condition. Centrifuge at 20000 rpm for 5 minutes and discard the supernatant. Add 25mL of stock solution and wash with shaking for 5 min/3 times. Centrifuge at 20000 rpm for 5 minutes and discard the supernatant. And 6mL of storage solution is added, and the microbeads are resuspended at 1000 rpm for 15 seconds to obtain the microbead suspension chip product. Storing at 4 ℃ in the dark.

The counting method comprises the following steps:

sucking appropriate amount of microbead, diluting, and counting with counting plate (0.1mm, l/400 mm)²) Counting under a normal microscope. Number of microbeads multiplied by 10 per large grid⁴X dilution times volume (mL).

Antibody coating efficiency detection:

the antibody-coated microbeads were resuspended at 1000 rpm for 30 seconds and the biotin-labeled goat anti-mouse IgG (purchased from CST Co.) was diluted to a concentration of 2ug/mL with PBS buffer. Subpackaged to 48 EP tubes, 50uL each. 50uL of PBS buffer was added to 1 EP tube as a blank. Add l uL of bead resuspension to each EP tube. Incubate at 1000 rpm for 30 min in the dark at room temperature, centrifuge for 5 min at 20000 rpm, and discard the supernatant. And (3) shaking and washing by using a proper amount of microbead washing liquid for 5 minutes/time and 3 times, centrifuging for 5 minutes at 20000 rpm, and removing a supernatant.

Each EP tube was filled with 50uL streptavidin-phycoerythrin at a concentration of 2 ug/mL. Incubate at 1000 rpm for 30 min in the dark at room temperature, centrifuge for 5 min at 20000 rpm, and discard the supernatant. Adding 100uL of microbead washing solution to resuspend microbeads, resuspending microbeads at 1000 rpm for 15 seconds, and detecting the fluorescence intensity by using a fluorescence spectrometer. The fluorescence intensity of the successfully coupled microbeads should be higher than 2000MFI, the fluorescence intensity of the negative control should be lower than 100MFI, and if the fluorescence intensity of the coated microbeads is lower than 2000MFI, the coating is unsuccessful. Through the optimization and detection of the microbead material, we find that: chloromethyl polystyrene resin is selected as the microbead of the microbead solid matrix, and the antibody coating efficiency of the microbead solid matrix is 1.64 times that of the traditional polystyrene resin. Therefore, chloromethyl polystyrene resin is selected as the bead material in the application.

Example 3

A kit for detecting a protein factor associated with pre-warning of myocardial infarction, the kit comprising the bead suspension chip product of embodiment 2, further comprising: antigen standard substances with different concentrations, a microbead solid matrix, a PBS buffer solution, a microbead activation buffer solution, a microbead washing solution, a confining liquid and the like.

Example 4

A method of detecting a protein factor associated with pre-warning of myocardial infarction, the method comprising:

the bead suspension chip product of example 2 was added to a 96-well suction filter plate at 50. mu.L per well and vacuum dried. Adding 200 mu L of microbead washing solution for 5 min/time and 3 times in total, and after washing, vacuumizing and drying. 100 mul of plasma sample to be tested (diluted by microbead washing solution according to a ratio of 1: 5000) and 100 mul of antigen standard substance with different concentrations are respectively added, and a sealing plate film is attached. Incubating for 30 min at 1000 rpm in the dark at room temperature, and vacuum filtering. Adding 200 mu L of microbead washing solution for 5 min/time and 3 times in total, and after washing, vacuumizing and drying. Adding 200 mu L of biotin-labeled goat anti-mouse IgG, incubating for 30 minutes at room temperature under the condition of keeping out of the sun at 1000 rpm, and carrying out vacuum filtration. Adding 200 mu L of microbead washing solution for 5 min/time and 3 times in total, and after washing, vacuumizing and drying. Streptavidin-phycoerythrin 50uL was added at a concentration of 2 ug/mL. Incubating for 30 minutes at the room temperature under the condition of keeping out of the light at 1000 rpm, and vacuumizing to dry. Adding 200 mu L of microbead washing solution for 5 min/time and 3 times in total, and after washing, vacuumizing and drying. Adding 100uL of confining liquid to resuspend the microbeads, resuspending the microbeads at 1000 rpm for 15 seconds, detecting the fluorescence intensity (wavelength 565nm) of the microbeads by using a fluorescence spectrometer to obtain the fluorescence intensity change curves of the standard products with different antigen concentrations, making a linear relation curve and obtaining the fluorescence intensity of the plasma sample to be detected, and then obtaining the content of 48 protein factors in the plasma sample to be detected according to the linear relation curve.

