Application of quantitative peripheral blood LRG1 reagent in the preparation of diagnostic reagents for dangerous carotid artery lesions

Abstract

This invention discloses the use of a quantitative peripheral blood LRG1 reagent in the preparation of a diagnostic reagent for dangerous carotid artery disease. The inventors discovered that the average LRG1 level in blood is higher in patients with carotid artery disease than in healthy individuals, and that as the disease worsens, the shedding of plaque in carotid vascular disease increases significantly. As a peripheral blood marker for dangerous carotid artery disease, this reagent exhibits high sensitivity and specificity and can be used in conjunction with ultrasound results and other diagnostic tools for carotid artery disease.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis, and in particular to the use of a reagent for quantifying the content of a peripheral blood marker LRG1 in the preparation of a diagnostic reagent for cervical major artery lesions. Background Art

[0002] The main arteries in the neck are the carotid arteries and vertebral arteries, which are responsible for the blood supply to the head and neck, especially the brain. When the neck artery disease is severe, the brain tissue will suffer from ischemia, resulting in serious problems such as stroke. Cervical artery disease is often caused by cervical arteriosclerosis and plaques. It is an arteriosclerotic disease caused by various factors, such as aging, long-term high blood lipids and hypertension, smoking, and other factors. The arteries of middle-aged patients aged 40 to 50 years old may slowly develop atherosclerosis. Among them, age >60 years old, male sex, long-term smoking history, history of hypertension, history of diabetes, and hyperlipidemia are risk factors for the formation of carotid artery plaques. Many patients with carotid arteriosclerotic disease do not have any clinical neurological symptoms or only have some non-specific manifestations, such as dizziness, headache, and syncope.

[0003] The existing methods for detecting carotid artery sclerosis are as follows:

[0004] Color Doppler Ultrasound: Color doppler ultrasound is currently the preferred noninvasive method for carotid artery examination. It is convenient and inexpensive, and is widely used for screening and follow-up of carotid artery atherosclerosis. It not only reveals the location and size of plaques and the location and severity of lumen stenosis, but also allows for hemodynamic analysis and plaque morphological evaluation.

[0005] Transcranial Doppler ultrasound (TCD): It can show the location, degree, blood flow velocity, blood flow direction and whether there is open collateral circulation of intracranial and extracranial arteries. It is often used in combination with Doppler ultrasound for the diagnosis and postoperative evaluation of carotid artery stenosis.

[0006] CT angiography (CTA): CTA is more accurate than Doppler ultrasound in diagnosing carotid artery stenosis, but its ability to visualize plaque morphology is less effective. It is currently widely used for diagnosing stenosis and serves as an important basis for preoperative diagnosis and treatment planning. CT angiography (CTA) can clearly demonstrate the location, extent, and severity of arterial lesions, confirming a diagnosis and aiding in the selection of treatment plans. However, due to the use of iodinated contrast agents, it may affect renal function and should be used with caution in patients with renal insufficiency.

[0007] Magnetic resonance angiography (MRA): It can clearly display the three-dimensional morphology and structure of the carotid artery and its branches, and reconstruct the image of the intracranial arteries. It has a high sensitivity in judging lesions with severe stenosis, but the price is relatively high. People with metal implants in their bodies (such as metal dentures, pacemakers or metal prostheses, etc.) are prohibited from undergoing this examination.

[0008] Digital Subtraction Angiography (DSA): DSA is the gold standard for diagnosing carotid artery stenosis. It allows for detailed assessment of lesion location, extent, and collateral formation, and delayed evaluation of distal outflow tract conditions. However, due to its invasive nature, high cost, and high risk, it is rarely used solely for clinical examination. DSA is crucial for lesion assessment and surgical selection. In hospitals with the necessary facilities, endovascular therapy can be performed simultaneously with angiography to address atherosclerosis.

