Markers of aortic aneurysm

Abstract

The present invention relates to the field of diagnosis and treatment of vascular diseases, and specifically to a marker for aortic aneurysm and a kit for auxiliary diagnosis of aortic aneurysm. Compared with a healthy control group, the miR663a content in the serum of patients with aortic aneurysm is significantly increased. And there is a correlation between miR663a and the time of onset. The shorter the onset time, the higher the miR663a content. Therefore, miR663a can be used as an aortic aneurysm marker for auxiliary diagnosis of aortic aneurysm. The kit for auxiliary diagnosis of aortic aneurysm described in the present invention can be used for clinical auxiliary diagnosis of aortic aneurysm quickly, sensitively and specifically, laying a foundation for clinicians to quickly and accurately grasp the patient's condition and evaluate the treatment effect.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis and treatment of vascular diseases, and in particular to a new aortic aneurysm marker and a method and a kit for assisting in diagnosing the occurrence of aortic aneurysm. Background Art

[0002] Aortic aneurysm refers to the local or diffuse abnormal expansion of the aortic wall, which compresses the surrounding organs and causes symptoms. Aneurysm rupture is the main danger. It often occurs in the ascending aorta, the aortic arch, the descending thoracic aorta, the thoracoabdominal aorta and the abdominal aorta. Aortic aneurysm develops slowly, with no symptoms and signs in the early stage. In the later stage, symptoms occur due to the compression of the surrounding tissues by the aneurysm. Its clinical manifestations vary depending on the size, shape, location and growth direction of the aneurysm. For example, aortic aneurysm compression of the trachea and bronchi can cause coughing, shortness of breath, pneumonia and atelectasis, compression of the esophagus can cause dysphagia, and compression of the recurrent laryngeal nerve can cause hoarseness. Compression of the phrenic nerve causes diaphragmatic paralysis, compression of the superior vena cava and brachiocephalic vein can cause edema of the upper limbs, neck, face and upper chest, and compression of the sternum can cause chest pain. Traditional methods for the examination of aortic aneurysms mainly include chest and abdominal X-rays, ultrasound, CT angiography (CTA) and magnetic resonance angiography (MRA), arteriography, etc.

[0003] In recent years, with the development of immunology and molecular biology technology, a class of substances related to cell growth and proliferation has been proposed - biomarkers. It can mark the biochemical indicators of changes or possible changes in the structure or function of systems, organs, tissues, cells and subcellular structures. Biomarkers can not only explore the pathogenesis at the molecular level, but also have unique advantages in accurately and sensitively evaluating early and low-level damage, and can provide early warnings, which to a large extent provide clinicians with a basis for auxiliary diagnosis.

[0004] At present, the most commonly used biomarker for aortic aneurysm is D-dimer, especially for acute aortic dissection, with a sensitivity of up to 99%. If D-dimer does not increase, the possibility of aortic dissection can be almost ruled out. However, the specificity is very low, and D-dimer will also increase in conditions such as pulmonary embolism and coronary thrombosis. Moreover, the increase in D-dimer occurs after the dissection occurs and cannot be used as a prediction. In addition, C-reactive protein, smooth muscle myosin heavy chain, troponin, etc. are also used as biomarkers for clinical auxiliary diagnosis of aortic aneurysm, but the sensitivity and specificity are not high, and further in-depth research and discussion are still needed.

[0005] A large number of studies have shown that many miRNAs are involved in the pathological process of aortic aneurysms. However, it is still unclear how to use miRNAs to quickly and accurately diagnose aortic aneurysms, and their predictive role in the occurrence of aortic aneurysms. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a new marker for aortic aneurysm and a kit for assisting in the diagnosis of aortic aneurysm in order to solve the problems in the prior art.

[0007] In some embodiments, the present invention collects clinical samples, detects the miRNA (microRNA) content in the serum of healthy people and aortic aneurysms, and analyzes the correlation between the expression of differentially expressed miRNAs in different characteristic populations (gender, risk factors, severity of the disease, degree of disease progression, etc.) and the characteristics. The results show that miR663a has the largest expression difference. Compared with the healthy control group, the miR663a content in the serum of aortic aneurysm patients is significantly increased. And there is a correlation between miR663a and the onset time. The shorter the onset time, the higher the miR663a content.

[0008] Therefore, the present invention provides the use of miR663a in preparing aortic aneurysm markers.

[0009] The sample for aortic aneurysm in the application of the present invention can be any sample known to those skilled in the art, including but not limited to blood, saliva, and urine. Preferably, the sample for aortic aneurysm is serum or plasma.

[0010] The present invention also provides the use of miR663a in preparing an aortic aneurysm auxiliary diagnosis kit.

[0011] The present invention also provides a kit for auxiliary diagnosis of aortic aneurysm, comprising a primer of miR663a.

[0012] Preferably, the primer sequences of miR663a are shown as SEQ ID No.1 and SEQ ID No.2.

[0013] In some embodiments, the kit of the present invention further comprises enzymes and reagents commonly used in PCR reactions, such as reverse transcriptase, Taq enzyme, dNTPs, MgCl2, DEPC water and buffer, etc.

[0014] Furthermore, in some embodiments, the kit of the present invention also includes a reference substance and a standard substance.

