Application of THBS1 as radioactive liver injury marker and detection kit of THBS1

Abstract

The invention discloses application of THBS1 as a radioactive liver injury marker and a detection kit of THBS1. The plasma concentration of the protein is less than 13ng / mL in a normal state and greater than 20ng / mL in a radioactive liver injury state, and the protein has the advantages of high specificity and strong sensitivity. The kit based on the sandwich ELISA principle can rapidly detect the THBS1 concentration in plasma, realizes early diagnosis, severity evaluation and prognosis monitoring of radioactive liver injury, and provides a reliable diagnostic tool for clinic.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomedical diagnosis, and particularly relates to application of THBS1 as a marker for radioactive liver injury and a detection kit thereof. BACKGROUND

[0002] Radiotherapy is one of the three core means of tumor treatment. Stereotactic body radiotherapy (SBRT) has become an important way for liver cancer (HCC) treatment, and is particularly suitable for patients with middle and advanced HCC who cannot undergo surgery, and plays a key role in controlling primary and metastatic lesions. However, radiotherapy inevitably causes radiotherapy side effects, and radioactive liver injury (RILI) is one of the most serious complications. It was first proposed by Ingold, and the main clinical manifestations are abdominal discomfort, hepatomegaly and anhydric ascites.

[0003] The liver has high sensitivity to radiation, and the radiation sensitivity of liver cancer cells is close to that of normal liver cells, so the liver is easily exposed to radiation during HCC radiotherapy and induces RILI. If the patient has cirrhosis or liver dysfunction, the risk of RILI is further increased. In addition, patients with other cancers who receive upper abdominal radiotherapy may also develop RILI due to the proximity of the liver to the irradiation field. The occurrence of RILI spans a wide range of time, and can occur as early as 2 weeks after radiotherapy, and as late as 7 months after radiotherapy, and its pathological process presents a phased feature: early stage is mainly characterized by hepatocyte edema, degeneration and necrosis, and late stage gradually progresses to liver fibrosis and even cirrhosis.

[0004] At present, the pathogenesis of RILI is not completely clear, and the mainstream theory believes that it is related to the direct and indirect effects of ionizing radiation (IR): the direct effect causes DNA breakage, and the indirect effect damages key proteins through active oxygen (ROS) generated by radiation and water reaction; the two effects together can start cell programmed apoptosis, but the specific mechanism leading to liver fibrosis still needs further research.

[0005] From the correlation between radiation dose and risk, the effective dose of radiotherapy for most solid tumors is 50-70Gy, but when the liver tissue is exposed to 30-35Gy, the risk of RILI is 5%-10%; when the radiation dose increases to 60Gy, the risk sharply rises to more than 50%. Severe RILI is prone to rapid complication of liver failure, which significantly affects the treatment process and quality of life of liver cancer patients. Therefore, it is of great clinical significance to clarify the mechanism of RILI and screen reliable biomarkers for early diagnosis and disease monitoring.

[0006] The pathological process of RILI involves the synergistic action of multiple factors: platelet-derived growth factor A (PDGFA) expressed by liver epithelial cells is activated and forms a dimer under IR stimulation, which plays a strong inflammatory chemotactic role; activated Kupffer cells (KC) release pro-inflammatory cytokines such as TGF-β, IL-6, and IL-1β, which increase collagen synthesis through activation of the p38 / MAPK and JAK / STAT signaling pathways. At the same time, damaged sinusoidal endothelial cells (SECs) initiate the coagulation cascade, and the TGF-β1-Smad2 / 3 signaling pathway is activated after radiation, further promoting collagen deposition and liver tissue remodeling. Previous studies have shown that inhibition of the JAK / STAT pathway can significantly reduce radiation-induced inflammatory response, thereby preventing liver fibrosis.

