Protein Biomarkers for Lymphangioleiomyomatosis and Their Applications

Abstract

The present invention discloses a protein biomarker for lymphangioleiomyomatosis and its application, belonging to the technical field of immunoassay. Specifically, it includes detecting the expression level of any one or a combination of two or more of ferritin, β2-microglobulin or transferrin in the plasma of a subject. Experiments have found that there are significant differences in the contents of ferritin, β2-microglobulin and transferrin in the plasma between patients with lymphangioleiomyomatosis and the healthy control group. Therefore, any one or a combination of two or more of ferritin, β2-microglobulin or transferrin can be used as a biomarker for the auxiliary diagnosis of LAM, providing a positive significance for assisting clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to a clinical diagnosis method for lymphangioleiomyomatosis, belonging to the technical field of immunoassay, and specifically relates to a protein biomarker for lymphangioleiomyomatosis and its application. Background Art

[0002] Lymphangioleiomyomatosis (LAM) is a rare, idiopathic, multi-system, low-grade malignant neoplastic disease, which is more common in women of childbearing age, extremely rare in children and men, and the female incidence is about 5 / 1 million. The lungs are the main affected organs, and the typical manifestation is diffuse cystic changes in both lungs. LAM has an insidious onset and often progresses slowly. The early symptoms are mild. As the disease progresses, progressive decline in lung function, dyspnea and even respiratory failure occur. The prognosis of this disease is poor, and there is currently no cure. Relevant studies have shown that the 5-year, 10-year, 15-year and 20-year survival rates of LAM patients are 94%, 85%, 75% and 64% respectively.

[0003] Currently, the clinical diagnosis of pulmonary LAM depends on characteristic chest HRCT changes and simultaneously meets any one of the following manifestations: lung biopsy confirmed as LAM; combined with tuberous sclerosis; combined with renal angiomyolipoma; serum vascular endothelial growth factor-D (VEGF-D) ≥ 800 ng / L; chylous pleural or peritoneal effusion; retroperitoneal or pelvic tumor pathology confirmed as LAM.

[0004] However, due to the diversity of its clinical symptoms, patients who do not meet the typical signs cannot be excluded from LAM, and invasive lung biopsy still needs to be performed. Therefore, finding non-invasive, economical, applicable, convenient and safe biomarkers is of great significance for assisting the diagnosis of LAM and subsequent treatment methods. Summary of the Invention

[0005] To solve the above technical problems, the present invention discloses a protein biomarker for lymphangioleiomyomatosis and its application. By detecting the expression level of any one or a combination of two or more of ferritin, β2-microglobulin or transferrin in the plasma of a subject, it is further used to assist in the diagnosis of LAM, providing a positive significance for assisting clinical diagnosis and treatment.

[0006] To achieve the above technology, the present invention discloses the application of any one or a combination of two or more of ferritin, β2-microglobulin or transferrin as a biomarker in the preparation of a detection agent for assisting the diagnosis of lymphangioleiomyomatosis.

[0007] Further, the biomarker includes a combination of ferritin, β2-microglobulin and transferrin.

[0008] Furthermore, the biomarker includes a combination of ferritin and β2-microglobulin.

[0009] Furthermore, the biomarker includes a combination of ferritin and transferrin.

[0010] Furthermore, the biomarker includes a combination of β2-microglobulin and transferrin.

[0011] Furthermore, it includes detecting the expression level of ferritin and / or β2-microglobulin and / or transferrin in the blood of the subject.

[0012] Furthermore, the lymphangioleiomyomatosis is pulmonary lymphangioleiomyomatosis.

[0013] The present invention also discloses a detection agent having the above application, and the detection agent includes a substance capable of specifically binding to ferritin and / or β2-microglobulin and / or transferrin.

[0014] Furthermore, the detection agent further includes a substance for detecting the expression level of ferritin and / or β2-microglobulin and / or transferrin.

[0015] Furthermore, the detection agent is an ELISA kit or a chemiluminescence kit.

[0016] In addition, the present invention also discloses a screening method for ferritin, β2-microglobulin or transferrin in the blood, as specifically described in the examples.

