A peripheral blood marker to assist in the diagnosis of lower extremity arterial injury

Abstract

The present invention discloses a peripheral blood marker for assisting in the diagnosis of lower extremity artery injury. The inventor has found through research that the content of peripheral blood LRG1 is closely related to lower extremity artery injury. By detecting the content of LRG1 in peripheral blood, lower extremity artery injury, especially lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion, can be well predicted. In particular, lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion caused by hypertension, hyperlipidemia or hyperglycemia can be well predicted. The inventor's invention can be well used for lower extremity arterial injury, especially the early diagnosis of lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion caused by hypertension, hyperlipidemia or hyperglycemia, and has the advantages of simple detection method and low cost.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis, and in particular to a peripheral blood marker for assisting in diagnosing lower extremity artery injury. Background Art

[0002] Common lower limb artery injuries include lower limb arteriosclerosis, plaque formation, and thrombosis, which cause lower limb arteriosclerosis and occlusion, leading to chronic limb ischemia and, in severe cases, lower limb arterial occlusive disease. With the improvement of the overall living standards of society and the aging of the population, the incidence of lower limb arteriosclerosis and occlusive disease has increased year by year.

[0003] Epidemiological surveys show that smoking, diabetes, hyperlipidemia, hypertension, hyperhomocysteinemia, hypercoagulable state, increased blood viscosity and advanced age are risk factors for lower extremity arteriosclerosis obliterans. Among them, smoking and diabetes are the most harmful. Both can increase the incidence of peripheral arterial disease by 3 to 4 times, and the risk is higher when they coexist. The second is hyperlipidemia, especially elevated low-density lipoprotein cholesterol, which is closely related to the occurrence of atherosclerosis in multiple parts of the body. Timely detection and control of risk factors leading to arteriosclerosis can delay the progression of arteriosclerosis and reduce the risk of lower extremity arteriosclerosis obliterans.

[0004] There are several theories about the main pathogenesis of lower limb artery injury.

[0005] 1. Injury and smooth muscle cell proliferation theory

[0006] Various damaging factors, such as hypertension, hemodynamic changes, thrombosis, hormone and chemical stimulation, immune complexes, bacteria and viruses, diabetes and hypoxemia, lead to endothelial cell damage. After endothelial cell damage, it secretes a variety of growth factors and chemokines, stimulating smooth muscle cells (SMC) to migrate to the intima, proliferate, secrete extracellular matrix and phagocytize lipids to form SMC-derived foam cells, and finally form atherosclerotic plaques.

[0007] 2. Lipid Infiltration Theory

[0008] This theory holds that lipids in plasma deposit in the arterial intima and stimulate connective tissue proliferation, causing atherosclerosis. In this process, endothelial cell damage, increased permeability and lipid transport disorders may play a major role.

[0009] 3. Hemodynamic theory

[0010] In the process of atherosclerosis, hemodynamic factors also play a role and are correlated with the location of atherosclerotic plaques. Studies have shown that atherosclerotic plaques are mainly located in the low shear area of ​​the blood vessel wall. Turbulence plays a role in plaque rupture or thrombosis.

[0011] 4. Genetic theory

[0012] Genetic surveys show that the incidence of this disease in people with a family history is 2 to 6 times higher than that of the general population. This may be due to genetic defects that cause abnormal feedback control of cellular cholesterol synthesis, resulting in excessive accumulation of cholesterol.

[0013] Lower extremity arteriosclerosis is generally seen in middle-aged and elderly people, often accompanied by risk factors such as smoking, diabetes, hypertension, and hyperlipidemia. The presence and severity of symptoms of lower extremity arteriosclerosis are affected by multiple factors such as the speed of lesion progression, the amount of collateral circulation, and individual tolerance. Symptoms generally develop gradually from mild to severe, but when acute thrombosis occurs on the basis of arteriosclerosis, symptoms may suddenly worsen.

[0014] There may be no obvious symptoms in the early stage, or only slight discomfort, such as chills and coldness. After that, intermittent claudication symptoms gradually appear, which is a characteristic symptom of lower extremity arteriosclerotic injury occlusive disease. It manifests as fatigue and soreness of the affected limb after walking a certain distance, and is forced to rest for a period of time; the symptoms can be completely relieved after resting, and the symptoms will reappear after walking again. The distance of each walk and the time of rest are generally fixed; in addition, the location of the soreness is correlated with the location of the vascular lesion. If the lesion develops further, rest pain will appear, that is, the patient will have pain in the extremities when resting, which is easy to occur when lying flat and resting at night. Eventually, ulcers and gangrene may occur in the limbs, which are mostly induced by minor extremity injuries.

[0015] The severity of clinical manifestations can be divided into Fontine staging or Rutherford staging to increase the objectivity of clinical evaluation and make the results of various clinical treatments more comparable. The Rutherford staging system is currently commonly used, which is divided into seven levels from 0 to 6 from mild to severe.