The experimental analysis shows that compared with the measured value of an ELISA method, the obtained result has no significant difference and higher accuracy.

Example 5

A myocardial infarction prognosis risk index detection device, the detection device at least comprising:

a protein factor detection system, wherein the detection system adopts the kit in the embodiment 3, and the kit can detect the content of Ang- (1-7), renin, Ang-II, CTNI, TNF-alpha and NT-proBNP in blood plasma;

a prognostic data processor for receiving values of Ang- (1-7), renin, Ang-II, CTNI, TNF- α and NT-proBNP; further, Δ EDV,. DELTA.LVEF,. DELTA.E '/A ' values were calculated, wherein. DELTA.edv ═ 137 to 0.412 × Ang- (1 to 7) +0.334 × TNF-. alpha., Δ LVEF ═ 85 to 0.358 × renin activity-0.315 × Log-CTNI, and. DELTA.E '/A ═ 32 to 0.415 × Ang-II to 0.304 × Log-NT-proBNP;

the detection device also comprises a prognosis result display for displaying the values of delta EDV, delta LVEF and delta E '/A'.

Embodiment 6 a method for constructing an early warning model for the prognosis of myocardial infarction, the method comprising:

1 test subject

Continuous acute ST elevation myocardial infarction (STEMI) 300 patients who successfully received percutaneous coronary intervention (PCI therapy) within 12 hours. The sample size is required to be at least 5 times of the number of variables participating in analysis, and is set to 300 cases in consideration of allowing a rate of missing visits of not more than 20%.

1.1 inclusion criteria

Patients receiving PCI therapy (drug-coated stents) within 12 hours of STEMI, who fell their forever into a culprit vessel. STEMI definition: elevation of ST segments of 2 or more precordial leads is more than or equal to 2mm, and is accompanied by elevation of three items (CTnI, CK-MB and Myo) of typical chest pain or myocardial infarction;

② the age is more than or equal to 18 years old;

and signing the informed consent by the patient or the authorized relatives.

1.2 exclusion criteria

(ii) suffers from non-ischemic cardiomyopathy;

planning to receive the cardiac surgery treatment within 6 months;

③ STEMI mechanical complications (perforation of the ventricular septum, rupture of the free wall and acute severe mitral regurgitation);

renal failure or liver failure;

malignant tumor, HIV infection or central nervous system disorder;

sixthly, the cardiopulmonary resuscitation is performed for more than 15 minutes and the consciousness is lost;

seventhly, cardiogenic shock;

-participating in other research projects involving intervention of drugs or instruments;

ninthly, not signing an informed consent;

the chloride has a history of myocardial infarction.

2 physical examination and specimen detection

Patient height, weight and blood pressure were measured by clinical professional researchers. After resting for 15 minutes in the sitting position, the blood pressure was measured 7 times, and the average of the last 3 times was taken.

Before PCI treatment, arterial blood is collected in a catheter chamber, the arterial blood is placed in a heparin anticoagulant tube pretreated by a protease inhibitor, plasma is immediately centrifugally separated at high speed (4 ℃, 4000 rpm and 15 minutes), the plasma is stored in a liquid nitrogen tank, and the plasma levels of 48 left ventricular remodeling related protein factors such as Ang- (1-7) and the like are measured by utilizing the microbead chip integrated detection system.

3 evaluation index

At follow-up visit 6 months, LVEDV, LVEF and E '/A' within 12 hours of STEMI onset and at the end of the 6 th month after PCI treatment were determined using a Philips SONOS IE33 model real-time three-dimensional echocardiography. Δ EDV, Δ LVEF, and Δ E '/A' were calculated.

The subject or the authorized consignor signs an informed consent, takes the left lateral lying position, and takes the dynamic images of the long axis beside the sternum of the left ventricle, the four-chamber heart of the apex of the heart, the two-chamber heart of the apex of the heart, the long axis of the apex of the heart, the level of the apex of the left ventricle, the level of the papillary muscle and the level of the mitral valve. Meanwhile, pulse wave Doppler is used for obtaining a mitral valve orifice blood flow frequency spectrum in diastole, and tissue Doppler is used for obtaining a mitral valve annulus motion frequency spectrum.