[0017] Beneficial effects:

[0018] Through experimental exploration, the present invention found that the expression levels of ferritin and β2-microglobulin in the plasma of LAM patients are significantly higher than those of normal people, and transferrin is significantly lower than that of normal people, and there are significant differences in each expression level. Therefore, any one or a combination of two or more of ferritin, β2-microglobulin or transferrin can be used as a biomarker for the auxiliary diagnosis of LAM, providing a reference basis for clinical diagnosis and treatment. Description of the drawings

[0019] Figure 1 It is a diagram of differential expression of ferritin between the LAM disease group and the healthy control group;

[0020] Figure 2 It is a diagram of differential expression of β2-microglobulin between the LAM disease group and the healthy control group;

[0021] Figure 3 It is a diagram of differential expression of transferrin between the LAM disease group and the healthy control group;

[0022] Figure 4 It is an ROC curve diagram for diagnosing LAM with ferritin;

[0023] Figure 5 ROC curve for diagnosing LAM with β2 microglobulin;

[0024] Figure 6 ROC curve for diagnosing LAM with transferrin;

[0025] Figure 7 ROC curve for diagnosing LAM with ferritin + β2 microglobulin;

[0026] Figure 8 ROC curve for diagnosing LAM with ferritin + transferrin;

[0027] Figure 9 ROC curve for diagnosing LAM with β2 microglobulin + transferrin;

[0028] Figure 10 ROC curve for diagnosing LAM with the combination of three indicators of ferritin + β2 microglobulin + transferrin. Detailed implementation methods

[0029] Terms and explanations

[0030] Lymphangioleiomyomatosis: Abbreviated as LAM in English, it is a systemic disease of unknown etiology. Due to abnormal proliferation of smooth muscle, it causes obstruction of bronchi, lymphatic vessels and small blood vessels, and develops progressively. Clinically, there are often manifestations such as dyspnea, spontaneous pneumothorax, chylothorax, etc. Typical chest imaging shows thin-walled small cysts diffusely distributed in both lungs.

[0031] Ferritin: Abbreviated as FRT in English, it is a widely existing iron storage protein with a hydrated iron oxide core of nanometer size and a protein shell with a cage-like structure. It mainly exists in the spleen, liver, bone marrow, serum, blood cells, etc.

[0032] β2 microglobulin: Abbreviated as B2M in English, it is a small molecule globulin produced by lymphocytes, platelets and polymorphonuclear leukocytes, with a molecular mass of 11,800 and a single-chain polypeptide composed of 99 amino acids. It is widely present in plasma, urine, cerebrospinal fluid, saliva and colostrum.

[0033] Transferrin: Abbreviated as TF in English, it is the main iron-containing protein in plasma, with a molecular weight of about 77,000, a single-chain glycoprotein, and a sugar content of about 6%. It is responsible for transporting iron absorbed by the digestive tract and iron released by the degradation of red blood cells. It enters the bone marrow in the form of a TF-Fe 3+ complex to supply the generation of mature red blood cells.

[0034] ELISA kit: That is, an enzyme-linked immunosorbent assay kit. The ELISA kit protected by the present invention may include a solid support, and further includes means for detecting biomarkers. For example, an antibody that can specifically bind to a biomarker. The kit includes a container for collecting blood samples, reagents for immunochemical detection of proteins collected in the container, and instructions. The instructions include a description of the use of the kit, as well as diagnostic criteria for assessing lymphangioleiomyomatosis associated with elevated or decreased protein levels.

[0035] Chemiluminescence kit: The chemiluminescence kit protected by the present invention may include a nucleic acid probe capable of specifically hybridizing with mRNA, and the mRNA is related to the gene of one or more biomarker proteins. The kit includes a container for collecting blood samples, reagents for extracting RNA from the blood in the container, and instructions for using the kit to analyze RNA by known methods. The instructions include, for example, diagnostic criteria for assessing lymphangioleiomyomatosis associated with elevated or decreased protein levels.

[0036] Regarding the use of "comprising", "including", "having", etc. in this article, they are all open-ended terms, that is, they are intended to include but not limited to.

