Proteomics-based spinal cord injury inflammation marker as well as screening method and application thereof

Abstract

The invention discloses a spinal cord injury inflammation marker based on proteomics as well as a screening method and application thereof, creatively provides establishment of a relation chain between a cerebrospinal fluid sample and spinal cord injury, verifies the reference value of the relation chain for identifying grade differentiation of spinal cord injury, finds out a key point of the relation chain, namely a biomarker, and further provides a screening method for the spinal cord injury inflammation marker based on proteomics. The biomarker, namely the inflammation marker, specifically comprises five core proteins: interleukin 16 (IL-16), tumor necrosis factor (TNF-alpha), complement component 5a (C5a), matrix metalloproteinase 8 (MMP-8) and interleukin 3 (IL-3), and is beneficial to effectively, quickly and accurately understanding the pathogenesis of spinal cord injury.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to proteomics-based spinal cord injury inflammatory markers and screening methods and applications thereof. Background Art

[0002] The spinal cord exists in the spine and is part of the central nervous system. The spinal cord has the function of conducting ascending and descending nerve impulses and performing reflex activities. It is an important component that supports the normal use of the spine. The importance of the spinal cord to the human body is self-evident.

[0003] The spinal cord connects the brain to the rest of the body and is one of the main channels for transmitting brain commands. The spinal cord is long and cylindrical, with a total length of 41 to 45 cm. The upper end is connected to the medulla oblongata in the skull, and the lower end is conical, ending at the lower edge of the first lumbar vertebra. The spinal cord is located in the vertebral canal, consistent with the curvature of the spine. The spinal cord has a distinct segmented structure, including four parts: cervical, thoracic, lumbar, and sacral. The spinal cord is protected by the vertebral canal and transmits information to the nervous system scattered in various parts, and transmits perception to the brain and limbs. It also has the functions of controlling muscle contraction and glandular secretion. At the same time, it is also responsible for regulating body movement, visceral activity, and the input and output of sensations, and maintaining the balance of the body's internal environment. Therefore, spinal cord injury can lead to varying degrees of paralysis and sensory disorders, and even life-threatening in severe cases. If the spinal cord is uncomfortable, it is recommended to seek medical attention in time.

[0004] Spinal cord injury refers to the damage of spinal cord function caused by external force or other diseases. There may be sensory, motor, reflex and other neurological dysfunctions below the injury plane. The level of spinal cord injury is divided into acute, subacute and chronic according to the injury period. According to the severity, it is divided into ASIA, A / B / C. The symptoms of spinal cord injury are related to the degree, location and age of injury. Patients may experience pain, swelling, muscle spasms and movement disorders at the injured site. Usually, symptoms of spinal cord injury appear within 24 hours to 6 days after injury, but some patients may not show symptoms until hours or days after injury. Some patients may even have minor injuries after injury and have no obvious changes from external observation. However, if this type of patients do not pay attention to protection after injury, the best treatment period will be delayed and even irreversible secondary damage will be caused.

[0005] There is a reaction time from the onset of spinal cord injury to the lesion and then to the irreversible stage. It is crucial to accurately determine the degree and type of injury, or even confirm whether there is an injury within this time frame, which is of great significance for restoring the patient's physical activities. Existing methods for determining spinal cord injury include diagnosing the condition through physical examinations, imaging examinations, electrophysiological examinations, laboratory tests, etc. However, there are many influencing factors in the diagnosis: on the one hand, patients often cannot cooperate with physical examinations due to coma or fractures, etc., and the ASIA score cannot be performed. On the other hand, the imaging findings often do not match the severity of the injury, resulting in doctors lacking a clear and objective assessment of the patient and being unable to adopt appropriate treatment methods and timely adjust the treatment dosage. On the one hand, patients often cannot cooperate with physical examinations due to coma or fractures, etc., and the ASIA score cannot be performed. On the other hand, the imaging findings often do not match the severity of the injury, resulting in doctors lacking a clear and objective assessment of the patient. There is a risk during sampling, which is likely to cause secondary harm to the body with unclear conditions, and also cannot adopt appropriate treatment methods and timely adjust the treatment dosage. This is time-consuming and not comprehensive and accurate enough. Therefore, other methods need to be found to replace them.

[0006] Currently, there are no reliable results for patients with different severity grades and at different time points after injury. Therefore, we included a cohort of cerebrospinal fluid samples from patients with different severities of spinal cord injury at different time points, sequenced them through a human cytokine array, and performed bioinformatics analysis.

