WO2018223832A1 - Use of plasma s100a12 in early diagnosis of st-segment elevation myocardial infarction - Google Patents
Use of plasma s100a12 in early diagnosis of st-segment elevation myocardial infarction Download PDFInfo
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- WO2018223832A1 WO2018223832A1 PCT/CN2018/087422 CN2018087422W WO2018223832A1 WO 2018223832 A1 WO2018223832 A1 WO 2018223832A1 CN 2018087422 W CN2018087422 W CN 2018087422W WO 2018223832 A1 WO2018223832 A1 WO 2018223832A1
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Definitions
- the invention belongs to the technical field of medical biology, in particular to the application of plasma S100A12 in the early diagnosis of ST-segment elevation myocardial infarction, and specifically relates to the application of S100A12 protein in the early diagnosis of ST-segment elevation myocardial infarction (STEMI).
- ST-segment elevation myocardial infarction ST-segment elevation myocardial infarction
- Acute myocardial infarction is one of the most important diseases that seriously threaten human health.
- Acute myocardial infarction, especially ST-segment elevation myocardial infarction, is onset, and the incidence and mortality are continuously increasing.
- Early diagnosis and reperfusion therapy are the key to improve the survival rate of acute myocardial infarction.
- the World Health Organization stipulates that the diagnosis of acute myocardial infarction is based on clinical symptoms of chest pain, changes in electrocardiogram, and elevation of biomarkers such as myocardial enzymology.
- CK-MB is the gold standard for diagnosing STEMI patients, it has poor sensitivity and does not increase in the early stage of the disease (within 4 hours before onset); in 1990, a large number of clinical studies confirmed that Troponin (TnT, TnI) can be used for early diagnosis and assessment of STEMI.
- the American College of Cardiology/American Heart Association has revised the diagnostic criteria for STEMI to include troponin as a myocardial marker for the diagnosis of STEMI.
- the STEMI definition is updated to a myocardial marker, especially a defined value of elevated troponin to myocardial damage, which can be diagnosed as STEMI.
- troponin begins to rise at 3-4h after onset, peaking at 10-24h, and has limited significance for early diagnosis of STEMI patients; CK-MB and troponin increase in the early stage of STEMI (3h after onset) Small, low diagnostic efficiency, myoglobin began to increase 1 h after the onset of STEMI patients, but the diagnostic specificity is low, and more need to be combined with other indicators to diagnose STEMI. Clinically, about one-third of STEMI patients have atypical clinical symptoms and electrocardiographic findings.
- STEMI markers with high sensitivity and specificity within 1-2 hours of onset is particularly important for the early diagnosis of STEMI patients.
- the object of the present invention is to provide the application of plasma S100A12 in the early diagnosis of ST-segment elevation myocardial infarction, specifically the application of S100A12 protein in the early diagnosis of ST-segment elevation myocardial infarction.
- S100A12 is shown in SEQ.ID.NO.1: Met Thr Lys Leu Glu Glu His Leu Glu Gly Ile Val Asn Ile Phe His Gln Tyr Ser Val Arg Lys Gly His Phe Asp Thr Leu Ser Lys Gly Glu Leu Lys Gln Leu Leu Thr Lys Glu Leu Ala Asn Thr Ile Lys Asn Ile Lys Asp Lys Ala Val Ile Asp Glu Ile Phe Gln Gly Leu Asp Ala Asn Gln Asp Glu Gln Val Asp Phe Gln Glu Phe Ile Ser Leu Val Ala Ile Ala Leu Lys Ala Ala His Tyr His Thr His Lys Glu.
- the S100A12 protein can be used as a biomarker for early diagnosis of ST-segment elevation myocardial infarction; specifically, the S100A12 protein is prepared for early diagnosis of ST-segment elevation myocardial infarction.
- a S100A12 protein detection kit provided by the present invention is directly purchased from CircuLexTM, Japan.
- the S100A12 protein detection kit is applied to the early diagnosis of ST-segment elevation myocardial infarction.
- the application of the S100A12 protein in the early diagnosis of ST-segment elevation myocardial infarction is as follows.
- Step 1 Plasma collection: The venous blood was taken immediately after the study was selected, and 5 ml of blood samples were collected with EDTA vacuum anticoagulation tube; venous blood of patients with STEMI less than 2 h was continuously taken, and the time of blood collection was 30 min, 1 h after STEMI. There were 10 time points in 2h, 4h, 6h, 12h, 24h, 3d, 7d and 30d.
- Step 2 Sample treatment: 10 minutes of venous blood was drawn, anticoagulation tube was centrifuged at 3000 rpm for 10 min, plasma was separated, and the treated plasma was stored in a -80 ° C refrigerator, and the S100A12 ELISA test kit was purchased. Japan CircuLexTM company) unified detection of plasma S100A12 concentration in the selected subjects; blood samples collected from the coagulation tube were immediately sent to our laboratory for detection of hscTnT, CK-MB and MYO concentrations.
- Step 3 ELISA method to detect the expression level of S100A12 in plasma: S100A12 ELISA kit (using double antibody sandwich enzyme-linked immunosorbent assay) purchased from CircuLexTM, Japan, to detect the change of plasma S100A12 concentration in the selected subjects, The average value of the sub-holes is the measured value of the detection target.
- Step 4 Standardization of data: The concentration of the standard product of S100A12 is prepared according to the instructions of the kit, and the standard product is sequentially added according to the concentration gradient according to the specification, and a standard curve is established.
- Step 5 Detection of plasma myocardial zymogram: The plasma hscTnT and MYO concentrations of the selected subjects were detected by electrochemiluminescence, and the plasma CK-MB activity of the selected subjects was detected by enzymatic method.
- Step 6 The expression level of S100A12 in plasma of patients with acute myocardial infarction: combined with other diagnostic parameters, according to the measured level of S100A12 in plasma, compared with the diagnostic threshold, greater than the critical value to diagnose myocardial infarction, less than the critical value is non- Patients with myocardial infarction.
- Step 7 Immunohistochemical detection of S100A12 expression in coronary artery (autopsy specimen) of patients with sudden cardiac death and expression of coronary thrombosis in STEMI patients.
- S100 is a newly discovered family of calmodulin proteins which are mainly expressed in neutrophils and also expressed in activated monocyte macrophages.
- S100A12 is a newly discovered member of the S100 family.
- S100A12 has a molecular weight of 12KD and is mainly secreted by cardiomyocytes, atherosclerotic smooth cells and blood neutrophils.
- S100A12 contains two EF-type calcium-binding regions; In atherosclerotic inflammatory lesions, neutrophils, macrophages, lymphocytes and endothelial cells in plaques have a large number of S100A12 expression, and S100A12 binds to its receptor for advanced glycation end products, which can up-regulate the expression of transcription factor ⁇ B in the nucleus.
- NF- ⁇ B-mediated release of cytokines and increased inflammatory response in atherosclerotic lesions S100A12 has a strong chemotactic effect, and chemotactic inflammatory cells migrate to localized atherosclerotic lesions, and Release of inflammatory factors through cascading amplification of inflammation, mediates and potentiates inflammatory responses and leads to the formation of unstable atherosclerotic plaques; peripheral blood mononuclear cells in patients with premature coronary artery disease (without diabetes) High expression of S100A12 leads to increased instability of mononuclear cells and promotes the conversion of mononuclear cells into macrophages; Coronary autopsy of diabetic patients found that S100A12 was abundantly expressed in coronary atherosclerotic plaques, indicating that S100A12 is associated with the formation of unstable atherosclerotic plaques; the basis of acute myocardial infarction is coronary porridge Rupture of atherosclerotic plaque and thrombosis,
- Figure 1 shows the changes in S100A12 protein concentration in the control group, UA group, NSTEMI group and STEMI group.
- Figure 2 is a ROC curve of plasma S100A12 concentration in STEMI patients.
- Figure 3 shows the phase changes of plasma concentrations of biomarkers in patients with STEMI; 3-1 is the phase change of plasma S100A12 concentration; 3-2 is the phase change of MYO concentration; 3-3 is CK-MB concentration The phase change of the phase; 3-4 is the phase change of the hscTnT concentration.
- Figure 4 shows the sensitivity of S100A12 and other plasma biomarkers for the diagnosis of STEM at various time points after STEMI onset; 4-1 is the sensitivity of plasma S100A12; 4-2 is the sensitivity of MYO; 4-3 is CK-MB Sensitivity; 4-4 is the sensitivity of hscTnT.
- Figure 5 shows the specificity of S100A12 and other plasma biomarkers for the diagnosis of STEMI at various time points after STEMI onset; 5-1 is the specificity of plasma S100A12; 5-2 is the specificity of MYO; 5-3 is CK-MB Specificity; 5-4 is the specificity of hscTnT.
- Figure 6 shows the expression of S100A12 in coronary atherosclerotic unstable plaques in patients with normal coronary artery and sudden cardiac death; coronary vascular tissue specimens from normal subjects in A, D and G; B, E, H, Coronary vascular tissue specimens of patients with C, F, and I death; AF was HE staining; GI was immunohistochemical staining.
- Figure 7 shows the expression of S100A12 in coronary artery thrombosis specimens of STEMI patients; A and D are peripheral blood clots in healthy people; B and E are non-criminal coronary clots; C and F are criminal crowns. Arterial thrombosis.
- a total of 1050 subjects were selected from May 2014 to December 2016 for chest pain and meeting the inclusion criteria. All of them were inpatients of Cardiovascular Department of Shenyang Military Region General Hospital; 820 males and 230 females.
- Non-ST segment elevation myocardial infarction group Non-ST-segment elevation 50 cases of myocardial infarction, NSTEMI; 930 cases of STEMI; coronary angiography showed left main trunk (LM), left anterior descending (LAD), left circumflex artery (LCX), right coronary artery (RCA), any extracardiac None of the subdural coronary arteries were considered as normal coronary arteries, and 20 patients were selected as normal control group.
- NSTEMI and UA group According to the World Health Organization UA diagnostic criteria: at least 1 angina pectoris within 48 hours before admission; ischemic chest pain time ⁇ 30min; ECG showed ST segment horizontal or down oblique depression ⁇ 1mm or ST segment elevation (limb lead ⁇ 1mm, chest lead ⁇ 2mm), ST segment returned to pre-onset level after onset; no changes in myocardial necrosis markers.
- the diagnostic criteria for NSTEMI a: The dynamic progression of ST-T is often longer than 24 hours (ST-T changes in transient myocardial ischemic attacks often recover in a few hours); b: chest pain For at least half an hour, it is consistent with the characteristics of chest pain in patients with myocardial infarction; c: the change of serum enzymology is consistent with the change of myocardial infarction and/or the increase of serum troponin T or I is not less than 2 times of the normal value, if any "a and c" or "b and c" can be diagnosed as NSTEMI.
- STEMI group patients with typical chest pain symptoms lasting more than 30 minutes; ECG dynamic evolution process, at least two limbs lead ST segment elevation ⁇ 0.1mv, or chest lead ST segment elevation ⁇ 0.2mv, Or a new left bundle branch block; serum myocardial enzymes have dynamic changes.
- the patient has the following diseases: active inflammatory disease; autoimmune disease; severe heart failure; hemodynamic instability; suspected myocarditis or pericarditis; hematopoietic diseases; advanced kidney or liver disease; malignant disease; Immunosuppressive drugs; previous coronary intervention or coronary artery bypass surgery.
- Treatment of the sample The venous blood was taken for 10 minutes, the anticoagulation tube was centrifuged at 3000 rpm for 10 minutes, the plasma was separated, and the treated plasma was stored in a -80 ° C refrigerator, and the double antibody sandwich enzyme-linked immunosorbent assay was used.