[0037] Unless otherwise specified, all technical and scientific terms used in this article have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0038] The specific embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, those of ordinary skill in the art can make various changes, modifications, substitutions, and variations to these embodiments without creative efforts, and still fall within the protection scope of the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels. Throughout the specification, unless otherwise specifically stated, the terms used in this article should be understood as having the meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used in this article have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. If there is a contradiction, the definition in this article takes precedence.

[0040] Example 1

[0041] Experimental subjects and materials:

[0042] 1. Experimental subjects

[0043] Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology included patients clinically diagnosed with LAM disease and healthy control groups from January 2018 to December 2021.

[0044] Among them, the inclusion criteria for the above LAM disease group are as follows:

[0045] (1) Meeting the diagnostic criteria of the 2018 Chinese LAM expert consensus;

[0046] (2) No infection within the past 2 weeks;

[0047] (3) No other respiratory diseases such as asthma and tuberculosis;

[0048] (4) No other chronic diseases such as hypertension and diabetes;

[0049] (5) Not receiving treatment.

[0050] The inclusion criteria for the above healthy control group are as follows:

[0051] Female, no infection within the past two weeks, no chronic respiratory diseases, no history of tumors such as uterine fibroids, no family history of tuberous sclerosis, and no smoking history.

[0052] In this example, 10 patients with LAM disease and 6 healthy control groups were collected. At the same time, the basic information of each experimental subject, including age, gender, BMI, etc., was recorded during sample collection. There were no statistical differences in age, gender, and BMI between the disease group and the healthy group.

[0053] 2. Sample collection

[0054] Collect 5 mL of peripheral blood from the experimental subjects in a fasting state into an anticoagulant collection tube. After standing at room temperature for 30 minutes, centrifuge at 4°C and 3000 rpm for 10 minutes. Then use a pipette to aspirate the upper plasma in the collection tube and aliquot it into 1.5 mL EP tubes, label the sample number and collection date, and store it in a -80°C refrigerator.

[0055] 3. Proteomics technology detection

[0056] (3.1) Protein extraction: Take out the sample from -80°C, thaw it on ice and then centrifuge at 4°C and 12000g for 10 minutes to remove cell debris. Transfer the supernatant to a new centrifuge tube and remove high-abundance proteins according to the usage requirements of the instructions of the relevant centrifuge tube or centrifuge column. In the present invention, the preferred centrifuge column is preferably Pierce TM Top 14 Abundant ProteinDepletion Spin Columns Kit (Thermo Scientific). Then use a BCA protein quantification kit to measure the protein concentration.

[0057] (3.2) Trypsin digestion: Take equal amounts of protein from each sample for digestion. Add an appropriate amount of standard protein, adjust the volume to be consistent with lysis buffer, then add dithiothreitol (DTT) to a final concentration of 5 mM, and reduce at 56 °C for 30 min. Then add iodoacetamide (IAA) to a final concentration of 11 mM, and incubate in the dark at room temperature for 15 min. Transfer the alkylated sample to an ultrafiltration tube, centrifuge at 12000 g at room temperature for 20 min, replace with 8 M urea three times, and then replace urea with replacement buffer three times. Add trypsin at a ratio of 1:50 (protease: protein, m / m), and digest overnight. Centrifuge at 12000 g at room temperature for 10 min to recover the peptide segments, and recover the peptide segments with ultrapure water once. Combine the two peptide segment solutions.

[0058] (3.3) TMT labeling: The peptide segments obtained by trypsin digestion are preferably desalted using Strata X C18 (Phenomenex) and then vacuum freeze-dried. Dissolve the peptide segments with 0.5 M tetraethylammonium bromide buffer (TEAB), and label the peptide segments according to the operation instructions of the TMT kit. The simple operation is as follows: After thawing, dissolve the labeling reagent with acetonitrile, mix it with the peptide segments, and incubate at room temperature for 2 h. After mixing the labeled peptide segments, desalt them and vacuum freeze-dry.