Left Ventricular Ejection Fraction (LVEF) was used to assess left ventricular systolic function: LVEF (%)/LVEDV × 100 [ Left Ventricular End Diastolic Volume (LVEDV) — diastole volume (lvadv) — Left Ventricular End Systolic Volume (LVESV) ]; normal contractile function is defined as EF ≧ 50%. In order to evaluate the diastolic function of the left ventricle, the diastolic early blood flow velocity (E), the diastolic late blood flow velocity (A) and the ratio of the two are measured on the four-chamber cardiac section of the apex of the heart by using the pulse wave Doppler technology; performing tissue Doppler examination on the apical quadric-luminal heart tangent plane and measuring the mitral valve annulus early diastole peak velocity (E '), late diastole peak velocity (A') and their ratios. The 3 cardiac cycles were measured consecutively and averaged. The ultrasound operator was blinded to the present study.

4 statistical analysis

Statistical processing was performed using SPSS v19 software.

(1) The 48-protein factor levels, Ang- (1-7), were analyzed for correlations with Δ EDV, Δ LVEF, and Δ E '/A' in baseline plasma using both Pearson and Spearman correlation assays.

(2) And determining independent early warning factors of the regression equation, left ventricular reconstruction and functional indexes such as delta EDV, delta LVEF and delta E '/A' and the like by using a multivariate stepwise regression analysis method, and establishing an early warning model.

5 construction of a predictive model

The selected subjects were 300, 186 male and 114 female. The patient was 61.4 + -5.6 years old, with a chest pain duration of 45.0 + -8.9 minutes and a onset-to-reperfusion time of 138.9 + -6.5 minutes.

And (3) bringing all the factors into stepwise regression analysis, constructing an early warning model, and predicting left ventricle reconstruction and cardiac function parameters.

An early warning model aiming at the left ventricle reconstruction of the acute myocardial infarction patient is constructed by utilizing the micro-bead suspension chip, and the result prompts: ang- (1-7), renin, Ang-II, CTNI, TNF-alpha and NT-proBNP are independent predictors of left ventricular remodeling, and can be used to predict a long-term prognosis. There was no significant correlation between any 2 factors.

Pearson correlation analysis results show that: Δ EDV is inversely correlated with Ang- (1-7) levels (FIG. 2); the Spearman rank correlation analysis results show that: Δ EDV was positively correlated with TNF- α levels (FIG. 3). Pearson correlation analysis results show that: Δ LVEF is inversely correlated with renin and Log-CTNI levels (fig. 4 and 5). Pearson correlation analysis results show that: Δ E '/A' is inversely correlated with Ang-II and Log-NT-proBNP levels (FIGS. 6 and 7).

The myocardial reconstruction early warning model is as follows:

ΔEDV＝137-0.412×Ang-(1-7)+0.334×TNF-α；

wherein Δ EDV units mL, Ang- (1-7) and TNF- α units are each pg/mL.

Δ LVEF ═ 85-0.358 × renin activity-0.315 × Log-CTNI;

ΔE'/A'＝32-0.415×Ang-II-0.304×Log-NT-proBNP；

wherein the Ang-II unit is pg/mL, and the NT-proBNP unit is pg/mL.

TABLE 1 Baseline clinical characteristics of enrolled patients

Results are expressed as number of cases (%), mean + standard error or median [ interquartile range ].

BMI: body mass index; SBP: contracting pressure; E/A: the ratio of the blood flow velocity of the mitral valve in the early diastole to the blood flow velocity of the mitral valve in the late diastole; E/Ea: a ratio of early diastolic mitral blood flow velocity to early diastolic mitral annulus motion velocity; LVEDV: left ventricular end diastolic volume; LVEF: left ventricular ejection fraction; NT-proBNP: n-terminal forebrain natriuretic peptide.

TABLE 2 prediction of left ventricular reconstruction and cardiac function indices using stepwise linear regression model

Example 8 methodological testing and generalization of the Pre-alarm System

The prognostic risk index detection device in the embodiment 5 is applied to carry out left ventricular reconstruction risk prediction on myocardial infarction subjects, all subjects are divided into a high-risk group and a low-risk group for clinical follow-up, and the prediction accuracy of the early warning system is tested after the follow-up is carried out for 3 years at present. The method is used in medical institutions for trial and is prepared for further large-scale popularization and application.

The median delta EDV predicted by 300 patients according to the early warning model is divided into two groups (high-risk group and low-risk group, 150 cases respectively), and the incidence rates of delta EDV, delta LVEF, delta E '/A', cardiac death and acute heart failure are compared with each other until the follow-up. The results prove that the incidence rates of delta EDV, delta LVEF, delta E '/A' and cardiac death and acute heart failure of two groups of people are all significantly different, and the early warning model has higher reliability and accuracy (Table 3).

