Immune Raman multicolor imaging kit for predicting and evaluating liver cancer

Abstract

The invention relates to an immune Raman multicolor imaging kit for liver cancer prediction and evaluation, the immune Raman multicolor imaging kit comprises five Raman probes, and the Raman probes are composed of Raman balls and antibodies connected to the surfaces of the Raman balls; wherein the Raman sphere is formed by polymerizing a triple bond monomer and (i) acrylic acid or (ii) styrene and acrylic acid, the particle size is 10-100 nm, and the Raman shift is 1800-2800 cm <-1 >; antibodies of the five Raman probes are respectively selected from one of an AFP antibody, an HBX antibody, a GPC3 antibody, a GS antibody and a CD34 antibody. The immune Raman multicolor imaging reagent and kit provided by the invention can realize one-step marking and multiple color development of five liver cancer related protein markers in the same liver tissue slice. Compared with a multiple fluorescence immunohistochemical technology, the method has remarkable advantages in efficiency and precision. Experiments show that the detection result is consistent with the clinical diagnosis result, and the application potential is huge.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical precision diagnosis, and particularly relates to an immune Raman multicolor imaging kit for liver cancer prediction and evaluation. BACKGROUND

[0002] In clinical practice, liver cancer is mostly in the advanced stage once diagnosed. Its early symptoms are not obvious, the malignancy is high, and the prognosis is poor, so it is called "silent killer" by the medical community. Studies have shown that the early cure rate of liver cancer can exceed 70%, but the current early diagnosis still mainly relies on imaging examination and routine histopathological examination. However, imaging examination is easily disturbed by factors such as hemangioma, non-uniform fatty liver, cirrhosis, and liver heterotypic hyperplastic nodules; routine histopathological examination faces the tedious and time-consuming processes of tissue sectioning and staining, and the diagnostic results often depend on the subjective judgment of pathologists. These uncertain results are easy to cause misdiagnosis, thereby affecting the timeliness and effectiveness of the treatment plan.

[0003] There are about 257 million chronic hepatitis B infected people in the world, of which 30% to 40% will develop into cirrhosis or liver cancer. In China, 80% of liver cancer patients have hepatitis B cirrhosis background, and hepatitis B infection related liver cancer accounts for as high as 84%. Hepatitis B virus infection is considered to be the core pathogenic factor of liver cancer, although antiviral treatment can reduce the risk of liver cancer, but still cannot eliminate the risk of its occurrence (the annual incidence of liver cancer is still more than 1%). Therefore, it is of great significance to carry out rapid and accurate detection of the causes of liver cancer. Revealing the deep mechanism of hepatitis B virus persistent carcinogenesis is the key to the radical cure of liver cancer. Liver cancer related marker detection can effectively improve the sensitivity and specificity of liver cancer diagnosis by feeding back tumor causes through multiple indicators, and provide strong guarantee for precise diagnosis of liver cancer. At present, there is no standardized method for simultaneous detection of multiple molecular markers related to liver cancer in clinical practice, the main difficulties are the determination of markers and the problem of signal overlap in simultaneous detection of multiple proteins, and the existing multiple imaging has high cost. SUMMARY

[0004] The application provides an immunoraman multicolor imaging kit for liver cancer prediction and evaluation. The kit simultaneously images and detects five new etiological proteins induced by hepatitis B virus to cause liver cancer and various proteins in the microenvironment during the development of liver cancer. The five liver cancer related proteins are alpha-fetoprotein (AFP), glutamine synthetase (GS), glypican 3 (GPC3), liver susceptibility virus protein (HBX), and highly glycosylated type I transmembrane glycoprotein (CD34). These proteins are closely related to the viral infection, malignancy degree and metastasis progression of primary liver cancer. Simultaneous detection can effectively realize the differential diagnosis of liver cancer etiology and evaluate the malignancy degree and progression of liver cancer. The application realizes the innovative detection of "one-step labeling and multiple color development" of key proteins of liver cancer etiology through the kit. Compared with traditional immunohistochemical detection, the application has significant advantages in precision and efficiency, and the experimental results fully meet the needs of clinical detection.