[0007] Thrombospondin-1 (THBS1) is a secreted glycoprotein mainly present in blood and extracellular matrix, which is involved in multiple biological processes related to RILI, including promoting liver inflammatory response, mediating acute and chronic liver failure, and fibrous protein deposition. The protein was first identified as a platelet membrane-sensitive protein in 1971, and was secreted into the extracellular matrix after platelet activation, involved in platelet degranulation and regulation of coagulation factor activity. Recent studies have revealed the key role of THBS1 in collagen deposition through single-cell RNA sequencing and experimental verification; in addition, various molecules have been shown to affect the synthesis and deposition of collagen in the extracellular matrix by targeting the expression of THBS1.

[0008] Proteins are the direct executors of biological processes, and after the occurrence of RILI, the protein network in the body will adaptively change to maintain a new balance. Therefore, it is of great value to explore the molecular mechanism of RILI from the protein level. Based on the tandem mass tag (TMT) proteomics technology, different samples can be labeled by chemical tags, and high-throughput quantitative comparison of multiple samples can be achieved in a single analysis, providing an effective tool for screening RILI-related differential proteins.

[0009] In summary, in the prior art, the pathogenesis of RILI (especially the fibrosis mechanism) has not been fully elucidated, and there is a lack of specific and sensitive biomarkers for early diagnosis and disease monitoring. Therefore, it is urgent to identify new key proteins and related mechanisms to provide new targets for clinical diagnosis and treatment of RILI. SUMMARY

[0010] The present application aims to at least partially solve the above technical problems in the art.

[0011] (I) Technical problems to be solved

[0012] The present application aims to provide a new application of THBS1 protein as a marker for evaluating radiation-induced liver injury, overcome the limitations of existing markers, and achieve accurate and early evaluation of radiation-induced liver injury.

[0013] (II) Technical solutions

[0014] 1. Marker

[0015] The THBS1 protein has an amino acid sequence as shown in SEQ ID NO: 1, or a variant thereof having the same function (such as conservative amino acid substitution, active fragment, homolog).

[0016] 2. Application

[0017] The protein can be used to prepare a reagent for evaluating radiation-induced liver injury, and the specific application scenarios include:

[0018] Diagnosing radiation-induced liver injury (distinguishing from healthy state and other types of liver injury);

[0019] Evaluating the severity of radiation-induced liver injury (based on the difference in plasma concentration);

[0020] Monitoring the treatment or recovery effect of radiation-induced liver injury.

[0021] 3. Evaluation criteria

[0022] The evaluation is realized based on the difference in THBS1 concentration in plasma:

[0023] In a normal state, the plasma THBS1 concentration is < 13 ng / mL;

[0024] In a state of radiation-induced liver injury, the plasma THBS1 concentration is > 20 ng / mL.

[0025] 4. Detection kit

[0026] A kit for evaluating radiation-induced liver injury, comprising:

[0027] (1) Pre-coated enzyme-labeled plate: coated with high-affinity human monoclonal anti-THBS1 antibody;

[0028] (2) Detection antibody: biotin-labeled rabbit anti-THBS1 antibody;

[0029] (3) Recombinant standard: human THBS1 recombinant protein freeze-dried powder;

[0030] (4) Auxiliary reagents: standard / sample diluent PBS+1% BSA, washing solution PBS+0.1% Tween-20, color developing solution TMB+ citric acid buffer+H2O2, stop solution 2NH2SO4, and plate sealing tape.

[0031] 5. Evaluation method

[0032] An evaluation method of radiation-induced liver injury, comprising the following steps:

[0033] (1) obtaining a plasma sample of a subject;

[0034] (2) detecting the concentration of THBS1 in the sample by sandwich ELISA method (see "detailed description" for specific steps);

[0035] (3) comparing the detection result with the reference value: if the concentration is > 20 ng / mL, it indicates the presence of radiation-induced liver injury, and if < 13 ng / mL, it indicates normal state.