TABLE 3 follow-up results of two groups of patients grouped according to the early warning model

Results are expressed as number of cases (%) or mean + standard error.

^§P＜0.05。

The above embodiments are preferred embodiments of the present disclosure, but the embodiments of the present disclosure are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present disclosure should be regarded as equivalent replacements within the scope of the present disclosure.

Claims

1. A myocardial infarction prognosis risk index detection device is characterized by at least comprising:

a protein factor detection system capable of detecting at least one of the following three groups of values in plasma: the content values of Ang- (1-7) and TNF-alpha in the sample, the content values of renin activity and CTNI in the sample, and the content values of Ang-II and NT-proBNP in the sample; a prognostic data processor for receiving values from at least one of the following three groups: the content values of Ang- (1-7) and TNF-alpha in the sample, the content values of renin activity and CTNI in the sample, and the content values of Ang-II and NT-proBNP in the sample; further calculating one of the EDV, LVEF, E '/A' values, wherein EDV =137-0.412 XAng- (1-7) +0.334 XTNF-alpha, Δ LVEF =85-0.358 Xrenin activity-0.315 XAng-CTNI, E '/A' =32-0.415 XAng-II-0.304 XLog-NT-BNP;

wherein, EDV represents the end-diastolic volume change value of the left ventricle; the Link status represents the change value of the left ventricular ejection fraction; Δ E '/A' represents the variation of the ratio of the peak diastolic early velocity to the peak diastolic late velocity of the mitral valve annulus.

2. The inspection device of claim 1, wherein: the protein factor detection system comprises a microbead chip or a microbead suspension chip product or a kit;

the microbead chip comprises a microbead solid matrix made of chloromethylated polystyrene resin, and the microbead solid matrix is coupled with a protein factor antibody related to myocardial infarction prognosis early warning through an amido bond;

the micro-bead suspension chip product contains 1 multiplied by 10 per 100 mu L⁶~2×10⁶A microbead comprising the microbead chip as claimed in claim 1 and an unconjugated activated microbead solid matrix, wherein the microbead chip accounts for 70-80%;

the kit at least comprises a microbead chip or a microbead suspension chip product, and also comprises: antigen standard substances with different concentrations, a microbead solid matrix, a PBS buffer solution, a microbead activation buffer solution, a microbead washing solution and a confining liquid.

3. The inspection device of claim 2, wherein: the preparation method of the micro-bead suspension chip product comprises the following steps:

(1) activation of microbeads:

adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxy thiosuccinimide into suspended microbeads to react, then adding PBS buffer solution, vibrating and then carrying out ultrasonic treatment to obtain carboxylated microbead solution;

(2) coupling antibody:

4. The inspection device of claim 1, wherein: the detection device also comprises an early warning result display for displaying the values of Δ EDV, Δ LVEF, Δ E '/A'.

5. A method for establishing an early warning model for myocardial infarction prognosis is characterized by comprising the following steps:

respectively obtaining the LVEDV, the LVEF and/or the E '/A' of a plurality of patients before and after treatment, and calculating the Δ EDV, the Δ LVEF and/or the Δ E '/A';

(2) and (3) processing data by adopting SPSS software:

the detection result is determined by the mean value. + -. standard error or median [ interquartile range ]]If the normal distribution is obeyed, performing comparison among groups by using variance analysis, and if the difference among the groups has statistical significance, further performing pairwise comparison by using a Bonferroni method; if not, performing comparison among groups by using Kruskal-Wallis rank sum test, and if the difference among the groups has statistical significance, further performing multiple comparison by using a Dwass-Steel-Critchlow-Fligner method; two sidesPIf the difference is less than 0.05, the difference is considered to have statistical significance;

(3) according to normalityThe result of the test selects a Pearson or Spearman correlation test method to analyze the correlation between the level of each protein factor and the Δ EDV, the Δ LVEF and the Δ E '/A' in the blood plasma; selecting Pearson correlation analysis if the sample accords with normal distribution; if the samples do not conform to normal distribution, Spearman rank correlation analysis is selected; two sidesPIf the difference is less than 0.05, the difference is considered to have statistical significance;

(4) determining an EDV, an LVEF and/or an E '/A' left chamber reconstruction and a regression equation of the function index by using a multiple stepwise regression analysis method, determining independent early warning factors of the EDV, the LVEF and/or the E '/A', and establishing an early warning model; two sidesPDifferences were considered statistically significant < 0.05.