Peripheral blood marker combination for identifying and diagnosing benign and malignant pulmonary nodules

Abstract

The invention discloses a peripheral blood marker combination for identifying and diagnosing benign and malignant pulmonary nodules, and relates to the technical field of medical detection, and the marker combination comprises mi croRNA-21, a carcino-embryonic antigen (CEA), a cytokeratin 19 fragment CYFRA21-1, a squamous cell carcinoma antigen (SCC) and a vascular endothelial growth factor (VEGF). The marker combination comprehensively considers the effects of different markers in identification of benign and malignant pulmonary nodules through combined detection of a plurality of markers, compared with single marker detection, benign and malignant pulmonary nodules are identified more accurately, the misdiagnosis and missed diagnosis rates are reduced, and the detection efficiency is improved based on peripheral blood marker detection. The marker combination can effectively identify the benign and malignant pulmonary nodules in patient groups with different clinical characteristics, is not obviously influenced by the factors, has a wide clinical application prospect, and can be widely applied to clinical diagnosis and treatment of the pulmonary nodules, such as pulmonary nodule benign and malignant pulmonary nodules, pulmonary nodule benign and malignant pulmonary nodules, pulmonary nodule benign and malignant pulmonary nodules. And a reliable basis can be provided for early diagnosis and clinical treatment of pulmonary nodules.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules. Background Art

[0002] Pulmonary nodules are an imaging diagnosis that refers to the appearance of round or irregular lesions with a diameter of less than 3 cm in the lungs. These lesions appear as increased density shadows in the lung area on chest X-ray or chest CT. They can be isolated or multiple. Pulmonary nodules do not refer to a specific disease. Many diseases can cause nodules in the lungs, including but not limited to: bacterial infections such as Staphylococcus aureus and Pneumocystis jiroveci, tuberculosis infection, fungal infection, such as inflammatory pseudotumors, granulomas, pulmonary adenomas, small atelectasis, small pulmonary emboli, etc., which may be nodules of lung cancer or other malignant tumors, such as lung cancer, metastases, etc. In addition, pulmonary nodules can be divided into solid nodules and subsolid nodules according to their density and composition. Among them, subsolid pulmonary nodules are further divided into partially solid pulmonary nodules and pure ground glass nodules. These classifications help doctors better understand the nature and possible risks of pulmonary nodules. Early and accurate identification of benign and malignant pulmonary nodules is crucial for patients' treatment selection and prognosis.

[0003] At present, the commonly used clinical diagnosis methods for lung nodules include imaging examinations (such as chest X-rays, CT, etc.), bronchoscopy, percutaneous lung puncture biopsy, etc. However, these methods have certain limitations. Although imaging examinations can detect lung nodules, their accuracy in judging benign or malignant is limited, especially for small nodules and ground glass nodules. Bronchoscopy and percutaneous lung puncture biopsy are invasive examinations that may cause certain pain and risks to patients, such as bleeding and pneumothorax. In some cases, due to factors such as the location of the nodules and the patient's physical condition, it may not be possible to obtain satisfactory tissue samples for pathological diagnosis.

[0004] In addition, as a non-invasive detection method, peripheral blood marker detection has the advantages of simplicity, ease, and repeatability, and has broad application prospects in the field of tumor diagnosis. Although some existing single peripheral blood markers have certain value in the differential diagnosis of benign and malignant pulmonary nodules, the diagnostic accuracy needs to be improved. Therefore, it is necessary to propose a combination of peripheral blood markers for the differential diagnosis of benign and malignant pulmonary nodules to solve the problems in the existing technology. Summary of the invention

[0005] The purpose of the present invention is to make up for the shortcomings of the prior art and to provide a combination of peripheral blood markers for the differential diagnosis of benign and malignant lung nodules. It can overcome the shortcomings of the existing differential diagnosis methods for benign and malignant lung nodules and provide a non-invasive, highly accurate differential diagnosis method based on the combination of peripheral blood markers to improve the accuracy of differential diagnosis of benign and malignant lung nodules, reduce unnecessary invasive examinations, and provide patients with a more accurate diagnostic basis.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: a peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules, the marker combination comprising microRNA-21, carcinoembryonic antigen CEA, cytokeratin 19 fragment CYFRA21-1, squamous cell carcinoma antigen SCC and vascular endothelial growth factor VEGF, wherein:

[0007] MicroRNA-21 is highly expressed in the peripheral blood of patients with malignant pulmonary nodules, and its expression level satisfies the following formula: M n -M b >0.5, where M m is the relative expression of microRNA-21 in the peripheral blood of patients with malignant pulmonary nodules determined by real-time fluorescence quantitative polymerase chain reaction qRT-PCR technology, ranging from 1.5 to 5.0, M b is the relative expression of microRNA-21 in the peripheral blood of patients with benign pulmonary nodules determined by the same qRT-PCR technique, ranging from 0.5 to 1.0;