[0007] Cerebrospinal fluid does not belong to spinal cord tissue, but general spinal cord injury will cause changes in cerebrospinal fluid. One form of change is specifically manifested as an increase in the protein content in cerebrospinal fluid. Therefore, proteomics can be used to study the proteins in cerebrospinal fluid as the research object. Proteomics is the science that studies the protein composition and its activity laws in cells, tissues, or the whole organism at the overall level. By studying the characteristics of proteins through proteomics, including the expression level of proteins, post-translational modifications, and protein-protein interactions, etc., a comprehensive understanding of the processes such as disease occurrence and disease development at the protein level can be obtained. Since the mechanism of spinal cord injury is a complex process involving many factors, using proteomics can help people understand the pathogenesis and treatment methods of spinal cord injury more deeply. Summary of the Invention

[0008] Aiming at the shortcomings of the existing technology, the present invention provides a spinal cord injury biomarker based on proteomics, its screening method, and application.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] Spinal cord injury markers, and the spinal cord injury inflammatory markers are selected from one or more of the following substances: IL-16, TNF-α, C5a, MMP-8, IL-3.

[0011] The present invention also provides a method for screening the spinal cord injury inflammatory markers, comprising the following steps:

[0012] 1) Sample selection;

[0013] 2) Filter the cerebrospinal fluid control sample and the reference sample;

[0014] 3) Take the filtered cerebrospinal fluid sample, spot the cerebrospinal fluid sample with a spotter and fix it in a 96-well plate for reaction for 1 hour; block the plate with a solution containing skim milk, take out the substances in the well plate, break the gel with a glass rod, dissolve and soak it overnight with a buffer solution, centrifuge and take the supernatant for concentration, subject the concentrated solution to ultrasonic-assisted enzymatic hydrolysis for 15 s, and filter the enzymatic hydrolysis solution with a 0.45 μm filter membrane;

[0015] 4) Identify the filtrate in step 3) by LC-MS / MS liquid chromatography-mass spectrometry; the liquid chromatography conditions are as follows: the chromatographic column is a MONO-Q column; the injection volume is 10 μL; mobile phase A is 10 mmol / L NaOH, and mobile phase B is: 10 mmol / L NaOH + 1.8 mol / L NaCl; the gradient is 0-5 min, 0% B, 5-23 min, 0-80% B, 23-35 min, 100% B, and the flow rate is: 0.2 mL / min; the detection wavelength is detected at UV230 nm; the mass spectrometry measurement parameters are set as follows: the mass-to-charge ratio range is 400-5000, the step size is 0.25, the scanning time is 6 s, the low OR voltage is 35 V, and the high OR voltage is 100 V; other parameter settings are all set according to the parameters after PPG calibrates the instrument;

[0016] 5) Use the labelfree quantification method based on MS1 and MS2 mass spectrometry data for data analysis; by comparing the number of mass spectrometry analysis times or the intensity of mass spectrometry peaks, analyze the abundance changes of sample proteins, based on MS1, calculate the integral of the signal intensity of each peptide segment on LC-MS, and use the identification results of MS2 as the quantitative basis to correct the data on a large scale;

[0017] 6) Screen out significantly up-regulated or down-regulated protein biomarkers through the corrected data analysis results;

[0018] The data analysis includes quality control of proteome sequencing data, differential gene analysis, GO / KEGG analysis, WGCNA, PPI interaction analysis, and screening out the final biomarkers;

[0019] The biomarkers are IL-16, TNF-α, C5a, MMP-8 and IL-3.

[0020] Preferably, the buffer is 0.25-0.3% bromophenol blue solution: 0.25-0.3% xylene cyanol FF: 40% sucrose aqueous solution = 15-25:15:60-70.

[0021] The present invention also provides the application of spinal cord injury inflammation markers based on proteomics in the preparation of products for diagnosing spinal cord injury.

[0022] Preferably, the product is a detection reagent, a kit, a microarray or a biochip.

[0023] Preferably, the biomarker is used to detect a sample by means of protein array sequencing and bioinformatics analysis, and the sample is from cerebrospinal fluid.