- the method (ELISA method) was used to detect the concentration of plasma S100A12; the blood samples were collected by the coagulation tube, and immediately sent to the laboratory of the hospital to detect the concentration of hscTnT, CK-MB and MYO.
- S100A12 The S100A12 ELISA kit used in this study was purchased from CircuLexTM, Japan. The components are as follows (see the instructions for the specific concentration and content of each component): washing buffer, dilution buffer, S100A12 Standard, 20 ⁇ horseradish peroxidase, substrate, termination reagent; detection range is 0-5000 ng/ml, detection sensitivity is 56 pg/ml, according to the sample specification, the following specific operations are performed: establishing a standard curve: according to the kit The specification is to prepare the concentration of S100A12 standard.
- the concentration gradient of the standard is 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 313pg/ml, 156pg/ml, 78pg/ml, 0pg/ml, according to the requirements.
- the gradient was sequentially added to the standard, 100 ⁇ l per well. After the plasma sample to be tested was diluted 100 times, 100 ⁇ l was added to the well of the reaction plate, and the reaction plate was placed at room temperature for 25 hours and washed for 1 hour. 100 ⁇ l of the enzyme-labeled antibody working solution was added to each well, and the reaction plate was placed at room temperature for 25 hours and washed for 1 hour.
- Detection of plasma myocardial zymogram The plasma hsTnT and MYO concentrations of the selected subjects were detected by electrochemiluminescence method; the plasma CK-MB activity of the selected subjects was detected by enzymatic method; the normal range was: TnT ⁇ 0.05ng/ml; MYO 28-72 ng/ml; CK-MB ⁇ 25 U/L.
- Coronary angiography The selected subjects underwent coronary angiography in the Department of Cardiology, Shenyang Military Region General Hospital within 1 week after admission. The Judkins method was used to select left and right coronary angiography in multiple positions. According to the diagnostic criteria for coronary heart disease, any of the subdural coronary artery LM, LAD, LCX, RCA without any stenosis is considered completely normal, such as diameter stenosis less than 50% for non-significant pathological lesions, such as diameter stenosis greater than 50 % is a pathologically significant lesion. The degree of coronary stenosis and the number of coronary lesions were evaluated by experienced clinicians, and were divided into single-vessel disease, double-vessel disease, and three-vessel disease.
- PCI percutaneous coronary intervention
- DES drug-eluting stent
- bare metal stent All patients who were scheduled for emergency PCI were given aspirin 300mg and clopidogrel 300-600mg before surgery; firstly, 5000U heparin was given. After the end of angiography, if PCI was performed, 70-100 U/kg should be used to make up the amount.
- the cardio-intensive care unit was monitored and given subcutaneous low-molecular-weight heparin, subcutaneously once every 12 hours (5-7d); clopidogrel 75mg (1 time/d), and long-term use of aspirin 100mg (1 time / d). All selected subjects were treated with secondary prevention of coronary heart disease in strict accordance with the guidelines.
- Immunohistochemical method was used to detect the expression changes of vascular thrombus aspiration specimen S100A12 in human coronary artery tissue and STEMI patients.
- the coronary arteries were obtained from the root of the left anterior descending coronary artery. 1.5cm, cut into 3 segments, each segment is about 0.5cm; all coronary arteries are fixed in 10% paraformaldehyde; all samples must be approved by the Ethics Committee of Shenyang Military Region General Hospital and signed informed consent.
- Dehydration The specific order is as follows: 70% alcohol 2h-80% alcohol 2h-90% alcohol 2h-95% alcohol I 4h-95% alcohol II overnight -100% alcohol I 1.5h-100% alcohol II 1.5h .
- tissue block was placed in xylene I for 1 h, taken out and placed in xylene II for 1 h.
- Dipping wax The specific order is as follows: 1 h in paraffin I - 1 h in paraffin II 1 hour in paraffin III.
- tissue block is embedded with paraffin and placed at room temperature.
- tissue block was sectioned using a paraffin slicer to a thickness of 3 ⁇ m and attached to a glass slide.
- Section dewaxing The specific sequence is as follows: xylene I in 20min-xylene II in 20min-95% alcohol I in 15min-95% alcohol II in 15min-90% alcohol 10min-80% alcohol 5min-70% alcohol 5min-distilled water 30min.
- Paraffin sections were placed in 1% hydrochloric acid for differentiation for 30 s, rinsed with running water.
- Cytoplasmic staining Paraffin sections were stained in water-soluble eosin solution for 5 min, rinsed with running water.
- Table 1 General clinical baseline status of the control group, UA group, NSTEMI group and STEMI group.
- CK-MB creatine kinase isoenzyme
- hscTnT hypersensitive troponin T
- hs-CRP hypersensitive C-reactive protein
- pro-BNP Brain natriuretic peptide precursor.
- Table 2 Binary logistic regression analysis of risk factors associated with STEMI.
- plasma S100A12 diagnosis of STEMI optimal threshold by comparing 930 STEMI patients with normal control group, UA group, NSTEMI group of plasma S100A12 concentration changes, determine which plasma S100A12 concentration is the best critical for the diagnosis of STEMI value.
- the normal control group, UA group, NSTEMI group were used as the negative reference group, and 930 STEMI patients were used as the positive reference group to plot the ROC curve of plasma S100A12 concentration in STEMI patients, as shown in Figure 2.
- STEMI occurs, the temporal changes of S100A12 and other biomarkers in plasma: 120 patients with STEMI from the time of onset to less than 2 hours, the basic clinical data of STEMI patients, see Table 3.
- S100A12 and myocardial markers MYO, CK-MB and hscTnT concentrations at different time points (30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 7 d, 30 d
- CK-MB creatine kinase isoenzyme
- hscTnT hypersensitive troponin T
- 17.hs-CRP hypersensitive C-reactive protein
- pro-BNP brain natriuretic peptide precursor.
- the concentration of S100A12 in plasma reached the peak at 1h-2h, which lasted for 10h, and the plasma concentration of S100A12 began to decrease until the onset.
- the concentration of plasma S100A12 decreased to normal in 3d; the concentration of MYO increased in the range of 1h-2h after the onset of STEMI, and the concentration of MYO in plasma reached the peak at 4h after the onset of disease, which lasted for 12h, and the pathogenesis decreased to the normal range at 24h; CK-MB was in STEMI.
- the concentration of plasma in the 4h onset was significantly increased.
- the concentration of CK-MB in plasma reached the peak at 10h-12h, and it lasted for 24h. Then the plasma concentration of CK-MB began to decrease until the plasma CK-MB concentration decreased to normal after 3d.
- the concentration of hscTnT in plasma from 1 h to 2 h after STEMI was significantly increased. The concentration of plasma hscTnT peaked at 12h-24h, which lasted for 7d-10d, and then the plasma concentration of hscTnT began to decrease until the plasma MYO concentration decreased to normal after 30 days. .
- plasma S100A12 concentrations increased earlier than classical myocardial enzymology markers.
- the plasma S100A12 concentration of 263.9 ng/ml was used as the optimal diagnostic threshold for STEMI (the optimal cutoff values of STYO for MYO, CK-MB and hscTnT were 72 ng/ml, 25 U/L, 0.05 ng/ml, respectively).
- the selected normal control group, UA group and NSTEMI group were used as the negative reference group, and STEMI patients as the positive reference group were drawn to the plasma S100A12, MYO, CK-MB and 30 min, 1 h, 2 h, 4 h, 6 h after STEMI.
- the ROC curve of hscTnT is shown in Figure 4 and Figure 5.
- the ROC curve analysis showed that the sensitivity of plasma S100A12, MYO, CK-MB and hscTnT in the diagnosis of STEMI was 76.3%, 35%, 1.8%, 30.3%, respectively, and the specificity was 84%, 21% after STEMI. %, 80.4%, 59.3%; 1 hour after the onset of STEMI, the sensitivity of plasma S100A12, MYO, CK-MB and hscTnT in the diagnosis of STEMI were 80%, 39%, 10%, 33%, respectively, and the specificity was 85.3%, respectively.
- S100A12 23%, 82.6%, 58.6%, S100A12 was the highest; 2 hours after the onset of STEMI, the sensitivity of plasma S100A12, MYO, CK-MB and hsTnT in the diagnosis of STEMI were 80%, 61%, 14.5%, 39.7%, respectively. The specificities were 85.5%, 24%, 84.2%, and 59.6%, respectively. S100A12 was the highest. At 4 hours after STEMI, the sensitivity of S100A12, MYO, CK-MB and hscTnT in the diagnosis of STEMI was 81.8%.
- Immunohistochemical detection of S100A12 expression in coronary arteries of normal subjects with coronary artery and sudden cardiac death HE staining results showed that the expression level of S100A12 in normal coronary vessels was low, and coronary artery vessels in patients with sudden cardiac death The expression of S100A12 was significantly higher in tissues than in normal coronary vascular tissue specimens. S100A12 is abundantly expressed in the vascular wall tissue under the fibrous cap of the atherosclerotic unstable plaque, and the S100A12 in the immediate plaque tissue occurs in the acute myocardial infarction. A large amount is released into the patient's plasma.
- Immunohistochemical detection of S100A12 expression in coronary artery thrombosis in STEMI patients HE staining results suggest that the components of the thrombus in the coronary artery include red blood cells, inflammatory cells, platelets and fibrin, and non-criminal agglutination in vitro.
- the components of coronary blood clots are mainly red blood cells and fibrin, and the content of platelets and inflammatory cells is small.
- Immunohistochemistry showed that the expression of CCL2 and CCR2 in coronary artery thrombosis of patients with STEMI was significantly higher than that of their own coronary blood clots.
- S100A12 in coronary thrombosis in patients with acute myocardial infarction was significantly higher than that in normal blood clot specimens and in non-criminal coronary clots, indicating that coronary atherosclerosis was unstable in the very early stage of acute myocardial infarction. Block rupture releases a large amount of S100A12 into the bloodstream. In the very early stage of acute myocardial infarction, high expression of S100A12 was detected in the serum of patients with acute myocardial infarction. Immunohistochemistry showed neutrophils in the specimens of coronary artery thrombosis in STEMI patients. The expression of S100A12 in (MPO positive) was significantly increased, as shown in Figure 7.
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Abstract
The present invention falls within the technical field of medical biology, and in particular relates to the use of plasma S100A12 in very early diagnosis of ST-segment elevation myocardial infarction (STEMI). S100A12 is a member of the calcium binding protein family which is mainly secreted by myocardial cells, vascular smooth muscle cells of vascular wall atherosclerosis plaque and blood neutrophils. S100A12 is a protein having a molecular weight of 12 KD, contains two EF chiral calcium ion binding areas, and can be rapidly released into circulating blood from myocardial cells, vascular smooth muscle cells and neutrophils after acute myocardial infarction outsets due to the relatively small molecular weight thereof, and an increase in the expression level of S100A12 can be detected early on (within 30 minutes of chest pain) during the occurrence of acute myocardial infarction, and S100A12 can be used as a biomarker for the early diagnosis of STEMI.
Description
本发明属于医学生物学技术领域，尤其涉及血浆S100A12在ST段抬高型心肌梗死早期诊断中的应用，具体为S100A12蛋白在ST段抬高型心肌梗死(STEMI)极早期诊断中的应用。The invention belongs to the technical field of medical biology, in particular to the application of plasma S100A12 in the early diagnosis of ST-segment elevation myocardial infarction, and specifically relates to the application of S100A12 protein in the early diagnosis of ST-segment elevation myocardial infarction (STEMI).
