Biomarkers for liver ischemia-reperfusion injury based on proteomics and applications thereof

Abstract

The application discloses a biomarker of liver ischemia-reperfusion injury based on proteomics, and the biomarker is neutrophil granule protein (NGP), which can be used for early diagnosis and prevention of post-hepatectomy complications, prognosis monitoring and judgment, etc.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a biomarker for liver ischemia-reperfusion injury based on proteomics and application thereof. BACKGROUND

[0002] Despite the progress in surgical techniques and perioperative care, the incidence of postoperative complications after hepatectomy remains high, especially in patients with cirrhosis who often have impaired liver function, abnormal coagulation function, and reduced liver reserve function, and the patient's tolerance to surgery is weakened. Therefore, surgeons urgently need to rely on different preoperative assessments to stratify risks, provide a basis for determining whether liver resection is feasible and the scope of liver resection, so as to reduce the risk of liver resection surgery.

[0003] At present, the identification of useful biomarkers for predicting the risk of postoperative complications after liver resection is still insufficient, and the available serum biomarkers show low sensitivity and heterogeneous specificity. It is urgent to find one or more serum markers with high sensitivity and high specificity to predict postoperative complications and mortality after liver resection, so as to provide a basis for clinical or medical researchers to determine whether liver resection is feasible and the scope of liver resection.

[0004] Proteomics is a science that studies the composition and activity of proteins in cells, tissues or entire organisms at the whole level by taking proteins as the research object. By studying the characteristics of proteins, including the expression level of proteins, post-translational modification and protein-protein interaction, a comprehensive understanding of the processes of disease occurrence and disease development at the protein level can be obtained. In proteomics, the key technology for protein identification of biological samples is mass spectrometry (Mass Spectrum, MS). Hepatic ischemia-reperfusion injury (HIRI) is an important pathological mechanism of postoperative complications after liver resection, and since the mechanism of hepatic ischemia-reperfusion injury is a complex process involving many factors, proteomics can help people better understand the pathogenesis and treatment of hepatic ischemia-reperfusion injury.

[0005] Neutrophil granule protein (NGP) is a protein with a molecular weight of about 19.33kD, mainly existing in the granules of neutrophils. At present, there are few direct functional studies on NGP, but according to the biological characteristics of neutrophils, it is speculated that NGP may be involved in antibacterial defense, inflammation regulation and other processes. However, the study of NGP protein in hepatic ischemia-reperfusion injury is still in its infancy, and its predictive value for postoperative complications in liver resection patients has not been determined. SUMMARY

[0006] In order to overcome the above-mentioned defects of the prior art, the present application aims to provide a biomarker for liver ischemia-reperfusion injury based on proteomics and application thereof, which can be used for early diagnosis and prevention of postoperative complications of liver resection, prognosis monitoring and judgment, etc.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] In a first aspect of the present application, a biomarker for liver ischemia-reperfusion injury based on proteomics is provided, and the marker is NGP. The present application first detects the liver tissue proteome at 6h and 24h after liver ischemia-reperfusion (IR) in mice, finds that neutrophil granule protein (NGP) is significantly up-regulated after IR, further detects serum NGP of patients after liver resection, finds that NGP on the first day after operation can be used as a marker for predicting postoperative complications of liver resection. The serum NGP level can well predict the risk of postoperative complications of liver resection patients.

[0009] Further, the biomarker is used for detecting a sample from liver tissue by proteomic analysis means.

[0010] In a second aspect of the present application, the application of the neutrophil granule protein (NGP) in preparing a reagent, a kit, a microarray or a biochip for predicting postoperative complications of clinical liver resection patients is provided.

[0011] Further, the neutrophil granule protein is human serum neutrophil granule protein.

[0012] Further, the postoperative complications of clinical liver resection patients are severe complications of grade III-V according to Clavien-Dindo classification, such as hemorrhage, liver failure, abdominal infection, bile leakage, pleural effusion and pulmonary complications, ascites, etc.

[0013] Further, the reagent or kit comprises an immunoglobulin G antibody of the neutrophil granule protein.

[0014] Compared with the prior art, the present application has the following beneficial effects:

[0015] The present application provides a biomarker for postoperative complication risk analysis of liver resection and application thereof. With these biomarkers, a postoperative complication risk analysis reagent or kit for liver resection can be prepared to predict the risk of postoperative complications of clinical liver resection.

[0016] The biomarker provided by the application helps to better understand the pathophysiology of postoperative complications of clinical liver resection, provides new opportunities for diagnosis and prognosis, and thus improves the clinical service of patients. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A box plot of the whole proteome data of Example 1 of the application.