[0036] As Figure 1 shown, Figure 1 the E part of the figure is the illustration of the immunohistochemical staining, and the immunohistochemical staining is analyzed by using imageJ software, and the results are visualized by bar chart;

[0037] The F part of the figure is a plasma Elisa experiment statistical chart, the concentration of THBS1 in rat plasma is determined, and ANOVA analysis is carried out, compared with the control group, the concentration of THBS1 in the experimental group increases (p<0.05), and the bar chart is visualized;

[0038] The H part of the figure is the result of immunohistochemical staining, which indicates that the liver tissue appears hydropic degeneration (i.e. early liver injury), and the expression level of THBS1 is significantly increased, which verifies the certainty of THBS1 in indicating liver injury and the stability of its expression at the tissue level.

[0039] Figure 1 The results in the E, F, G and H parts of the figure are verified by Western blot and ELISA method for the change of key biomarkers, and the two methods are widely used in marker screening, and the existing research uses WB and Elisa and IHC (immunohistochemistry) to screen biomarkers at the protein level and the tissue level respectively, and the results of these studies support the rationality of using WB and ELISA as verification means in the present invention and the authenticity of the results in the present work.

[0040] 6. The PDGF / PDGFR signaling pathway and extracellular matrix tissue-related differential genes in the present invention mainly include PLA2G2A, STAT3, LUM, COL6A2, THBS1 and BGN, and the P value and log2FC (log2 fold change) value are shown in the following table 1.

[0041] Table 1 Difference analysis of protein expression

[0042]

[0043] (Three) THBS1 is used for multi-dimensional verification experiment of radiation-induced liver injury RILI

[0044] 1. Animal model construction and modeling verification

[0045] 33 healthy male rats (body weight 160-230g) were selected and randomly divided into control group (3), 20Gy group (15) and 30Gy group (15); all rats were fasted for 2 hours before the experiment and inhaled isoflurane anesthesia, and the experimental groups received single 20Gy, 30Gy irradiation respectively, and the target area of the rat liver was accurately outlined during irradiation to ensure that the irradiation field was aligned with the positioning contour. The liver tissue was stained with HE, and the results showed that the liver cells in the 30Gy group appeared cell swelling and inflammatory infiltration, confirming that the radiation-induced liver injury rat model was successfully constructed, providing a reliable model basis for subsequent verification experiments.

[0046] 2. Proteomics screening and difference verification

[0047] Tandem mass tag (TMT) proteomics technology was used to screen the differential proteins of the control group (no radiation-induced liver injury) and the experimental group (radiation-induced liver injury) liver tissue; principal component analysis (PCA) showed that the gene expression profiles of the 30Gy group and the control group were significantly separated in three-dimensional space, the intra-group sample clustering consistency was high, and the variability was low; the CV cumulative curve showed that the RILI group and the control group sample had good repeatability. Volcano plot analysis showed that THBS1 was significantly up-regulated under the condition of |log2FC|≥1 and P<0.05 (Log2FC=0.633, P=0.0437); hierarchical clustering heatmap can clearly divide all samples into two groups according to the differential protein expression profile of THBS1, showing a synergistic up-regulation module, confirming that the difference between the groups is stable and the consistency between the repetitions is good.

[0048] 3. Multi-method verification of THBS1 expression level

[0049] (1) Tissue level verification

[0050] THBS1 expression in liver tissue was detected by immunohistochemistry (IHC): ImageJ software was used to analyze the staining results, and it was found that when the liver tissue appeared hydropic degeneration (early liver injury), the expression level of THBS1 was significantly increased; Western blot (WB) experiment further verified that the expression of THBS1 protein in the liver tissue of the RILI group was significantly up-regulated compared with the control group, and it was also confirmed that the expressions of PDGFA, p-JAK2, p-STAT3 and collagen (COL5A1, COL6A1, COL6A2) were up-regulated synchronously.

[0051] (2) Plasma level verification

[0052] The ELISA method was used to detect the plasma THBS1 concentration in rats: ANOVA analysis showed that compared with the control group, the plasma THBS1 concentrations in the 20 Gy and 30 Gy groups were significantly increased (P<0.05); and the concentration showed an increasing trend with increasing radiation dose (plasma concentration in the 0 Gy group was 10.73-13.86 ng / mL, in the 20 Gy group was 16.20-17.76 ng / mL, and in the 30 Gy group was 19.21-20.40 ng / mL), meeting the evaluation criteria of "normal state <13 ng / mL, RILI state >20 ng / mL".