[0005] The technical scheme provided by the application is as follows:

[0006] In a first aspect, the application provides an immunoraman multicolor imaging kit for liver cancer prediction and evaluation, comprising five Raman probes, wherein the Raman probe is composed of a Raman sphere and an antibody connected to the surface of the Raman sphere; wherein:

[0007] The Raman sphere is polymerized from a three-key monomer and (i) acrylic acid or (ii) styrene and acrylic acid, and has a particle size of 10-100 nm and a Raman shift of 1800-2800 cm -1 The three-key monomers of the five Raman probes are selected from compounds 1-7, wherein compounds 1 and 2 are not selected at the same time, compounds 3 and 4 are not selected at the same time, and the structures of compounds 1-7 are as shown below:

[0008] ;

[0009] The antibodies of the five Raman probes are selected from one of AFP antibodies, HBX antibodies, GPC3 antibodies, GS antibodies and CD34 antibodies.

[0010] In combination with the first aspect of the application, in some embodiments: the three-key monomers in the five Raman probes are compounds 2, 3, 5, 6 and 7, respectively;

[0011] The characteristic peak of the Raman probe with the three-key monomer of compound 2 is 2160 cm -1 ;

[0012] The characteristic peak of the Raman probe with the three-key monomer of compound 3 is 2186 cm -1 ;

[0013] The characteristic peak of the Raman probe of the triple bond monomer as compound 5 is 2227 cm -1 ;

[0014] The characteristic peak of the Raman probe of the triple bond monomer as compound 6 is 2241 cm -1 ;

[0015] The characteristic peak of the Raman probe of the triple bond monomer as compound 7 is 2260 cm -1 .

[0016] In combination with the first aspect of the present application, in some embodiments:

[0017] The antibody on the Raman probe of the triple bond monomer as compound 2 is an AFP antibody;

[0018] The antibody on the Raman probe of the triple bond monomer as compound 3 is a GPC3 antibody;

[0019] The antibody on the Raman probe of the triple bond monomer as compound 5 is an HBX antibody;

[0020] The antibody on the Raman probe of the triple bond monomer as compound 6 is a GS antibody;

[0021] The antibody on the Raman probe of the triple bond monomer as compound 7 is a CD34 antibody.

[0022] In combination with the first aspect of the present application, in some embodiments:

[0023] The Raman ball is connected to the antibody by a chemical bond; and / or,

[0024] The particle size of the Raman ball is 30-70 nm.

[0025] In a second aspect, the present application provides an immunoraman multicolor imaging kit for liver cancer prediction and evaluation, comprising the above-mentioned immunoraman multicolor imaging reagent; when used, liver tissue slices are co-incubated with the immunoraman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL.

[0026] In combination with the second aspect of the present application, in some embodiments: the immunoraman multicolor imaging kit further comprises PBS, wherein the total volume of the PBS is configured so that after mixing with all the immunoraman multicolor imaging reagents, the concentration of each Raman probe is not less than 0.5 mg / mL.

[0027] In a third aspect, the present application provides a liver cancer prediction and evaluation system, comprising a sample processing module, a collection module, and an acquisition module, which are physically connected or communicatively connected; wherein:

[0028] The sample processing module is configured to co-incubate one liver tissue slice of a subject with the above-mentioned immunoraman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL.

[0029] The acquisition module is configured to acquire Raman imaging information of the liver tissue slice.

[0030] The acquisition module is configured to analyze the expression levels of alpha-fetoprotein, liver susceptibility virus protein, glypican 3, glutamine synthetase and highly glycosylated type I transmembrane glycoprotein according to the Raman imaging information.

[0031] In combination with the third aspect of the present application, in some embodiments, the liver cancer prediction and evaluation system further comprises an evaluation module:

[0032] The evaluation module is configured to evaluate the risk of liver cancer, the cause of liver cancer, the degree of malignancy and the metastasis of the subject according to the expression levels.

[0033] In combination with the third aspect of the present application, in some embodiments, the evaluation module is specifically configured to take the average level of healthy liver tissue as a reference, and realize multidimensional evaluation based on the expression level difference of each marker, including:

[0034] According to the expression level of alpha-fetoprotein, the risk of liver cancer of the subject is evaluated;

[0035] According to the expression level of liver susceptibility virus protein, it is judged whether the liver cancer is caused by hepatitis B virus;

[0036] According to the expression level of glypican 3, the degree of malignancy and the risk of deterioration of liver cancer are evaluated;

[0037] According to the combined expression level of glutamine synthetase and highly glycosylated type I transmembrane glycoprotein, the metastasis of liver cancer is evaluated.

[0038] In the fourth aspect, the present application provides a liver cancer prediction and evaluation instrument, which comprises the above-mentioned liver cancer prediction and evaluation system.