[0018] Figure 2 A PCA plot of the whole proteome of Example 1 of the application.

[0019] Figure 3 A volcano plot of DEPs of Example 1 of the application, red marks up-regulated proteins, gray marks proteins with no differential changes, and blue marks down-regulated proteins.

[0020] Figure 4 A clustering heat map analysis of DEPs of Example 1 of the application, blue is up-regulated protein, and cyan is down-regulated protein.

[0021] Figure 5 A bubble plot of G0 analysis of differential proteins of the whole proteome of Example 1 of the application.

[0022] Figure 6 A whole proteome level NGP Western Blot of Example 1 of the application.

[0023] Figure 7 An effect diagram of Western Blot for expression of NGP in liver tissue according to the application.

[0024] Figure 8 A diagnostic effect diagram of a mouse liver ischemia-reperfusion injury model according to the application. DETAILED DESCRIPTION

[0025] The following further illustrates the design, positive sample verification and result analysis of the application by combining with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. In addition, it should be understood that after reading the content taught by the application, those skilled in the art can make various modifications or changes to the application, and these equivalent forms also fall within the scope defined by the appended claims of the application.

[0026] Example 1

[0027] 1. Experimental materials and instruments

[0028] 1.1 Animal source

[0029] All experiments used 8-10 week old, 20-23 g adult male SPF C57BL / 6 mice (purchased from Xi'an Jiaotong University Animal Center).

[0030] 1.2 Main reagents for experiment

[0031] BCA Protein Assay Kit (Thermo); Western and IP cell lysis buffer (Meilunbio); Protease inhibitor cocktail (PD) (Roche); Phosphatase inhibitor (PPI) (Roche); Formic acid (FA) (mass spectrometry grade) (Sigma-Aldrich); Trifluoroacetic acid (TFA) (Sigma-Aldrich); Acetonitrile ACN (mass spectrometry grade) (Thermo Fisher Science); Anhydrous methanol (Guangzhou Chemical Reagent Factory); Iodoacetamide (IAA) (Sigma-Aldrich); Dithiothreitol (DTT) (Sigma-Aldrich); Urea (UREA) (Sigma-Aldrich); Trypsin (Sigma protein V5280); Anhydrous ethanol (Guangzhou Chemical Reagent Factory); Tetraethylammonium bromide (TEAB) (Sigma-Aldrich); iRT Kit (Biognosys); Tween (Sigma-Aldrich); ECL chemiluminescence substrate luminous liquid (Bio-Rad); PVDF membrane; 5x Loading buffer (Bio-Rad); Protein Marker (Bio-Rad); Skim milk powder (Genebase); Ammonium persulfate (APS) (Sigma-Aldrich); Sodium dodecyl sulfate (SDS) (Sigma-Aldrich); Tetramethyl ethylenediamine (TEMED) (Sigma Aldrich); Acrylamide (Sigma-Aldrich); 1.5M Tris-HCl buffer (Sigma-Aldrich).

[0032] 1.3 Main antibodies for experiment

[0033] Rabbit anti-NGP monoclonal antibody (1:1000) (Proteintech); GAPDH polyclonal antibody (1:2000) (Proteintech); Rabbit secondary antibody (1:5000) (Proteintech).

[0034] 1.4 Main instruments for experiment

[0035] Ice maker (Henan Tianchi Instrument and Equipment Co., Ltd.); electronic balance instrument (Dolence Science Instrument (Beijing) Co., Ltd.); low-temperature tissue grinder (Dinghaoyuan (Tianjin) Biological Technology Co., Ltd.); non-contact ultrasonic instrument (Ningbo Xinzhi Biological Technology Co., Ltd.); refrigerated centrifuge (Germany Eppendorf); mass spectrometer (Thermo Fisher Scientific); microplate reader (Xi'an Saiya Technology Co., Ltd.); vacuum desalination pump (Zhengzhou Great Wall Science and Technology Co., Ltd.); ELGA PURELAB Classic UV pure water machine (Guangzhou Shenhua Biological Technology Co., Ltd.); constant temperature culture shaker (Shanghai Zhichu Instrument Co., Ltd.); water bath (Beijing Changfeng Instrument Co., Ltd.); vortex shaker (Haimen Qilunbiel Instrument Manufacturing Co., Ltd.); freeze dryer (Hunan Hesi Instrument Equipment Co., Ltd.); protein gel electrophoresis device (Xi'an Tengling Biological Technology Co., Ltd.); multicolor fluorescence chemiluminescence gel imaging system (Time and Wisdom Biological Technology Co., Ltd.).