[0024] Advantages of the present invention:

[0025] The present invention creatively proposes to replace the direct reference sample of spinal cord tissue, establish the relationship chain between cerebrospinal fluid samples and spinal cord injury, and verify the reference value of such a relationship chain for differentiating the grades of spinal cord injury, find out the key points of the relationship chain - biomarkers, which is objectively beneficial to effectively, quickly and accurately understand the pathogenesis of spinal cord injury and can thus find targeted treatment methods for patients with spinal cord injury. Description of the Drawings

[0026] Figure 1 It is a schematic diagram of proteomic analysis at different injury degrees and time points;

[0027] Figure 2 It is a schematic diagram of differential analysis between acute phase and subacute phase;

[0028] Figure 3 It is a schematic diagram of differential analysis between acute phase and subacute phase;

[0029] Figure 4 It is a schematic diagram of differential analysis between subacute phase and subacute phase;

[0030] Figure 5 It is a differential analysis and expression difference heat map between acute phase and control;

[0031] Figure 6 It is a differential analysis and expression difference heat map between subacute phase and control;

[0032] Figure 7 It is a differential analysis and expression difference heat map between subacute phase and control;

[0033] Figure 8Schematic diagram for differential analysis between grade A and grade B;

[0034] Figure 9 Schematic diagram for differential analysis between grade A and grade C;

[0035] Figure 10 Schematic diagram for differential analysis between grade B and grade C;

[0036] Figure 11 Schematic diagram for weighted gene co-expression network analysis - screening of co-expressed core proteins. Detailed implementation mode

[0037] The following further elaborates on the design, positive sample verification, and result analysis of the present invention in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0038] In the embodiments of the present invention, the raw materials, samples, reagents, equipment, etc. used in the preparation process of the tea beverage are all commercially available products and can be purchased in the market.

[0039] The following further describes the present invention through specific embodiments, but it does not limit the scope of the present invention.

[0040] Example 1:

[0041] Set basic grading conditions

[0042] The most commonly used quantitative diagnostic method for neurological function assessment is the neurological function score of the American Spinal Cord Injury Association (ASIA). The grading and conditions are shown in the following table:

[0043]

[0044] The content of the above table can be interpreted as follows: According to the ASIA score, patients with different severities of grade A, grade B, and grade C are selected respectively; and according to the injury time, three time points of the acute phase, subacute phase, and subchronic phase after injury of the same patient are selected respectively.

[0045] Preliminary screening

[0046] 1) Sample selection;

[0047] 2) Filter the cerebrospinal fluid control sample and the reference sample;

[0048] 3) Take the filtered cerebrospinal fluid sample, spot the cerebrospinal fluid sample using a spotter and fix it in a 96-well plate respectively for reaction for 1 hour; block the plate with a solution containing skim milk, take out the substances in the well plate, crush the gel with a glass rod, dissolve and soak it overnight with a buffer solution, take the supernatant after centrifugation and concentrate it, subject the concentrated solution to ultrasonic-assisted enzymatic hydrolysis for 15 s, and filter the enzymatic hydrolysate through a 0.45 μm filter membrane;

[0049] 4) Identify the filtrate in step 3) by LC-MS / MS liquid chromatography-mass spectrometry; the liquid chromatography conditions are as follows: the chromatographic column is a MONO-Q column; the injection volume is 10 μL; mobile phase A is 10 mmol / L NaOH, and mobile phase B is: 10 mmol / L NaOH + 1.8 mol / L NaCl; the gradient is 0 - 5 min, 0% B, 5 - 23 min, 0 - 80% B, 23 - 35 min, 100% B, and the flow rate is: 0.2 mL / min; the detection wavelength is detected at UV230 nm; the mass spectrometry measurement parameters are set as follows: the mass-to-charge ratio range is 400 - 5000, the step size is 0.25, the scanning time is 6 s, the low OR voltage is 35 V, and the high OR voltage is 100 V; other parameter settings are all set according to the parameters after calibrating the instrument with PPG;

[0050] 5) Adopt the labelfree quantification method and conduct data analysis based on MS1 and MS2 mass spectrometry data; by comparing the number of mass spectrometry analyses or the intensity of mass spectrometry peaks, analyze the abundance changes of sample proteins, based on MS1, calculate the integral of the signal intensity of each peptide segment on LC-MS, and take the identification result of MS2 as the quantification basis to correct the data on a large scale;

[0051] 6) Screen out significantly up-regulated or down-regulated protein biomarkers through the corrected data analysis results; precise data analysis

[0052] The data analysis includes quality control of proteome sequencing data, differential gene analysis, GO / KEGG analysis, WGCNA, PPI interaction analysis, and screening out the final biomarkers:

[0053] Chip pretreatment: After the chip is taken out of the package, it needs to be equilibrated at room temperature for 20 - 30 minutes to avoid condensation caused by temperature difference in subsequent experiments. After opening the package and removing the sealing strip, place the chip in a vacuum dryer for drying for at least 1 hour, or dry it at room temperature for 1 - 2 hours to minimize background interference.