[0039] Compared with the prior art, the present application has at least the following beneficial effects:

[0040] 1. The immunoraman multicolor imaging reagent and kit provided by the present application can realize one-step labeling and multiple color development of five liver cancer-related protein markers in the same liver tissue slice, and are simple to operate, low in cost and high in labeling efficiency.

[0041] 2. Compared with the existing liver cancer diagnosis method, the immunoraman multicolor imaging kit provided by the application realizes simultaneous imaging of five liver cancer related markers of AFP, HBX, GPC3, GS and CD34 for the first time, constructs a liver cancer prediction and progress evaluation method according to the combined imaging results for the first time, and realizes risk evaluation of hepatitis B liver cancer, deterioration degree and metastasis condition of the patient's tumor tissue for the first time, so as to guide the clinician to provide scientific and personalized treatment methods for liver cancer patients, and provide the latest basis for the prognosis treatment of the patients.

[0042] 3. The kit provided by the application is simple to prepare, has good stability, a long storage period and is easy to mass produce. BRIEF DESCRIPTION OF DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0044] Figure 1 It is a Raman spectrum of monomer 1-7.

[0045] Figure 2 It is a transmission electron microscope image of the nanospheres prepared in Example 2 of the application, a is the nanospheres polymerized from compound 2; b is the nanospheres polymerized from compound 3; c is the nanospheres polymerized from compound 5; d is the nanospheres polymerized from compound 6; e is the nanospheres polymerized from compound 7.

[0046] Figure 3 It is a Raman spectrum of each compound before and after polymerization in Example 2 of the application, M1 is the Raman spectrum of compound 2; P1 is the Raman spectrum of the nanospheres of compound 2; M2 is the Raman spectrum of compound 3; P2 is the Raman spectrum of the nanospheres of compound 3; M3 is the Raman spectrum of compound 5; P3 is the Raman spectrum of the nanospheres of compound 5; M4 is the Raman spectrum of compound 6; P4 is the Raman spectrum of the nanospheres of compound 6; M5 is the Raman spectrum of compound 7; P5 is the Raman spectrum of the nanospheres of compound 7.

[0047] Figure 4 It is an infrared spectrum of each compound before and after polymerization in Example 2 of the application, M1 is the infrared spectrum of compound 2; P1 is the infrared spectrum of the nanospheres of compound 2; M2 is the infrared spectrum of compound 3; P2 is the infrared spectrum of the nanospheres of compound 3; M3 is the infrared spectrum of compound 5; P3 is the infrared spectrum of the nanospheres of compound 5; M4 is the infrared spectrum of compound 6; P4 is the infrared spectrum of the nanospheres of compound 6; M5 is the infrared spectrum of compound 7; P5 is the infrared spectrum of the nanospheres of compound 7.

[0048] Figure 5 Preparation and use flow chart of the immunoraman multicolor imaging kit provided by the present application.

[0049] Figures 6a to 6j The immunoraman probe prepared for example 5 proves the raman imaging graph of hepatitis B infection and liver cancer diagnosis; wherein:

[0050] Figure 6a For the raman imaging of AFP protein in example 5, 2160 cm -1 The raman imaging graph with specific raman signal as characteristic peak;

[0051] Figure 6b The corresponding 2160 cm -1 The raman spectrum graph with characteristic peak;

[0052] Figure 6c For the raman imaging of HBX protein in example 5, 2227 cm -1 The raman imaging graph with specific raman signal as characteristic peak;

[0053] Figure 6d The corresponding 2227 cm -1 The raman spectrum graph with characteristic peak;

[0054] Figure 6e For the raman imaging of GPC3 protein in example 5, 2186 cm -1 The raman imaging graph with specific raman signal as characteristic peak;

[0055] Figure 6f The corresponding 2186 cm -1 The raman spectrum graph with characteristic peak;

[0056] Figure 6g For the raman imaging of GS protein in example 5, 2241 cm -1 The raman imaging graph with specific raman signal as characteristic peak;

[0057] Figure 6h The corresponding 2241 cm -1 The raman spectrum graph with characteristic peak;

[0058] Figure 6i For the raman imaging of CD34 protein in example 5, 2260 cm -1 The raman imaging graph with specific raman signal as characteristic peak;

[0059] Figure 6jRaman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0060] Figures 7a to 7j Raman spectrum corresponding to 2260 cm

[0061] Figure 7a Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0062] Figure 7b Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0063] Figure 7c Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0064] Figure 7d Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0065] Figure 7e Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0066] Figure 7f Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0067] Figure 7g Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0068] Figure 7h Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0069] Figure 7i Raman spectrum corresponding to 2260 cm -1 Raman spectrum corresponding to 2260 cm

[0070] Figure 7j Raman spectrum corresponding to 2260 cm-1 Raman spectrum with characteristic peaks.