[0036] 1.5 Preparation of main reagents

[0037] (1) Reagent formula related to mass spectrometry experiment

[0038] a) 8M Urea: 24.024g Urea was dissolved in 50nL water, and concentrated hydrochloric acid was used to adjust the pH value to 8.0.

[0039] b) 1M DTT: 154.2mg DTT was dissolved in 1nL water.

[0040] c) 1M IAA: 184.96mg IAA was dissolved in 1mL water.

[0041] d) Conditioning Buffer: 0.1% TFA was dissolved in 20% acetonitrile solution.

[0042] e) Washing Buffer: 0.1% TFA was dissolved in 5% acetonitrile solution.

[0043] f) Elution Buffer: 0.1% TFA was dissolved in 60% acetonitrile solution.

[0044] (2) Reagent formula related to Western Blot

[0045] a) 5% concentrated gel, ultrapure water 2.1mL, 30% monomer 0.5nL, 1.5M Tris-HCl pH 6.80.38mL, 10% SDS 30μL, 10% APS 30μL, TEMED 3μL.

[0046] b) 10% Resolving Gel, Ultra-pure water 6.9 mL, 30% monomer 4 mL, 1.5 M Tris-HCI pH 8.8 0.75 mL, 10% SDS 60 μL, 10% APS 60 μL, TEMED 9 μL.

[0047] c) Electrophoresis Buffer (10x), Tris-base 30.3 g, Glycine 144.4 g, SDS 10 g, pure water 1000 mL.

[0048] d) Transfer Buffer (10x), Tris-base 37.9 g, Glycine 187.7 g, pure water 1000 mL.

[0049] e) Transfer Buffer 1x, 10x Transfer Buffer 80 mL, anhydrous methanol 200 mL, pure water 720 mL.

[0050] f) TBS Buffer (10x), Sodium Chloride 80 g, Potassium Chloride 2 g, Tris-base 30 g, pure water 1000 mL.

[0051] g) TBST Buffer (1x), 10x TBS Buffer 50 mL, Tween 20 500 μL, pure water 450 μL.

[0052] h) 5% Skim Milk, Skim Milk Powder 2.5 g, 1x TBST Buffer 50 L.

[0053] 2 Experimental Methods

[0054] 2.1 Establishing a Model of Liver Ischemia-reperfusion Injury in Mice

[0055] (1) In this example, 8-10 week old, 20-23 g adult male: SPF C57BL / 6 mice were used to construct a model of liver ischemia-reperfusion injury. Six mice were randomly divided into two groups: sham operation group (Sham) and injury group (Injury).

[0056] (2) Injury group mice as experimental group, ① Mice were anesthetized by intraperitoneal injection of anesthetic (such as 3% isoflurane gas or pentobarbital sodium). ② A midline incision was made in the abdomen, the abdominal cavity was opened, and the hepatic peduncle of the left and middle lobes of the liver (including the portal vein and hepatic artery) was carefully separated. ③ The portal vein and hepatic artery of the middle and left lobes were clamped with a non-invasive vascular clamp to cause about 70% liver ischemia. ④ After 0.5 min, compared with the non-blocked right lobe, the blocked lobe was obviously white under naked eye, indicating that the blocking was successful. The skin incision was clamped with a hemostat to temporarily close the abdominal cavity, and the mouse was placed on a 37°C constant temperature heating pad for warming. The incision was covered with a wet cotton pad during the operation interval to prevent fluid loss. ⑤ After 1 h of continuous ischemia, the vascular clamp was quickly removed, and the liver tissue was observed to be red, indicating that the blood flow was restored. After successful modeling, the subcutaneous tissue and skin were sutured in turn. The mice were deeply anesthetized by intraperitoneal injection of pentobarbital sodium at 6 h and 24 h after reperfusion, respectively, the ischemic liver tissue of the mice was extracted, washed with PBS for 3 times, and then placed into 2 mL cryogenic tubes, quickly frozen with liquid nitrogen, and stored in a refrigerator at a temperature of -80°C.

[0057] (3) The sham operation group mice as control group, only skin incision was performed, and the liver was not treated, and the subcutaneous tissue and skin were sutured in turn.

[0058] 2.3 Tissue grinding and lysis

[0059] (1) The mouse liver tissue was taken out from the -80°C refrigerator and placed on dry ice, and the liver tissue was cut into small pieces on the clean bench and placed into 2 mL grinding tubes. The grinding beads were washed with anhydrous ethanol and dried with a water-absorbing paper. A small amount of grinding beads was added to each grinding tube, and a pre-cooled tissue grinder was used for grinding (working conditions: 70 Hz, 2 min, -50°C grinding to powder

[0060] (2) 1 mL of cell lysis solution containing protease inhibitors and phosphatase inhibitors was added to the tissue grinding tube. The grinding beads in the tissue grinding tube were taken out with tweezers, and the non-contact ultrasonic was pre-cooled to 4C. The water circulation of the pre-cooled system was started, and the tissue in the grinding tube was ultrasonically broken (4C, ultrasonic 5s, interval 10s, 10min continuously). The tissue particles were completely lysed.