[0053] 4. Specificity and sensitivity verification

[0054] (1) Specificity verification

[0055] The experiment compared the expression of THBS1 in different liver injury models: in the drug-induced liver injury model, there was no significant change in THBS1 expression; only in the radiation-induced liver injury model, THBS1 was significantly overexpressed in both liver tissue and plasma (P<0.05), and the difference compared with the healthy control group was statistically significant, confirming that it can distinguish radiation-induced liver injury from normal state and other types of liver injury.

[0056] (2) Sensitivity verification

[0057] In the early stages of RILI (10 days after radiation, before obvious fibrosis changes appeared on imaging), WB and ELISA tests revealed that THBS1 was already significantly overexpressed. Furthermore, plasma THBS1 concentrations increased with increasing radiation dose (20 Gy to 30 Gy) and severity of liver damage (hepatocyte hydropic degeneration and Kupffer cell proliferation), demonstrating that THBS1 can be detected early in the course of RILI and possesses value for early diagnosis.

[0058] 5. Correlation and mechanism verification

[0059] (1) Correlation analysis

[0060] Pearson correlation analysis showed that the relative expression of THBS1 in rat liver tissue was strongly positively correlated with the plasma THBS1 concentration (R=0.8973), and the immunohistochemistry and plasma ELISA results were highly consistent, confirming the stability of THBS1 expression and its reliability as a marker.

[0061] (2) Mechanism Verification

[0062] The protein-protein docking (PPD) model (using HDOCK1.1 software) was constructed to analyze the interaction of THBS1 (PDBID: 2ES3) and PDGFA (PDBID: 3MJK): the binding energy reached-674.79 kcal / mol, the binding interface formed hydrogen bonds and electrostatic interactions through key amino acid residues such as TYR-103 and SER-108, and the binding was stable; WB experiments verified that THBS1 can activate the PDGF / PDGFR signaling pathway and the downstream JAK2 / STAT3 pathway, ultimately mediate the deposition of collagen proteins such as COL5A1 and COL6A1, and clarify the mechanism of its participation in the process of RILI liver fibrosis, further supporting the rationality of its serving as a RILI marker.

[0063] (Four) Beneficial Effects

[0064] 1. High specificity, can distinguish between radiation-induced liver injury and normal state and other liver injuries

[0065] The experimental results show that THBS1 is significantly up-regulated in the liver tissue and serum of the RILI model rats (Log2FC = 0.633, P = 0.0437), and there is no significant change in the drug-induced liver injury model, and the difference with the healthy control group is statistically significant (P < 0.05). This indicates that THBS1 has specific recognition ability for radiation-induced liver injury, and can effectively distinguish RILI from normal physiological state and other types of liver injury, overcoming the defect of insufficient specificity of existing markers (such as ALT, AST).

[0066] 2. High sensitivity, can achieve early diagnosis

[0067] Based on TMT proteomics screening and WB verification, THBS1 has been significantly highly expressed in the early stage of RILI (10 days after radiation, when no obvious fibrosis changes have appeared in imaging), and serum ELISA detection shows that its concentration increases with the increase of radiation dose (20Gy-30Gy), suggesting that it can be detected in the early stage of RILI, providing a time window for early intervention, and solving the problem of insufficient sensitivity of existing diagnostic methods for early damage.

[0068] 3. Positive correlation with injury degree, can be used for disease monitoring and severity assessment

[0069] The serum ELISA results show that the concentration of THBS1 increases with the increase of radiation dose (20 Gy group-30 Gy group) and the severity of liver damage (hydropic degeneration of hepatocytes, the degree of Kupffer cell proliferation), and the Pearson correlation analysis confirms that the serum concentration is consistent with the expression of the tissue (positive correlation). This means that by detecting the level of THBS1, the progress of RILI can be dynamically monitored, the severity of the damage can be quantitatively evaluated, and the basis for adjusting the clinical treatment plan can be provided. The original data of the Pearson correlation of the application is shown in the following table 2.

