Gall bladder malignant tumor protein diagnosis biomarker combination, kit and application

Abstract

The invention discloses a gallbladder malignant tumor protein diagnosis biomarker combination, a kit and application. The protein marker provided by the invention comprises CA1 and / or IGFBP6 (insulin-like growth factor binding protein 6). According to the invention, an LC-MS / MS mass spectrometry method is adopted, mass spectrometry is carried out on a large number of clinical plasma samples, and by calculating log2 difference multiples of corresponding protein molecule contents in the plasma of a gallbladder cancer patient and the plasma of a normal person, it is determined that two protein molecules (CA1 protein and IGFBP6 protein) have good inspection benefits; the kit can be used as a marker for early diagnosis of gallbladder cancer; therefore, the invention provides a non-invasive screening means based on plasma, and the non-invasive screening means is used for predicting the cholecystic malignant tumor and distinguishing the cholecystic carcinoma crowd from the non-cancer crowd. And materials are convenient to take, the blood sample amount is small, and the sensitivity is high. The invention can fill the blank that no protein marker combination with high diagnostic efficiency exists at present for the cholecystic malignant tumor, and has great clinical significance.

Description

TECHNICAL FIELD

[0001] The application relates to a gallbladder malignant tumor protein diagnostic biomarker combination, a kit and application, and belongs to the technical field of biomedical detection. BACKGROUND

[0002] Gallbladder cancer is a common malignant tumor of the biliary system, accounting for 80-95% of the total biliary system malignant tumors. At present, surgery is the only effective treatment for gallbladder cancer, but more than 85% of gallbladder cancer patients have metastasis when diagnosed and cannot be treated by surgery. The overall median survival time of gallbladder cancer patients is 13.2 to 19 months, and the overall survival time of patients with advanced gallbladder cancer is shortened to less than half a year. Gallbladder cancer patients are often accompanied by gallbladder stones, cholecystitis, gallbladder polyps and other benign diseases of the gallbladder. Although the probability of canceration of patients with benign diseases is less than 1 / 100,000, according to the data of the pathology center of Zhongshan Hospital Affiliated to Fudan University, after diagnosis, as high as 60% of gallbladder cancer patients are classified as TNM staging of T2b and above, and the overall pathological grading shows a trend of late. Despite this, the incidence of gallbladder cancer is still increasing every year. How to diagnose precancerous lesions and gallbladder cancer in advance is a great challenge to improve the survival rate of gallbladder cancer patients. Therefore, finding a high-efficiency biomarker that can help early diagnosis of precancerous lesions and gallbladder cancer is of great significance for early diagnosis, early surgical intervention and early treatment of gallbladder cancer patients in clinical work.

[0003] Non-invasive diagnostic techniques such as liquid biopsy can detect nucleic acids derived from tumors in blood or other body fluid samples. Nucleic acids derived from circulating tumor cells, such as circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), circulating messenger RNA (mRNA) and long non-coding RNA (lncRNA), provide a reliable source for this detection. Circulating tumor cell nucleic acid testing has shown high specificity in cancer diagnosis, which can effectively reduce the frequency of false positive results. In addition, methylation of tumor suppressor genes or cancer gene groups also has good sensitivity and specificity in distinguishing gallbladder cancer patients from non-cancer populations, and has certain clinical application potential.

[0004] Compared with the extensive biomarker research targeting nucleic acids, the research on plasma proteins as early biomarkers of gallbladder cancer is relatively less. The development of a gallbladder malignant tumor protein diagnostic biomarker with high specificity and sensitivity based on plasma proteins as early biomarkers of gallbladder cancer is of great significance due to its small trauma and large potential research space. SUMMARY

[0005] The application aims to provide a gallbladder malignant tumor protein diagnostic biomarker combination, kit and application, the plasma protein biomarker has higher sensitivity and specificity for distinguishing gallbladder cancer patients, gallbladder benign diseases, healthy people, and can be used for preparing diagnostic and therapeutic products related to gallbladder cancer.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0007] The application provides an application of a biomarker or a detection reagent thereof in preparation of a gallbladder cancer diagnostic product, wherein the biomarker at least includes at least one of CA1 protein and IGFBP6 protein.

[0008] Preferably, the product includes a reagent, a kit or a diagnostic system.

[0009] Preferably, the biomarker further includes a tumor marker.

[0010] Preferably, the tumor marker includes CEA and / or CA199.