[0061] (3) The tissue grinding tube was placed on ice for lysis for 30 min, and shaken vigorously once every 10 min. The lysed tissue was centrifuged in a pre-cooled centrifuge (working conditions: 4°C, 12000 rpm, 30 min), and the supernatant was transferred to a 1.5 mL centrifuge tube with a pipette.

[0062] 2.4 High-efficiency protein digestion

[0063] (1) The liver protein sample extracted in the previous step is detected for protein concentration by BCA kit: dilute the protein standard with ultrapure water to prepare standard samples with final concentrations of 0 μg / μL, 0.0625 μg / μL, 0.125 μg / μL, 0.25 μg / μL, 0.5 μg / μL, 1 μg / μL, and 2 μg / μL. Prepare BCA working solution according to the ratio of 200 μL B liquid to 4 μL A liquid, incubate in a 37°C incubator for 30 minutes, and use a microplate reader to read the absorbance value at 570 nm. According to the protein concentration detected by the BCA kit, take 3 mg of the corresponding volume of the protein sample and place it in a 15 mL centrifuge tube for enzymolysis.

[0064] (2) Add appropriate volume of 8M Urea to the 15 mL centrifuge tube to make the final concentration greater than 4M (i.e. 1:1), that is, use the lowest concentration sample as the reference, and supplement the remaining higher concentration samples with 8M Urea to the same volume, and shake well.

[0065] (3) Prepare 1M DTT (gold water preparation), add appropriate volume to make the working concentration 50mM (i.e. 1:20), shake well, and incubate in a 37°C water bath for 1h to reduce the disulfide bond of the protein.

[0066] (4) Prepare 1M IAA (gold water preparation), add appropriate volume to make the working concentration 135mM, shake well, and place in the dark at room temperature for 30min.

[0067] (5) Rinse the ultrafiltration tube with 1mL TEAB, centrifuge (working conditions: 20°C, 4000rpm, 20min), if the liquid does not completely separate, change the centrifugation angle to 90° to avoid damage to the ultrafiltration tube membrane, and centrifuge until the liquid completely separates, each time not more than 15min to avoid damage to the ultrafiltration tube membrane.

[0068] (6) Add the sample to the rinsed ultrafiltration tube, the volume of each tube should not exceed 1mL, centrifuge (working conditions: 20C, 4000rpm, 20min), if the liquid does not completely separate, change the centrifugation angle to 90° to avoid damage to the ultrafiltration tube membrane, and centrifuge until the liquid completely separates, each time not more than 15min to avoid damage to the ultrafiltration tube membrane.

[0069] (7) Add 1mL 8M Urea to the ultrafiltration tube, shake well, and centrifuge until the liquid completely separates (working conditions: 4°C, 4000rpm, 20min), repeat 2 times.

[0070] (8) Add 1 mL of TEAB to the ultrafiltration tube, shake thoroughly, and centrifuge (working conditions: 4℃, 4000 rpm, 20 min) until all the liquid has separated. Repeat 5 times. Discard the collection tube and replace it with a new collection tube.

[0071] (9) Dissolve the pancreatic enzyme in the company's HCl solution to prepare a pancreatic enzyme concentration of 1 μg / μL. Then add 500 μL of 50 mM TEAB and 75 μL of the prepared mass spectrometry grade pancreatic enzyme solution to the ultrafiltration tube.

[0072] (10) Use a clean pipette tip to blow away the flocculent protein in the ultrafiltration tube to make the solution turbid, shake it thoroughly for 5 minutes, seal the ultrafiltration tube with plastic wrap, and place it on a shaker at 37℃ and 200rpm for 16-18 hours. After 8 hours, use a clean pipette tip to blow away the flocculent protein again to make the solution turbid, and shake it thoroughly for 5 minutes.

[0073] (11) Centrifuge the ultrafiltration tube from the previous step into a collection tube (working conditions: 4℃, 4000rpm, 20min), then add 1mL of gold solution, use a clean pipette tip to disperse the flocculent protein to make the solution turbid, shake thoroughly for 5min, and centrifuge (working conditions: 4℃, 4000rpm, 20min) to collect the enzymatically digested peptide solution.

[0074] (12) The concentration of the peptide solution after enzymatic hydrolysis was detected by BCA kit, and the enzymatic hydrolysis efficiency of the protein sample was calculated.