[0008] The concentration of carcinoembryonic antigen CEA in the peripheral blood of patients with malignant pulmonary nodules is higher than that of patients with benign pulmonary nodules. The concentration relationship satisfies: C m -C b >5ng / mL, where C m is the concentration of CEA in the peripheral blood of patients with malignant pulmonary nodules determined by chemiluminescent immunoassay, ranging from 10-100 ng / mL. b is the concentration of CEA in the peripheral blood of patients with benign pulmonary nodules determined by the chemiluminescent immunoassay, which ranges from 0 to 5 ng / mL;

[0009] The content of cytokeratin 19 fragment CYFRA21-1 in the peripheral blood of patients with malignant pulmonary nodules is increased, and the relationship between the content is: Yx-Y b >3.3 ng / mL, where Y m is the CYFRA21-1 content in the peripheral blood of patients with malignant pulmonary nodules determined by electrochemiluminescence immunoassay, ranging from 5-20 ng / mL, Y hThe content of CYFRA21-1 in the peripheral blood of patients with benign pulmonary nodules determined by the electrochemiluminescence immunoassay is in the range of 0-2 ng / mL;

[0010] Squamous cell carcinoma antigen (SCC) can be detected in the peripheral blood of patients with malignant pulmonary nodules at abnormal levels, and its level characteristics are: S m -S b ≠0, where S m The SCC detection value in the peripheral blood of patients with malignant pulmonary nodules is determined by enzyme-linked immunosorbent assay (ELISA), and the range is 1.5-5.0 ng / mL i , S b The detection value of SCC in the peripheral blood of patients with benign pulmonary nodules determined by this ELISA method ranges from 0 to 1.0 ng / mL;

[0011] The expression of vascular endothelial growth factor VEGF in the peripheral blood of patients with malignant pulmonary nodules increases, and the expression relationship is: V m -V b >50pg / mL, where V m is the expression value of VEGF in the peripheral blood of patients with malignant pulmonary nodules determined by immunoturbidimetry, ranging from 150 to 500 pg / mL. b The expression level of VEGF in the peripheral blood of patients with benign pulmonary nodules determined by the immunoturbidimetric method is in the range of 50-100 pg / mL, and there are differences between the expression levels of the above markers in the peripheral blood of patients with malignant pulmonary nodules and those in the peripheral blood of patients with benign pulmonary nodules, which can be reflected by specific detection technology based on their chemical nature and molecular structure characteristics.

[0012] Furthermore, the real-time fluorescence quantitative polymerase chain reaction qRT-PCR technology for detecting microRNA-21 has a specific primer sequence for amplifying microRNA-21, and its forward primer sequence is 5'-ACACTCCAGCTGGGAGCTG-3', and the reverse primer sequence is 5'-CTCAACTGGTGTCGTGGA-3'. The reaction system for qRT-PCR reaction using this primer pair includes: 10 μL of 2×SYBR Green PCR Master Mix, 0.5 μL of each upstream and downstream primer with a concentration of 10 μM, 2 μL of template RNA, and 7 μL of enzyme-free water. The reaction conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 40 cycles; and finally extension at 72°C for 10 minutes.

[0013] Furthermore, the chemiluminescent immunoassay for detecting carcinoembryonic antigen CEA, the detection reagent used in the method comprises: a specific anti-CEA monoclonal antibody with a concentration of 1-5 μg / mL, a sheep anti-mouse IgG antibody labeled with a chemiluminescent substance, and a buffer with a pH value of 7.0-7.5, comprising a phosphate buffer and a bovine serum albumin component, and the detection process comprises: incubating the sample to be tested with the specific anti-CEA monoclonal antibody at 37° C. for 1-2 hours, adding a sheep anti-mouse IgG antibody labeled with a chemiluminescent substance after washing, incubating at 37° C. for 30-60 minutes, adding a chemiluminescent substrate solution after washing again, measuring the luminescence intensity on a chemiluminescence detector, and calculating the CEA concentration according to the standard curve.

[0014] Furthermore, the electrochemiluminescence immunoassay for detecting the cytokeratin 19 fragment CYFRA21-1, the detection kit comprises: magnetic beads coated with anti-CYFRA21-1 monoclonal antibodies at a concentration of 2-5 μg / mL, anti-CYFRA21-1 polyclonal antibodies labeled with terpyridine ruthenium, and electrochemiluminescence substrates. During the detection, the sample reacts immunologically with the reagents in the kit, the sample and the magnetic beads are first incubated at 37° C. for 1-1.5 hours, and then washed and then the anti-CYFRA21-1 polyclonal antibodies labeled with terpyridine ruthenium are added, and incubated at 37° C. for 30-45 minutes. After washing again, the electrochemiluminescence substrate is added, and the luminescence signal is measured on an electrochemiluminescence detector. The reaction temperature during the detection process is controlled at 37° C., and the total reaction time is 2-2.5 hours.