[0011] Preferably, the reagent or the kit at least includes a reagent for detecting a) expression amount and / or b) concentration and / or c) content of CA1 and / or IGFBP6 (protein or nucleic acid).

[0012] Preferably, the reagent for detecting CA1 includes a reagent (such as an Elisa kit, an antibody specifically recognizing CA1 protein) used for detecting CA1 protein by an Elisa method or a reagent used for detecting CA1 protein content by LC-MS / MS mass spectrometric analysis; and the reagent for detecting IGFBP6 includes a reagent (such as an Elisa kit, an antibody specifically recognizing IGFBP6 protein) used for detecting IGFBP6 protein by an Elisa method or a reagent used for detecting IGFBP6 protein content by LC-MS / MS mass spectrometric analysis.

[0013] Preferably, the reagent for detecting CA1 expression amount includes a primer or a probe specifically recognizing CA1 nucleic acid; and the reagent for detecting IGFBP6 expression amount includes a primer or a probe specifically recognizing IGFBP6 nucleic acid.

[0014] Preferably, the reagent or the kit further includes a reagent for detecting a tumor marker CEA and / or CA199.

[0015] The reagent for detecting the tumor marker CEA and / or CA199 is a detection reagent known in the art.

[0016] In some embodiments of the application, the kit further includes an instruction or a carrier recording a diagnostic standard, and the diagnostic standard is:

[0017] When the protein expression intensity value of CA1 protein in the sample is less than 89.28 detected by LC-MS / MS mass spectrometry, it is judged as a gallbladder cancer patient;

[0018] and / or, when the protein expression intensity value of IGFBP6 protein in the sample is less than 77.27 detected by LC-MS / MS mass spectrometry, it is judged as a gallbladder cancer patient.

[0019] Preferably, the reagent or kit is for detecting a blood sample of a subject.

[0020] The present application also provides a gallbladder cancer diagnostic kit, which comprises at least a reagent for detecting the expression amount and / or concentration and / or content of CA1 and / or IGFBP6 (protein or nucleic acid).

[0021] In some embodiments of the present application, the kit further comprises a reagent for detecting tumor marker CEA and / or CA199.

[0022] In some embodiments of the present application, the reagent or method for detecting tumor marker CEA and / or CA199 is known in the art.

[0023] The present application also provides a gallbladder cancer diagnostic system, which comprises a sample collection device, a separation device, a sample detection device and a diagnostic device; wherein:

[0024] The sample collection device is configured as a device for collecting a blood sample of a subject; the separation device is configured as a device for separating plasma from the blood sample; the sample detection device comprises a device capable of detecting the content of the marker in the plasma; the diagnostic device is configured to include a memory and a processor, the memory stores a computer program, and the processor is configured to execute the computer program stored in the memory to realize: according to the detection value of the content of the marker in the plasma of the subject, judge whether the subject contains gallbladder cancer; the marker is: i) at least one of CA1 protein and IGFBP6 protein, ii): the marker in i) combined with tumor marker CEA and / or CA199.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) The application adopts LC-MS / MS mass spectrum analysis method to detect the sample to be detected, a large number of clinical samples are subjected to mass spectrum analysis, and the log difference multiple (greater than 1.5 or less than -1.5) of the corresponding protein molecular content in the plasma of gallbladder cancer patients and normal people is determined, so that two protein molecules (CA1 protein and IGFBP6 protein) have good test benefits; and the two protein molecules can be used as markers for early diagnosis of gallbladder cancer;

[0027] (2) The application uses CA1 protein and / or IGFBP6 protein as biomarkers for early diagnosis of gallbladder cancer in subjects, has the advantages of easy detection and non-invasive detection, the diagnosis process is safe and effective and easy to be accepted by patients, the diagnosis standard is unified, and is less affected by personal subjective factors;

[0028] (3) The two protein biomarkers screened out by the application are helpful for the diagnosis of gallbladder cancer, improve the screening efficiency of gallbladder cancer, reduce the screening cost, are helpful for providing a direction for individualized treatment, and improve the survival rate and quality of life of patients;

[0029] (4) The two proteins as plasma protein biomarkers of gallbladder cancer in the application have good sensitivity and specificity for identification and screening of gallbladder cancer samples, have good diagnostic ability, and have great significance for the diagnosis and treatment of gallbladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a three-line table of gallbladder cancer (GBC) and normal (Normal) clinical information;

[0031] Figure 2 It is a volcano plot of differential proteins of gallbladder cancer group and normal group;