[0075] 2.5 High-flux desalination

[0076] (1) Connect the vacuum desalination instrument and check its airtightness. Clean the desalination column connector with pure water.

[0077] (2) Activate the desalination column: Add methanol to activate the Sep-pak C18 desalination column for 10 min. The vacuum pump pressure should not exceed 300 kPa.

[0078] (3) Equilibrium desalting column: Prepare 2% acetonitrile containing 0.1% FA as equilibrium buffer and pass it through the column twice, 1 mL each time, and let it stand for 1 min for the first time.

[0079] (4) Sample aspiration: Place the waste liquid tube on the rack of the negative pressure tank, and pass the enzymatically digested peptide solution through the column 3-5 times, 1 mL each time. The first time, the solution should be left to stand for 1 minute. Use a vacuum pump to desalt the solution. Be careful not to make the pressure difference too large, which will cause the flow rate to be too fast and generate bubbles, resulting in leakage.

[0080] (5) Cleaning the sample: Prepare a gold solution containing 0.1% FA as the washing buffer, 1 mL each time: repeat the column flushing 5 times.

[0081] (6) Elution Buffer: 0.1% FA in 40% acetonitrile, 200 μL each, 3 times.

[0082] (7) Elution Buffer: 0.1% FA in 80% acetonitrile, 200 μL each, 3 times. 200 μL of the peptide sample was used for total protein detection. The remaining sample was lyophilized and desalted.

[0083] 2.7 Liquid chromatography-mass spectrometry (LC-MS / MS)

[0084] (1) The total protein sample was dissolved in 20 μL of 0.1% FA (prepared with gold water) and the concentration was determined using a BCA kit. The amount of 0.1% FA was adjusted to achieve a final concentration of 0.5 μg / μL.

[0085] (2) De-particle: 15 μL of each sample was centrifuged (working conditions: 4°C, 12000g, 20 min) into a new EP tube. 12 μL of the supernatant was centrifuged (working conditions: 4°C, 12000g, 20 min) into a new EP tube. 9.5 μL of the supernatant was added to a new EP tube, 0.5 μL of the marker peptide (Irt) was added, and the mixture was vortexed and centrifuged (working conditions: 4°C, 12000g, 10 min).

[0086] (3) 5 μL of each sample was taken into a new loading tube. Note that the loading tube should not have air bubbles.

[0087] (4) 3 μL of each sample after de-particle was taken into the same centrifuge tube and mixed. 15 μL was taken into the loading tube (be careful not to generate air bubbles) for DDA library construction. DDA was performed for 3 injections, each requiring 5 μL.

[0088] (5) The loading tube was placed in the mass spectrometer instrument, and the total proteome was analyzed by DIA model.

[0089] 2.8 Search database

[0090] Spectronaut software was used for data quantitative analysis of DIA. After comparison with the mouse protein database, the atlas data comparison results of the total proteome were exported.

[0091] 2.9 Bioinformatics statistical analysis method

[0092] 2.9.1 Analysis software

[0093] The R software (4.2.0) is mainly used for bioinformatics analysis in this embodiment. In this embodiment, the treatment of missing values of data is considered to be a stable method: the protein quantitative value offset is too large (i.e., less than 500) is defined as a missing value, and the missing value of protein quantification is filled with the average value.

[0094] 2.9.2 Sample repeatability evaluation

[0095] In this embodiment, biological repeat samples are taken during protein expression extraction. Therefore, principal component analysis (PCA) and box plot visualization are used to evaluate the protein repeatability between samples to test whether the experimental results of biological repeat samples have statistical consistency, which helps to improve the accuracy and reliability of the experiment.

[0096] 2.9.3 Whole proteome difference analysis

[0097] Based on the quality control of whole proteome data, previous studies have shown that the power law global analysis (PLGEW) model is beneficial to the statistical analysis of proteome data, and is also one of the commonly used proteome analysis models. In this embodiment, the PLGEM model is used to fit the whole proteome quantitative results and evaluate the data quality. The PLGEM model is used for difference analysis, and the differential proteins are visualized by volcano plot and heat map based on R language using the ggplot2 package.

[0098] 2.9.4 Protein function enrichment analysis

[0099] In this embodiment, the differential proteins are annotated for function, and the enrichment test significance P value is less than 0.05.

[0100] GO (Gene Ontology) enrichment analysis is a method of classifying and annotating genes to reveal the biological functions and relationships of different genes and gene sets. The function of proteins or genes is mainly analyzed from three levels: molecular function, cellular component and biological process.