急性心肌梗死，是严重威胁人类健康的常见的重要疾病之一。急性心肌梗死尤其是ST段抬高型心肌梗死发病急，发病率和死亡率呈持续升高趋势，早期诊断并进行再灌注治疗是提高急性心肌梗死生存率的关键。世界卫生组织规定急性心肌梗死的诊断基于胸痛的临床症状，心电图的改变及生物标记物如心肌酶学的升高。Acute myocardial infarction is one of the most important diseases that seriously threaten human health. Acute myocardial infarction, especially ST-segment elevation myocardial infarction, is onset, and the incidence and mortality are continuously increasing. Early diagnosis and reperfusion therapy are the key to improve the survival rate of acute myocardial infarction. The World Health Organization stipulates that the diagnosis of acute myocardial infarction is based on clinical symptoms of chest pain, changes in electrocardiogram, and elevation of biomarkers such as myocardial enzymology.
目前，传统的生物标记物各有缺陷，CK-MB虽然是诊断STEMI患者的金标准，但是敏感性差，在发病早期(发病前4h内)不升高；在1990年，大量的临床研究证实，肌钙蛋白(TnT、TnI)可以用于STEMI的早期诊断及对病情的评估，美国心脏病学学会/美国心脏病协会修改了对STEMI诊断标准，把肌钙蛋白纳入为诊断STEMI的心肌标记物，STEMI定义更新为心肌标记物，特别是肌钙蛋白升高达到心肌损伤的界定值，即可诊断为STEMI。但是，肌钙蛋白在发病后3-4h开始升高，10-24h才能达到高峰，对STEMI患者早期诊断意义有限；CK-MB及肌钙蛋白在STEMI患者发病早期(发病后3h)升高幅度小，诊断效能低，肌红蛋白在STEMI患者发病后1h开始升高，但是诊断特异性低，多需要联合其他指标对STEMI进行诊断。临床上约有1/3的STEMI患者早期临床症状及心电图表现不典型，目前在临床使用的心肌损伤标记物又存在上述不足，CK-MB升高较迟，且诊断敏感性较低；肌钙蛋白亦不能在STEMI发病2h内被检出；肌红蛋白虽然在STEMI发病后的1-2h开始升高，但特异性差，不具有单独诊断价值。At present, traditional biomarkers are defective. Although CK-MB is the gold standard for diagnosing STEMI patients, it has poor sensitivity and does not increase in the early stage of the disease (within 4 hours before onset); in 1990, a large number of clinical studies confirmed that Troponin (TnT, TnI) can be used for early diagnosis and assessment of STEMI. The American College of Cardiology/American Heart Association has revised the diagnostic criteria for STEMI to include troponin as a myocardial marker for the diagnosis of STEMI. The STEMI definition is updated to a myocardial marker, especially a defined value of elevated troponin to myocardial damage, which can be diagnosed as STEMI. However, troponin begins to rise at 3-4h after onset, peaking at 10-24h, and has limited significance for early diagnosis of STEMI patients; CK-MB and troponin increase in the early stage of STEMI (3h after onset) Small, low diagnostic efficiency, myoglobin began to increase 1 h after the onset of STEMI patients, but the diagnostic specificity is low, and more need to be combined with other indicators to diagnose STEMI. Clinically, about one-third of STEMI patients have atypical clinical symptoms and electrocardiographic findings. The current clinical use of myocardial injury markers has the above-mentioned deficiencies, CK-MB rises later, and the diagnostic sensitivity is low; Protein could not be detected within 2 hours of STEMI; myoglobin increased at 1-2h after the onset of STEMI, but the specificity was poor and did not have a separate diagnostic value.
因此，研发在发病内1-2h内即可被检测敏感性及特异性均较高的STEMI 标志物，对于STEMI患者的极早期诊断,显得尤为重要。Therefore, the development of STEMI markers with high sensitivity and specificity within 1-2 hours of onset is particularly important for the early diagnosis of STEMI patients.
发明内容Summary of the invention
针对上述问题，本发明的目的在于提供血浆S100A12在ST段抬高型心肌梗死早期诊断中的应用，具体为S100A12蛋白在ST段抬高型心肌梗死早期诊断中的应用。In view of the above problems, the object of the present invention is to provide the application of plasma S100A12 in the early diagnosis of ST-segment elevation myocardial infarction, specifically the application of S100A12 protein in the early diagnosis of ST-segment elevation myocardial infarction.
所述的S100A12的氨基酸序列如SEQ.ID.NO.1所示：Met Thr Lys Leu Glu Glu His Leu Glu Gly Ile Val Asn Ile Phe His Gln Tyr Ser Val Arg Lys Gly His Phe Asp Thr Leu Ser Lys Gly Glu Leu Lys Gln Leu Leu Thr Lys Glu Leu Ala Asn Thr Ile Lys Asn Ile Lys Asp Lys Ala Val Ile Asp Glu Ile Phe Gln Gly Leu Asp Ala Asn Gln Asp Glu Gln Val Asp Phe Gln Glu Phe Ile Ser Leu Val Ala Ile Ala Leu Lys Ala Ala His Tyr His Thr His Lys Glu。The amino acid sequence of S100A12 is shown in SEQ.ID.NO.1: Met Thr Lys Leu Glu Glu His Leu Glu Gly Ile Val Asn Ile Phe His Gln Tyr Ser Val Arg Lys Gly His Phe Asp Thr Leu Ser Lys Gly Glu Leu Lys Gln Leu Leu Thr Lys Glu Leu Ala Asn Thr Ile Lys Asn Ile Lys Asp Lys Ala Val Ile Asp Glu Ile Phe Gln Gly Leu Asp Ala Asn Gln Asp Glu Gln Val Asp Phe Gln Glu Phe Ile Ser Leu Val Ala Ile Ala Leu Lys Ala Ala His Tyr His Thr His Lys Glu.
所述的S100A12蛋白可以作为ST段抬高型心肌梗死早期诊断的生物标志物；具体的，具体为S100A12蛋白制备ST段抬高型心肌梗死早期诊断试剂盒。The S100A12 protein can be used as a biomarker for early diagnosis of ST-segment elevation myocardial infarction; specifically, the S100A12 protein is prepared for early diagnosis of ST-segment elevation myocardial infarction.
为了实现上述问题，本发明提供的一种S100A12蛋白检测试剂盒直接购于日本CircuLexTM公司。In order to achieve the above problems, a S100A12 protein detection kit provided by the present invention is directly purchased from CircuLexTM, Japan.
所述的S100A12蛋白检测试剂盒应用于ST段抬高型心肌梗死极早期诊断。The S100A12 protein detection kit is applied to the early diagnosis of ST-segment elevation myocardial infarction.
所述的S100A12蛋白在ST段抬高型心肌梗死患者早期诊断中的应用，方法如下。The application of the S100A12 protein in the early diagnosis of ST-segment elevation myocardial infarction is as follows.
步骤1、血浆的采集：研究对象入选后即刻抽取其静脉血，用含EDTA真空抗凝管收集血标本5ml；连续抽取发病小于2h STEMI患者的静脉血，采血时间点为STEMI发病30min、1h、2h、4h、6h、12h、24h、3d、7d、30d共10个时间点。Step 1. Plasma collection: The venous blood was taken immediately after the study was selected, and 5 ml of blood samples were collected with EDTA vacuum anticoagulation tube; venous blood of patients with STEMI less than 2 h was continuously taken, and the time of blood collection was 30 min, 1 h after STEMI. There were 10 time points in 2h, 4h, 6h, 12h, 24h, 3d, 7d and 30d.
步骤2、样本的处理：抽取静脉血10分钟内，抗凝管3000转/min离心处理10min，分离血浆，将处理好的血浆储藏于-80℃冰箱保存，用S100A12 ELISA检测试剂盒(购于日本CircuLexTM公司)统一检测入选研究对象血浆S100A12浓度；促凝管采集血标本即刻送本院检验科，检测其hscTnT、CK-MB及MYO浓度。Step 2: Sample treatment: 10 minutes of venous blood was drawn, anticoagulation tube was centrifuged at 3000 rpm for 10 min, plasma was separated, and the treated plasma was stored in a -80 ° C refrigerator, and the S100A12 ELISA test kit was purchased. Japan CircuLexTM company) unified detection of plasma S100A12 concentration in the selected subjects; blood samples collected from the coagulation tube were immediately sent to our laboratory for detection of hscTnT, CK-MB and MYO concentrations.
步骤3、ELISA方法检测血浆中S100A12表达含量：S100A12 ELISA检测试剂盒(采用双抗体夹心酶联免疫试验法)购于日本CircuLexTM公司，检测入选研究对象的血浆中S100A12浓度的变化，以实验孔和副孔的平均值作为检测对象的测定值。Step 3: ELISA method to detect the expression level of S100A12 in plasma: S100A12 ELISA kit (using double antibody sandwich enzyme-linked immunosorbent assay) purchased from CircuLexTM, Japan, to detect the change of plasma S100A12 concentration in the selected subjects, The average value of the sub-holes is the measured value of the detection target.
步骤4、数据的标准化处理：按照试剂盒说明书配制S100A12标准品浓度，根据说明书要求按浓度梯度依次加入标准品，建立标准曲线。Step 4: Standardization of data: The concentration of the standard product of S100A12 is prepared according to the instructions of the kit, and the standard product is sequentially added according to the concentration gradient according to the specification, and a standard curve is established.
步骤5、血浆心肌酶谱的检测：用电化学发光法检测入选对象血浆hscTnT和MYO浓度，用酶法检测入选对象血浆CK-MB活性。Step 5: Detection of plasma myocardial zymogram: The plasma hscTnT and MYO concentrations of the selected subjects were detected by electrochemiluminescence, and the plasma CK-MB activity of the selected subjects was detected by enzymatic method.
步骤6、急性心肌梗死患者血浆中S100A12的表达水平：结合其它诊断参数，根据测得的血浆中的S100A12的水平，与诊断临界值比较，大于临界值则诊断心肌梗死，小于临界值则为非心肌梗死的患者。Step 6. The expression level of S100A12 in plasma of patients with acute myocardial infarction: combined with other diagnostic parameters, according to the measured level of S100A12 in plasma, compared with the diagnostic threshold, greater than the critical value to diagnose myocardial infarction, less than the critical value is non- Patients with myocardial infarction.
步骤7、免疫组织化学检测S100A12在心源性猝死的患者冠状动脉(尸检标本)的表达及STEMI患者罪犯冠状动脉内血栓组织的表达。Step 7. Immunohistochemical detection of S100A12 expression in coronary artery (autopsy specimen) of patients with sudden cardiac death and expression of coronary thrombosis in STEMI patients.
本发明的有益效果。The beneficial effects of the present invention.