[0032] Figure 3 It is the expression amount of CA1 protein in the plasma protein of gallbladder cancer patients (GBC) and normal people (Normal) and gallbladder benign disease (Benign) in an independent discovery set;

[0033] Figure 4 It is the expression amount of CA1 protein in the plasma protein of gallbladder cancer patients (GBC) and normal people (Normal) and gallbladder benign disease (Benign) in an independent verification set;

[0034] Figure 5 It is the expression amount of IGFBP6 protein in the plasma protein of gallbladder cancer patients (GBC) and normal people (Normal) and gallbladder benign disease (Benign) in an independent discovery set;

[0035] Figure 6To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of;

[0036] Figure 7 To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of;

[0037] Figure 8 To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of;

[0038] Figure 9 To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of;

[0039] Figure 10 To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of;

[0040] Figure 11 To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of;

[0041] Figure 12 To verify the independent set of gallbladder cancer patients (GBC) and normal people (Normal), gallbladder benign disease (Benign) plasma protein IGFBP6 protein expression in the amount of; DETAILED DESCRIPTION

[0042] In order to make the present application more apparent and easy to understand, the preferred embodiments are described in detail below with the help of the accompanying drawings.

[0043] Example 1 Screening of plasma protein biomarkers of gallbladder cancer

[0044] A method for screening plasma protein biomarkers of gallbladder cancer, comprising the following steps:

[0045] (1) Protein sample screening: 30 cases of normal human plasma protein, 15 cases of gallbladder cancer patient plasma protein, the clinical information of gallbladder cancer (GBC) and normal people (Normal) three-line table is as followsFigure 1 As shown;

[0046] (2) Sample preprocessing:

[0047] ① Blood samples were collected from gallbladder cancer patients and healthy subjects. Dipotassium salt of ethylenediaminetetraacetic acid was added to the collected blood samples as an anticoagulant. The samples were centrifuged at 4°C, and the supernatant was collected to obtain patient plasma samples and healthy control plasma samples.

[0048] ② The plasma samples from gallbladder cancer patients and healthy controls were pretreated individually to obtain mass spectrometry analysis samples from gallbladder cancer patients and healthy controls, respectively. Here, 50 μg of protein was dissolved in 50 mM TEAB, 10 mM DTT was added to a final concentration and reacted at 37°C for one hour, 20 mM IAA was added to a final concentration and reacted at room temperature in the dark for 0.5 hours, trypsin was added at a ratio of 1:50 and enzymatically digested overnight at 37°C, and the enzymatic digestion was terminated by adding formic acid to a final concentration of 5%. The samples were centrifuged, and the supernatant peptides were collected. The final samples were desalted using a Waters Sepak desalting column before being prepared for mass spectrometry analysis.

[0049] (3) LC-MS / MS&4D-timsTOF Pro

[0050] ① Separation was performed using a Bruker nanoElute (Bruker Daltonics) liquid chromatograph with a 25cm x 75μm column (Inopticks) and 1.6μm C18 packing material. The column oven was set to 50℃. The flow rate was 300 nl / min, and the analysis time was 1 hour.

[0051] ② A spectral library was established using fractions from the mixed sample. All fractions were analyzed using a TIMS-TOF Pro mass spectrometer (Bruker Daltonics) with nanoESI, a scan range of 100-1700 m / z, and a spectral density range of 0.7-1.3 Vs / cm. 2 The single-cycle acquisition time was 1.16 s, with one ms scan followed by ten Pasef secondary scans. The intensity threshold was 5000, the accumulation and release time was 100 ms, the ion source voltage was 1500 V, the auxiliary gas was set to 3 L / min, and the ion source temperature was set to 180 °C.

[0052] ③ The sample was scanned using the DDA method to obtain the MS spectrum; the top 20 abundant peptide ions were selected from the MS spectrum obtained from each frame of scanning to obtain the MS2 spectrum; the MS2 spectrum was analyzed and identified, and the identified protein peptides were used to construct the DIA library; the mass spectrometry analysis samples included the mass spectrometry analysis samples of the gallbladder cancer patient and the mass spectrometry analysis samples of the healthy control.