[0015] Furthermore, the enzyme-linked immunosorbent assay (ELISA) method for detecting squamous cell carcinoma antigen SCC, the microplate used in the ELISA method is coated with an anti-SCC monoclonal antibody with a concentration of 1-3 μg / mL, the enzyme-labeled secondary antibody used for detection is a goat anti-mouse IgG antibody labeled with horseradish peroxidase HRP, and its dilution is 1:5000-1:10000, the substrate solution is a 3',5,5'-tetramethylbenzidine TMB solution with a concentration of 0.1-0.5 mg / mL, the sample reacts with the coated antibody and the enzyme-labeled secondary antibody in the microplate in sequence, incubated at 37° C. for 1-1.5 hours, washed, added with TMB substrate solution, and color developed in the dark at room temperature for 15-30 minutes, finally added with a stop solution, and the absorbance is measured at a wavelength of 450 nm to quantify the level of SCC.

[0016] Furthermore, the immunoturbidimetric method for detecting vascular endothelial growth factor VEGF, the antibody used in the immunoturbidimetric method is a rabbit anti-human VEGF polyclonal antibody, the concentration of which is 5-10 μg / mL, the composition of the reaction buffer includes 0.01-0.1M, pH 7.2-7.6 phosphate buffer, 0.1-0.5M sodium chloride, 0.5%-2% polyethylene glycol, during the detection process, VEGF in the sample forms an immune complex with the antibody and is incubated at 37°C for 30-60 minutes.

[0017] Furthermore, when testing the patient's peripheral blood, the sample collection and processing process is as follows: 3-5 mL of the patient's peripheral venous blood is collected, placed in an anticoagulant tube containing ethylenediaminetetraacetic acid EDTA anticoagulant with a concentration of 1-2 mg / mL, stored at 4-8°C, and processed within 2-4 hours. The processing process includes centrifugation at 4°C and 3000-4000 rpm for 10-15 minutes, and the supernatant is transferred to a new sterile centrifuge tube. If it cannot be tested immediately, the supernatant can be stored below -80°C.

[0018] Furthermore, the method for determining the test results is as follows: respectively determining the levels of microRNA-21, CEA, CYFRA21-1, SCC and VEGF in the patient's peripheral blood; when the relative expression of microRNA-21 meets the expression relationship of patients with malignant pulmonary nodules, the concentration of CEA meets the concentration relationship of patients with malignant pulmonary nodules, the content of CYFRA21-1 meets the content relationship of patients with malignant pulmonary nodules, the detection value of SCC meets the abnormal level relationship, and the expression of VEGF meets the expression relationship of patients with malignant pulmonary nodules, it is determined that the possibility of the patient's pulmonary nodule being malignant is high; otherwise, if the level relationship of the above markers does not meet the characteristics of patients with malignant pulmonary nodules, it is determined that the possibility of the patient's pulmonary nodule being benign is high.

[0019] Compared with the existing technology, the peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules has the following beneficial effects:

[0020] The marker combination of the present invention comprehensively considers the role of different markers in distinguishing benign and malignant lung nodules through the joint detection of multiple markers. Compared with single marker detection, it can more accurately distinguish the benign and malignant lung nodules, reduce the misdiagnosis and missed diagnosis rates, and avoid the pain and risks brought to patients by invasive operations based on peripheral blood marker detection, which is easier to be accepted by patients. This marker combination can effectively distinguish the benign and malignant lung nodules in patient groups with different clinical characteristics, is not significantly affected by these factors, has broad clinical application prospects, and can provide a reliable basis for the early diagnosis and clinical treatment of lung nodules.

[0021] Other advantages, objectives and features of the present invention will be set forth in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is a flowchart of a combination of peripheral blood markers for differential diagnosis of benign and malignant pulmonary nodules. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Embodiment 1

[0026] A 55-year-old male patient had a long history of smoking (20 cigarettes a day, smoking for 30 years). During the physical examination, a chest CT scan revealed a nodule with a diameter of 1.8 cm in his lungs. The patient had no obvious symptoms such as cough, sputum, or chest pain, but given the presence of the nodule, further differentiation of its benign or malignant nature was required.

[0027] According to the sample collection method of the present invention, 4 mL of peripheral venous blood is collected from the patient using a disposable blood collection needle, and the blood is injected into an anticoagulant tube containing ethylenediaminetetraacetic acid (EDTA) anticoagulant (concentration of 1.5 mg / mL), gently inverted to mix, and then stored in an environment of 4-8°C and subsequently processed within 3 hours.

[0028] The collected anticoagulation tube samples were centrifuged at 4°C and 3500 rpm for 12 minutes.

[0029] After centrifugation, carefully transfer the supernatant to a new sterile centrifuge tube. If not tested immediately, quickly store the supernatant in a -80°C refrigerator.

[0030] Marker detection:

[0031] For microRNA-21 detection (qRT-PCR method), the stored supernatant was taken out, the total RNA therein was extracted, and then the extracted RNA was used as a template for real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) to detect microRNA-21.

[0032] The reaction system was configured as follows: 10 μL of 2×SYBR Green PCR Master Mix, 0.5 μL of upstream and downstream primers (the forward primer sequence was 5'-ACACTCCAGCTGGGAGCTG-3', the reverse primer sequence was 5'-CTCAACTGGTGTCGTGGA-3', both concentrations were 10 μM), 2 μL of template RNA, and 7 μL of enzyme-free water.