[0054] Experimental procedure: This experiment adopts the quantitative antibody chip technology to quantitatively analyze the expression level of the target protein in the biological sample by detecting the fluorescence signal intensity.

[0055] Blocking and sample incubation: Add 100 μL of sample diluent to each chip well and incubate on a shaker at room temperature for 1 hour for blocking. After removing the blocking solution, pre-treat according to the sample type: serum samples are diluted with sample diluent at a ratio of 1:2, and cerebrospinal fluid samples are directly used with the original solution. Add 60 μL of pre-treated sample to each well and incubate at 4°C overnight (12-16 hours) to promote the full binding of the target protein in the sample with the antibody on the chip.

[0056] 3 washes: This protocol includes three identical washes, all performed using the ThermoScientific Wellwash Versa microplate washer, to remove unbound material. Each wash consists of the following two sub-steps:

[0057] Wash 10 times with 1× washing solution I (stock solution is 20×) diluted with deionized water, 250 μL per well each time, and shake at high intensity for 10 seconds.

[0058] Replace with 1× washing solution II (the original solution is 20×) diluted with deionized water and wash 6 times with the same parameters as above.

[0059] Incubation of detection antibody with Cy3-streptavidin: After centrifugation of the detection antibody mixture and the Cy3-streptavidin tubes, 1.4 mL of sample diluent was added to each well to mix thoroughly, and then centrifuged again quickly to remove possible aggregates. First, 80 μL of the diluted detection antibody mixture was added to each well and incubated on a shaker at room temperature for 2 hours to allow the detection antibody to specifically bind to the target protein bound to the chip. After the incubation is completed, wash once. Then, add 80 μL of diluted Cy3-streptavidin to each well, wrap the chip with aluminum foil for light-proof incubation, and incubate on a shaker at room temperature for 1 hour. This step allows Cy3-streptavidin to bind to the biotin label on the detection antibody in preparation for subsequent fluorescence detection. After the incubation is completed, wash again.

[0060] Fluorescence scanning: The chip was scanned at an excitation wavelength of 532 nm (Cy3 / green channel) and a resolution of 10 μm using an InnoScan 300 Microarray Scanner laser scanner, produced by Innopsys (Carbonne, France), to detect the fluorescence signal.

[0061] Bioinformatics analysis

[0062] Analysis software: This example mainly uses R software (4.2.0) for bioinformatics analysis.

[0063] Sample repeatability assessment: To evaluate whether the experimental results of biological replicate samples are statistically consistent and to improve the accuracy and reliability of the experiment, this study used principal component analysis (PCA) and box plots for visual analysis. PCA can show the distribution of samples in a multi-dimensional space, observe whether the samples cluster by group, and thus evaluate the within-group and between-group variations. Box plots can visually compare the dispersion and median of sample data in each group, and thus evaluate the repeatability between samples.

[0064] Differential expression analysis: On the basis of data quality control, this study considered the following differential expression analysis methods according to the experimental design and data characteristics: differential expression analysis based on the PLGEM model, and the ggplot2 package in R language was used to draw volcano plots and heatmaps to visually display the differentially expressed proteins. Volcano plots show the fold change and statistical significance (usually using the adjusted p-value, such as FDR), and heatmaps show the overall change trend of protein expression profiles in different samples.

[0065] Protein function enrichment analysis: To deeply understand the biological functions of differentially expressed proteins, this study conducted function enrichment analysis and set the significance P-value threshold for enrichment test to 0.05. Multiple testing correction methods are usually used to adjust the P-value to control the false positive rate.

[0066] GO enrichment analysis: The clusterProfiler was used to classify and annotate genes, revealing the biological functions and interrelationships of different genes and gene sets from three aspects: molecular function, cellular component, and biological process.

[0067] KEGG pathway enrichment analysis: The clusterProfiler was used to perform KEGG pathway enrichment analysis on differentially expressed proteins to understand the functions and interactions of genes and proteins in metabolic pathways, providing important clues for studying biological processes, discovering potential biomarkers, and developing drugs.