[0016] 1. Rutherford Level 0

[0017] No clinical symptoms, normal treadmill test or reactive hyperemia test, no hemodynamic manifestations of arterial occlusion;

[0018] 2. Rutherford Level 1

[0019] Mild intermittent claudication, complete a treadmill test, ankle artery pressure > 50 mmHg after exercise, but less than about 20 mmHg at rest;

[0020] 3. Rutherford Level 2

[0021] Moderate intermittent claudication, between 1 and 3;

[0022] 4. Rutherford Level 3

[0023] Severe intermittent claudication, inability to complete the treadmill test, ankle artery pressure <50 mmHg after exercise;

[0024] 5. Rutherford Level 4

[0025] Ischemic rest pain, ankle artery pressure <40 mmHg at rest, dorsalis pedis and posterior tibial arteries barely palpable, toe artery pressure <30 mmHg;

[0026] 6. Rutherford Level 5

[0027] Small tissue loss, non-healing ulcers, focal gangrene with diffuse ischemic changes on the sole of the foot, ankle artery pressure <60 mmHg at rest, dorsalis pedis and posterior tibial arteries barely palpable, toe artery pressure <40 mmHg;

[0028] 7. Rutherford Level 6

[0029] Large tissue loss, exceeding the metatarsal plane, foot function cannot be preserved, and the rest of the criteria are the same as Rutherford grade 5. (Standard treadmill test on a 15-degree slope, at a speed of 2 miles per hour, for 5 minutes).

[0030] In the prior art, the inspection methods for lower extremity artery injuries are mainly as follows:

[0031] Because most patients are elderly people who may have multiple comorbidities and risk factors for atherosclerosis, a comprehensive examination is required, including blood pressure, blood sugar, blood lipid measurements, and cardiovascular and cerebrovascular assessments.

[0032] 2. Special inspection

[0033] (1) Segmental arterial systolic pressure measurement measures the pressure levels at different planes of the lower limb arteries and compares them bilaterally. If there is obvious stenosis in the artery, the distal pressure will be significantly reduced, which can preliminarily determine whether the artery has lesions and their location.

[0034] (2) Ankle-brachial index (ABI): The ratio of ankle artery systolic pressure to brachial artery systolic pressure is measured using Doppler blood flow meter and pressure gauge. In the resting state, ABI is generally between 0.91 and 1.30. A value higher than 1.30 indicates that the arterial wall is stiff and not easy to collapse; ABI between 0.90 and 0.41 indicates mild to moderate ischemia; ABI ≤ 0.40 indicates severe ischemia. In addition, there is a toe-brachial index (TBI) to understand the condition of terminal arterial lesions.

[0035] (3) Transcutaneous oxygen partial pressure measurement: By measuring the oxygen partial pressure of local tissues, we can indirectly understand the blood perfusion of local tissues and evaluate the degree of ischemia. It can also be used to determine the healing trend of acral ulcers and wounds. If the transcutaneous oxygen partial pressure is too low, it indicates that the wound is difficult to heal.

[0036] (4) Color Doppler ultrasound is a commonly used screening method, which can detect arteriosclerotic plaques, luminal stenosis, occlusion, etc. This method is non-invasive, convenient and low-cost.

[0037] (5) CT angiography (CTA) has become the preferred examination method for lower extremity arteriosclerosis obliterans. It can clearly show the location, range, and degree of arterial lesions, make a clear diagnosis, and help determine the treatment plan. The disadvantage is that it requires the use of iodine-containing contrast agents, which may affect renal function. It should be used with caution in patients with renal insufficiency.

[0038] (6) Magnetic resonance angiography (MRA) is similar to CTA and can also provide a clear imaging diagnosis of lower extremity arteriosclerosis obliterans. Its advantage is that it does not require the use of iodinated contrast agents, but its ability to distinguish calcification is poor and may overestimate the severity of the lesion.

[0039] (7) Digital subtraction angiography (DSA) is the gold standard for diagnosing lower extremity arteriosclerotic occlusive disease. It can accurately show the location, range, degree, and collateral circulation of the lesion. The delayed phenomenon can evaluate the distal outflow tract. DSA is of great significance for the evaluation of lesions and the selection of surgical methods. At the same time, in hospitals with conditions, endovascular treatment can be performed at the same time as angiography to solve arterial lesions at the same time.

[0040] The typical clinical manifestations of lower extremity arteriosclerotic occlusive disease can be used to diagnose the disease in combination with non-invasive or invasive vascular examinations. Differential diagnosis of lumbar spinal stenosis

[0041] It may manifest as intermittent claudication symptoms, which can be easily confused with the early and middle stage symptoms of lower limb arteriosclerosis obliterans. However, the symptoms of this disease are obviously related to body position. Changing body position can alleviate or relieve the symptoms. At the same time, the limb arterial pulsation is normal, which can be used for identification.