本发明中，S100是新发现的钙调节蛋白家族，其主要在中性粒细胞内表达，并且在激活的单核巨噬细胞内也有表达。S100A12是S100家族新发现的成员，S100A12分子量为12KD，主要由心肌细胞、血管壁动脉粥样硬化的平滑细胞、血液中性粒细胞分泌，S100A12含有2个EF手型钙离子结合区；在动脉粥样硬化炎症病灶处即斑块内中性粒细胞、巨噬细胞、淋巴细胞及内皮细胞有大量S100A12表达，S100A12与其糖基化终产物受体结合，可引起细胞核内转录因子κB的表达上调，从而导致NF-κB介导的细胞因子的释放，动脉粥样硬化病变部位炎症反应的加剧；S100A12具有较强的趋化作用，趋化炎症细胞游走到动脉粥样硬化炎症病变局部，并通过炎症的级联放大效应引起炎性因子的释放，介导并加强炎症反应和导致动脉粥样硬化不稳 定斑块的形成；早发冠状动脉疾病患者(不合并糖尿病)的外周血单个核细胞内S100A12高表达，导致单个核细胞的不稳定性增加，并促进单个核细胞转化成巨噬细胞；猝死的糖尿病患者的冠状动脉尸检发现，患者的冠状动脉粥样硬化斑块内S100A12大量表达，说明S100A12与动脉粥样硬化不稳定斑块的形成存在相关关系；急性心肌梗死的发病基础是冠状动脉内粥样硬化斑块破裂和血栓形成，造成持久而严重的心肌缺血，导致心肌急性坏死，约90％-95％透壁性心肌梗死是由冠状动脉斑块破裂导致局部血栓形成所致。在猝死的冠状动脉尸检标本中，我们发现冠状动脉不稳定斑块破裂口处S100A12的表达明显增强，同时在STEMI患者罪犯血管的血栓标本中S100A12表达明显增强。因此，血浆S100A12可以在早期精确的诊断出ST段抬高型心肌梗死疾病。In the present invention, S100 is a newly discovered family of calmodulin proteins which are mainly expressed in neutrophils and also expressed in activated monocyte macrophages. S100A12 is a newly discovered member of the S100 family. S100A12 has a molecular weight of 12KD and is mainly secreted by cardiomyocytes, atherosclerotic smooth cells and blood neutrophils. S100A12 contains two EF-type calcium-binding regions; In atherosclerotic inflammatory lesions, neutrophils, macrophages, lymphocytes and endothelial cells in plaques have a large number of S100A12 expression, and S100A12 binds to its receptor for advanced glycation end products, which can up-regulate the expression of transcription factor κB in the nucleus. Thus, NF-κB-mediated release of cytokines and increased inflammatory response in atherosclerotic lesions; S100A12 has a strong chemotactic effect, and chemotactic inflammatory cells migrate to localized atherosclerotic lesions, and Release of inflammatory factors through cascading amplification of inflammation, mediates and potentiates inflammatory responses and leads to the formation of unstable atherosclerotic plaques; peripheral blood mononuclear cells in patients with premature coronary artery disease (without diabetes) High expression of S100A12 leads to increased instability of mononuclear cells and promotes the conversion of mononuclear cells into macrophages; Coronary autopsy of diabetic patients found that S100A12 was abundantly expressed in coronary atherosclerotic plaques, indicating that S100A12 is associated with the formation of unstable atherosclerotic plaques; the basis of acute myocardial infarction is coronary porridge Rupture of atherosclerotic plaque and thrombosis, resulting in persistent and severe myocardial ischemia, leading to acute myocardial necrosis, about 90%-95% of transmural myocardial infarction caused by local thrombosis caused by rupture of coronary plaque. In the coronal autopsy specimens of the dying, we found that the expression of S100A12 was significantly increased in the rupture of coronary artery plaque, and the expression of S100A12 was significantly enhanced in the thrombus specimens of the patients with STEMI. Therefore, plasma S100A12 can accurately diagnose ST-segment elevation myocardial infarction disease at an early stage.
图1为对照组、UA组、NSTEMI组及STEMI组S100A12蛋白浓度变化。Figure 1 shows the changes in S100A12 protein concentration in the control group, UA group, NSTEMI group and STEMI group.
图2为STEMI患者血浆S100A12浓度的ROC曲线。Figure 2 is a ROC curve of plasma S100A12 concentration in STEMI patients.
图3为STEMI患者发病后各生物标记物血浆浓度的时相性改变；其中3-1为血浆S100A12浓度的时相性改变；3-2为MYO浓度的时相性改变；3-3为CK-MB浓度的时相性改变；3-4为hscTnT浓度的时相性改变。Figure 3 shows the phase changes of plasma concentrations of biomarkers in patients with STEMI; 3-1 is the phase change of plasma S100A12 concentration; 3-2 is the phase change of MYO concentration; 3-3 is CK-MB concentration The phase change of the phase; 3-4 is the phase change of the hscTnT concentration.
图4为S100A12与其它血浆生物标记物在STEMI发病后各时间点诊断STEM的敏感性；其中4-1为血浆S100A12的敏感性；4-2为MYO的敏感性；4-3为CK-MB的敏感性；4-4为hscTnT的敏感性。Figure 4 shows the sensitivity of S100A12 and other plasma biomarkers for the diagnosis of STEM at various time points after STEMI onset; 4-1 is the sensitivity of plasma S100A12; 4-2 is the sensitivity of MYO; 4-3 is CK-MB Sensitivity; 4-4 is the sensitivity of hscTnT.
图5为S100A12与其它血浆生物标记物在STEMI发病后各时间点诊断STEMI的特异性；其中5-1为血浆S100A12的特异性；5-2为MYO的特异性；5-3为CK-MB的特异性；5-4为hscTnT的特异性。Figure 5 shows the specificity of S100A12 and other plasma biomarkers for the diagnosis of STEMI at various time points after STEMI onset; 5-1 is the specificity of plasma S100A12; 5-2 is the specificity of MYO; 5-3 is CK-MB Specificity; 5-4 is the specificity of hscTnT.
图6为S100A12在人正常冠状动脉及心源性猝死患者冠状动脉粥样硬化不稳定斑块的标本中的表达；A、D、G正常人的冠状动脉血管组织标本；B、E、H、C、F、I猝死患者冠状动脉血管组织标本；A-F为HE染色；G-I为免疫组化染色。Figure 6 shows the expression of S100A12 in coronary atherosclerotic unstable plaques in patients with normal coronary artery and sudden cardiac death; coronary vascular tissue specimens from normal subjects in A, D and G; B, E, H, Coronary vascular tissue specimens of patients with C, F, and I death; AF was HE staining; GI was immunohistochemical staining.
图7为S100A12在STEMI患者罪犯冠状动脉内血栓组织标本中的表达；A、D为健康人外周血凝块组织；B、E为自身非罪犯冠状动脉血凝块组织；C、F为罪犯冠状动脉内血栓组织。Figure 7 shows the expression of S100A12 in coronary artery thrombosis specimens of STEMI patients; A and D are peripheral blood clots in healthy people; B and E are non-criminal coronary clots; C and F are criminal crowns. Arterial thrombosis.
下面结合具体实施例对本发明作进一步的说明。The invention will now be further described in conjunction with specific embodiments.
实施例1。Example 1.
本发明的研究基础。The research basis of the present invention.
1、酶联免疫吸附实验检测STEMI患者血清S100A12表达变化。1. The expression of serum S100A12 in STEMI patients was detected by enzyme-linked immunosorbent assay.
1.1、研究对象。1.1, the research object.
连续入选2014年5月至2016年12月因胸痛就诊并符合入选标准的研究对象共1050例，全部为沈阳军区总医院心血管内科住院病人；其中男性820例，女性230例。A total of 1050 subjects were selected from May 2014 to December 2016 for chest pain and meeting the inclusion criteria. All of them were inpatients of Cardiovascular Department of Shenyang Military Region General Hospital; 820 males and 230 females.
根据美国心脏病学学会/欧洲心脏病学会诊断指南，将入选患者分为不稳定型心绞痛组(Unstable angina pectoris，UA)50例；非ST段抬高型心肌梗死组(Non-ST-segment elevation myocardial infarction，NSTEMI)50例；STEMI组930例；冠状动脉造影显示左主干(LM)、左前降支(LAD)、左回旋支(LCX)、右冠状动脉(RCA)中，任何一支心外膜下冠状动脉均无任何狭窄者视为冠状动脉正常，将其入选为正常对照组20例。According to the American College of Cardiology/European Society of Cardiology diagnostic guidelines, 50 patients were included in the unstable angina pectoris group (Unstable angina pectoris, UA); non-ST segment elevation myocardial infarction group (Non-ST-segment elevation 50 cases of myocardial infarction, NSTEMI; 930 cases of STEMI; coronary angiography showed left main trunk (LM), left anterior descending (LAD), left circumflex artery (LCX), right coronary artery (RCA), any extracardiac None of the subdural coronary arteries were considered as normal coronary arteries, and 20 patients were selected as normal control group.
1.2、入选标准。1.2, inclusion criteria.
1.2.1、本研究经沈阳军区总医院医学伦理委员会同意，研究中所有符合入选标准的研究对象均知情并签署知情同意书。1.2.1. The study was approved by the Medical Ethics Committee of the General Hospital of Shenyang Military Region. All subjects in the study who met the inclusion criteria were informed and signed informed consent.
1.2.2、入选年龄35岁-75岁。1.2.2, selected age 35 years old -75 years old.
1.2.3、正常对照组：因胸痛就诊于胸痛中心，冠脉造影阴性，排除冠心病的患者。1.2.3, normal control group: due to chest pain in the center of chest pain, coronary angiography negative, exclude patients with coronary heart disease.
1.2.4、NSTEMI及UA组：根据世界卫生组织UA的诊断标准为：入院前 48h内心绞痛发作至少1次；缺血性胸痛时间≤30min；心电图表现为ST段水平型或下斜型压低≥1mm或ST段抬高(肢体导联≥1mm，胸导联≥2mm)，发作后ST段又恢复到发作前的水平；无心肌坏死标记物的改变。1.2.4, NSTEMI and UA group: According to the World Health Organization UA diagnostic criteria: at least 1 angina pectoris within 48 hours before admission; ischemic chest pain time ≤ 30min; ECG showed ST segment horizontal or down oblique depression ≥ 1mm or ST segment elevation (limb lead ≥ 1mm, chest lead ≥ 2mm), ST segment returned to pre-onset level after onset; no changes in myocardial necrosis markers.
根据ACC/ESC诊断指南，NSTEMI的诊断标准：a:ST-T的动态衍变持续时间较长往往超过24h(一过性心肌缺血发作的ST-T改变常在数小时恢复)；b:胸痛持续至少半小时以上，符合心肌梗死的胸痛特点；c:血清酶学的改变符合心肌梗死的变化规律和(或)血清肌钙蛋白T或I升高不小于正常值的2倍以上，如有“a和c”或“b和c”两条，即可诊断为NSTEMI。According to the ACC/ESC diagnostic guidelines, the diagnostic criteria for NSTEMI: a: The dynamic progression of ST-T is often longer than 24 hours (ST-T changes in transient myocardial ischemic attacks often recover in a few hours); b: chest pain For at least half an hour, it is consistent with the characteristics of chest pain in patients with myocardial infarction; c: the change of serum enzymology is consistent with the change of myocardial infarction and/or the increase of serum troponin T or I is not less than 2 times of the normal value, if any "a and c" or "b and c" can be diagnosed as NSTEMI.
1.2.5、STEMI组：患者出现持续30分钟以上的典型胸痛症状；心电图出现动态演变过程，至少两个肢体导联ST段抬高≥0.1mv，或者胸导联ST段抬高≥0.2mv，或者新发的左束支传导阻滞；血清心肌酶有动态变化。1.2.5, STEMI group: patients with typical chest pain symptoms lasting more than 30 minutes; ECG dynamic evolution process, at least two limbs lead ST segment elevation ≥ 0.1mv, or chest lead ST segment elevation ≥ 0.2mv, Or a new left bundle branch block; serum myocardial enzymes have dynamic changes.
1.3、排除标准。1.3, exclusion criteria.