[0101] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a systems biology database that integrates information on genomes, biochemical reactions, metabolic pathways, etc. Enrichment analysis of differential proteins can help researchers understand the functions and interactions of genes and proteins in metabolic pathways, providing important clues for studying biological processes, discovering new biomarkers, and developing drugs.

[0102] 2.10 Western blot (immunoblotting)

[0103] (1) Preparation of samples: The protein sample concentration was detected by BCA kit, and equal amounts of protein (about 30-100 μg) were taken into a new EP tube, and the volume was adjusted with cell lysis buffer, 5x loading buffer was added, and the sample was heated at 95°C for 10 min and then cooled on ice.

[0104] (2) Preparation of electrophoresis gel: The 1.5 m thin glass plate and thick glass plate were washed with water and then air-dried at room temperature and fixed with a rack. The airtightness of the gel preparation rack was tested with pure water. First, 10% separation gel was added, and anhydrous ethanol was added to remove bubbles, and the separation gel was flattened. After waiting for 20-30 min for the separation gel to solidify, 5% concentrated gel was added, and the comb was inserted horizontally. After waiting for 20-30 min for the concentrated gel to solidify.

[0105] (3) Sample loading: The glass plate was clamped with the electrophoresis tank, and the prepared electrophoresis liquid was filled into the tank. The comb was taken out horizontally to avoid tilting of the sample loading hole, and the sample was added to the sample loading hole using a pipette.

[0106] (4) Electrophoresis: The power supply was inserted, and the parameters were adjusted to 80V. After the sample was run through the gel, the voltage was adjusted to 120V. The electrophoresis was stopped according to the molecular weight of the target protein and the internal reference protein to avoid the sample running off the gel.

[0107] (5) Membrane transfer: The pre-cooled 1L transfer solution was placed in the operation tray, and then the "sandwich clip" was soaked in the transfer solution, with the black side down. The sponge and filter paper were placed in turn, and a small piece of 8x5cm PVDF membrane was cut with scissors, with the corners cut on the right upper corner to avoid the direction being reversed after transfer. After electrophoresis, the thin glass plate was peeled off, the concentrated gel was cut off, and the separation gel was gently separated and placed on the filter paper. The prepared PVDF membrane was soaked in methanol for 1 min to achieve activation, and then the PVDF membrane was placed on the separation gel, and the position was adjusted to ensure that the PVDF membrane covered the entire separation gel without air bubbles. The whole process should be gentle to avoid breaking the separation gel. The "sandwich clip" was placed in the electrotransfer tank, and then the transfer solution in the operation tray was poured into the electrotransfer tank to ensure that the PVDF membrane was fully soaked in the electrotransfer solution. A piece of ice box was placed in the remaining empty position to ensure the safety of the electrotransfer. The parameters were adjusted to 100V constant voltage, 235mA current, and 120 min to start the electrotransfer.

[0108] (6) Blocking: After the completion of the transfer film, the PVDF membrane is placed in a small box containing TBST (containing Tween) and washed twice to remove TBST (containing Tween). The prepared 5% blocking solution is added to the PVDF membrane, and the PVDF membrane is immersed in the blocking solution. The PVDF membrane is placed on a shaker for 1 hour.

[0109] (7) Unblocking primary antibody: After the completion of the blocking, the blocking solution is removed, and the PVDF membrane is washed with TBST (containing Tween) for 5 minutes on a shaker. The TBST (containing Tween) is removed, and this washing step is repeated 3 times to remove all the blocking solution. The PVDF membrane is immersed in the prepared primary antibody solution, and then placed on a 4°C shaker for slow shaking overnight.

[0110] (8) Incubation of secondary antibody: The primary antibody solution is recovered, and the PVDF membrane is washed with TBST (containing Tween) for 5 minutes on a shaker. The TBST (containing Tween) is removed, and this washing step is repeated 3 times. The prepared secondary antibody is added to the PVDF membrane, and the PVDF membrane is immersed in the secondary antibody solution. The PVDF membrane is placed on a shaker for 1 hour.

[0111] (9) Development: The secondary antibody solution is removed, and the PVDF membrane is washed with TBST (containing Tween) for 5 minutes on a shaker. The TBST (containing Tween) is removed, and this washing step is repeated 3 times. The prepared luminescent solution, forceps, paper towel, and PVDF membrane are taken to the biomolecular imager. The PVDF membrane is gently picked up with forceps, and the front side is placed on the paper towel. The remaining TBST (containing Tween) is absorbed as much as possible, and then the PVDF membrane is placed on the biomolecular imager. The luminescent solution is dropped on the target protein band for development and preservation of the results.