[0071] Figures 8a to 8j Raman imaging images of liver cancer deterioration and cancer metastasis proved by the immunoraman probes prepared in Example 7; wherein:

[0072] Figure 8a For Raman imaging of AFP protein in Example 7, 2160 cm -1 Raman imaging image with specific Raman signals as characteristic peaks;

[0073] Figure 8b For Raman imaging in Example 7, the corresponding 2160 cm -1 Raman spectrum with characteristic peaks;

[0074] Figure 8c For Raman imaging of HBX protein in Example 7, 2227 cm -1 Raman imaging image with specific Raman signals as characteristic peaks;

[0075] Figure 8d For Raman imaging in Example 7, the corresponding 2227 cm -1 Raman spectrum with characteristic peaks;

[0076] Figure 8e For Raman imaging of GPC3 protein in Example 7, 2186 cm -1 Raman imaging image with specific Raman signals as characteristic peaks;

[0077] Figure 8f For Raman imaging in Example 7, the corresponding 2186 cm -1 Raman spectrum with characteristic peaks;

[0078] Figure 8g For Raman imaging of GS protein in Example 7, 2241 cm -1 Raman imaging image with specific Raman signals as characteristic peaks;

[0079] Figure 8h For Raman imaging in Example 7, the corresponding 2241 cm -1 Raman spectrum with characteristic peaks;

[0080] Figure 8i For Raman imaging of CD34 protein in Example 7, 2260 cm -1 Raman imaging image with specific Raman signals as characteristic peaks;

[0081] Figure 8j For Raman imaging in Example 7, the corresponding 2260 cm -1 Raman spectrum with characteristic peaks. DETAILED DESCRIPTION

[0082] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below in connection with embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0083] In the present application, the term "immunoraman multicolor imaging reagent" is defined as a reagent containing five raman probes, wherein the raman probe is composed of a raman sphere and a surface-attached antibody; the raman sphere is polymerized from a tri-functional monomer and (i) acrylic acid or (ii) styrene and acrylic acid, with a particle size of 10-100 nm and a Raman shift of 1800-2800 cm -1 ; the antibodies of the five probes are AFP antibody, HBX antibody, GPC3 antibody, GS antibody and CD34 antibody, respectively.

[0084] In the present application, the term "raman probe" is defined as a detection unit composed of a raman sphere and a surface-attached specific antibody, each probe having a unique raman characteristic peak and being capable of specifically recognizing a liver cancer related protein marker.

[0085] In the present application, the term "raman sphere" is defined as a nanoparticle polymerized from a tri-functional monomer and (i) acrylic acid or (ii) styrene and acrylic acid, with a particle size of 10-100 nm and a Raman shift of 1800-2800 cm -1 , which is the core structure of the raman probe.

[0086] In the present application, the term "tri-functional monomer" is defined as a compound (compound 1-7) used for synthesizing the raman sphere, having a specific raman characteristic peak, wherein compound 2, 3, 5, 6, 7 correspond to 2160 cm -1 , 2186 cm -1 , 2227 cm -1 , 2241 cm -1 , 2260 cm -1 characteristic peaks, respectively.

[0087] In the present application, the term "AFP antibody" is defined as an alpha-fetoprotein antibody attached to the surface of the raman sphere, which is used for detecting the expression level of alpha-fetoprotein (AFP) and evaluating the risk of liver cancer.

[0088] In the present application, the term "HBX antibody" is defined as a hepatitis B virus protein antibody attached to the surface of the raman sphere, which is used for detecting hepatitis B virus X protein (HBX) and judging whether the liver cancer is caused by hepatitis B virus.

[0089] In this article, the term "GPC3 antibody" is defined as a phosphatidylinositol protein glycan 3 antibody attached to the surface of the Raman sphere, used to detect the expression level of GPC3 protein, evaluate the malignancy degree and risk of deterioration of liver cancer.

[0090] In this article, the term "GS antibody" is defined as a glutamine synthetase antibody attached to the surface of the Raman sphere, used to detect the expression level of GS protein, combined with CD34 to evaluate the metastasis of liver cancer.