[0070] Table 2 Original data of Pearson correlation

[0071]

[0072] As Figure 1 shown in the A, B, C and D parts of the figure, it is verified at the protein level that THBS1 is up-regulated after radiation-induced liver injury, activates the downstream signaling pathway, and ultimately leads to collagen deposition, and mediates the process of liver fibrosis.

[0073] In the following table, the original data of the Pearson correlation of the application is shown:

[0074] 4. Convenient detection, suitable for clinical promotion

[0075] THBS1 can be detected by serum samples (ELISA method), without invasive liver biopsy, and the operation is simple, fast, and the reagent kit composition is clear (including specific antibodies, standard products and auxiliary reagents), the detection conditions are easy to standardize, suitable for primary medical institutions and large-scale screening, and improve the feasibility of clinical application.

[0076] 5. Clear mechanism, providing potential target for treatment

[0077] Molecular docking experiments confirm that THBS1 and PDGFA have strong interaction (binding energy =-674.79 kcal / mol), which can activate the PDGF / PDGFR signaling pathway and the downstream JAK2 / STAT3 pathway, and ultimately mediate the deposition of collagen proteins such as COL5A and COL6A, and participate in the process of liver fibrosis. This mechanism not only provides new insights into the pathogenesis of RILI, but also suggests that THBS1 can be used as a potential therapeutic target, providing a direction for the intervention treatment of RILI, and has the dual value of diagnosis and treatment research.

[0078] As Figure 2 shown in the interface, there are a large number of hydrogen bonds and salt bridges, which indicates that the binding interface not only has a large area but also has stable binding energy.

[0079] It should be noted that PPD is one of the key tools for studying protein interactions, which can reveal the binding mode, binding site and affinity between proteins, and predict their function in biological processes (BP);

[0080] In the present application, a PPD model is constructed to analyze the interaction between THBS1 (PDBID: 2ES3) and PDGFA (PDBID: 3MJK) in depth;

[0081] PPD calculation is performed using HDOCK1.1 software, and the results with the best binding energy are visualized accordingly, and the zoomed-in area in FIG. Figure 2 The interaction between THBS1 and PDGFA involves multiple key amino acid residues, including TYR-103, SER-108, SER-136, ASP-55, GLU-44 and ARG-28, which promote the binding of the two through hydrogen bonds and electrostatic interactions, forming a stable binding surface;

[0082] According to the calculation, the binding energy of THBS1 and PDGFA reaches-674.79 kcal / mol, indicating that there is strong affinity between the two, and in addition to the strong interaction between THBS1 and PDGFA, this PPD analysis also indicates that THBS1 plays a key role in the PDGFA / PDGFRα signaling pathway and extracellular matrix remodeling process.

[0083] Additional contents and advantages of the present application will be given in the following description, or can be understood through the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0084] The technical solutions and advantages of the present application will become apparent and easy to understand from the following content combined with the accompanying drawings, in which:

[0085] Figure 1 It is a schematic diagram of the Pearson correlation experiment of the present application;

[0086] Figure 2 It is a protein and protein docking simulation diagram of the present application;

[0087] Figure 3 It is a schematic diagram of rat modeling used in the experiment of the present application;

[0088] Figure 4 It is a schematic diagram of the comparison between the control group and the 30Gy group of liver tissue HE staining of the present application;

[0089] Figure 5 It is a schematic diagram of the principal component analysis PCA and repeatability analysis of THBS1 screened by proteomics determination of the present application;

[0090] Figure 6 A schematic diagram of the differential analysis of protein expression of THBS1 screened by proteomics determination of the present application;

[0091] Figure 7 A ROC curve diagram of the present application;

[0092] Figure 8 A schematic diagram of the reverse verification result of the present application. DETAILED DESCRIPTION

[0093] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments.