[0059] (3.4) HPLC fractionation: The peptide segments are fractionated by high pH reversed-phase HPLC. The chromatographic column is preferably Agilent 300 Extend C18 (particle size 5 μm, inner diameter 4.6 mm, length 250 mm). The operation is as follows: The peptide segment fractionation gradient is 8%-32% acetonitrile, pH 9, and 60 components are separated in 60 min. Subsequently, the peptide segments are combined into 9 components. After the combined components are vacuum freeze-dried, follow-up operations are carried out.

[0060] (3.5) LC-MS / MS analysis: The peptide segments are dissolved with mobile phase A of liquid chromatography and then separated using an EASY-nLC 1200 ultra-high performance liquid system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient settings are as follows: 0-38 min, 4%-20% B; 38-52 min, 20%-32% B; 52-56 min, 32%-80% B; 56-60 min, 80% B, and the flow rate is maintained at 500 nL / min. After the peptide segments are separated by the ultra-high performance liquid system, they are injected into the NSI ion source for ionization and then enter Q Exactive TMThe analysis was performed in an HF-X mass spectrometer. The ion source voltage was set to 2.1 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap mass spectrometer. The primary mass spectrometer scanning range was set to 350-1600 m / z, and the scanning resolution was set to 120,000; the secondary mass spectrometer scanning range was fixed at 100 m / z, and the secondary scanning resolution was set to 45,000. The data acquisition mode used a data-dependent scanning program (DDA), that is, after the primary scan, the top 20 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometer, the automatic gain (AGC) control was set to 1E5, the signal threshold was set to 8.3E4 ions / s, the maximum injection time was set to 60 ms, and the dynamic exclusion time of the tandem mass spectrometer scan was set to 30 seconds to avoid repeated scanning of the parent ion.

[0061] (3.6) Data analysis: The raw data from the mass spectrometer was imported into the search software, and the secondary mass spectrometry data was preferably retrieved using the proteomics analysis software Proteome Discoverer (v2.4.1.15). The accuracy FDR of identification at the three levels of spectrum, peptide, and protein was set to 1%; the identified protein must contain at least one specific peptide. The relative quantitative value of the protein was calculated based on the signal intensity value of each peptide in different samples.

[0062] 4. Differential protein screening

[0063] This experiment was divided into LAM disease group and healthy control group. After TMT labeling mass spectrometry analysis, a total of 543 quantifiable proteins were identified. For the identified proteins, the ratio of the mean relative quantitative values of the LAM disease group and the healthy control group was calculated as the difference multiple. In order to judge the significance of the difference, the relative quantitative value of each protein in the comparison group sample was subjected to T test, and the corresponding P value was calculated as the significance index. In order to make the test data conform to the normal distribution required by the T test, the protein relative quantitative value needs to be transformed by Log2 before the test. The calculation formula is as follows: Pik = T.test (Log2 (Rik, i∈A), Log2 (Rik, i∈B)), where R represents the relative quantitative value of the protein, i represents the sample, k represents the protein, A represents the LAM disease group, and B represents the healthy control group. Through the above difference analysis, when P value <0.05, the difference expression change of more than 1.5 is used as the threshold of significant upregulation, and less than 1 / 1.5 is used as the threshold of significant downregulation. Among them, the expression of ferritin (FRT) and β2-microglobulin (B2M) was significantly upregulated in LAM patients, and the expression of transferrin (TF) was significantly downregulated in LAM patients. The specific expression is shown in Table 1.

[0064] Table 1 Expression of each protein

[0065]

[0066] As can be seen from Table 1 above, the fold change of ferritin (FRT) is 2.354, and the fold change of β2-microglobulin (B2M) is 1.782, satisfying p ≤ 0.05 and fold change ≥ 1.5; the fold change of transferrin (TF) is 0.659, satisfying fold change ≤ 0.666 and p ≤ 0.05. Therefore, we chose to further explore the relationship between these three proteins and LAM disease.