[0091] In this article, the term "CD34 antibody" is defined as a highly glycosylated type I transmembrane glycoprotein antibody attached to the surface of the Raman sphere, used to detect the expression level of CD34 protein, combined with GS to evaluate the metastasis of liver cancer.

[0092] In this article, the term "immunoraman multicolor imaging kit" is defined as a detection tool containing immunoraman multicolor imaging reagents and PBS, which is used to co-incubate liver tissue sections with reagents in PBS (the concentration of each probe is not less than 0.5 mg / mL) to achieve synchronous detection of five markers.

[0093] In this article, the term "liver cancer prediction and evaluation system" is defined as a system containing sample processing, collection, acquisition and evaluation modules, used for liver tissue section incubation, Raman imaging acquisition, marker expression analysis, and multidimensional evaluation of liver cancer risk, etiology, malignancy degree and metastasis.

[0094] In this article, "positive expression" is defined as the expression level significantly higher than the average expression level of healthy liver tissue.

[0095] The application selects HBX, GS, GPC3, AFP and CD34 as core markers of liver cancer caused by hepatitis B, which is derived from the specificity and complementarity of the five markers to the carcinogenic mechanism of hepatitis B. HBx, as hepatitis B virus X protein, is the "etiological marker" of hepatitis B-related hepatocellular carcinoma. HBx protein can be detected in 85% of hepatitis B-related hepatocellular carcinoma tissues, which is directly related to the source of virus-driven carcinogenesis. GPC3 is a specific membrane protein of liver cancer, and the positive rate of GPC3 in hepatitis B-related hepatocellular carcinoma is as high as 82%. GPC3 is almost not expressed in benign lesions outside the background of hepatitis B cirrhosis, and can accurately distinguish between hepatitis B-related malignant proliferation and benign liver cell regeneration. Although AFP is a classic marker of liver cancer, its dynamic changes in hepatitis B-related hepatocellular carcinoma have special characteristics. The increase of AFP in chronic hepatitis B patients is mostly related to liver damage, and the simple AFP index will cause misjudgment. GS, as glutamine synthetase, is diffusely and strongly expressed in hepatitis B-related hepatocellular carcinoma due to abnormal activation of the Wnt / β-catenin pathway, which reflects the virus-induced metabolic reprogramming of hepatocytes, which is a key metabolic feature of hepatitis B carcinogenesis. CD34 marks tumor blood vessel endothelium. Hepatitis B-related hepatocellular carcinoma shows a "complete positive" blood vessel pattern (full lobular sinusoidal blood vessel staining) due to high expression of virus-induced vascular growth factors, and the positive rate is as high as 89%. CD34 can reflect the activity of virus-driven tumor angiogenesis and is related to the invasiveness of liver cancer. Therefore, the above five indexes respectively lock the key links in the etiological chain. AFP is used for early warning of liver cancer, HBx is used for virus integration, GS is used for metabolic adaptation, GPC3 / AFP is used for malignant transformation, and CD34 is used for microenvironment change. The application first proposes a hepatitis B-related hepatocellular carcinoma etiology and pathology evaluation system based on the simultaneous detection of HBX, GS, GPC3, AFP and CD34.

[0096] The application mixes five kinds of Raman probes with different Raman characteristic peaks together to form an immune Raman multicolor imaging reagent for immune detection point marking of liver tissue sections. The five kinds of Raman probes realize simultaneous multicolor imaging of the tissue based on the Raman characteristic peaks of the five kinds of Raman probes, and the imaging result analysis is a liver cancer etiology diagnosis method.

[0097] The method for preparing the above Raman probes comprises: polymerizing a triple bond monomer with (i) acrylic acid or (ii) styrene and acrylic acid to form Raman spheres with a particle size of 10-100 nm; activating the carboxyl on the surface of the Raman spheres by using 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and connecting the carboxyl on the surface of the Raman spheres with the amino group of a targeting molecule by using NHS, which comprises: adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide to the water dispersion of the Raman spheres at room temperature to activate the carboxyl on the surface of the Raman spheres; adding N-hydroxysuccinimide ester to the water dispersion of the Raman spheres, and adding the targeting molecule after stirring for at least 30 minutes and reacting for at least 2 hours. When preparing each Raman probe, the targeting molecule is taken from one of AFP antibody, HBX antibody, GPC3 antibody, GS antibody and CD34 antibody.

[0098] In some embodiments, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the reaction solution is 10-50 mg / mL, the mass fraction of N-hydroxysuccinimide ester is 10-50 mg / mL, and the mass ratio of the Raman sphere to the targeting molecule is 50-100:1 when the Raman probe is prepared.