[0112] 3. Experimental results and statistical analysis

[0113] P<0.05, Fold change >1.5 is considered statistically significant change. All data analysis was performed in R 4.2.0 software.

[0114] 4. Serum NGP in patients undergoing hepatectomy to predict postoperative complications

[0115] Serum samples were obtained from patients undergoing hepatectomy. The occurrence of postoperative complications was collected, and according to the Clavien-Dindo classification, severe complications of grade III-V were considered as positive results.

[0116] Detection of serum samples

[0117] b1) Quantitative chip dry weight

[0118] The quantitative chip is taken out of the box and placed at room temperature for 20-30 minutes. The packaging bag is opened, the seal is removed, and then the quantitative chip is placed in a desiccator or dried at room temperature for 1-2 hours.

[0119] b2) Preparation of standard solution

[0120] b21) Add 500 μL of sample diluent to the small tube of standard solution mixture, re-dissolve the standard solution mixture, and quickly centrifuge before opening the small tube, gently blow up and down to dissolve the powder, and mark the small tube as Std 1.

[0121] b22) Mark 6 clean centrifuge tubes as std2, std3, std4, std5, std6, and std7 respectively, and add 200 μL of sample diluent to each centrifuge tube.

[0122] b23) Take 100 μL of sample diluent from the centrifuge tube marked as Std 1 and add it to the centrifuge tube marked as std2, mix gently, then take 100 μL of sample diluent from the centrifuge tube marked as std2 and add it to the centrifuge tube marked as std3, and so on, and finally to the centrifuge tube marked as std7, to obtain the standard solution.

[0123] b24) Take 100 μL of standard solution from the centrifuge tube marked as std7 and add it to another new centrifuge tube, marked as CVTRL, as a negative control.

[0124] b3) Operation process of the quantification chip

[0125] b31) Add 100 μL of sample diluent to each well of the quantification chip, and incubate at room temperature on a shaking table for 1 h to seal the quantification chip.

[0126] b32) Remove the sample diluent from each well, take 80 μL of standard solution from the centrifuge tube marked as CNTRL, and add it to the well together with the serum sample, and incubate overnight at 4°C.

[0127] b33) Wash the quantification chip, first wash with 1× wash solution I, 250 μL of 1× wash solution I per well, wash 10 times, each time shake for 10 s, and the shaking intensity is selected as high, dilute 20× wash solution I with deionized water, then use 1× wash solution II to wash the channel, 250 μL of 1× wash solution II per well, wash 6 times, each time shake for 10 s, and the shaking intensity is selected as high, dilute 20× wash solution II with deionized water.

[0128] b34) Incubate the NGP protein immunoglobulin G antibody, centrifuge the small tube containing the NGP protein immunoglobulin G antibody, then add 1.4 mL of sample diluent, mix well, and then quickly centrifuge again, then add 80 μL of NGP protein immunoglobulin G antibody to each well, and incubate at RT on a shaking table for 2 h.

[0129] (b35) Cleaning, same as step (b33).

[0130] b36) Incubation of Cy3-streptavidin: Centrifuge the tube containing Cy3-streptavidin, then add 1.4 mL of sample diluent, mix well, and centrifuge again quickly. Add 80 g of Cy3-streptavidin to each well, wrap the quantitative chip with aluminum foil to protect it from light, and incubate on an RT shaker for 1 hour.

[0131] (b37) Cleaning, same as step (b33).

[0132] b38) Fluorescence detection is performed using a laser scanner to scan the signal, employing either Cy3 or the green channel.

[0133] b39) The QAHI-CUST data analysis software was used to analyze the data and obtain the NGP protein content in each serum sample.

[0134] As used in this invention, the term "ROC curve" or "ROC plot" refers to a graphical representation of the performance of a binary classifier system as a function of its discrimination threshold. This curve is created by plotting the true positive rate against the false positive rate at various threshold settings. The true positive rate is also referred to as sensitivity. The false positive rate is calculated as 1 - specificity. Therefore, the ROC curve is a graphical representation of the true positive rate against the false positive rate (sensitivity vs. (1 - specificity)) over a range of cutoff values, and a way to select the optimal cutoff value for clinical use. Accuracy is expressed as the area under the ROC curve (AUC), providing a useful parameter for comparing test performance. An AUC close to 1 indicates that the test is highly sensitive and highly specific, while an AUC close to 0.5 indicates that the test is neither sensitive nor specific.