[0033] The reaction system was placed in a qRT-PCR instrument and amplified according to the following reaction conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 40 cycles; and finally extension at 72°C for 10 minutes. After the reaction was completed, the relative expression level of microRNA-21 was calculated to be 3.2 according to the standard curve (within the relative expression level range of 1.5-5.0 of microRNA-21 in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention).

[0034] CEA test (chemiluminescent immunoassay) uses chemiluminescent immunoassay to detect carcinoembryonic antigen (CEA). The detection reagent contains specific anti-CEA monoclonal antibody (concentration of 3μg / mL), sheep anti-mouse IgG antibody labeled with chemiluminescent substance (acridinium ester) and buffer (pH 7.2, containing phosphate buffer, bovine serum albumin and other components).

[0035] The sample to be tested was incubated with specific anti-CEA monoclonal antibody at 37°C for 1.5 hours, washed after the incubation, and then added with goat anti-mouse IgG antibody labeled with chemiluminescent substance, and incubated at 37°C for another 45 minutes. After washing again, chemiluminescent substrate solution was added, and the luminescence intensity was immediately measured on a chemiluminescence detector.

[0036] The concentration of CEA was calculated based on the pre-established standard curve to be 25 ng / mL (within the CEA concentration range of 10-100 ng / mL in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention and meeting the CEA concentration range of 10-100 ng / mL). m -C b >5 ng / mL, C b The CEA concentration in the peripheral blood of patients with benign pulmonary nodules is usually lower (range 0-5 ng / mL).

[0037] CYFRA21-1 detection (electrochemiluminescent immunoassay) uses electrochemiluminescent immunoassay to detect cytokeratin 19 fragment (CYFRA21-1). The detection kit contains magnetic beads coated with anti-CYFRA21-1 monoclonal antibodies (concentration of 3.5μg / mL), anti-CYFRA21-1 polyclonal antibodies labeled with terpyridine ruthenium, and electrochemiluminescent substrates (including tripropylamine and other components).

[0038] The sample and the magnetic beads in the kit were incubated at 37°C for 1.2 hours, and after washing, an anti-CYFRA21-1 polyclonal antibody labeled with terpyridine ruthenium was added, and incubated at 37°C for 35 minutes. After washing again, an electrochemiluminescent substrate was added, and the luminescent signal was measured on an electrochemiluminescence detector. During the detection process, the reaction temperature was strictly controlled at 37°C, and the total reaction time was about 2.2 hours. It was determined that the content of CYFRA21-1 was 12 ng / mL (within the range of 5-20 ng / mL of CYFRA21-1 content in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention, and met Y m -Y b >3.3 ng / mL, Y b The CYFRA21-1 level in the peripheral blood of patients with benign pulmonary nodules is lower (range 0-2 ng / mL).

[0039] SCC detection (ELISA method): Squamous cell carcinoma antigen (SCC) was detected by enzyme-linked immunosorbent assay (ELISA). The microplate was pre-coated with anti-SCC monoclonal antibody (concentration of 2 μg / mL), the enzyme-labeled secondary antibody used for detection was horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (dilution of 1:8000), and the substrate solution was 3,3',5,5'-tetramethylbenzidine (TMB) solution (concentration of 0.3 mg / mL).

[0040] The sample was incubated with the coated antibody at 37°C for 1.2 hours in a microplate, washed, and then the enzyme-labeled secondary antibody was added, incubated at 37°C for 1 hour, washed again, and then the TMB substrate solution was added. The color was developed at room temperature in the dark for 20 minutes, and finally the stop solution (2M sulfuric acid) was added. The absorbance was measured at a wavelength of 450nm. The SCC detection value was calculated according to the standard curve to be 3.5ng / mL (within the SCC detection value range of 1.5-5.0ng / mL in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention, and the SCC detection value meets the SCC detection value of 1.5-5.0ng / mL). m -S b ≠0, S b The lower SCC detection value range is 0-1.0 ng / mL in the peripheral blood of patients with benign pulmonary nodules).

[0041] VEGF detection (immunoturbidimetry) was performed to detect vascular endothelial growth factor (VEGF) using the immunoturbidimetry method, using rabbit anti-human VEGF polyclonal antibody (concentration of 8 μg / mL), and the reaction buffer contained phosphate buffer (0.05 M, pH 7.4), sodium chloride (0.3 M), and polyethylene glycol (1%).

[0042] Result judgment:

[0043] The VEGF in the sample was incubated with the antibody at 37°C for 45 minutes, and then the turbidity change of the reaction system at a wavelength of 700nm was measured to determine the expression of VEGF. After detection, the expression of VEGF was 300pg / mL (within the range of 150-500pg / mL of VEGF expression in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention, and meeting V m -V b >50pg / mL, V b The VEGF expression level in the peripheral blood of patients with benign pulmonary nodules is lower (range 50-100 pg / mL).