[0042] Leucine-rich α2-glycoprotein 1 (LRG1) is a member of the leucine-rich repeat family. LRG1 belongs to the LRR protein family. The leucine-rich repeat family proteins include eight leucine-rich repeat sequences (LRR). Studies have shown that LRR proteins are transmembrane proteins, which mainly participate in normal physiological activities as cell adhesion molecules or ligand binding proteins. Past studies have shown that LRG1 participates in important physiological and pathological processes of the body, such as protein interactions, signal transduction, and cell adhesion. LRG1 can also be expressed during granulocyte differentiation. No study has found that LRG1 is related to the early diagnosis of lower extremity artery injury. Summary of the invention

[0043] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a peripheral blood marker for assisting in diagnosing lower extremity artery injury.

[0044] The technical solution adopted by the present invention is:

[0045] The first aspect of the present invention provides:

[0046] Application of reagents for quantifying peripheral blood LRG1 content in the preparation of diagnostic reagents for lower limb artery injury.

[0047] In some application examples, the reagent for quantifying the LRG1 content in peripheral blood is an ELISA reagent.

[0048] In some application examples, the lower limb artery injury is selected from lower limb arteriosclerosis or plaque, lower limb arterial stenosis, thrombosis or occlusion.

[0049] The second aspect of the present invention provides:

[0050] A lower limb artery injury diagnosis system, comprising:

[0051] LRG1 quantification device, used to quantify the amount of LRG1 in the peripheral blood of the sample;

[0052] An analytical device for determining the risk of lower extremity artery damage based on the content of LRG1;

[0053] The result output device is used to output the analysis result of the analysis device.

[0054] In some examples of the lower extremity artery injury diagnosis system, the LRG1 quantification device is an ELISA detection device.

[0055] In some examples of lower extremity artery injury diagnostic systems, a high level of LRG1 indicates a high risk of lower extremity artery injury.

[0056] In some examples of the lower extremity artery injury diagnostic system, a high LRG1 content refers to a content of LRG1 in a peripheral blood sample that is higher than 5 ng / mL.

[0057] In some examples of the lower limb artery injury diagnosis system, the lower limb artery injury is selected from lower limb arteriosclerosis or plaque, lower limb artery stenosis, thrombosis or occlusion.

[0058] In some examples of the lower limb artery injury diagnosis system, the lower limb artery injury is accompanied by hypertension, hyperlipidemia or hyperglycemia.

[0059] In some examples of the lower limb artery injury diagnosis system, a B-ultrasound device is also included.

[0060] The beneficial effects of the present invention are:

[0061] The inventors have found through research that the content of peripheral blood LRG1 is closely related to lower extremity artery damage. By detecting the content of peripheral blood LRG1, lower extremity artery damage, especially lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion, can be well predicted. In particular, lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion caused by hypertension, hyperlipidemia or hyperglycemia can be well predicted. The inventor's invention can be well used for lower extremity arterial damage, especially lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion caused by hypertension, hyperlipidemia or hyperglycemia. The invention of the inventors can be well used for early diagnosis of lower extremity arteriosclerosis or plaques, lower extremity arterial stenosis, thrombosis or occlusion caused by hypertension, hyperlipidemia or hyperglycemia, and has the advantages of simple detection method and low cost. Implementation

[0062] The technical solution of the present invention is further explained below in conjunction with experiments.

[0063] Fifty cases of lower extremity arterial injury in the past two years were collected, and gender and age were recorded. All patients were tested for blood pressure, blood lipids, blood sugar, liver function, kidney function, four coagulation tests, ECG and other examinations. All cases were confirmed by bilateral lower extremity vascular ultrasound, and some cases were performed lower extremity artery CTA. At the same time, 60 normal subjects with matching age, gender and other conditions were selected as controls.

[0064] Screening and collection of clinical samples:

[0065] Specimen collection: 5 mL of venous blood was collected from patients who met the inclusion criteria, allowed to stand for 15 min, and centrifuged at 3000 r / min for 15 min to extract serum, which was placed in sterile cryopreservation tubes and stored at -80°C for research.

[0066] Study of blood samples from case patients

[0067] Separate serum. The inspection method is as follows: Separate serum and cells under the action of a high-speed centrifuge.