[0053] ④ Perform DIA mass spectrometry analysis on each mass spectrometry analysis sample one by one to obtain the raw DIA files of all mass spectrometry analysis samples; use peaks onlines software with default parameters to analyze the raw DIA files of all the above mass spectrometry analysis samples, and match the raw DIA files based on the DIA library constructed in step (2), while combining with existing databases to identify and annotate the proteins in each mass spectrometry analysis sample; the mass spectrometry analysis samples include the mass spectrometry analysis samples of gallbladder cancer patients and the mass spectrometry analysis samples of healthy controls;

[0054] (4) Data Analysis:

[0055] ① Data were analyzed using Peaks Online (Bioinformatics Solutions Inc). A self-built serum chromatogram database was used, with a first-order error of 15 ppm and a second-order error of 0.05 Daltons. Trypsin digestion was set to partial digestion. Carbamidomethylation on C was set to fixed modification, while Acetylation (Protein N-term), Oxidation (M), and Deamidation (NQ) were set to variable modifications. The peak areas of the retrieved proteins were used for subsequent statistical analysis. The false positive rate for both chromatograms and proteins was set to 1%. Proteins annotated above underwent preprocessing: proteins with more than 50% missing values ​​in all mass spectrometry samples were excluded, and the remaining missing values ​​were filled.

[0056] ② Perform a t-test on the preprocessed dataset to calculate the P-value of the expression level of each protein; calculate the Log2(FC) value of each protein, and screen out all differentially expressed proteins based on the criteria of P-value < 0.0001 and |log2(FC)| > 1.5;

[0057] ③ Receiver operating characteristic (ROC) analysis was performed on the above differentially expressed proteins, and the area under the curve (AUC) was calculated. Using the criteria of an AUC ROC greater than 0.6 and Log2(FC) greater than 1.5, two proteins, CA1 and IGFBP6, were selected as biomarkers for gallbladder cancer detection. Here, log2(Fold Change) is abbreviated as Log2(FC).

[0058] (5) Statistical analysis

[0059] ①Data quality control was performed on the protein spectrum data of the normal group, benign gallbladder disease group, and gallbladder cancer group. Protein items with 30% or more missing values were excluded. The normal group and the benign gallbladder disease group were compared with the gallbladder cancer group, respectively. Data were uniformly logarithmically processed, normalized, and missing values were filled. Finally, the significance and fold change were calculated by difference analysis. The results included the intensity ratio and P-value, FDR (false positive rate) of each comparison group, thereby preliminarily screening the differential proteins between groups. Further verification of the significance of the differential proteins was performed according to P-value and FDR. Proteins with fold change > 1.5 or < -1.5 were selected as proteins with significant differences in the content of gallbladder cancer patient plasma and normal plasma. Proteins with P value and FDR less than 0.0001 were selected as proteins with significant differences. Thus, the differential protein molecules were obtained.

[0060] ②Then, the ROC curve of the differential proteins was drawn using R language and Prism, and the area under the curve (AUC) was calculated to determine the diagnostic value. The specific determination method was that the area under the AUC line was greater than 0.8, and P was less than 0.0001. The threshold value (cut off value) at the maximum Youden index was used as the threshold value (fold change greater than 1.5 was considered greater than the threshold value for gallbladder cancer detection positive, and fold change less than -1.5 was considered less than the threshold value for gallbladder cancer detection positive), thereby obtaining higher sensitivity and specificity.

[0061] (6) Bioinformatics analysis

[0062] ① GO function annotation: Blast2GO was used to annotate the target protein set with GO. The process can be roughly divided into four steps: sequence alignment (Blast), GO entry extraction (Mapping), GO annotation (Annotation), and InterProScan supplementary annotation (Annotation Augmentation).

[0063] ② KEGG pathway annotation: KAAS (KEGG Automatic Annotation Server) software was used to annotate the target protein set with KEGG pathway.

[0064] ③ Enrichment analysis of GO annotation and KEGG annotation: Fisher's Exact Test was used to compare the distribution of each GO category or KEGG pathway in the target protein set and the total protein set. Enrichment analysis of GO annotation or KEGG pathway annotation was performed on the target protein set.

[0065] IV. Protein clustering analysis: First, the quantitative information of the target protein set was normalized (normalized to the interval (-1, 1)). Then, the Complexheatmap R package (R Version 3.4) was used to classify both the sample and protein expression dimensions simultaneously (distance algorithm: Euclidean, connection method: Average linkage), and a hierarchical clustering heat map was generated.

[0066] V. Protein interaction network analysis: Based on the information in the STRING (http: / / string-db.org / ) database, the interaction relationships between the target proteins were found, and the CytoScape software (version number: 3.2.1) was used to generate and analyze the interaction network.