[0015] It can be seen from the above technical scheme that the present invention provides a marker for aortic aneurysm and a kit for auxiliary diagnosis of aortic aneurysm. Compared with the healthy control group, the miR663a content in the serum of patients with aortic aneurysm is significantly increased. And there is a correlation between miR663a and the time of onset. The shorter the onset time, the higher the miR663a content. Therefore, miR663a can be used as an aortic aneurysm marker for auxiliary diagnosis of aortic aneurysm. The kit for auxiliary diagnosis of aortic aneurysm described in the present invention can be used for clinical auxiliary diagnosis of aortic aneurysm quickly, sensitively and specifically, laying a foundation for clinicians to quickly and accurately grasp the patient's condition and evaluate the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0017] Figure 1 The t test result diagram of miR663a expression level in aortic aneurysm patients and healthy subjects in Example 1 is shown;

[0018] Figure 2 The ROC curve diagram of miR663a expression level in aortic aneurysm patients and healthy subjects in Example 1 is shown;

[0019] Figure 3 The graph showing the results of the detection of miR663a expression in patients with acute and chronic aortic aneurysms in Example 2;

[0020] Figure 4 The linear correlation analysis results of miR663a expression levels in patients with aortic aneurysm at different disease courses in Example 2 are shown. DETAILED DESCRIPTION

[0021] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Embodiment 1,

[0023] 1. Collect serum from patients with aortic aneurysm and healthy subjects and store at -80℃

[0024] 2. Matching gender and age, select 37 healthy subjects as controls and 50 patients with aortic aneurysms;

[0025] 3. Use QIAGEN's microRNA extraction kit to extract microRNA from serum according to the instructions, send it to the company, and use liquid chips to detect microRNA expression. Two independent sample t-tests and ROC curve statistical methods are used for detection.

[0026] The results showed that the expression of miR663a was the most different. Compared with the healthy control group, the serum miR663a content in patients with aortic aneurysm was significantly increased (Table 1, Figure 1 and Figure 2 ). The average expression level of the healthy control group was -0.051, and the average expression level of the aortic aneurysm group was 2.624, and the area under the curve of the subjects (Area=0.879) was the best. This showed that the content of miR663a in serum was related to aortic aneurysm.

[0027] Table 1 miR663a expression

[0028]

[0029]

[0030] Embodiment 2,

[0031] The patients with aortic aneurysm described in Example 1 were divided into groups according to acute and chronic conditions. The grouping criteria were as follows: chronic conditions were asymptomatic or symptoms lasted for more than 2 weeks, and acute conditions were acute onset and the course of the disease was within 2 weeks. The expression of miR663a was analyzed by two independent sample t-tests. The results are shown in (Table 2, Figure 3 ).

[0032] Table 2 Expression of miR663a in patients with aortic aneurysm according to acute and chronic groups

[0033]

[0034]

[0035] The results showed that compared with the control group or chronic group, the expression level of miR663a in the acute onset group was higher, and the difference was statistically significant.

[0036] The course of aortic aneurysm patients described in Example 1 was further subdivided into the following groups: onset time more than 5 years, 1-5 years, 2 months-1 year, 1 month-2 months, 7 days-1 month, 1-7 days, less than 1 day. The expression of miR663a was analyzed by linear correlation analysis, and the results are shown in (Table 3, Figure 4 ).

[0037] Table 3 Expression of miR663a in patients with aortic aneurysm according to the time of onset

[0038]

[0039]

[0040] The results showed that miR663a was correlated with the onset time, that is, the shorter the onset time, the higher the miR663a content.

[0041] The above results indicate that miR663a is related to the acute and chronic onset of aortic aneurysm.

[0042] Example 3, Aortic Aneurysm Detection Kit

[0043] It includes miR663a upstream primer (SEQ ID No.1) and miR663a downstream primer (SEQ ID No.2), TaqDNA polymerase, MgCl2SYBRGreen and the like.

[0044] Example 4: Aortic aneurysm detection method

[0045] 1. Extract miRNA using serum / plasma miRNA extraction kit. The specific steps are as follows:

[0046] (1) Serum / plasma + lysis buffer (serum / plasma: lysis buffer = 1:5) and extract at room temperature for 5 min;

[0047] (2) Add 3.5 μL of internal control, miR39;

[0048] (3) Add chloroform (serum / plasma:chloroform = 1:1), shake for 15 seconds, and leave at room temperature for 3 minutes;

[0049] (4) Centrifuge at 12000g for 15 min at 4°C, transfer the supernatant to a new collection tube, add 1.5 times the volume of anhydrous ethanol, and mix well;

[0050] (5) Pipette 700 μL of the mixture into a new collection tube, centrifuge at ≥8000 g for 15 s at room temperature, and repeat this step with the remaining liquid;

[0051] (6) Add 700 μL of eluent RWT and centrifuge at ≥8000 g for 15 s at room temperature;

[0052] (7) Add 500 μL of eluent RPE and centrifuge at ≥8000 g for 15 s at room temperature;

[0053] (8) Add 500 μL of 80% ethanol and centrifuge at ≥8000 g for 2 min at room temperature; (9) Replace with a new collection tube and centrifuge at full speed for 5 min with the lid open;

[0054] (10) Replace the collection tube, add 14 μL of RNase-free water, and centrifuge at full speed for 1 min;

[0055] (11) NanoDrop was used to measure sample concentration. RNA samples can be stored at -20°C for a long time.

[0056] 2. RT-PCR was used to detect the expression of miR663a.

[0057] (1) miRNA reverse transcription: reverse transcribe the total RNA containing miRNA using a miRNA reverse transcription kit;

[0058] (2) RT-PCR: The expression of miR663a was detected using SYBR Green and miR663a-specific primers (SEQ ID No. 1 and SEQ ID No. 2).

Claims

1. Application of miR663a in the preparation of a diagnostic kit for detecting aortic aneurysm, wherein the diagnostic kit is a diagnostic kit for distinguishing acute aortic aneurysm from chronic aortic aneurysm.

2. The use according to claim 1, characterized in that: (1) The sample tested in the kit is serum or plasma; (2) The kit includes primers for quantifying miR663a as shown in SEQ ID No. 1 and SEQ ID No. 2.

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