[0099] When the immune detection point marker is labeled, the immune Raman multicolor imaging reagent is co-incubated with the biological tissue sample for at least 12 hours, and the unbound Raman probe is removed by washing; the labeled biological sample is imaged by using a Raman spectrometer, and the Raman signals at 2160 cm -1 , 2186 cm -1 , 2227 cm -1 , 2241 cm -1 , and 2260 cm -1 are collected.

[0100] The liver cancer prediction and evaluation system provided by the application comprises a sample processing module, a collection module and an acquisition module, and is physically connected or communicatively connected between the modules.

[0101] The sample processing module is configured to co-incubate a liver tissue slice of a subject with the immune Raman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL.

[0102] The collection module is configured to collect Raman imaging information of the liver tissue slice.

[0103] The acquisition module is configured to analyze the expression levels of alpha-fetoprotein, hepatitis B X protein, glypican 3, glutamine synthetase and highly glycosylated type I transmembrane glycoprotein according to the Raman imaging information.

[0104] In some embodiments, the liver cancer prediction and evaluation system further comprises an evaluation module configured to evaluate the risk of liver cancer, the cause of liver cancer, the degree of malignancy and the metastasis of the subject according to the expression levels. For example, the positive co-expression of AFP and HBX predicts that liver cancer is prone to occur and determines that the cause of liver cancer is mainly caused by hepatitis B virus. The existing data shows that the diagnosis rate and accuracy can reach 100%. When AFP, HBX and GPC3 are positive at the same time, the patient's liver cancer is evaluated as having a high degree of malignancy, and the tumor size may be large, the patient may have complications such as cirrhosis, and the patient may have liver cancer metastasis. When AFP, HBX, GPC3, GS and CD34 are positive at the same time, the patient's liver cancer is evaluated as having a high degree of malignancy and having developed metastasis.

[0105] The technical solutions provided by the application will be described in detail below with reference to the embodiments.

[0106] The following are examples of the present application, the examples described below are exemplary, only for the purpose of explaining the present application, and can not be understood as a limitation of the present application. In the examples, the specific technology or conditions are not noted, according to the technology or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used are not noted by the manufacturer, which are conventional products that can be obtained by purchase. Compound 6 acrylonitrile was purchased from Aldrich Reagent Company, purity 99%; compounds 1-5, 7 were synthesized in the laboratory. The antibody information used in the examples is shown in Table 1.

[0107] Table 1 Antibody information used in the examples

[0108]

[0109] Clinical sample information: The liver tissue paraffin blocks used in Examples 5-7 of the present application were provided by Hubei People's Hospital, and the preparation method of the liver tissue paraffin blocks was as follows: after the liver tissue was taken, it was fixed in 4% paraformaldehyde or neutral formalin fixing solution, then dehydrated, transparent and embedded with wax and embedding treatment; the wax block was cut into 5 μm thick slices with a microtome; then the prepared tissue sections were treated with deparaffinization and hydration; the clinical information of the patient was confirmed by the pathology department and the clinician, as follows:

[0110] Example 5: The patient was diagnosed with hepatitis B related liver cancer (early stage), and no cirrhosis and distant metastasis of tumor was found.

[0111] Example 6: The patient was diagnosed with hepatitis B and cirrhosis, and the tumor size was more than 10 cm.

[0112] Example 7: The patient was diagnosed with hepatitis B and cirrhosis, and the liver cancer had distant metastasis (bone metastasis).

[0113] Example 1: Synthesis of monomer

[0114] 1. Synthesis of compound 1

[0115]

[0116] To a solution of 3-bromo-4-iodobenzoic acid methyl ester (340.94 mg, 1.0 mmol) in tetrahydrofuran (15 mL) was added triisopropylsilyl acetylene (218.4 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol) and dichlorobis(triphenylphosphine)palladium (14 mg, 0.2 mmol). The reaction system was frozen by liquid nitrogen, then 420 μL of dry triethylamine (TEA) was added and the system was protected by nitrogen, and then the reaction was carried out at 65 °C for 24 h. After the temperature decreased to room temperature, the solvent was evaporated under reduced pressure, and the mixture was purified by column chromatography using a mixture of petroleum ether (PE) and ethyl acetate (EA) (10:1, by volume) as eluent to obtain 300 mg of white solid intermediate, with a yield of 94.2%.