[0094] In the embodiments of the present application, from September 2022 to September 2025, 85 serum and plasma samples of patients with radioactive liver damage were randomly collected from the General Hospital of the People's Liberation Army of the Northern Theater Command in Shenyang, Liaoning Province, as shown in the following Table 3.

[0095] Table 3 Serum and plasma samples of patients with radioactive liver damage

[0096]

[0097] At the same time, 85 healthy volunteers were recruited to have blood samples taken on an empty stomach at 9 am. They had no underlying liver disease and no history of lipid-lowering drugs. The plasma samples were stored at -80°C. The concentration of THBS1 in the samples was detected. The data were verified for normal distribution using Kolmogorov-Smirnov. The 95% confidence interval (mean ± 1.96 x standard deviation, 5-13 ng / ml) of the protein in the normal population was calculated. The statistical results are as follows: the lower limit of the normal human plasma THBS1 range is 9.2514-1.96x2.1358 = 5.0652 ng / ml ≈ 5 ng / ml, and the upper limit is 9.2514+1.96x2.1358 = 13.4376 ng / ml ≈ 13 ng / ml. The Kolmogorov-Smirnov verification results of 85 patients without underlying liver disease are shown in Table 4.

[0098] Table 4 K-S verification results

[0099]

[0100] As Figure 7As shown in the ROC curve, the threshold is calculated by the Youden index (J), the Youden index (J) = sensitivity + specificity - 1, which is the most commonly used statistical method to find the threshold, and the point on the ROC curve that makes the Youden index reach the maximum is found by using the Youden index method, which is the point with the maximum sensitivity and specificity value, which is equivalent to minimizing the misdiagnosis and missed diagnosis rate, and the critical value corresponding to the point is the best critical value (diagnosis) in the statistical sense, that is, 20.375 ng / ml ≈ 20 ng / ml.

[0101] As shown in Figure 8 , the reverse verification of the application was carried out, three cell lines of rat normal hepatocyte BRL, BRL-3A and human normal hepatocyte THLE-2 were selected for THBS1 knockdown experiment verification, and the protein was extracted 48h after giving 10Gy radiation dose, and western blot experiment verification was carried out. The western blot experiment verification result can be seen that after knocking down THBS1, the three cell lines all showed obvious down-regulation of collagen expression, indicating that THBS1 as a starting factor plays a key role in the whole process of radiation-induced liver injury.

[0102] As shown in Figure 3 , a total of 33 healthy male rats (body weight 160-230g) were used, and randomly divided into control group (3), 20Gy group (15) and 30Gy group (15), before subsequent treatment, all rats were fasted for 2 hours and then inhaled isoflurane for anesthesia, the rats in the experimental group received single 20Gy and single 30Gy irradiation respectively, and the rats were well aligned with the positioning contour during irradiation.

[0103] It should be noted that in Figure 3 , part A is a schematic diagram of simulated positioning and ray beam arrangement of the rat's radiation field, part B is a dose distribution curve, part C is a schematic diagram of in vivo distribution of radiation dose, part D is a schematic diagram of local dose distribution and structure of the radiation device, and part E is the standard setting parameter of the radiotherapy machine. Parts A, C and D together show the accurate delineation of multiple latitudes for rat liver target area, which avoids radiation from affecting the accuracy of experimental results by causing radiation-induced intestinal injury.

[0104] It should also be noted that in Figure 3 , parts F and G are related experimental pictures of protein extraction from fresh tissue of rats killed after exposure to single 10Gy dose of radiation and pathway verification.

[0105] As shown in Figure 4 , the comparison of liver tissue HE staining between the control group and the 30Gy group showed that the liver cells appeared swollen (yellow arrow) and inflammatory infiltration (black arrow), among which PA was the portal area and CV was the central vein.

[0106] It should be noted that, in Figure 4 , the area indicated by the yellow arrow is the swelling of the hepatocytes, the area indicated by the black arrow is the inflammatory cell infiltration, and the area marked as PA is the portal vein area, and the area marked as CV is the central vein, which indicates that the rat modeling is successful, and the subsequent pathway verification result is guaranteed.