[0068] WGCNA analysis: Weighted gene co-expression network analysis - screening co-expressed core proteins: To more comprehensively analyze the data and obtain protein co-expression modules related to clinical information, weighted gene co-expression network analysis (WGCNA) was performed.

[0069] Results found:

[0070] Grouped based on the injury level (ASIA classification) and injury time point, and further analyzed the dynamic changes in cerebrospinal fluid protein expression: in the cerebrospinal fluid of patients with ASIA-C grade compared to those with grades A and B, the expression of inflammation-related proteins such as interleukin IL-16, IL-18, IL-6, IL-1F10, etc. was more significantly upregulated; at the same time, some upregulated proteins were shared between the 3-day acute-phase and subacute-phase samples, such as IL-33, IL-3, C5a, IL-32α, etc., and these proteins were significantly different from the 1-day acute-phase samples. It is worth noting that tumor necrosis factor α (TNFα) and matrix metalloproteinase 8 (MMP-8) were only significantly upregulated at 1 day of the acute phase, for reference Figure 1 .

[0071] The emerald green module had a stronger correlation with the subacute phase. Further analysis identified 5 core proteins, namely inflammation biomarkers: interleukin 16 (IL-16), tumor necrosis factor (TNF-α), complement component 5a (C5a), matrix metalloproteinase 8 (MMP-8), and interleukin 3 (IL-3). The results can be seen in Figure 11 .

Claims

1. Spinal cord injury inflammatory markers, characterized in that The spinal cord injury inflammatory marker is selected from one or more of the following substances: IL-16, TNF-α, C5a, MMP-8, and IL-3.

2. A method for screening the spinal cord injury inflammatory marker according to claim 1, characterized in that: The steps include: 1) Sample selection; 2) filtering the CSF control sample and the reference sample; 3) taking the filtered cerebrospinal fluid samples, spotting the cerebrospinal fluid samples with a spotting instrument and fixing them in a 96-well plate for reaction for 1 hour; sealing the plate with a solution containing skim milk, taking out the substances in the well plate, breaking the gel with a glass rod, dissolving and soaking in a buffer solution overnight, concentrating the supernatant after centrifugation, performing ultrasound-assisted enzymatic hydrolysis on the concentrate for 15 seconds, and filtering the enzymatic hydrolyzate with a 0.45 μm filter membrane; 4) The filtrate of step 3) was subjected to protein identification by LC-MS / MS; the liquid chromatography conditions were as follows: the chromatographic column was a MONO-Q column; the injection volume was 10 μL; the mobile phase A was 10 mmol / L NaOH, and the mobile phase B was: 10 mmol / L NaOH + 1.8 mol / L NaCl; the gradient was 0-5 min, 0% B, 5-23 min, 0-80% B, 23-35 min, 100% B, and the flow rate was 0.2 mL / min; the detection wavelength was UV230 nm; the mass spectrometry parameters were set as follows: mass-to-charge ratio range 400-5000, step size 0.25, scan time 6 s, low OR voltage 35 V, high OR voltage 100 V; other parameter settings were all in accordance with the parameter settings after the PPG calibration instrument; 5) Using labelfree quantitative method to analyze data based on MS1 ​​and MS2 mass spectrometry data; analyzing the abundance changes of sample proteins by comparing the number of mass spectrometry analysis or mass spectrometry peak intensity, calculating the integral of the signal intensity of each peptide on LC-MS based on MS1, and correcting the data on a large scale based on the identification results of MS2; 6) Screening out significantly up-regulated or down-regulated protein biomarkers through the revised data analysis results; The data analysis includes quality control of proteomic sequencing data, differential gene analysis, GO / KEGG analysis, WGCNA, and PPI interaction analysis; The protein biomarkers are IL-16, TNF-α, C5a, MMP-8 and IL-3.

3. The screening method according to claim 2, characterized in that The buffer solution is 0.25-0.3% bromophenol blue solution: 0.25-0.3% xylene cyanol FF: 40% sucrose aqueous solution = 15-25:15:60-70.

4. Application of proteomics-based spinal cord injury inflammatory markers in the preparation of products for diagnosing spinal cord injury.

5. The use according to claim 4, characterized in that: The product is a detection reagent, a test kit, a microarray or a biochip.

6. The use according to claim 4, characterized in that: The biomarker is used to detect a sample by means of protein array sequencing and bioinformatics analysis, and the sample is from cerebrospinal fluid.

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