[0117] The above white solid intermediate (955 mg, 2.5 mmol) was dissolved in 20 mL of toluene, and tributylvinyltin (0.95 mL, 3 mmol) and tetrakis(triphenylphosphine)palladium (57.7 mg, 0.05 mmol) were added. The reaction system was frozen by liquid nitrogen, then protected by nitrogen, and then the reaction was carried out at 115 °C for 24 h. The solvent was then evaporated under reduced pressure, and the mixture was purified by column chromatography using a mixture of PE and EA (10:1, by volume) as eluent to obtain a yellow liquid intermediate (0.7 g, 81.8%).

[0118] The yellow liquid intermediate (342 mg, 1 mmol) was dissolved in a mixture of methanol (MeOH, 5 mL) and water (1 mL), and lithium hydroxide (LiOH, 224 mg, 4 mmol) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and the mixture was purified by column chromatography using a mixture of dichloromethane (DCM) and MeOH (10:1, by volume) as eluent to obtain 319 mg of compound 1 as a white solid, with a yield of 97.2%.1H NMR (400 MHz, CDCl3): δ 8.30 (d, J = 4.0 Hz, 1H), 7.91 (q, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 5.94 (d, J = 16.0 Hz, 1H), 5.54 (d, J = 12.0 Hz, 1H), 1.15 (s, 2H).

[0119] 2. Synthesis of compound 4

[0120]

[0121] To a solution of 3-bromo-4-iodobenzoic acid methyl ester (340.94 mg, 1.0 mmol) in tetrahydrofuran (15 mL) was added 4-pentyn-1-ol (100.94 mg, 1.2 mmol), cuprous iodide (8 mg, 0.04 mmol) and dichlorobis(triphenylphosphine)palladium (14 mg, 0.2 mmol). The reaction system was frozen by liquid nitrogen, then 420 μL of dry TEA was added, and N2 was introduced to protect. The reaction was refluxed in an oil bath at 65 °C for 24 h. After the temperature decreased to room temperature, the solvent was evaporated under reduced pressure. The eluent was a mixture of DCM and MeOH (volume ratio 100:1) to obtain 300 mg of yellow liquid intermediate with a yield of 97.6%.

[0122] The above yellow liquid intermediate (891 mg, 3 mmol) was dissolved in 20 mL of toluene, and tributylvinyltin (1.14 mL, 3.6 mmol) and tetrakis(triphenylphosphine)palladium (69 mg, 0.06 mmol) were added. The reaction system was frozen by liquid nitrogen, then N2 was introduced to protect. The reaction was refluxed in an oil bath at 115 °C for 24 h, and then the solvent was evaporated under reduced pressure. The eluent was a mixture of DCM and MeOH (volume ratio 100:1) to obtain a yellow liquid intermediate (0.4 g, 54.6%).

[0123] The yellow liquid intermediate (244 mg, 1 mmol) was dissolved in a mixture of MeOH (5 mL) and water (1 mL), and LiOH (96 mg, 4 mmol) was added. The mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and the eluent was a mixture of DCM and MeOH (volume ratio 10:1) to obtain 83 mg of compound 4 in the form of a yellow solid with a yield of 36.0%.1H NMR (400 MHz, CDCl3): δ 7.78 (d, J = 4.0 Hz, 1H), 7.78 (q, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.13 (dd, J = 20.0 Hz, 12 Hz, 1H), 5.98 (d, J = 20.0 Hz, 1H), 5.46 (d, J = 12.0 Hz, 1H), 3.53 (t, J = 8.0 Hz, 2H), 2.55 (t, J = 8.0 Hz, 2H), 1.72 (m, 2H).

[0124] 3. Synthesis of compound 5

[0125]

[0126] 4-Bromobenzonitrile (1820.2 mg, 10 mmol) and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 231 mg, 0.2 mmol) were added to a 250 mL three-necked flask. After removing all O2 from the flask using a double-row tube, tributylvinyltin (3804 mg, 12 mmol) was dissolved in 10 mL of toluene and injected into the flask using a syringe. The mixture was cooled with liquid nitrogen, completely frozen, and then O2 was removed three times. The mixture was refluxed and reacted at 120 °C and 600 rpm for 24 h. After quenching with saturated potassium fluoride solution, the reaction solution was extracted with EA and deionized water, washed three times with deionized water, and three times with saturated NaCl solution. The organic phase was dried over anhydrous NaSO4, concentrated by rotary evaporation, and purified by column chromatography using a mixture of PE and EA (20:1 v / v) as the eluent to give 435 mg of compound 5 as a clear liquid, with a yield of 34.0%. 1H NMR (400 MHz, CDCl3): δ 7.61 (d, J = 8.0 Hz, 2H), 7.48 (d, J =8.0 Hz, 2H), 7.26 (s, 1H), 6.73 (q, J = 16.0 Hz, 1H), 5.88 (d, J = 20.0 Hz, 1H), 5.45 (d, J = 12.0 Hz, 1H), 1.60 (s, 1H).