[0067] (7) Differential expression protein screening

[0068] I. Unpaired t-test was performed on the above pre-processed data set to calculate whether each variable (protein expression level) had a significant difference between the gallbladder cancer group and the normal group, expressed as P value (P value). The corrected P value was calculated using the Benjamini & Hochberg correction method.

[0069] II. The standard of P value less than 0.0001 and |log2(Fold Change)|>1.5 was used to select differential proteins. Log2(Fold Change) is abbreviated as Log2(FC), which is the difference in concentration after log2 transformation. Fold change (Fold Change) refers to the average value of a single protein in the GBC group divided by its average value in the Normal group. Figure 2 In order to show the volcano plot of all proteins in the above pre-processed data set in gallbladder cancer and normal people (or differences). The volcano plot can be visualized by the R language ggplot package. Figure 2 DOWN represents down-regulated proteins, UP represents up-regulated proteins, and NS represents proteins with no significant difference. From the volcano plot, it can be seen that there are significant up-regulated and down-regulated proteins in the gallbladder cancer group relative to the normal group. Figure 2 It can be seen that there are significant up-regulated and down-regulated proteins in the gallbladder cancer group relative to the normal group. 36 proteins meet the standard of P value less than 0.0001 and |log2(Fold Change)|>1.5 (i.e., Log2(FC)>2 or Log2(FC) less than 2), which are selected as differential proteins. Among the 36 differential proteins, 6 proteins are up-regulated and 30 proteins are down-regulated.

[0070] (8) Basic principles of the experiment

[0071] ①Data-independent acquisition (DIA) has become an important mass spectrometry quantification method in recent years. The core principle is to use the identification results of multiple by ions of MS2 as the basis for qualitative and quantitative analysis. The difference between various methods lies in the modification of high-throughput data by the post-search algorithm.

[0072] ②ELISA is the abbreviation of Enzyme-Linked Immunosorbnent Assay. It is an immunoenzyme technique developed after immunofluorescence and radioimmunoassay. During detection, the specimen to be tested (determining the antigen therein) reacts with the antibody on the surface of the solid carrier. After washing, the enzyme-labeled antibody is combined on the solid carrier through reaction. After adding the substrate for enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is directly related to the amount of the tested substance in the specimen. Qualitative or quantitative analysis can be performed according to the color depth.

[0073] (9) ELISA verification method: corresponding protein kit

[0074] ①Sample addition: Take out the pre-coated and sealed 96-well plate, equilibrate to room temperature, and add 100 μL of diluted standard and diluted sample, respectively, and incubate at 37℃ for 90 min.

[0075] ②Plate aspiration: aspirate the reaction solution in the enzyme-labeled plate and tap a few times against the water-absorbing paper without washing.

[0076] ③Add biotin-labeled antibody: add 100 μL of biotin-labeled antibody diluted at 1:100, and incubate at 37℃ for 60 min.

[0077] ④Wash the plate: wash with 1X washing buffer for 3 times, each time for 1 min.

[0078] ⑤Add peroxidase-labeled avidin (ABC): add 100 μL of ABC diluted at 1:100, and incubate at 37℃ for 30 min.

[0079] ⑥Wash the plate: wash with 1X washing buffer for 5 times, each time for 90 s.

[0080] ⑦Add substrate (TMB): add 90 μL of TMB per well, and incubate at 37℃ for 20 min in the dark.

[0081] ⑧Add stop solution: add 100 μL of stop solution per well, and mix gently.

[0082] ⑨Plate reading: according to the instrument operation method of the enzyme-labeled instrument, detect the OD value (at 450 nm), and calculate the concentration of the measured substance by software (ELISA Calc).

[0083] Experimental results:

[0084] Through the analysis of the mass spectrometry data, the protein molecules of the plasma of the gallbladder cancer patient and the normal person are compared, and finally two protein molecules (i.e. CA1 protein and IGFBP6 protein) can be used as the molecular markers related to the early diagnosis of the gallbladder cancer. The subsequent verification process is verified by ELISA.

[0085] In order to evaluate the protein expression intensity value of the protein molecule for the early diagnosis efficiency of the gallbladder malignant tumor, the present application adopts the ROC curve analysis, the AUC is the area under the ROC curve, which is the most commonly used parameter for evaluating the characteristics of the ROC curve and is also an important test accuracy index. If the AUC is less than 0.6, the accuracy of the diagnosis is low; if the AUC is greater than 0.6, the requirement of the clinical diagnosis can be met.