[0135] 5.1 Quality Control of Whole Proteomics Mass Spectrometry Data

[0136] Because the quality of proteomic data affects subsequent analysis and validation, quality control of proteomic data is necessary. This embodiment assesses proteomic data quality through data distribution comparison and PCA analysis. Figure 1 As shown, the horizontal axis represents the sample name, and the vertical axis represents the intensity value after Log10 transformation. The color of the bins represents different groups. Horizontal comparison can roughly show the dispersion of data distribution within and between groups. The sample means are at the same level, and the protein expression distribution trends are similar, indicating good sample quality. Principal component analysis was performed based on the relative quantitative values ​​of all samples, and a visualized PCA plot was plotted. The horizontal and vertical axes show the explanatory power of PC1 and PC2; the larger the value, the higher the explanatory power. Figure 2 The data shows that duplicate samples in each group tend to cluster together.

[0137] The proteins with P<0.05 and Fold change>1.5 in the PLGEM model were defined as the differential proteins (liver injury group vs control group; DEPs) in this embodiment. In the whole proteome data, there were 142 differential proteins in T1 group relative to the control group, including 81 up-regulated differential proteins and 61 down-regulated differential proteins; there were 298 differential proteins in T2 group relative to the control group, including 141 up-regulated differential proteins and 157 down-regulated differential proteins. As shown in the volcano plot in Figure 3 and the heat map in Figure 4 , the selected DEPs can clearly distinguish the control group and the liver injury group.

[0138] 5.2 Whole proteome differential protein enrichment analysis

[0139] In order to further explore the biological processes and cell functions affected by liver injury, GO enrichment analysis was performed on the differential proteins, as shown in Figure 5 . It can be seen from the results that, relative to the control group, the biological processes (BP) of T1 group are enriched in neutrophil aggregation, vascular endothelial cell signaling pathway, positive regulation of macrophage activation, negative regulation of epidermal cell apoptosis, regulation of keratinocyte proliferation, etc. Relative to the control group, the biological processes of T2 group are enriched in induction of bacterial coagulation, vascular endothelial cell differentiation, regulation of TH1 type immune response, fibrinolysis and acute phase response, etc.

[0140] In addition, the KEGG enrichment analysis results of the differential proteins suggest that the differential proteins of T1 / C group are significantly aggregated in tumor transcription disorder, acute myeloid leukemia, hematopoietic cell line, tuberculosis and IL-17 signaling pathway, and the differential proteins of T2 / C group are significantly aggregated in intestinal IgA synthesis immune network, sphingomyelin biosynthesis, complement and coagulation cascade, Staphylococcus aureus infection and mineral element absorption signaling pathway, as shown in Figure 6 .

[0141] In summary, the enrichment analysis results of the differential proteins show that the pathophysiological mechanism of liver injury is particularly complex, which affects the treatment and prognosis of liver injury.

[0142] 5.4 Western Blot verification of mass spectrometry data accuracy

[0143] Further, Western Blot was used to verify the expression of NGP in liver tissue. At the whole protein level in liver tissue, it was shown that NGP was up-regulated after liver ischemia-reperfusion, as shown in Figure 7 .

[0144] In summary, this invention utilizes mass spectrometry to obtain proteomics data related to liver injury and identifies biological signaling pathways associated with liver injury. Furthermore, it identifies NGP as closely related to liver injury, and Western blotting confirms that NGP expression is upregulated in the liver injury group, consistent with the mass spectrometry analysis results. This invention innovatively provides the application of the aforementioned biomarkers in the prognosis of liver injury and offers new targets for drug research on the treatment of liver injury. This invention has significant clinical, research, and drug translational value.

[0145] The diagnostic performance of the model is as follows Figure 8 As shown, the AUC of serum NGP level on postoperative day 1 was 0.787, as Figure 8 As shown, the sensitivity and specificity of serum NGP levels on postoperative day 1 under a specific cutoff were 64.4% and 72.9%, respectively. This indicates that serum NGP levels on postoperative day 1 have a good predictive value for postoperative complications after hepatectomy.

[0146] The preferred embodiments and examples of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments and examples. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the concept of the present invention.

Claims

1. The use of neutrophil granulule protein (NGP) in the preparation of reagents or kits for predicting postoperative complications in patients undergoing clinical hepatectomy, wherein the neutrophil granulule protein is human serum neutrophil granulule protein, and the postoperative complications in the patients undergoing clinical hepatectomy are severe complications of grade III-V according to the Clavien-Dindo classification.

2. The application according to claim 1, characterized in that: The reagent is a microarray or biochip.

3. The application according to claim 1, characterized in that: Postoperative complications in patients undergoing clinical hepatectomy include bleeding, liver failure, abdominal infection, bile leakage, pleural effusion and pulmonary complications, and ascites.

4. The application according to claim 1, characterized in that: The reagent or kit includes an immunoglobulin G antibody against neutrophilic granules.

Images