患者患有以下疾病：活动性炎性疾病；自身免疫病；严重的心衰；血流动力学不稳定；可疑心肌炎或心包炎；造血系统疾病；进展期肾脏或肝脏疾病；恶性肿瘤疾病；服用免疫抑制药物；既往曾行冠状动脉介入治疗或冠状动脉搭桥手术。The patient has the following diseases: active inflammatory disease; autoimmune disease; severe heart failure; hemodynamic instability; suspected myocarditis or pericarditis; hematopoietic diseases; advanced kidney or liver disease; malignant disease; Immunosuppressive drugs; previous coronary intervention or coronary artery bypass surgery.
1.4、实验流程。1.4. Experimental procedure.
1.4.1、胸痛患者就诊即刻抽取静脉血，按照入选标准、排除标准及冠状动脉造影结果入选研究对象共1050例，并分为对照组20例、UA组50例、NSTEMI组50例及STEMI组930例。1.4.1 Patients with chest pain immediately received venous blood. A total of 1050 patients were enrolled according to the inclusion criteria, exclusion criteria and coronary angiography results, and were divided into 20 patients in the control group, 50 patients in the UA group, 50 patients in the NSTEMI group, and the STEMI group. 930 cases.
1.4.2、从上述930例STEMI患者中选出发病小于2h的STEMI患者120例，并采集120例STEMI患者发病后30min、1h、2h、4h、6h、12h、24h、3d、7d及30d共10个时间点的血浆样品。同时将待检的血样送到检验科检测患者血浆 中S100A12、心肌酶hscTnT、CK-MB及肌红蛋白(Myoglobin，MYO)的浓度表达变化。1.4.2 Select 120 patients with STEMI who had less than 2 hours of onset from the above 930 STEMI patients, and collect 120 patients with STEMI 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 7 d, and 30 d after onset. Plasma samples at 10 time points. At the same time, the blood samples to be tested were sent to the laboratory for detection of changes in the expression levels of S100A12, myocardial enzymes hscTnT, CK-MB and myoglobin (MYO).
1.4.3、询问研究对象病史，对其进行常规体格检查及血常规、血液生化、血脂分析、肝肾功能等常规的临床检查。1.4.3, ask the patient's medical history, routine physical examination and routine clinical examination of blood routine, blood biochemistry, blood lipid analysis, liver and kidney function.
1.5、标本的采集及样本的处理。1.5. Collection of specimens and processing of samples.
1.5.1、标本的采集：入选1050例研究对象，入选后即刻抽取其静脉血，用含EDTA真空抗凝管收集血标本5ml；连续抽取120例发病小于2h STEMI患者的静脉血，采血时间点为STEMI发病30min、1h、2h、4h、6h、12h、24h、3d、7d、30d共10个时间点，每个时间点抽取两管血标本。1.5.1 Collection of specimens: 1050 subjects were enrolled. Immediately after enrollment, venous blood was drawn, and 5 ml of blood specimens were collected with EDTA vacuum anticoagulation tube; 120 consecutive venous blood samples from patients with STEMI less than 2 h were collected, and blood collection time was taken. For the STEMI, there were 10 time points at 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 7 d, 30 d, and two blood samples were taken at each time point.
1.5.2、样本的处理：抽取静脉血10分钟内，抗凝管3000转/min离心处理10min，分离血浆，将处理好的血浆储藏于-80℃冰箱保存，采用双抗体夹心酶联免疫试验法(ELISA法)检测血浆S100A12浓度；促凝管采集血标本，即刻送本院检验科，检测其hscTnT、CK-MB及MYO浓度。1.5.2. Treatment of the sample: The venous blood was taken for 10 minutes, the anticoagulation tube was centrifuged at 3000 rpm for 10 minutes, the plasma was separated, and the treated plasma was stored in a -80 ° C refrigerator, and the double antibody sandwich enzyme-linked immunosorbent assay was used. The method (ELISA method) was used to detect the concentration of plasma S100A12; the blood samples were collected by the coagulation tube, and immediately sent to the laboratory of the hospital to detect the concentration of hscTnT, CK-MB and MYO.
1.5.3、S100A12的测定：本研究所采用的S100A12 ELISA检测试剂盒购置于日本CircuLexTM公司，其组分如下(各组分的具体浓度和含量参见说明书)：洗涤缓冲液、稀释缓冲液、S100A12标准品、20×辣根过氧化酶、底物、终止试剂；其检测范围为0-5000ng/ml，检测灵敏度为56pg/ml，按样本说明书进行下述具体操作：建立标准曲线：按照试剂盒说明书配制S100A12标准品浓度，标准品浓度梯度分别为5000pg/ml、2500pg/ml、1250pg/ml、625pg/ml、313pg/ml、156pg/ml、78pg/ml、0pg/ml，根据说明书要求按浓度梯度依次加入标准品，每孔100μl。待测血浆标本稀释100倍后，取100μl加入反应板孔内，将反应板置于室温25℃1h后洗板。每孔加入酶标抗体工作液100μl，将反应板置于室温25℃1h后洗板。每孔加入底物工作液100μl，室温25℃避光孵育20min。每孔加入100μl终止液，混匀后，用450nm光波检测OD值。1.5.3. Determination of S100A12: The S100A12 ELISA kit used in this study was purchased from CircuLexTM, Japan. The components are as follows (see the instructions for the specific concentration and content of each component): washing buffer, dilution buffer, S100A12 Standard, 20× horseradish peroxidase, substrate, termination reagent; detection range is 0-5000 ng/ml, detection sensitivity is 56 pg/ml, according to the sample specification, the following specific operations are performed: establishing a standard curve: according to the kit The specification is to prepare the concentration of S100A12 standard. The concentration gradient of the standard is 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 313pg/ml, 156pg/ml, 78pg/ml, 0pg/ml, according to the requirements. The gradient was sequentially added to the standard, 100 μl per well. After the plasma sample to be tested was diluted 100 times, 100 μl was added to the well of the reaction plate, and the reaction plate was placed at room temperature for 25 hours and washed for 1 hour. 100 μl of the enzyme-labeled antibody working solution was added to each well, and the reaction plate was placed at room temperature for 25 hours and washed for 1 hour. 100 μl of the substrate working solution was added to each well, and the mixture was incubated at 25 ° C for 20 min at room temperature in the dark. 100 μl of the stop solution was added to each well, and after mixing, the OD value was measured with a 450 nm light wave.
1.5.4、血浆心肌酶谱的检测：用电化学发光法检测入选对象血浆hsTnT和 MYO浓度；用酶法检测入选对象血浆CK-MB活性；正常值范围为：TnT<0.05ng/ml；MYO 28-72ng/ml；CK-MB<25U/L。1.5.4. Detection of plasma myocardial zymogram: The plasma hsTnT and MYO concentrations of the selected subjects were detected by electrochemiluminescence method; the plasma CK-MB activity of the selected subjects was detected by enzymatic method; the normal range was: TnT<0.05ng/ml; MYO 28-72 ng/ml; CK-MB <25 U/L.
1.5.5、冠状动脉造影检查：入选的研究对象入院1周内在沈阳军区总医院心内科导管室接受冠状动脉造影检查。采用Judkins法，选择多体位行左右冠状动脉造影。根据冠心病诊断标准，心外膜下冠状动脉LM、LAD、LCX、RCA中任一支均无任何狭窄者视为完全正常，如直径狭窄小于50％为无意义病理病变，如直径狭窄大于50％为有病理意义的病变。由有经验的临床医师分别评估冠状狭窄程度及冠脉病变支数，分为单支病变、双支病变、三支病变。1.5.5. Coronary angiography: The selected subjects underwent coronary angiography in the Department of Cardiology, Shenyang Military Region General Hospital within 1 week after admission. The Judkins method was used to select left and right coronary angiography in multiple positions. According to the diagnostic criteria for coronary heart disease, any of the subdural coronary artery LM, LAD, LCX, RCA without any stenosis is considered completely normal, such as diameter stenosis less than 50% for non-significant pathological lesions, such as diameter stenosis greater than 50 % is a pathologically significant lesion. The degree of coronary stenosis and the number of coronary lesions were evaluated by experienced clinicians, and were divided into single-vessel disease, double-vessel disease, and three-vessel disease.
1.5.6、急诊经皮冠状动脉介入治疗(PCI)及PCI前后用药：急诊PCI时，原则上只干预梗死相关血管，对相关病变植入药物洗脱支架(DES)或金属裸支架。所有拟行急诊PCI的患者术前嚼服阿司匹林300mg、氯吡格雷300-600mg；首先给予5000U的肝素，造影结束后如行PCI手术需要按照70-100U/kg补足用量，术后均转入冠心病监护室进行监护治疗，给予皮下注射低分子肝素，每12h皮下给予1次(5-7d)；服用氯吡格雷75mg(1次/d)，并长期服用阿司匹林100mg(1次/d)。所有入选的研究对象均严格按照指南要求进行冠心病的二级预防。1.5.6, emergency percutaneous coronary intervention (PCI) and before and after PCI: In emergency PCI, in principle only intervention in infarct-related blood vessels, implanted drug-eluting stent (DES) or bare metal stent. All patients who were scheduled for emergency PCI were given aspirin 300mg and clopidogrel 300-600mg before surgery; firstly, 5000U heparin was given. After the end of angiography, if PCI was performed, 70-100 U/kg should be used to make up the amount. The cardio-intensive care unit was monitored and given subcutaneous low-molecular-weight heparin, subcutaneously once every 12 hours (5-7d); clopidogrel 75mg (1 time/d), and long-term use of aspirin 100mg (1 time / d). All selected subjects were treated with secondary prevention of coronary heart disease in strict accordance with the guidelines.
2、免疫组化方法检测人冠状动脉组织及STEMI患者罪犯血管血栓抽吸标本S100A12的表达变化。2. Immunohistochemical method was used to detect the expression changes of vascular thrombus aspiration specimen S100A12 in human coronary artery tissue and STEMI patients.
2.1、正常人冠状动脉和心源性猝死患者的冠状动脉标本的收集。2.1. Collection of coronary specimens from normal coronary arteries and sudden cardiac death patients.
收集无CAD病史，因意外死亡的年轻尸检者冠状动脉3例；同时收集生前有CAD病史，心源性猝死患者的冠状动脉标本3例；冠状动脉均在左冠状动脉前降支根部取得，约1.5cm，截取为3段，每段约0.5cm；所有的冠状动脉均放入10％多聚甲醛中固定；入选样本均需经过沈阳军区总医院伦理委员会同意，并签署知情同意书。Three patients with coronary artery disease who had no history of CAD and died of accidental death were collected. At the same time, 3 cases of coronary artery specimens with history of CAD and sudden cardiac death were collected. The coronary arteries were obtained from the root of the left anterior descending coronary artery. 1.5cm, cut into 3 segments, each segment is about 0.5cm; all coronary arteries are fixed in 10% paraformaldehyde; all samples must be approved by the Ethics Committee of Shenyang Military Region General Hospital and signed informed consent.
2.2、抽吸导管技术收集STEMI患者罪犯冠状动脉内的血栓组织。2.2, aspiration catheter technology to collect thrombosis in the coronary artery of STEMI patients.
入选STEMI患者后，首先行冠状动脉造影明确患者的罪犯病变血管，在导丝的指引下将血栓抽吸导管送至病变部位，反复抽吸4-6次直至血栓消失，血栓抽 吸完成后直接在病变血管中置入支架，将一部分血栓组织放入4％多聚甲醛溶液中固定；另外一部分放至1.5ml EP管中，-80℃保存备用。入选样本均需经过沈阳军区总医院伦理委员会同意，并签署知情同意书。After enrolling in STEMI patients, coronary angiography was performed to confirm the patient's diseased blood vessels. The thrombus aspiration catheter was delivered to the lesion under the guidance of the guide wire, and repeated suction for 4-6 times until the thrombus disappeared. A stent was placed in the diseased blood vessel, and a part of the thrombus tissue was fixed in a 4% paraformaldehyde solution; the other part was placed in a 1.5 ml EP tube and stored at -80 ° C until use. All samples must be approved by the Ethics Committee of the Shenyang Military Region General Hospital and signed informed consent.