[0009] Proteins are the executors of gene functions. Research on protein structure, localization, and protein-protein interactions will provide a direct basis for clarifying the essence of life phenomena. Almost all physiological and pathological processes, as well as the effects of drugs and environmental factors, rely on proteins and cause changes in the proteome. Before any disease manifests any noticeable symptoms, certain proteins must have changed. The greatest harm of cervical artery disease is the carotid artery. Vertebral artery stenosis suddenly worsens or arterial plaques detach and block the cerebral artery, leading to thrombosis. In this process, there must be changes in the gene proteins of related cells at the site of carotid artery disease. Therefore, finding key proteins and marker proteins for various diseases is of great significance for disease diagnosis, pathological research, and drug screening.

[0010] Leucine-rich α2-glycoprotein 1 (LRG1) is a member of the leucine-rich repeat family and belongs to the LRR protein family. Previous studies have shown that LRG1 is involved in important physiological and pathological processes such as protein interactions, signal transduction, and cell adhesion. LRG1 is also expressed during granulocyte differentiation. However, no studies have shown that LRG1 is associated with the early diagnosis of major carotid artery disease. Summary of the Invention

[0011] The object of the present invention is to overcome at least one deficiency of the prior art and provide a reagent for quantifying the content of the peripheral blood marker LRG1 for use in preparing a diagnostic reagent for cervical major artery lesions.

[0012] The technical solution adopted by the present invention is:

[0013] The present invention provides use of a reagent for quantifying the content of a peripheral blood marker LRG1 in preparing a diagnostic reagent for cervical major artery lesions.

[0014] In some examples, the reagent for quantifying the level of the peripheral blood marker LRG1 is an ELISA reagent.

[0015] In some instances, the major artery in the neck is the carotid artery or the vertebral artery.

[0016] In some examples, the major arterial lesions in the neck are selected from major arterial sclerosis or plaques in the neck, plaque shedding in neck vascular lesions, and major arterial stenosis, thrombosis or occlusion in the neck.

[0017] In another aspect, the present invention provides a system for diagnosing lesions in the main carotid arteries, comprising:

[0018] LRG1 quantification device, used to quantify the amount of LRG1 in peripheral blood of a sample;

[0019] An analytical device that determines the risk of major arterial disease in the neck based on the level of LRG1;

[0020] The result output device is used to output the analysis result of the analysis device.

[0021] In some examples, the LRG1 quantification device is an ELISA detection device.

[0022] In some instances, high levels of LRG1 indicate a higher risk of disease in the major arteries of the neck.

[0023] In some embodiments, a high level of LRG1 means a level of LRG1 greater than 5 ng / mL in a peripheral blood sample.

[0024] In some examples, the major arterial disease in the neck is accompanied by hypertension, hyperlipidemia, or hyperglycemia.

[0025] In some embodiments, an ultrasound device is also included.

[0026] The beneficial effects of the present invention are:

[0027] Through research, the inventors found that the average LRG1 level in the blood of patients with carotid artery disease is higher than that of normal people, and as the disease worsens, the shedding of plaques in carotid vascular disease increases more significantly. As a peripheral blood marker for dangerous carotid artery disease, it has high sensitivity and specificity and can be used in combination with B-ultrasound results to diagnose carotid artery disease. DETAILED DESCRIPTION

[0028] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0029] Screening and collection of clinical samples:

[0030] Sample selection: 60 patients with carotid artery disease were recruited over the past two years. Gender and age were recorded. All patients underwent blood pressure, lipid profile, blood sugar, liver function, renal function, coagulation tests, and electrocardiogram (ECG). All patients underwent carotid vascular ultrasound, and some underwent carotid CT angiography (CTA). Sixty healthy controls matched for age and gender were also recruited.

[0031] Specimen collection: 5 mL of venous blood was collected from patients who met the inclusion criteria. The blood was allowed to stand for 15 minutes and then centrifuged at 3000 r / min for 15 minutes. Serum was extracted and placed in sterile cryovials and stored at -80°C for research.

[0032] Study of blood samples from case patients

[0033] The inspection method is as follows: Separate serum and cells using a high-speed centrifuge.