[0107] As shown in Figure 5 and Figure 6 , the application of a THBS1 protein as a marker in the preparation of a reagent for evaluating radioactive liver damage, the amino acid sequence of the THBS1 protein is shown as SEQ ID NO: 1.

[0108] It should be noted that, in Figure 5 , the principal component analysis (PCA) of the left area shows that there is a significant separation between the gene expression profiles of the 30Gy group and the control group in the three-dimensional space, especially in the principal component 1 (PC1) direction, so there may be significant differences in gene expression or proteomic characteristics between the two groups, and the relative intra-group clustering of the three samples indicates that the intra-group samples have relative spatial consistency in the main characteristics, which indicates that the intra-group samples have lower variability and good reproducibility.

[0109] The CV cumulative curve of the right area can be used to evaluate the difference in variability between groups, where a lower CV on the X-axis indicates a higher concentration of sample values, and a higher CV indicates greater variability between samples, while the Y-axis represents the cumulative fraction, i.e. the proportion of samples with different CV values and below, as the CV value increases, the cumulative fraction gradually increases and eventually approaches 1, indicating 100% of the sample data, and the CV cumulative curve of the RILI group and the control group is steeper, indicating that the sample variability is lower at 100%, i.e. the reproducibility of the overall sample is better.

[0110] It should also be noted that, in Figure 6 , the left area is the screening of differential proteins (DEPs) in the present application as biomarkers for guiding the diagnosis of radioactive liver damage, and the present application performs a differential analysis on the liver tissues of the control group without radioactive liver damage and the liver tissues of the experimental group with radioactive liver damage, Group 2 represents the control group, and Group 1 represents the experimental group, the horizontal axis (Log2FoldChange) is closer to the right side (> 0), indicating that the expression of this part of the protein is higher (up-regulated), otherwise it is closer to the left side (0), Group 2 is lower (down-regulated), the vertical axis (-log10Pvalue) indicates that the smaller the p value, the higher the vertical coordinate, indicating that the difference is more significant, it should be noted that in the color, red represents up-regulation and reaches the threshold, green represents down-regulation and reaches the threshold, and black represents not reaching the significant threshold.

[0111] The right region is a heat map of the differential proteins, the behavior proteins are listed as sample names, and the color bars of different depths are the Z-scores of the row normalization (blue represents low expression, and red represents high expression). The tree diagram on the left is a gene clustering, which reveals the common up / down-regulated gene modules. The tree diagram on the top clusters the samples according to the expression profile, and tests whether the groups are naturally separated.

[0112] In the difference analysis of Group 2 relative to Group 1, the volcano plot shows that a batch of genes are significantly changed under the condition of |log2FC|≥1 and p<0.05, indicating that there is an expression difference with biological significance. The hierarchical clustering heat map based on the significant DEGs can clearly divide all samples into two clusters (i.e., the left / right two heat regions are transposed), and present the coordinated up / down-regulated modules of the groups, which means that Group 2 and Group 1 can be stably distinguished by the expression profile, indicating that the consistency between repetitions is good and the difference between groups is stable.

[0113] The evaluation includes diagnosing radiation-induced liver injury, evaluating the severity of radiation-induced liver injury, or monitoring the treatment effect of radiation-induced liver injury.

[0114] The reagent is used for detecting the concentration of THBS1 protein in the plasma sample of the subject, wherein: the concentration is <13 ng / mL under normal conditions, and the concentration is >20 ng / mL under the condition of radiation-induced liver injury.

[0115] The THBS1 protein includes a functional variant formed by one or more amino acid substitutions, deletions, or additions of the amino acid sequence shown in SEQ ID NO: 1.

[0116] A kit for evaluating radiation-induced liver injury comprises a reagent for specifically detecting the THBS1 protein of claim 1, and the reagent comprises:

[0117] (1) A pre-coated enzyme-labeled plate coated with a human monoclonal anti-THBS1 antibody;

[0118] (2) A biotin-labeled rabbit anti-THBS1 detection antibody;

[0119] (3) A human THBS1 recombinant protein standard.