2.3、体外实验制备STEMI患者非罪犯冠状动脉的血凝块。2.3. Preparation of blood clots in non-criminal coronary arteries of STEMI patients in vitro.
(1)收集冠脉造影阴性对照组的动脉血；同时收集STEMI患者非罪犯冠状动脉的动脉血2ml。(1) Collecting arterial blood from the negative control group of coronary angiography; and collecting 2 ml of arterial blood of non-criminal coronary artery in STEMI patients.
(2)将500μl冠状动脉血加到1.5ml EP管中，加入20μl凝血酶溶液，充分混匀。(2) 500 μl of coronary blood was added to a 1.5 ml EP tube, and 20 μl of thrombin solution was added and mixed well.
(3)室温放置2h，观察血凝块形成情况。(3) Place at room temperature for 2 hours to observe the formation of blood clots.
(4)将一部分冠状动脉血凝块放到4％多聚甲醛溶液中固定，另外一部分放至1.5ml EP管中，-80℃保存备用。(4) A part of the coronary blood clot was placed in a 4% paraformaldehyde solution, and the other part was placed in a 1.5 ml EP tube and stored at -80 °C until use.
2.4、石蜡切片的制备。2.4. Preparation of paraffin sections.
取材：将STEMI患者血栓组织及血凝块组织放置4％多聚甲醛溶液中过夜，并修剪组织。Materials: The thrombotic tissue and blood clot tissue of STEMI patients were placed in a 4% paraformaldehyde solution overnight, and the tissue was trimmed.
脱水：具体顺序如下：70％酒精2h-80％酒精2h-90％酒精2h-95％酒精I中4h-95％酒精II中过夜-100％酒精I中1.5h-100％酒精II中1.5h。Dehydration: The specific order is as follows: 70% alcohol 2h-80% alcohol 2h-90% alcohol 2h-95% alcohol I 4h-95% alcohol II overnight -100% alcohol I 1.5h-100% alcohol II 1.5h .
透明：将组织块放入二甲苯I中浸泡1h，取出后放入二甲苯II中浸泡1h。Transparent: The tissue block was placed in xylene I for 1 h, taken out and placed in xylene II for 1 h.
浸蜡：具体顺序如下：石蜡I中过夜-石蜡II中1h-石蜡III中1h。Dipping wax: The specific order is as follows: 1 h in paraffin I - 1 h in paraffin II 1 hour in paraffin III.
包埋：应用石蜡对组织块进行包埋，室温放置。Embedding: The tissue block is embedded with paraffin and placed at room temperature.
切片：使用石蜡切片机对组织块进行切片，厚度为3μm，贴于载玻片上。Sectioning: The tissue block was sectioned using a paraffin slicer to a thickness of 3 μm and attached to a glass slide.
烘片及烤片：将载玻片置于60℃烘片机上，放置1h；之后将载玻片放于65℃烤箱中，放置48h。Bake and baking sheets: The slides were placed on a 60 ° C dryer for 1 h; the slides were placed in a 65 ° C oven and allowed to stand for 48 h.
2.5、组织HE染色。2.5, tissue HE staining.
切片脱蜡：具体顺序如下：二甲苯I中20min-二甲苯II中20min-95％酒精I中15min-95％酒精II中15min-90％酒精10min-80％酒精5min-70％酒精5min-蒸馏水30min。Section dewaxing: The specific sequence is as follows: xylene I in 20min-xylene II in 20min-95% alcohol I in 15min-95% alcohol II in 15min-90% alcohol 10min-80% alcohol 5min-70% alcohol 5min-distilled water 30min.
细胞核染色：将石蜡切片放置苏木素中染色10min，流水冲洗。Nuclear staining: Paraffin sections were placed in hematoxylin for 10 min and rinsed with running water.
分化：将石蜡切片放于1％盐酸酒精中进行分化30s，流水冲洗。Differentiation: Paraffin sections were placed in 1% hydrochloric acid for differentiation for 30 s, rinsed with running water.
返蓝：将石蜡切片放于氨水中进行细胞核返蓝30s，流水冲洗。Return to the blue: Put the paraffin section in ammonia water for the core to return to the blue for 30s, rinse with water.
细胞质染色：将石蜡切片放于水溶性伊红溶液中染色5min，流水冲洗。Cytoplasmic staining: Paraffin sections were stained in water-soluble eosin solution for 5 min, rinsed with running water.
透明：将石蜡切片放于80％酒精5min-90％酒精5min-100酒精I中5min-100％酒精II中5min-二甲苯I中5min-二甲苯II中5min。Transparent: Paraffin sections were placed in 80% alcohol 5min-90% alcohol 5min-100 alcohol I in 5min-100% alcohol II in 5min-xylene I in 5min-xylene II for 5min.
封片：将石蜡切片放于通风橱中晾干，应用中性树脂进行封片。Cover: The paraffin sections were placed in a fume hood to dry and sealed with a neutral resin.
2.6、组织免疫组织化学染色。2.6, tissue immunohistochemical staining.
(1)石蜡切片脱蜡：同HE染色。(1) Paraffin section dewaxing: same as HE staining.
(2)抗原修复：将石蜡切片放于200ml抗原修复液中，100℃煮40min，自然降温。(2) Antigen retrieval: Paraffin sections were placed in 200 ml of antigen-repairing solution, boiled at 100 ° C for 40 min, and naturally cooled.
(3)滴加50μl试剂A至石蜡切片上，室温孵育10min，PBS冲洗3次/5min。(3) Add 50 μl of reagent A to paraffin sections, incubate for 10 min at room temperature, and rinse 3 times for 5 min in PBS.
(4)滴加50μl试剂B至石蜡切片上，室温孵育10min，去除血清。(4) 50 μl of reagent B was added dropwise to paraffin sections, and incubated for 10 min at room temperature to remove serum.
(5)滴加50μl稀释后的一抗溶液至石蜡切片上(1:100稀释，用PBS进行稀释)，4℃孵育过夜。(5) 50 μl of the diluted primary antibody solution was added dropwise to the paraffin section (diluted 1:100, diluted with PBS), and incubated overnight at 4 °C.
(6)次日将石蜡切片在室温条件下进行复温30min；PBS冲洗3次/5min。(6) Paraffin sections were rewarmed for 30 min at room temperature on the next day; PBS was washed 3 times/5 min.
(7)滴加50μl试剂C溶液至石蜡切片上(1:100稀释，用PBS进行稀释)，室温孵育10min；用PBS冲洗3次/5min。(7) Add 50 μl of reagent C solution to paraffin sections (diluted 1:100, diluted with PBS), incubate for 10 min at room temperature; rinse 3 times with PBS for 5 min.
(8)滴加50μl试剂D溶液至石蜡切片上，室温孵育10min；用PBS冲洗3次/5min。(8) Add 50 μl of reagent D solution to paraffin sections, incubate for 10 min at room temperature; rinse 3 times with PBS for 5 min.
(9)应用DAB溶液进行显色，显微镜下观察染色效果。(9) The DAB solution was used for color development, and the staining effect was observed under a microscope.
(10)细胞核染色，步骤同HE染色。(10) Nuclear staining, the procedure is the same as HE staining.
3、统计分析方法。3. Statistical analysis methods.
采用SPSS 19.0软件包进行统计分析；统计检验均采用双侧检验，检验水准为0.05；对各组的人口学信息和临床变量进行统计；定量指标采用均数、标准差表示；采用成组t检验或单变量ANOVA进行组间比较；定性指标采用例数和百分数表示；采用卡方检验或确切概率检验进行组间比较；依变量发生时间定义为：如发生终点事件，时间为STEMI发病时间至终点事件发生的时间。多因 素Logistic回归分析血浆S100A12浓度在STEMI发病中的危险度。ROC曲线分析血浆S100A12浓度诊断STEMI的最佳临界值、敏感性及特异性。两组间比较，均采用双侧检验(two-side test)，取α＝0.05；P≤0.05者，认为有统计学意义；所有可信区间的可信度采用95％。Statistical analysis was performed using SPSS 19.0 software package; the statistical test was performed by double-sided test, the test level was 0.05; the demographic information and clinical variables of each group were counted; the quantitative indicators were expressed by mean and standard deviation; using group t test Or univariate ANOVA for comparison between groups; qualitative indicators are expressed by number of cases and percentages; chi-square test or exact probability test is used for comparison between groups; time of occurrence of dependent variable is defined as: when an end point event occurs, time is STEMI onset time to end point The time when the event occurred. Multivariate logistic regression analysis of the risk of plasma S100A12 concentration in the pathogenesis of STEMI. ROC curve analysis of plasma S100A12 concentration to determine the optimal threshold, sensitivity and specificity of STEMI. Comparison between the two groups was performed by two-side test, taking α=0.05; P≤0.05, which was considered statistically significant; 95% of the confidence of all confidence intervals was used.
4、实验结果。4. Experimental results.
4.1、正常对照组、UA组、NSTEMI组及STEMI组研究对象临床基线资料及血浆S100A12浓度的比较：比较四组入选研究对象临床基线资料显示，年龄、性别、体重、血压、心率、近期吸烟史、糖尿病史、卒中病史、心梗病史、载脂蛋白A、甘油三酯、总胆固醇、低密度脂蛋白胆固醇、血尿素氮、肌酐、血小板计数、血糖、hsCRP、pro-BNP在四组间的差异无统计学意义(P>0.05)。而高血压史、PCI史、高密度脂蛋白胆固醇、白细胞计数、CK-MB峰值、hscTnT峰值及血浆S100A12浓度在四组研究对象间存在明显差异，具有统计学意义(P<0.05)，详见表1。4.1. Comparison of clinical baseline data and plasma S100A12 concentration in the normal control group, UA group, NSTEMI group and STEMI group: Compare the clinical baseline data of the four selected subjects, age, sex, weight, blood pressure, heart rate, recent smoking history History of diabetes, history of stroke, history of myocardial infarction, apolipoprotein A, triglycerides, total cholesterol, low density lipoprotein cholesterol, blood urea nitrogen, creatinine, platelet count, blood glucose, hsCRP, pro-BNP among the four groups The difference was not statistically significant (P>0.05). Hypertension history, PCI history, high-density lipoprotein cholesterol, white blood cell count, CK-MB peak, hscTnT peak and plasma S100A12 concentration were significantly different among the four groups (P<0.05). Table 1.
表1 对照组、UA组、NSTEMI组及STEMI组一般临床基线情况。Table 1 General clinical baseline status of the control group, UA group, NSTEMI group and STEMI group.