[0086] The specific results and analysis are as follows:

[0087] ①LC-MS / MS mass spectrometry analysis is adopted to detect the difference of CA1 protein in the plasma samples of the gallbladder cancer patient and the normal person. It is found through the research that the CA1 protein expression level of the plasma sample of the gallbladder cancer patient is significantly reduced by 4 times compared with the plasma sample of the normal person, and the p value is less than 0.0001. Through the ELISA retest, when the protein expression intensity value of the CA1 protein (the protein expression intensity value refers to the relative intensity of ions (Relative intensity of ions) of the mass spectrum graph when the protein is detected by LC-MS / MS mass spectrometry, and the base peak intensity value is 100) is less than 89.28, the sensitivity is 79.2% and the specificity is 83.33%; when the individual detection is carried out, if the protein expression intensity value of the protein is less than 89.28, it is judged as the gallbladder cancer patient, otherwise it is judged as the population without gallbladder cancer (the false positive rate is 16.66%). It can be seen from the above that the plasma samples of the gallbladder cancer patients are mainly distributed below the detection threshold (the dotted line in the figure), and the normal plasma samples are mainly distributed above the detection threshold, which indicates that the protein expression intensity values of the plasma of the gallbladder cancer patient and the normal person are quite different, and the detection threshold has good detection effect. Figure 3 Figure 4 Figure 8 It can be seen that the AUC of the CA1 protein is 0.8121>0.6, which indicates that the CA1 protein can be used as the molecular marker for diagnosing the gallbladder cancer.

[0088] In summary, the CA1 protein can be used as the early molecular marker for diagnosing the gallbladder cancer.

[0089] ​​② LC-MS / MS mass spectrometry analysis was used to detect the differences in IGFBP6 protein between the plasma samples of gallbladder cancer patients and normal people. It was found that the expression level of IGFBP6 protein in the plasma samples of gallbladder cancer patients was significantly down-regulated by 4 times compared with that in the plasma samples of normal people (p<0.0001). ELISA retesting found that when the protein expression intensity value of IGFBP6 protein was less than 77.27, the sensitivity was 62% and the specificity was 65.4%; when individual detection was performed, if the protein expression intensity value of the protein was less than 77.27, it was judged as a gallbladder cancer patient, otherwise it was judged as a person without gallbladder cancer (false positive rate was 34.6%). It can be seen from Figure 5 that the plasma samples of gallbladder cancer patients are mainly distributed below the detection threshold (dashed line in the figure), and the plasma samples of normal people are mainly distributed above the detection threshold (repeated test Figure 6 ), which shows that the protein expression intensity values of the plasma of gallbladder cancer patients and normal people are quite different, and the detection threshold has good detection effect. It can be seen from Figure 9 that the AUC of IGFBP6 protein is 0.622>0.6, which shows that IGFBP6 protein can be used as a molecular marker for diagnosing gallbladder cancer.

[0090] In summary, IGFBP6 protein can be used as an early molecular marker for diagnosing gallbladder cancer.

[0091] ③ LC-MS / MS mass spectrometry analysis was used to detect the differences in IGFBP6 protein and CA1 protein between the plasma samples of gallbladder cancer patients and normal people. It can be seen from Figure 3 、 4 , 5, 6 that the plasma samples of gallbladder cancer patients are mainly distributed below the detection threshold (dashed line in the figure), and the plasma samples of normal people are mainly distributed above the detection threshold, which shows that the protein expression intensity values of the plasma of gallbladder cancer patients and normal people are quite different, and the detection threshold has good detection effect. In summary, CA1 protein combined with IGFBP6 protein can be used as an early molecular marker combination for diagnosing gallbladder cancer. It can be seen from Figure 10 that the AUC of IGFBP6 protein+CA1 protein is 0.809>0.6, which shows that CA1 protein combined with IGFBP6 protein can be used as a high-efficiency molecular marker combination for diagnosing gallbladder cancer.