[0044] The relative expression level of microRNA-21 in the patient's peripheral blood was 3.2, the CEA concentration was 25 ng / mL, the CYFRA21-1 content was 12 ng / mL, the SCC detection value was 3.5 ng / mL, and the VEGF expression level was 300 pg / mL, all of which met the expression characteristics of each marker in the peripheral blood of patients with malignant pulmonary nodules in the present invention (i.e., the relationship between the above formula and the numerical range). Comprehensively judging, the patient's pulmonary nodules are likely to be malignant.

[0045] According to the test result determination method of the present invention, the levels of each marker obtained by the above detection are analyzed, and the result determination table is as follows:

[0046]

[0047] The effects brought by this embodiment are as follows:

[0048] The peripheral blood marker combination and detection method of the present invention can accurately differentiate the benign and malignant nature of lung nodules. In this embodiment, after the patient's lung nodules are discovered through chest CT, the marker detection of the present invention confirms that there is a high possibility that they are malignant, which provides an important basis for subsequent further clinical diagnosis and treatment, and avoids delays in the disease due to unclear diagnosis.

[0049] The entire detection process only requires the collection of peripheral venous blood, which is a non-invasive operation. It avoids the pain and risks that may be caused by invasive examinations such as bronchoscopy or percutaneous lung puncture biopsy, such as complications such as bleeding and pneumothorax. Patients do not need to bear additional physical burdens when undergoing the test, which improves patient compliance. It is especially suitable for patients like those in this embodiment who have no obvious symptoms but the nature of lung nodules is unknown.

[0050] From sample collection to final result determination, the entire process is relatively simple and efficient. The sample collection process is simple and easy, and can be carried out in the outpatient clinics or physical examination centers of regular medical institutions. Although the various detection technologies used in the detection process are somewhat professional, they can be carried out relatively smoothly in laboratories with corresponding equipment and technicians, and the time required for detection is relatively short (such as the detection of each marker can be completed within a few hours), which can provide patients with diagnostic results in a timely manner, helping clinicians to quickly formulate treatment plans or further examination plans.

[0051] Embodiment 2

[0052] A 45-year-old female patient with no smoking history was found to have a ground-glass nodule with a diameter of about 1.2 cm in her lungs through low-dose spiral CT screening during the annual physical examination organized by her unit. The patient had no other discomfort symptoms except occasional slight fatigue. In order to clarify the nature of the nodule, the doctor decided to use the peripheral blood marker detection method of the present invention to assist in diagnosis.

[0053] According to standard operating procedures, a sterile blood collection needle was used to collect 3.5 mL of peripheral venous blood from the patient. The blood was slowly injected into an anticoagulant tube containing ethylenediaminetetraacetic acid (EDTA) anticoagulant (concentration of 1.2 mg / mL). The anticoagulant tube was gently inverted 8-10 times to fully mix the blood and anticoagulant. The anticoagulant tube was then temporarily stored in a cold chain box at 4-8°C and transported to the laboratory for processing within 2.5 hours.

[0054] After the sample arrived at the laboratory, the anticoagulant tube was taken out from the cold chain box and centrifuged in a centrifuge at 4°C and 3200 rpm for 10 minutes.

[0055] After centrifugation, use a pipette to carefully transfer the supernatant to a pre-labeled sterile centrifuge tube. Avoid touching the precipitate during the transfer. If the supernatant cannot be tested immediately, quickly place it in a -80°C ultra-low temperature refrigerator for storage. Be sure to mark and record the samples during storage.

[0056] For microRNA-21 detection (qRT-PCR method), the supernatant stored in the -80°C refrigerator was taken out, and total RNA was extracted using an appropriate method according to the kit instructions. During the extraction process, care was taken to prevent RNA degradation. The extracted RNA was used as a template for real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) to detect microRNA-21.

[0057] The reaction system was prepared as follows: 2×SYBR Green PCR Master Mix 10 μL, upstream and downstream primers (forward primer sequence is 5'-ACACTCCAGCTGGGAGCTG-3', reverse primer sequence is 5'-CTCAACTGGTGTCGTGGA-3', concentration of both is 10 μM) 0.5 μL each, template RNA 2 μL, enzyme-free water 7 μL.

[0058] The reaction tube was placed in a qRT-PCR instrument, and the reaction conditions were set as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 40 cycles; and finally extension at 72°C for 10 minutes. After the reaction was completed, the amplification curve and the melting curve were analyzed to ensure good reaction specificity. The relative expression level of microRNA-21 was calculated to be 2.0 based on the standard curve (within the relative expression level range of 1.5-5.0 of microRNA-21 in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention).

[0059] CEA test (chemiluminescent immunoassay), use chemiluminescent immunoassay to detect carcinoembryonic antigen (CEA), prepare the detection reagent, which includes specific anti-CEA monoclonal antibody (concentration of 2μg / mL), sheep anti-mouse IgG antibody labeled with chemiluminescent substance (acridinium ester) and buffer (pH 7.0, including phosphate buffer, bovine serum albumin and other components).