[0034] Main equipment, materials, and reagents: homogenizer, high-speed centrifuge, 3kDa ultrafiltration centrifuge tubes, ICP-MS. 18.2 MΩ / cm ultrapure water was used throughout the experiment. Sample pretreatment methods: Fully automatic biochemical analyzer (one each of Beckman DXC800 and AU400), 1 / 10,000 electronic analytical balance (Shimadzu), microplate reader, Roche electrochemiluminescence automated immunoassay analyzer (Elecsys 2010), specific protein analyzer (imported), water purification machine (MEDICA60), electric constant temperature water bath (DK-600), low-temperature high-speed centrifuge (Sigma), ultra-low temperature freezer (Thermo Fisher Scientific), low-temperature freezer (HFC350, Germany). The above equipment conditions are fully sufficient for the implementation of this project.

[0035] Statistical methods

[0036] The mean values ​​of each group were compared with T test using SPSS statistical software, and P < 0.05 was considered statistically significant.

[0037] result

[0038] Sensitivity = number of true positives / (number of true positives + number of false negatives)*100%. The rate of correctly diagnosing patients

[0039] Specificity = number of true negatives / (number of true negatives + number of false positives)*100%. The rate of correctly identifying non-patients

[0040] False positive rate = number of false positives / number of gold standard negatives

[0041] False negative rate = number of false negatives / number of gold standard positives

[0042] The changes of peripheral blood LRG1 in patients with carotid artery lesions are shown in Table 1.

[0043] Table 1

[0044] N LRG1 (Huizhou) ng / ml Normal people 60 3.2±1.1 Carotid artery disease 60 8.2±1.0

[0045] Comparison of carotid artery lesions with normal controls, P<0.001

[0046] Normal values ​​are defined as the mean plus two standard deviations. The upper limit of normal for the Huizhou-produced LRG1 detection kit (LRG1 Huizhou) is 5 ng / ml. In a test of 60 patients with carotid artery lesions using the Huizhou-produced reagent, the cutoff value was 5 ng / ml. Results exceeded the cutoff value in 55 patients, with 5 false negatives, resulting in a sensitivity of 91.66%. In a test of 60 healthy individuals, 12 exceeded the cutoff value, resulting in a false positive rate of 20% and a specificity of 80%. The sensitivity and specificity of LRG1 for diagnosing carotid artery lesions are shown in Table 2.

[0047] Table 2

[0048] Positive coincidence rate False positive Negative coincidence rate False negative LRG1 (Huizhou) 91.66% 20% 80% 8.33%

[0049] Among them, Table 3 shows the changes of LRG1 in different degrees of carotid artery lesions (X±SD, mean±standard deviation unit ng / ml). It can be found that compared with normal people, LRG1 is increased in carotid artery lesions from mild to severe, P<0.05.

[0050] Table 3

[0051] Normal people Neck arteriosclerosis or arterial plaque Neck artery stenosis, thrombosis, arterial occlusion, and detachment of vascular lesion plaques Number of cases N=60 N=39 N=21 LRG1 value 3.2±1.1 7.6±1.8 9.3±2.5

[0052] The results showed that the average LRG1 level in the blood of patients with carotid artery disease was higher than that of normal people, and as the disease worsened, the shedding of plaques in carotid vascular disease increased more significantly. As a peripheral blood marker for dangerous carotid artery disease, it has high sensitivity and specificity, and can be used in combination with B-ultrasound results to diagnose carotid artery disease.

[0053] References:

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[0063]

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[0064] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. Application of reagents for quantifying the content of peripheral blood marker LRG1 in the preparation of diagnostic reagents for cervical artery disease.

2. The use according to claim 1, characterized in that The reagent for quantifying the content of the peripheral blood marker LRG1 is an ELISA reagent.

3. A carotid artery disease diagnosis system, comprising: LRG1 quantification device, used to quantify the amount of LRG1 in peripheral blood of a sample; An analytical device for determining the risk of carotid artery disease based on the level of LRG1; The result output device is used to output the analysis result of the analysis device.

4. The carotid artery disease diagnosis system according to claim 3, characterized in that: The LRG1 quantitative device is an ELISA detection device.

5. The carotid artery disease diagnosis system according to claim 3, characterized in that: High levels of LRG1 indicate a high risk of carotid artery disease.

6. The carotid artery disease diagnosis system according to claim 5, characterized in that: High LRG1 levels refer to levels higher than 5 ng / mL in peripheral blood samples.

7. The carotid artery disease diagnosis system according to claim 3, characterized in that: Also includes a B-ultrasound device.