注：1.连续正态分布变量用均数±标准差表示；2.分类变量用例数(百分数％)表示；3.UA：不稳定性心绞痛；4.NSTEMI：急性非ST段抬高型心肌梗死；5.STEMI：急性ST段抬高型心肌梗死；6.MI：心肌梗死；7.PCI：经皮冠状动脉介入治疗；8.ApoA：载脂蛋白A；9.TG：甘油三酯；10.TC：总胆固醇；11.LDL-C：低密度脂蛋白胆固醇；12.HDL-C：高密度脂蛋白胆固醇；13.BUN：尿素氮；14.Crea：肌酐；15.WBC：白细胞计数；16.PLT：血小板计数；17.CK-MB：肌酸激酶同工酶；18.hscTnT：超敏肌钙蛋白T；19.hs-CRP：超敏C反应蛋白；20.pro-BNP：脑利钠肽前体。Note: 1. Continuous normal distribution variables are expressed as mean ± standard deviation; 2. categorical variable use cases (%); 3. UA: unstable angina; 4. NSTEMI: acute non-ST-segment elevation myocardial Infarction; 5. STEMI: acute ST-segment elevation myocardial infarction; 6. MI: myocardial infarction; 7. PCI: percutaneous coronary intervention; 8. ApoA: apolipoprotein A; 9. TG: triglyceride; 10.TC: total cholesterol; 11. LDL-C: low density lipoprotein cholesterol; 12. HDL-C: high density lipoprotein cholesterol; 13. BUN: urea nitrogen; 14. Crea: creatinine; 15. WBC: white blood cell count ; 16. PLT: platelet count; 17. CK-MB: creatine kinase isoenzyme; 18. hscTnT: hypersensitive troponin T; 19. hs-CRP: hypersensitive C-reactive protein; 20. pro-BNP: Brain natriuretic peptide precursor.
结果表明，随着心肌损伤程度的加重，白细胞计数、CK-MB峰值、hsTnT峰值及血浆S100A12浓度逐渐升高；具有高血压史、PCI史的病例数逐渐增多；高密度脂蛋白胆固醇浓度则逐渐降低，说明这些因素与心肌损伤坏死的严重程度有一定相关关系。The results showed that with the increase of myocardial damage, white blood cell count, CK-MB peak, hsTnT peak and plasma S100A12 concentration gradually increased; the number of cases with history of hypertension and PCI increased gradually; the concentration of high-density lipoprotein cholesterol gradually increased. Decrease, indicating that these factors are related to the severity of myocardial injury and necrosis.
将STEMI作为因变量，以高血压史、PCI病史、HDL-C、白细胞计数、CK-MB峰值、hscTnT峰值及血浆S100A12浓度作为协变量进行Logistic回归分析，分析结果提示STEMI患者血浆中S100A12浓度(OR＝2.007，P＝0.009)及hscTnT峰值(OR＝3.712，P＝0.034)是STEMI发病的独立危险因素，见表2。Using STEMI as a dependent variable, logistic regression analysis was performed with hypertension history, history of PCI, HDL-C, white blood cell count, CK-MB peak, hscTnT peak and plasma S100A12 concentration as covariates. The results showed that plasma S100A12 concentration in STEMI patients ( OR=2.007, P=0.009) and hscTnT peak (OR=3.712, P=0.034) were independent risk factors for STEMI incidence, as shown in Table 2.
表2.二元Logistic回归分析与STEMI相关的危险因素。Table 2. Binary logistic regression analysis of risk factors associated with STEMI.
| OR值OR value | 95％CI95% CI | P值P value | |
| S100A12S100A12 | 2.0072.007 | 2.002-2.0122.002-2.012 | 0.0090.99 |
| hscTnThscTnT | 3.7123.712 | 1.105-12.4721.105-12.472 | 0.0340.034 |
利用Spearman’s rho分析显示STEMI患者血浆中的S100A12表达水平与血浆中hscTnT浓度存在相关关系(OR＝0.356，P＝0.000)，上述分析显示STEMI患者血浆中的S100A12浓度增高是STEMI发生的独立危险因素。Spearman's rho analysis showed that there was a correlation between the expression of S100A12 in plasma and the concentration of hscTnT in plasma (OR=0.356, P=0.000). The above analysis showed that the increase of S100A12 concentration in plasma of STEMI patients was an independent risk factor for STEMI.
结果显示，入选的对照组、UA组、NSTEMI组及STEMI组研究对象血浆中的S100A12浓度分别为135±75ng/ml、112±80ng/ml、144±70ng/ml及490±271ng/ml。STEMI组研究对象血浆中S100A12浓度明显高于正常对照组(P<0.000)、UA组(P<0.000)及NSTEMI组(P<0.000)，而在对照组、UA组及NSTEMI组3组间血浆S100A12浓度无统计学差异，见图1，说明S100A12浓度变化与STEMI的发生存在相关关系。The results showed that the concentrations of S100A12 in the selected control group, UA group, NSTEMI group and STEMI group were 135±75 ng/ml, 112±80 ng/ml, 144±70 ng/ml and 490±271 ng/ml, respectively. Plasma concentrations of S100A12 in the STEMI group were significantly higher than those in the normal control group (P<0.000), UA group (P<0.000), and NSTEMI group (P<0.000), while plasma in the control group, UA group, and NSTEMI group. There was no statistical difference in the concentration of S100A12, as shown in Figure 1, indicating that there is a correlation between the concentration of S100A12 and the occurrence of STEMI.
4.2、血浆S100A12诊断STEMI的最佳临界值：通过比较930例STEMI组患者与正常对照组、UA组、NSTEMI组患者血浆中的S100A12浓度变化，确定何种血浆S100A12浓度为诊断STEMI的最佳临界值。4.2, plasma S100A12 diagnosis of STEMI optimal threshold: by comparing 930 STEMI patients with normal control group, UA group, NSTEMI group of plasma S100A12 concentration changes, determine which plasma S100A12 concentration is the best critical for the diagnosis of STEMI value.
以正常对照组、UA组、NSTEMI组研究对象作为阴性参考群体，930例STEMI患者作为阳性参考群体，绘制STEMI患者血浆S100A12浓度的ROC曲线，见图2。The normal control group, UA group, NSTEMI group were used as the negative reference group, and 930 STEMI patients were used as the positive reference group to plot the ROC curve of plasma S100A12 concentration in STEMI patients, as shown in Figure 2.
结果显示，血浆中的S100A12浓度为263.9ng/ml时，可作为诊断STEMI的最佳临界值，血浆中的S100A12诊断STEMI发生的特异性为81.7％、敏感性为87％、AUC为0.904，说明在以血浆中S100A12的浓度为263.9ng/ml作为最佳临界值时，其诊断STEMI的诊断效能较高。The results showed that when the concentration of S100A12 in plasma was 263.9 ng/ml, it could be used as the optimal threshold for the diagnosis of STEMI. The specificity of S100A12 in plasma for the diagnosis of STEMI was 81.7%, the sensitivity was 87%, and the AUC was 0.904. When the concentration of S100A12 in plasma is 263.9 ng/ml as the optimal threshold, the diagnostic efficiency of STEMI is higher.
4.3、STEMI发生时，血浆中S100A12及其他生物标记物的时相性改变：收集STEMI发病至就诊时间小于2h的患者共120例，入选STEMI患者的基本临床资料，见表3。检测STEMI患者发病后不同时间点(30min、1h、2h、4h、6h、12h、24h、3d、7d、30d)血浆S100A12及心肌标记物(MYO、CK-MB及hscTnT)浓度及时相性的改变，见图3。4.3. When STEMI occurs, the temporal changes of S100A12 and other biomarkers in plasma: 120 patients with STEMI from the time of onset to less than 2 hours, the basic clinical data of STEMI patients, see Table 3. To detect the changes of plasma S100A12 and myocardial markers (MYO, CK-MB and hscTnT) concentrations at different time points (30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 7 d, 30 d) after STEMI. See Figure 3.
表3. 120例发病2h内STEMI患者一般临床基线情况。Table 3. General clinical baseline of 120 patients with STEMI within 2 hours of onset.
| 变量variable | STEMI(N＝120)STEMI (N=120) | 变量variable | STEMI(N＝120)STEMI (N=120) |
| 年龄,(y)Age, (y) | 56±1256±12 | ApoA mg/LApoA mg/L | 204±195204±195 |
| 男性,[n(％)]Male, [n(%)] | 98(82)98(82) | TG mmol/dlTG mmol/dl | 1.4±1.41.4±1.4 |
| 体重,(kg)Weight, (kg) | 72±1172±11 | TC mmol/dlTC mmol/dl | 4.9±1.04.9±1.0 |
| 血压(mmHg)Blood pressure (mmHg) | 127±21127±21 | LDL-C mmol/dlLDL-C mmol/dl | 3.2±0.83.2±0.8 |
| 心率(bpm)Heart rate (bpm) | 83±1883±18 | HDL-C mmol/dlHDL-C mmol/dl | 1.1±0.21.1±0.2 |
| 病史,[n(％)]Medical history, [n(%)] | -- | 血糖mmol/dlBlood sugar mmol/dl | 8.6±3.88.6±3.8 |
| 近期吸烟史Recent smoking history | 77(64)77(64) | BUN mmol/dlBUN mmol/dl | 5.7±1.55.7±1.5 |
| 糖尿病病史History of diabetes | 12(10)12(10) | Crea umol/dlCrea umol/dl | 72.8±21.972.8±21.9 |
| 高血压病史History of hypertension | 58(48)58(48) |
WBC 10
9/L
|
12.1±3.512.1±3.5 |
| 卒中病史History of stroke | 10(8)10(8) |
PLT 10
9/L
|
213±47213±47 |
| MI病史History of MI | 5(4)5(4) | CK-MB峰值U/LCK-MB peak U/L | 309±204309±204 |
| PCI史PCI history | 10(8)10(8) | hsTnT峰值ng/mlhsTnT peak ng/ml | 5.6±3.25.6±3.2 |
| 生化指标Biochemical Indicators | -- | hs-CRP ng/mlhs-CRP ng/ml | 2.5±3.02.5±3.0 |
| -- | -- | pro-BNP pg/mlpro-BNP pg/ml | 149±149149±149 |
注：1.连续正态分布变量用均数±标准差表示；2.分类变量用例数(百分数％)表示；3.STEMI：急性ST段抬高型心肌梗死；4.MI：心肌梗死；5.PCI：经皮冠状动脉介入治疗；6.ApoA：载脂蛋白A；7.TG：甘油三酯；8.TC：总胆固醇；9.LDL-C：低密度脂蛋白胆固醇；10.HDL-C：高密度脂蛋白胆固醇；11.BUN：尿素氮；12.Crea：肌酐；13.WBC：白细胞计数；14.PLT：血小板计数；15.CK-MB：肌酸激酶同工酶；16.hscTnT：超敏肌钙蛋白T；17.hs-CRP：超敏C反应蛋白；18.pro-BNP：脑利钠肽前体。Note: 1. Continuous normal distribution variables are expressed as mean ± standard deviation; 2. categorical variable use cases (%); 3. STEMI: acute ST-segment elevation myocardial infarction; 4. MI: myocardial infarction; . PCI: percutaneous coronary intervention; 6. ApoA: apolipoprotein A; 7. TG: triglyceride; 8. TC: total cholesterol; 9. LDL-C: low density lipoprotein cholesterol; 10. HDL- C: high density lipoprotein cholesterol; 11. BUN: urea nitrogen; 12. Crea: creatinine; 13. WBC: white blood cell count; 14. PLT: platelet count; 15. CK-MB: creatine kinase isoenzyme; hscTnT: hypersensitive troponin T; 17.hs-CRP: hypersensitive C-reactive protein; 18. pro-BNP: brain natriuretic peptide precursor.