[0067] Example 2

[0068] Relationship between the protein expression levels of ferritin (FRT), β2-microglobulin (B2M), and transferrin (TF) and LAM

[0069] 1. Experimental samples:

[0070] Blood samples were collected from 25 LAM patients and 20 healthy controls. There were no statistical differences in age, gender, and BMI between the two groups. 5 mL of peripheral blood from the research subjects in the fasting state was collected into an anticoagulant blood collection tube, left to stand at room temperature for 30 minutes, and then centrifuged at 4°C and 3000 rpm for 10 minutes. Then, the upper plasma in the blood collection tube was aspirated with a pipette and aliquoted into 1.5 mL EP tubes, labeled with the sample number and collection date, and stored frozen at -80°C in a refrigerator.

[0071] 2. Detection of proteins by immunoturbidimetry

[0072] The above samples were taken out from -80°C and thawed on ice. The contents of FRT, B2M, and TF proteins were detected by immunoturbidimetry on a Roche cobas c701 biochemical analyzer in the clinical laboratory. The Mann Whitney test was used to compare whether there were differences in FRT, B2M, and TF between the LAM disease group and the healthy control group.

[0073] 3. Experimental results

[0074] Combined Figure 1-3 It can be seen that compared with the healthy control group, the levels of FRT and B2M in the plasma samples of the LAM disease group were significantly increased (p < 0.05), and the level of TF was significantly decreased (p < 0.05). This indicates that the protein expression levels of FRT, B2M, and TF can be used for the auxiliary diagnosis of LAM.

[0075] Example 3

[0076] Authenticity determination of ferritin (FRT), β2-microglobulin (B2M), and transferrin (TF) for the auxiliary diagnosis of LAM

[0077] According to the measured expression levels of FRT, B2M, and TF in the LAM group and the healthy control group, the ROC curve was plotted using SPSS software to analyze the diagnostic efficacy of FRT, B2M, and TF for LAM, and Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 . Combining with the ROC curve shown in Figure 4-10 , taking the OD value with the largest Youden index as the cut-off value, and simultaneously calculating the corresponding area under the curve (AUC), as well as specificity and sensitivity, Table 2 was obtained.

[0078] Table 2 Evaluation list of the ROC curve

[0079]

[0080] As can be seen from Table 2 above, the areas under the curves of FRT, B2M, TF, FRT + B2M, FRT + TF, B2M + TF, and FRT + B2M + TF are 0.699, 0.705, 0.689, 0.768, 0.690, 0.756, and 0.776 respectively.

[0081] In summary, the protein expression levels of FRT, B2M, and TF in the blood have good diagnostic efficacy for the auxiliary diagnosis of LAM. Especially the combination of the three indicators has high accuracy, sensitivity, and specificity.

[0082] The present invention also discloses and protects a detection agent, which is used for the directional or quantitative detection of ferritin and / or β2-microglobulin and / or transferrin in plasma. When in use, only the detection agent needs to be contacted with the blood of the subject, and the detection result can be displayed after a certain period of time. It is convenient to use and the operation is fast.

[0083] Specifically, the detection agent includes substances that can specifically bind to ferritin and / or β2-microglobulin and / or transferrin, and also includes substances for detecting the expression levels of ferritin and / or β2-microglobulin and / or transferrin. For example, the detection agent can be an ELISA kit or a chemiluminescence kit, etc.

[0084] In summary, the present invention explores and discovers that ferritin, β2-microglobulin, or transferrin in the plasma of LAM patients can be used as biomarkers for the auxiliary diagnosis of LAM, providing positive significance for assisting clinical diagnosis and treatment.

Claims

1. Use of β2-microglobulin as a biomarker in the preparation of a detection agent for assisting in the diagnosis of lymphangioleiomyomatosis.

2. Use of ferritin as a biomarker in the preparation of a detection agent for assisting in the diagnosis of lymphangioleiomyomatosis.

3. Use of the combination of ferritin and β2-microglobulin as a biomarker in the preparation of a detection agent for assisting in the diagnosis of lymphangioleiomyomatosis.

4. Use of the combination of transferrin and β2-microglobulin as a biomarker in the preparation of a detection agent for assisting in the diagnosis of lymphangioleiomyomatosis.

5. Use of the combination of ferritin and transferrin as a biomarker in the preparation of a detection agent for assisting in the diagnosis of lymphangioleiomyomatosis.

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