[0060] The sample to be tested was incubated with the specific anti-CEA monoclonal antibody at 37°C for 1 hour, and washed 3 times with a special washing solution after the incubation. Then, a sheep anti-mouse IgG antibody labeled with a chemiluminescent substance was added, and incubated at 37°C for 30 minutes. After washing 3 times again, a chemiluminescent substrate solution was added, and the reaction tube was quickly placed in a chemiluminescence detector to measure the luminescence intensity. The CEA concentration was calculated to be 8 ng / mL according to the pre-established standard curve (within the CEA concentration range of 10-100 ng / mL in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention, and the CEA concentration of 8 ng / mL was satisfied. m -C b >5 ng / mL, C b The CEA concentration in the peripheral blood of patients with benign pulmonary nodules is usually lower (range 0-5 ng / mL).

[0061] CYFRA21-1 detection (electrochemiluminescent immunoassay) uses electrochemiluminescent immunoassay to detect cytokeratin 19 fragment (CYFRA21-1). The detection kit contains magnetic beads coated with anti-CYFRA21-1 monoclonal antibodies (concentration of 2.5μg / mL), anti-CYFRA21-1 polyclonal antibodies labeled with terpyridine ruthenium, and electrochemiluminescent substrates (including tripropylamine and other components).

[0062] The sample and the magnetic beads in the kit were incubated at 37°C for 1 hour, washed 3 times with a special washing solution after the incubation, and then an anti-CYFRA21-1 polyclonal antibody labeled with terpyridine ruthenium was added, incubated at 37°C for 30 minutes, washed again 3 times, and then an electrochemiluminescent substrate was added. The luminescent signal was measured on an electrochemiluminescence detector. During the detection process, the reaction temperature was strictly controlled to be 37°C, and the total reaction time was about 2 hours. It was determined that the content of CYFRA21-1 was 7 ng / mL (within the range of CYFRA21-1 content in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention of 5-20 ng / mL, and met Y m -Y b >3.3 ng / mL, Y b The CYFRA21-1 level in the peripheral blood of patients with benign pulmonary nodules is lower (range 0-2 ng / mL).

[0063] SCC detection (ELISA method): Squamous cell carcinoma antigen (SCC) was detected by enzyme-linked immunosorbent assay (ELISA). The microplate was pre-coated with anti-SCC monoclonal antibody (concentration of 1.5 μg / mL), the enzyme-labeled secondary antibody used for detection was horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (dilution of 1:5000), and the substrate solution was 3,3',5,5'-tetramethylbenzidine (TMB) solution (concentration of 0.2 mg / mL).

[0064] The sample was incubated with the coated antibody at 37°C for 1 hour in a microplate, washed 3 times with a washing solution after the incubation, and then the enzyme-labeled secondary antibody was added, incubated at 37°C for 1 hour, washed again 3 times, and then the TMB substrate solution was added. The color was developed at room temperature in the dark for 15 minutes, and finally the stop solution (2M sulfuric acid) was added. The absorbance was measured at a wavelength of 450nm, and the SCC detection value was calculated according to the standard curve to be 2.0ng / mL (within the SCC detection value range of 1.5-5.0ng / mL in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention, and the SCC detection value meets the SCC detection value of 1.5-5.0ng / mL). m -S b ≠0, S b The lower SCC detection value range is 0-1.0 ng / mL in the peripheral blood of patients with benign pulmonary nodules).

[0065] VEGF detection (immunoturbidimetry) was performed to detect vascular endothelial growth factor (VEGF) using the immunoturbidimetry method, using rabbit anti-human VEGF polyclonal antibody (concentration of 6 μg / mL), and the reaction buffer contained phosphate buffer (0.03 M, pH 7.3), sodium chloride (0.2 M), and polyethylene glycol (0.8%).

[0066] The VEGF in the sample was incubated with the antibody at 37°C for 30 minutes, and then the reaction tube was placed in a turbidity meter to measure the turbidity change of the reaction system at a wavelength of 650nm to determine the expression of VEGF. After testing, the expression of VEGF was 200pg / mL (within the range of 150-500pg / mL of VEGF expression in the peripheral blood of patients with malignant pulmonary nodules specified in the present invention, and meeting V m -V b >50pg / ml, V b The VEGF expression level in the peripheral blood of patients with benign pulmonary nodules is lower (range 50-100 pg / mL).

[0067] According to the result judgment standard of the present invention, the detection results of each marker in the patient's peripheral blood are comprehensively evaluated, and the result judgment table is as follows:

[0068]

[0069] The relative expression of microRNA-21 in the patient's peripheral blood was 2.0, the CEA concentration was 8 ng / mL, the CYFRA21-1 content was 7 ng / mL, the SCC detection value was 2.0 ng / mL, and the VEGF expression was 200 pg / mL, all of which were consistent with the expression characteristics of each marker in the peripheral blood of patients with malignant pulmonary nodules in the present invention (i.e., the relationship between the above formula and the numerical range). Comprehensively judging, the patient's pulmonary nodules are likely to be malignant.