结果发现，发现在入选的STEMI患者中，S100A12在STEMI发病30min血浆中的浓度明显增高，发病1h-2h其血浆中S100A12浓度达到峰值，可持续至发病10h，而后S100A12血浆浓度开始下降，直到发病3d血浆S100A12浓度基本降至正常；MYO浓度在STEMI发病的1h-2h左右增高，发病4h其血浆中MYO浓度达到峰值，可持续至发病12h，发病24h基本降至正常范围；CK-MB在STEMI发病4h血浆中的浓度明显增高，发病10h-12h其血浆中CK-MB浓度达到峰值，可持续至发病24h，而后CK-MB血浆浓度开始下降，直到发病3d 血浆CK-MB浓度基本降至正常；hscTnT在STEMI发病1h-2h血浆中的浓度明显增高，发病12h-24h血浆hscTnT浓度达到峰值，可持续至发病7d-10d，而后hscTnT血浆浓度开始下降，直到发病30d血浆MYO浓度基本降至正常。The results showed that in the selected STEMI patients, the concentration of S100A12 in the plasma of STEMI 30min was significantly increased. The concentration of S100A12 in plasma reached the peak at 1h-2h, which lasted for 10h, and the plasma concentration of S100A12 began to decrease until the onset. The concentration of plasma S100A12 decreased to normal in 3d; the concentration of MYO increased in the range of 1h-2h after the onset of STEMI, and the concentration of MYO in plasma reached the peak at 4h after the onset of disease, which lasted for 12h, and the pathogenesis decreased to the normal range at 24h; CK-MB was in STEMI. The concentration of plasma in the 4h onset was significantly increased. The concentration of CK-MB in plasma reached the peak at 10h-12h, and it lasted for 24h. Then the plasma concentration of CK-MB began to decrease until the plasma CK-MB concentration decreased to normal after 3d. The concentration of hscTnT in plasma from 1 h to 2 h after STEMI was significantly increased. The concentration of plasma hscTnT peaked at 12h-24h, which lasted for 7d-10d, and then the plasma concentration of hscTnT began to decrease until the plasma MYO concentration decreased to normal after 30 days. .
在STEMI发病的早期，血浆S100A12浓度升高较经典的心肌酶学标志物出现的更早。以血浆中S100A12浓度263.9ng/ml作为STEMI的最佳诊断临界值(MYO、CK-MB及hscTnT诊断STEMI的最佳界值分别为72ng/ml、25U/L、0.05ng/ml)。以入选的正常对照组、UA组、NSTEMI组研究对象作为阴性参考群体，STEMI患者作为阳性参考群体，分别绘制STEMI患者发病后30min、1h、2h、4h、6h血浆S100A12、MYO、CK-MB及hscTnT的ROC曲线，见图4和图5。In the early stages of STEMI, plasma S100A12 concentrations increased earlier than classical myocardial enzymology markers. The plasma S100A12 concentration of 263.9 ng/ml was used as the optimal diagnostic threshold for STEMI (the optimal cutoff values of STYO for MYO, CK-MB and hscTnT were 72 ng/ml, 25 U/L, 0.05 ng/ml, respectively). The selected normal control group, UA group and NSTEMI group were used as the negative reference group, and STEMI patients as the positive reference group were drawn to the plasma S100A12, MYO, CK-MB and 30 min, 1 h, 2 h, 4 h, 6 h after STEMI. The ROC curve of hscTnT is shown in Figure 4 and Figure 5.
通过ROC曲线分析结果显示，在STEMI发病后30min，血浆S100A12、MYO、CK-MB及hscTnT诊断STEMI的敏感性分别为76.3％、35％、1.8％、30.3％，特异性分别为84％、21％、80.4％、59.3％；在STEMI发病后1h，血浆S100A12、MYO、CK-MB及hscTnT诊断STEMI的敏感性分别为80％、39％、10％、33％，特异性分别为85.3％、23％、82.6％、58.6％，均以S100A12为最高；在STEMI发病后2h，血浆S100A12、MYO、CK-MB及hsTnT诊断STEMI的敏感性分别为80％、61％、14.5％、39.7％，特异性分别为85.5％、24％、84.2％、59.6％，均以S100A12为最高；在STEMI发病后4h，血浆中的S100A12、MYO、CK-MB及hscTnT诊断STEMI的敏感性分别为81.8％、78％、33％、61％，特异性分别为82.1％、40％、80％、59.6％，均以S100A12为最高；在STEMI发病后6h，血浆中的S100A12、MYO、CK-MB及hscTnT诊断STEMI的敏感性分别为82％、92.5％、72.2％、86.7％，特异性分别为84％、56％、80.9％、60％，S100A12的综合诊断优势仍为最高。上述研究结果显示在STEMI发病小于2h内，尤其在STEMI发病极早期(<30min)，S100A12诊断STEMI的敏感性明显优于经典的心肌生化标注物，特异性与经典的心肌酶学标志物相当；而在STEMI发病4h、6h这两个时间点，S100A12诊断STEMI的敏感性和特异性均与经典的心肌酶学标志物相当。The ROC curve analysis showed that the sensitivity of plasma S100A12, MYO, CK-MB and hscTnT in the diagnosis of STEMI was 76.3%, 35%, 1.8%, 30.3%, respectively, and the specificity was 84%, 21% after STEMI. %, 80.4%, 59.3%; 1 hour after the onset of STEMI, the sensitivity of plasma S100A12, MYO, CK-MB and hscTnT in the diagnosis of STEMI were 80%, 39%, 10%, 33%, respectively, and the specificity was 85.3%, respectively. 23%, 82.6%, 58.6%, S100A12 was the highest; 2 hours after the onset of STEMI, the sensitivity of plasma S100A12, MYO, CK-MB and hsTnT in the diagnosis of STEMI were 80%, 61%, 14.5%, 39.7%, respectively. The specificities were 85.5%, 24%, 84.2%, and 59.6%, respectively. S100A12 was the highest. At 4 hours after STEMI, the sensitivity of S100A12, MYO, CK-MB and hscTnT in the diagnosis of STEMI was 81.8%. 78%, 33%, 61%, specificity were 82.1%, 40%, 80%, 59.6%, respectively, with S100A12 as the highest; 6h after STEMI onset, plasma S100A12, MYO, CK-MB and hscTnT diagnosis The sensitivity of STEMI was 82%, 92.5%, 72.2%, 86.7%, and the specificity was 84%, 56 respectively. %, 80.9%, 60%, the comprehensive diagnostic advantage of S100A12 is still the highest. The above findings show that the STEMI sensitivity of S100A12 is significantly better than that of classical myocardial biochemical markers, which is comparable to classical myocardial enzymology markers, in patients with STEMI less than 2 hours, especially in the early stage of STEMI (<30 min); At the two time points of STEMI at 4h and 6h, the sensitivity and specificity of S100A12 in the diagnosis of STEMI were comparable to those of classical myocardial enzymology markers.
免疫组化检测正常人冠状动脉和心源性猝死患者的冠状动脉组织中S100A12的表达：HE染色结果显示，正常的冠状动脉血管中S100A12的表达水平较低，在心源性猝死患者的冠状动脉血管组织中可以看到S100A12的表达明显高于正常的冠状动脉血管组织标本。在猝死患者冠状动脉粥样硬化不稳定斑块破裂口周围、粥样硬化不稳定斑块薄的纤维帽下的血管壁组织中S100A12大量表达，故在急性心肌梗死发生即刻斑块组织中的S100A12大量释放到患者的血浆中，因此，在急性心肌梗死发病的极早期，由于斑块组织破裂导致S100A12的大量释放，导致急性心肌梗死患者血清中S100A12表达增高，见图6。Immunohistochemical detection of S100A12 expression in coronary arteries of normal subjects with coronary artery and sudden cardiac death: HE staining results showed that the expression level of S100A12 in normal coronary vessels was low, and coronary artery vessels in patients with sudden cardiac death The expression of S100A12 was significantly higher in tissues than in normal coronary vascular tissue specimens. S100A12 is abundantly expressed in the vascular wall tissue under the fibrous cap of the atherosclerotic unstable plaque, and the S100A12 in the immediate plaque tissue occurs in the acute myocardial infarction. A large amount is released into the patient's plasma. Therefore, in the very early stage of acute myocardial infarction, the release of S100A12 due to rupture of plaque tissue leads to an increase in the expression of S100A12 in the serum of patients with acute myocardial infarction, as shown in Fig. 6.
免疫组织化学检测S100A12在STEMI患者罪犯冠状动脉内血栓组织的表达：HE染色结果提示，罪犯冠状动脉内血栓组织的成分主要包括红细胞、炎症细胞、血小板和纤维蛋白等成分，而体外凝集的非罪犯冠状动脉血凝块的成分主要为红细胞和纤维蛋白，血小板及炎症细胞的含量较少。免疫组织化学发现CCL2及CCR2在STEMI患者罪犯冠状动脉内血栓组织的表达明显高于自身冠状动脉血凝块。在急性心肌梗死的患者的冠状动脉血栓中S100A12的表达明显高于正常血凝块标本、自身非罪犯冠状动脉血凝块组织，说明在急性心肌梗死的极早期，冠状动脉粥样硬化不稳定斑块破裂释放大量的S100A12入血导致在急性心肌梗死的极早期，在急性心肌梗死患者的血清中检测到S100A12的高表达，免疫组织化学显示STEMI患者罪犯冠状动脉内血栓组织标本的中性粒细胞(MPO阳性)中的S100A12表达明显增高，见图7。Immunohistochemical detection of S100A12 expression in coronary artery thrombosis in STEMI patients: HE staining results suggest that the components of the thrombus in the coronary artery include red blood cells, inflammatory cells, platelets and fibrin, and non-criminal agglutination in vitro. The components of coronary blood clots are mainly red blood cells and fibrin, and the content of platelets and inflammatory cells is small. Immunohistochemistry showed that the expression of CCL2 and CCR2 in coronary artery thrombosis of patients with STEMI was significantly higher than that of their own coronary blood clots. The expression of S100A12 in coronary thrombosis in patients with acute myocardial infarction was significantly higher than that in normal blood clot specimens and in non-criminal coronary clots, indicating that coronary atherosclerosis was unstable in the very early stage of acute myocardial infarction. Block rupture releases a large amount of S100A12 into the bloodstream. In the very early stage of acute myocardial infarction, high expression of S100A12 was detected in the serum of patients with acute myocardial infarction. Immunohistochemistry showed neutrophils in the specimens of coronary artery thrombosis in STEMI patients. The expression of S100A12 in (MPO positive) was significantly increased, as shown in Figure 7.
Claims (4)
- 血浆S100A12在ST段抬高型心肌梗死早期诊断中的应用。Application of plasma S100A12 in the early diagnosis of ST-segment elevation myocardial infarction.
- 如权利要求1所述的S100A12蛋白在ST段抬高型心肌梗死早期诊断中的应用，其特征在于，所述的S100A12蛋白可以作为ST段抬高型心肌梗死早期诊断的生物标志物。The use of the S100A12 protein according to claim 1 for early diagnosis of ST-segment elevation myocardial infarction, characterized in that the S100A12 protein can be used as a biomarker for early diagnosis of ST-segment elevation myocardial infarction.
- 如权利要求1所述的S100A12蛋白在ST段抬高型心肌梗死早期诊断中的应用，其特征在于，所述的S100A12蛋白可以用于制备ST段抬高型心肌梗死早期诊断试剂盒。The use of the S100A12 protein according to claim 1 for early diagnosis of ST-segment elevation myocardial infarction, characterized in that the S100A12 protein can be used for preparing an early diagnosis kit for ST-segment elevation myocardial infarction.
- 如权利要求1所述的S100A12蛋白在ST段抬高型心肌梗死早期诊断中的应用，其特征在于，所述的试剂盒的组分如下：洗涤缓冲液、稀释缓冲液、S100A12标准品、20×辣根过氧化酶、底物和终止试剂。The use of the S100A12 protein according to claim 1 for early diagnosis of ST-segment elevation myocardial infarction, characterized in that the components of the kit are as follows: washing buffer, dilution buffer, S100A12 standard, 20 × horseradish peroxidase, substrate and termination reagent.
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