[0092] Example 2 Application of CA1 protein as a biomarker in the preparation of a gallbladder cancer diagnostic reagent

[0093] The application of CA1 protein as a biomarker in the preparation of a gallbladder cancer diagnostic reagent, process and results are as follows:

[0094] In the present application, the expression amount of CA1 protein in the sample to be detected can be detected by Elisa method or LC-MS / MS mass spectrometry. The expression amount of protein is detected by measuring the absorbance (OD value) by Elisa method. The higher the absorbance value, the higher the expression amount of protein. The specific calculation method adopts the standard curve method: the corresponding protein concentration of the sample to be detected is found on the standard curve according to the OD value of the sample to be detected, that is, the content of CA1 protein in the sample; specifically, the intensity value (intensity) of the expression amount of CA1 protein in the sample to be detected is detected by LC-MS / MS mass spectrometry in the present embodiment. When the intensity value of the expression amount of CA1 protein in the sample to be detected is less than 89.28, it is judged as a gallbladder cancer patient. When the expression amount of the protein of CA1 is less than 89.28, the sensitivity is 79.2%, and the specificity is 83.33%; when individual detection is carried out, the intensity value of the expression amount of the protein of the protein is less than 89.28, which is judged as a gallbladder cancer patient, otherwise it is judged as a population without gallbladder cancer (the false positive rate is 16.66%). Compared with the prior art, the present application has the advantages that the CA1 protein is a new biomarker for the diagnosis of gallbladder cancer, and the sensitivity and specificity of the CA1 protein are higher than those of the prior art. Figure 7 The CA1 protein is a new biomarker for the diagnosis of gallbladder cancer, and the sensitivity and specificity of the CA1 protein are higher than those of the prior art. Figure 8 As shown in the single factor ROC curve of the CA1 protein in the independent verification set, the AUC of the CA1 protein is greater than that of the commonly used tumor marker, and the AUC of the CA1 protein is 0.8121>0.76, which indicates that the CA1 protein is more effective than the commonly used tumor marker as a molecular marker for the diagnosis of gallbladder cancer.

[0095] The present embodiment also provides a kit and its use in the early diagnosis, treatment guidance and prognosis of gallbladder cancer. The kit includes a reagent for specifically detecting CA1 protein; the reagent for specifically detecting CA1 protein is an antibody that specifically recognizes CA1 protein; and the reagent is used for detecting tissue samples.

[0096] Example 3 Application of IGFBP6 protein as a biomarker in the preparation of a gallbladder cancer diagnostic reagent

[0097] The application of IGFBP6 protein as a biomarker in the preparation of a gallbladder cancer diagnostic reagent, process and results are as follows:

[0098] In the present application, the expression amount of IGFBP6 protein in the sample to be tested can be detected by Elisa method or LC-MS / MS mass spectrometry. Specifically, the present embodiment uses LC-MS / MS mass spectrometry to detect the intensity of the expression amount of IGFBP6 protein in the sample to be tested. When the intensity of the expression amount of IGFBP6 protein in the sample to be tested is less than 77.27, it is judged to be a gallbladder cancer patient. When the expression amount of the protein is less than 77.27, the sensitivity is 62.5%, and the specificity is 65.4%. When an individual is detected, if the intensity of the expression amount of the protein is less than 77.27, it is judged to be a gallbladder cancer patient, otherwise it is judged to be a non-gallbladder cancer population (the false positive rate is 34.6%). Compared with Figure 7 the commonly used tumor marker levels in the prior art, such as Figure 8 As shown in the single factor ROC curve of IGFBP6 in the independent verification set, the AUC is close to the commonly used tumor marker CA1 protein, and the AUC of CA1 protein is 0.622>0.61, which indicates that IGFBP6 protein has the advantage of becoming a potential specific marker for diagnosing gallbladder cancer compared with non-specific gallbladder cancer tumor markers.

[0099] The present embodiment also provides a kit and its use in the early diagnosis, treatment guidance and prognosis of gallbladder cancer. The kit includes a reagent for specifically detecting IGFBP6 protein; the reagent for specifically detecting IGFBP6 protein is an antibody that specifically recognizes IGFBP6 protein; and the reagent is used for detecting tissue samples.

[0100] Example 4: Application of IGFBP6 protein combined with CA1 protein as a biomarker in the preparation of a gallbladder cancer diagnostic reagent

[0101] The application of IGFBP6 protein combined with CA1 protein as a biomarker in the preparation of a gallbladder cancer diagnostic reagent, the process and results are as follows:

[0102] LC-MS / MS mass spectrometry is used to detect the intensity of the expression amount of IGFBP6 protein and CA1 protein in the sample to be tested. When the intensity of the expression amount of IGFBP6 protein in the sample to be tested is less than 77.27 and the intensity of the expression amount of CA1 protein is less than 89.28, it is judged to be a gallbladder cancer patient. Compared with Figure 7 the commonly used tumor marker levels in the prior art, such as Figure 10 As shown in the independent verification set, the AUC of CA1 protein combined with IGFBP6 protein is 0.809>0.6, which is significantly better than the single protein diagnostic efficiency, indicating that CA1 protein combined with IGFBP6 protein can be used as a high-efficiency molecular marker combination for diagnosing gallbladder cancer.