[0068] Main equipment, materials and reagents: homogenizer, high-speed centrifuge, 3kDa ultrafiltration centrifuge tube, ICP-MS. The water used in the experiment is 18.2 MΩ / cm ultrapure water. Sample pretreatment method:) Fully automatic biochemical analyzer (one each of Beckman DXC800 and AU400), 1 / 10,000 electronic analytical balance (Shimadzu), microplate reader, Roche electrochemiluminescence automatic immunoassay analyzer (Elecsys2010), specific protein analyzer (imported), purified water machine (MEDICA60), electric constant temperature water bath (DK-600), low-temperature high-speed centrifuge (sigma), ultra-low temperature refrigerator (Thermoelectric, USA), low-temperature refrigerator (HFC350, Germany), etc. The above equipment conditions can fully guarantee the implementation of this project.

[0069] Statistical methods:

[0070] The mean values ​​of each group were compared with T test using SPSS statistical software, and P < 0.05 was considered statistically significant.

[0071] result:

[0072] Sensitivity = number of true positives / (number of true positives + number of false negatives)*100%. The rate of correctly judging patients

[0073] Specificity = number of true negatives / (number of true negatives + number of false positives)*100%. Rate of correctly identifying non-patients

[0074] False positive rate = number of false positives / number of gold standard negatives

[0075] False negative rate = number of false negatives / number of gold standard positives

[0076] 3.1 Changes in peripheral blood LRG1 in patients with lower extremity artery injury. The results are shown in Table 1

[0077] Table 1: Changes of peripheral blood LRG1 in patients with lower extremity artery injury (X±SD)

[0078] N LRG1 (Wuhan) ng / ml Normal people 60 3.2±1.1 Lower extremity arterial injury 50 8.1±1.0

[0079] Compared with normal controls, patients with lower limb artery injury had a P<0.001.

[0080] The normal value is determined by adding 2 standard deviations to the mean. The upper limit of normal for the LRG1 test kit produced in Wuhan (LRG1 Wuhan) is 5ng / ml. The Wuhan-produced reagent tested 50 cases of lower extremity artery injury, with a CUTOFF value of 5ng / ml. The results exceeded CUTOFF in 45 cases, 5 false negatives, and a sensitivity of 90%. Among 60 normal people, 12 exceeded the CUTOFF value, with a false positive of 20% and a specificity of 80%.

[0081] 3.2 The sensitivity and specificity of LRG1 in diagnosing acute cerebral hemorrhage are shown in Table 2.

[0082] Table 2: Sensitivity and specificity of LRG1 in diagnosing lower extremity vascular injury

[0083] Positive coincidence rate False Positive Negative compliance rate False Negative LRG1 (Wuhan) 90% 20% 80% 10%

[0084] The normal upper limit of the LRG1 detection kit produced in Wuhan (LRG1 Wuhan) is 5ng / ml. The Wuhan-produced reagent was used to detect 50 cases of lower limb artery injury, with a CUTOFF value of 5ng / ml. The results exceeded the CUTOFF value in 45 cases, with 5 false negatives. Among 60 normal people, 12 exceeded the CUTOFF value, with a false positive rate of 20%.

[0085] Table 3: Changes of LRG1 in different degrees of lower limb vascular injury (X±SD, unit ng / ml)

[0086] Normal people Atherosclerosis or arterial plaque Arterial stenosis, thrombosis and lower limb arterial occlusion N=60 N=32 N=18 LRG1 value 3.2±1.1 5.2±1.2 9.5±3.6

[0087] Compared with normal subjects, LRG1 was increased in patients with lower limb artery injury from mild to severe, P<0.05.

[0088] discuss:

[0089] This article proposes and verifies for the first time that LRG1 is a peripheral blood marker for lower limb artery injury, which can be used in combination with B-ultrasound results to diagnose lower limb artery injury.

[0090] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

[0091] References:

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Claims

1. Use of a reagent for quantifying the content of LRG1 in peripheral blood in the preparation of a diagnostic reagent for lower limb artery injury, wherein the lower limb artery injury is selected from lower limb artery plaque or thrombus, and the lower limb artery injury is accompanied by hypertension and hyperlipidemia. A high content of LRG1 indicates a high risk of lower limb artery injury, and a high content of LRG1 means that its content in a peripheral blood sample is higher than 5 ng / mL.

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

3. A lower limb artery injury diagnosis system, wherein the lower limb artery injury is selected from lower limb artery plaque or thrombus, and the lower limb artery injury is accompanied by hypertension and hyperlipidemia, comprising: LRG1 quantification device, used to quantify the amount of LRG1 in the peripheral blood of the sample; An analytical device for determining the risk of lower extremity artery injury based on the content of LRG1, wherein a high content of LRG1 indicates a high risk of lower extremity artery injury, wherein a high content of LRG1 means that the content of LRG1 in a peripheral blood sample is higher than 5 ng / mL; The result output device is used to output the analysis result of the analysis device.

4. The lower extremity artery injury diagnosis system according to claim 3, characterized in that: The LRG1 quantification device is an ELISA detection device.

5. The lower extremity artery injury diagnosis system according to claim 3 or 4, characterized in that: Also includes a B-ultrasound device.