[0070] The effects brought by this embodiment are as follows:

[0071] This example once again demonstrates the effectiveness and accuracy of the peripheral blood marker combination and detection method of the present invention in the differential diagnosis of benign and malignant lung nodules. For the ground glass nodules found in the lungs of this female patient, by detecting peripheral blood markers, it can be more accurately judged that the possibility of malignancy is high, which provides strong support for further clinical diagnosis and formulation of personalized treatment plans, and helps to improve the cure rate and survival rate of patients.

[0072] When the patient has no obvious symptoms and the nodules are small (1.2 cm in diameter), the detection method of the present invention can detect the potential risk of malignant nodules at an early stage. Early diagnosis is crucial for the treatment of malignant tumors such as lung cancer. It can enable patients to receive timely intervention in the early stages of the disease, avoid the disease from progressing to the late stage, and reduce the difficulty of treatment and the patient's suffering.

[0073] The patient in this embodiment is a middle-aged non-smoking female, and has different clinical characteristics from the smoking male patient in Example 1, which shows that the marker combination and detection method of the present invention are not significantly affected by factors such as the patient's gender and smoking history, and have a wide range of applicability and can be applied to different types of lung nodule patient groups, providing clinicians with a reliable and universal auxiliary means for diagnosing lung nodules. At the same time, the detection method is based on peripheral blood and is relatively simple to operate. It can be promoted and applied in primary medical institutions or physical examination centers, which helps to improve the early screening and diagnosis of lung nodules, and is of great significance for the prevention and treatment of lung cancer.

[0074] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the same elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules, characterized in that: The marker combination includes microRNA-21, carcinoembryonic antigen CEA, cytokeratin 19 fragment CYFRA21-1, squamous cell carcinoma antigen SCC and vascular endothelial growth factor VEGF, wherein: MicroRNA-21 is highly expressed in the peripheral blood of patients with malignant pulmonary nodules, and its expression level satisfies the following formula: M m -M b >0.5, where M m is the relative expression of microRNA-21 in the peripheral blood of patients with malignant pulmonary nodules determined by real-time fluorescence quantitative polymerase chain reaction qRT-PCR technology, ranging from 1.5 to 5.0, M b is the relative expression of microRNA-21 in the peripheral blood of patients with benign pulmonary nodules determined by the same qRT-PCR technique, ranging from 0.5 to 1.0; The concentration of carcinoembryonic antigen CEA in the peripheral blood of patients with malignant pulmonary nodules is higher than that of patients with benign pulmonary nodules. The concentration relationship satisfies: C m -C b >5ng / mL, of which C m is the concentration of CEA in the peripheral blood of patients with malignant pulmonary nodules determined by chemiluminescent immunoassay, ranging from 10-100 ng / mL. b is the concentration of CEA in the peripheral blood of patients with benign pulmonary nodules determined by the chemiluminescent immunoassay, which ranges from 0 to 5 nng / mL; The content of cytokeratin 19 fragment CYFRA21-1 in the peripheral blood of patients with malignant pulmonary nodules is increased, and the relationship between the content is: Y m -Y b >3.3ng / mL, of which Y m is the CYFRA21-1 content in the peripheral blood of patients with malignant pulmonary nodules determined by electrochemiluminescence immunoassay, ranging from 5-20 ng / mL, Y h The content of CYFRA21-1 in the peripheral blood of patients with benign pulmonary nodules determined by the electrochemiluminescence immunoassay is in the range of 0-2 ng / mL; Squamous cell carcinoma antigen (SCC) can be detected in the peripheral blood of patients with malignant pulmonary nodules at abnormal levels, and its level characteristics are: S m -S b ≠0, where S m The SCC detection value in the peripheral blood of patients with malignant pulmonary nodules is determined by enzyme-linked immunosorbent assay (ELISA), and the range is 1.5-5.0 ng / mL i , S b The detection value of SCC in the peripheral blood of patients with benign pulmonary nodules determined by this ELISA method ranges from 0 to 1.0 ng / mL; The expression of vascular endothelial growth factor VEGF in the peripheral blood of patients with malignant pulmonary nodules is increased, and the expression relationship is: V m -V b >50pg / mL, of which V m is the expression value of VEGF in the peripheral blood of patients with malignant pulmonary nodules determined by immunoturbidimetry, ranging from 150 to 500 pg / mL. b The expression level of VEGF in the peripheral blood of patients with benign pulmonary nodules determined by the immunoturbidimetric method is in the range of 50-100 pg / mL, and there are differences between the expression levels of the above markers in the peripheral blood of patients with malignant pulmonary nodules and those in the peripheral blood of patients with benign pulmonary nodules, which can be reflected by specific detection technology based on their chemical nature and molecular structure characteristics.

2. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: The real-time fluorescence quantitative polymerase chain reaction qRT-PCR technology for detecting microRNA-21 has a specific primer sequence for amplifying microRNA-21, wherein the forward primer sequence is 5'-ACACTCCAGCTGGGAGCTG-3', and the reverse primer sequence is 5'-CTCAACTGGTGTCGTGGA-3'. The reaction system for qRT-PCR reaction using the primer pair comprises: 10 μL of 2×SYBR Green PCR Master Mix, 0.5 μL of each upstream and downstream primer with a concentration of 10 μM, 2 μL of template RNA, and 7 μL of enzyme-free water. The reaction conditions are: pre-denaturation at 95° C. for 5 minutes; denaturation at 95° C. for 15 seconds, annealing at 60° C. for 30 seconds, and extension at 72° C. for 30 seconds, for a total of 40 cycles; and finally extension at 72° C. for 10 minutes.

3. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: The chemiluminescent immunoassay for detecting carcinoembryonic antigen CEA, the detection reagent used in the method comprises: a specific anti-CEA monoclonal antibody with a concentration of 1-5 μg / mL, a goat anti-mouse IgG antibody labeled with a chemiluminescent substance, and a buffer with a pH value of 7.0-7.5, comprising a phosphate buffer and bovine serum albumin components, and the detection process comprises: incubating the sample to be tested with the specific anti-CEA monoclonal antibody at 37°C for 1-2 hours, adding the goat anti-mouse IgG antibody labeled with a chemiluminescent substance after washing, incubating at 37°C for 30-60 minutes, adding the chemiluminescent substrate solution after washing again, measuring the luminescence intensity on a chemiluminescent detector, and calculating the CEA concentration according to a standard curve.

4. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: The electrochemiluminescence immunoassay for detecting the cytokeratin 19 fragment CYFRA21-1, wherein the detection kit comprises: magnetic beads coated with anti-CYFRA21-1 monoclonal antibodies at a concentration of 2-5 μg / mL, anti-CYFRA21-1 polyclonal antibodies labeled with terpyridine ruthenium, and electrochemiluminescence substrates. During the detection, the sample reacts with the reagents in the kit by immunohistochemistry. The sample and the magnetic beads are first incubated at 37° C. for 1-1.5 hours, and then washed and then the anti-CYFRA21-1 polyclonal antibodies labeled with terpyridine ruthenium are added. The samples are incubated at 37° C. for 30-45 minutes, and then washed again and then the electrochemiluminescence substrates are added. The luminescence signal is measured on an electrochemiluminescence detector. The reaction temperature during the detection process is controlled at 37° C., and the total reaction time is 2-2.5 hours.

5. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: The enzyme-linked immunosorbent assay (ELISA) method for detecting squamous cell carcinoma antigen SCC, the microplate used in the ELISA method is coated with an anti-SCC monoclonal antibody with a concentration of 1-3 μg / mL, the enzyme-labeled secondary antibody used for detection is a goat anti-mouse IgG antibody labeled with horseradish peroxidase HRP with a dilution of 1:5000-1:10000, the substrate solution is a 3',5,5'-tetramethylbenzidine TMB solution with a concentration of 0.1-0.5 mg / mL, the sample reacts with the coated antibody and the enzyme-labeled secondary antibody in the microplate in sequence, incubated at 37° C. for 1-1.5 hours, washed, added with TMB substrate solution, color developed at room temperature in the dark for 15-30 minutes, finally added with a stop solution, and the absorbance is measured at a wavelength of 450 nm to quantify the level of SCC.

6. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: The immunoturbidimetric method for detecting vascular endothelial growth factor (VEGF) uses a rabbit anti-human VEGF polyclonal antibody with a concentration of 5-10 μg / mL. The reaction buffer comprises 0.01-0.1 M phosphate buffer with a pH value of 7.2-7.6, 0.1-0.5 M sodium chloride, and 0.5%-2% polyethylene glycol. During the detection process, VEGF in the sample forms an immune complex with the antibody and is incubated at 37° C. for 30-60 minutes.

7. A peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: When testing the patient's peripheral blood, the sample collection and processing process is as follows: collect 3-5mL of the patient's peripheral venous blood, place it in an anticoagulant tube containing ethylenediaminetetraacetic acid EDTA anticoagulant with a concentration of 1-2mg / mL, store it at 4-8℃, and process it within 2-4 hours. The processing process includes centrifugation at 4℃, 3000-4000rpm for 10-15 minutes, and transfer the supernatant to a new sterile centrifuge tube. If it cannot be tested immediately, the supernatant can be stored below -80℃.

8. The peripheral blood marker combination for differential diagnosis of benign and malignant pulmonary nodules according to claim 1, characterized in that: The method for determining the test results is as follows: respectively determine the levels of microRNA-21, CEA, CYFRA21-1, SCC and VEGF in the patient's peripheral blood; when the relative expression of microRNA-21 meets the expression relationship of patients with malignant pulmonary nodules, the concentration of CEA meets the concentration relationship of patients with malignant pulmonary nodules, the content of CYFRA21-1 meets the content relationship of patients with malignant pulmonary nodules, the detection value of SCC meets the abnormal level relationship, and the expression of VEGF meets the expression relationship of patients with malignant pulmonary nodules, it is determined that the possibility of the patient's pulmonary nodule being malignant is higher; otherwise, if the level relationship of the above markers does not meet the characteristics of patients with malignant pulmonary nodules, it is determined that the possibility of the patient's pulmonary nodule being benign is higher.

Images