[0120] The reagent further comprises:

[0121] The sample diluent is PBS+1% BSA, the washing solution is PBS+0.1% Tween-20, the TMB color developing solution, and the 2N H2SO4 stopping solution.

[0122] Specifically:

[0123] (I) Detection materials and sample sources

[0124] 1. Sample: human plasma sample of radioactive liver injury (exposed to 60 Gy radiation environment);

[0125] 2. Reagents: THBS1 monoclonal antibody, biotin-labeled polyclonal antibody, and recombinant THBS1 standard (all configured according to "kit composition").

[0126] (II) Plasma sample extraction steps

[0127] 1. For the person to be tested, after disinfection with 75% alcohol, collect blood with a 5 mL syringe (21G needle) and inject it into a centrifuge tube containing sodium citrate anticoagulant, and mix slowly for 5-10 times;

[0128] 2. Pre-cool the centrifuge at 4°C, centrifuge at 3000 rpm for 15 min, and transfer the upper plasma to a sterile EP tube, store at 4°C for short-term or at -80°C for long-term storage (avoid repeated freezing and thawing).

[0129] (III) Steps for ELISA detection of THBS1 concentration

[0130] 1. Preparation of pre-coated enzyme-labeled plate:

[0131] Dilute THBS1 monoclonal antibody to 2 μg / mL with carbonate buffer (pH 9.6), add 100 μL per well, and incubate at 4°C overnight; wash the plate 3 times with PBST (300 μL per well), add 300 μL blocking solution (PBS + 1% BSA) at 4°C overnight, dry and seal for storage.

[0132] 2. Detection process:

[0133] (1) Add 100 μL of standard (0-30 ng / mL gradient dilution) or plasma sample (3 replicate wells per group) to each well of the pre-coated plate, and incubate at 37°C for 1 hour;

[0134] (2) Wash the plate 3-5 times with PBST, add 100 μL of biotin-labeled detection antibody (1 μg / mL) per well, and incubate at room temperature for 1 hour;

[0135] (3) Wash the plate again 3-5 times, add 100 μL of TMB color developing solution, and incubate in the dark for 10-15 minutes, and add 50 μL of stop solution (2N H2SO4);

[0136] (4) Read the OD450 value on the enzyme-labeled instrument, draw the standard curve, and calculate the THBS1 concentration in the sample.

[0137] (IV) Comparison of detection results with reference values

[0138] If the concentration is >20 ng / mL, it indicates the presence of radioactive liver injury, and if it is <13 ng / mL, it indicates normal status.

[0139] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A method for preparing a reagent for evaluating radiation-induced liver injury using a THBS1 protein as a marker, characterized in that: The amino acid sequence of the THBS1 protein is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The evaluation includes diagnosing radiation-induced liver injury, evaluating the severity of radiation-induced liver injury, or monitoring the therapeutic effect of radiation-induced liver injury.

3. The use according to claim 1, characterized in that The reagent is used to detect the concentration of THBS1 protein in a subject's plasma sample, wherein: the concentration under normal conditions is <13 ng / mL, and the concentration under radiation-induced liver damage conditions is >20 ng / mL.

4. The use according to claim 1, wherein The THBS1 protein includes the amino acid sequence shown in SEQ ID NO:

1.

5. A kit for evaluating radiation-induced liver injury, characterized in that: A reagent for specifically detecting the THBS1 protein according to claim 1, wherein the reagent comprises: (1) Pre-coated ELISA plate with human monoclonal anti-THBS1 antibody; (2) Biotin-labeled rabbit anti-THBS1 detection antibody; (3) Human THBS1 recombinant protein standard.

6. The kit according to claim 5, wherein Also includes: Sample diluent: PBS + 1% BSA, washing solution: PBS + 0.1% Tween-20, TMB color development solution and 2NH2SO4 stop solution.

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