[0103] In addition, in the independent verification set, as shown in Table 2,Figure 11 As shown, the AUC of CA1 protein, IGFBP6 protein combined with tumor markers CEA and CA199 in the independent verification set is 0.887>0.8, which shows that CA1 and IGFBP6 combined with CEA and CA199 can be used as a high-performance molecular marker combination for the diagnosis of gallbladder cancer in clinic, and there is a significant performance advantage in the diagnosis of gallbladder cancer compared with common single tumor markers. Figure 12 As shown, the AUC of CA1 protein, IGFBP6 protein combined with tumor markers CEA and CA199 in the total verification set is 0.873>0.8, and the conclusion is repeatedly verified in the total verification set.

[0104] The embodiment also provides a kit and its use in the early diagnosis, treatment guidance and prognosis of gallbladder cancer, which comprises reagents for specifically detecting IGFBP6 and / or CA1 protein; the reagents for specifically detecting IGFBP6 and CA1 protein can be antibodies specifically recognizing IGFBP6 and CA1 protein; and the reagents are used for detecting tissue samples.

[0105] The present application uses CA1 protein, IGFBP6 protein or a combination of proteins as biomarkers or combined tumor markers (CEA, CA199) for early diagnosis of gallbladder cancer, which is simple, safe and effective in the diagnosis process, easy to be accepted by patients, has unified diagnostic criteria, and is less affected by personal subjective factors.

[0106] The above is only a preferred embodiment of the present application, and is not a limitation on the form and substance of the present application. It should be noted that those skilled in the art can make some improvements and supplements without departing from the present application, and these improvements and supplements should also be considered as the protection scope of the present application.

Claims

1. The application of a biomarker or its detection reagent in the preparation of gallbladder cancer diagnostic products, characterized in that, The biomarkers include at least one of the CA1 protein and the IGFBP6 protein.

2. The application according to claim 1, characterized in that, The products include reagents, kits, or diagnostic systems.

3. The application according to claim 1, characterized in that, The biomarkers also include tumor markers.

4. The application according to claim 3, characterized in that, The tumor markers include CEA and / or CA199.

5. The application according to claim 2, characterized in that, The reagents or kits described herein include at least reagents for specifically detecting a) expression level and / or b) concentration and / or c) content of CA1 and / or IGFBP6 (protein or nucleic acid).

6. The application according to claim 5, characterized in that, The reagents for detecting CA1 include those used for detecting CA1 protein by ELISA or those used for detecting CA1 protein content by LC-MS / MS mass spectrometry analysis; the reagents for detecting IGFBP6 include those used for detecting IGFBP6 protein by ELISA or those used for detecting IGFBP6 protein by LC-MS / MS mass spectrometry analysis.

7. The application according to claim 5, characterized in that, The reagent for detecting CA1 expression includes primers or probes that specifically recognize CA1 nucleic acid; the reagent for detecting IGFBP6 expression includes primers or probes that specifically recognize IGFBP6 nucleic acid.

8. The application according to claim 2, characterized in that, The reagents or kits also include reagents for detecting tumor markers CEA and / or CA199.

9. The application according to any one of claims 1 to 8, characterized in that, The product is designed for testing blood samples from test subjects.

10. A diagnostic kit for gallbladder cancer, characterized in that, The kit includes at least one reagent for detecting the expression levels of CA1 and / or IGFBP6 (protein or nucleic acid).

11. The gallbladder cancer diagnostic kit as described in claim 10, characterized in that, The kit also includes reagents for detecting tumor markers CEA and / or CA199.

12. A gallbladder cancer diagnostic system, characterized in that, The system includes a sample collection device, a separation device, a sample detection device, and a diagnostic device; wherein: The sample collection device is configured to collect blood samples from a subject; the separation device is configured to separate plasma from the blood sample; the sample detection device includes a means for detecting the content of biomarkers in the plasma; the diagnostic device is configured to include a memory and a processor, the memory storing a computer program, and the processor being configured to execute the computer program stored in the memory to: determine whether a subject has gallbladder cancer based on the detected value of the biomarker content in the subject's plasma; the biomarker is: i) at least one of CA1 protein and IGFBP6 protein, ii) the biomarker described in i) in combination with the tumor biomarker CEA and / or CA199.

Images