[0127] 4. Synthesis of compound 2: 2-Methacrylic acid and p-iodobenzyl alcohol undergo esterification to produce p-iodobenzyl 2-methacrylic acid. The p-iodobenzyl 2-methacrylic acid then reacts with trimethylsilylacetylene under the catalysis of cuprous iodide and palladium dichloride to produce compound 2.

[0128] 5. Synthesis of compound 3: 2-Methylacrylic acid and trimethylsilylpropynol undergo esterification to generate compound 3.

[0129] 6. Synthesis of compound 7: 2-methacrylic acid and trimethylsilylpropynol undergo esterification to generate compound 3; compound 3 and propynol are coupled via Sonogashira under Pd / Cu catalysis to obtain compound 7.

[0130] Example 2: Preparation of Raman spheres

[0131] like Figure 1 As shown, the Raman characteristic peak of compound 1 is at 2154 cm⁻¹. -1 The Raman characteristic peak of compound 2 is 2160 cm⁻¹. -1 The Raman characteristic peak of compound 4 is 2214 cm⁻¹. -1 The Raman characteristic peak of compound 5 is 2227 cm⁻¹. -1There is a significant spectral band overlap phenomenon. In order to improve the feature peak recognition, five kinds of monomers are selected for the preparation of Raman spheres: 2160 cm -1 of compound 2, 2186 cm -1 of compound 3, 2227 cm -1 of compound 5, 2241 cm -1 of compound 6, and 2260 cm -1 of compound 7, respectively, to construct Raman spheres with unique Raman fingerprint signals.

[0132] 1. Preparation of compound 2 Raman spheres: 0.06 g of compound 2 with Raman characteristic peak of 2160 cm -1 , 0.02 g of acrylic acid, 0.006 g of sodium dodecyl benzene sulfonate, and 5 mL of deionized water were added to a reaction vessel, and after bubbling nitrogen to remove oxygen, the temperature was raised to 70°C, and stirring was continued for 30 min, 0.008 g of potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 3 h. After the reaction was completed, the unreacted compound 2 and emulsifier were removed by dialysis (the pore size of the dialysis membrane was 8-14 kD) to obtain a pure water dispersion of compound 2 Raman spheres with Raman characteristic peak of 2160 cm -1 .

[0133] 2. Preparation of compound 3 Raman spheres: 0.2 g of compound 3 with Raman characteristic peak of 2186 cm -1 , 0.1 g of styrene, 0.05 g of acrylic acid, 0.008 g of sodium dodecyl benzene sulfonate, and 5 mL of deionized water were added to a reaction vessel, and after bubbling nitrogen to remove oxygen, the temperature was raised to 70°C, and stirring was continued for 30 min, 0.03 g of potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 4 h. After the reaction was completed, the unreacted compound 3 and emulsifier were removed by dialysis (the pore size of the dialysis membrane was 8-14 kD) to obtain a pure water dispersion of compound 3 Raman spheres with Raman characteristic peak of 2186 cm -1 .

[0134] 3. Preparation of compound 5 Raman spheres: 0.1 g of compound 5 with Raman characteristic peak of 2227 cm -1 , 0.02 g of acrylic acid, 0.005 g of sodium dodecyl benzene sulfonate, and 5 mL of deionized water were added to a reaction vessel, and after bubbling nitrogen to remove oxygen, the temperature was raised to 70°C, and stirring was continued for 30 min, 0.015 g of potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 3 h. After the reaction was completed, the unreacted compound 5 and emulsifier were removed by dialysis (the pore size of the dialysis membrane was 8-14 kD) to obtain a pure water dispersion of compound 5 Raman spheres with Raman characteristic peak of 2227 cm -1 .

[0135] 4. Preparation of compound 6 Raman spheres: 0.2 g of styrene, 0.15 g of acrylic acid, 0.015 g of sodium dodecyl benzene sulfonate, 10 mL of deionized water were added to a reaction vessel, after bubbling nitrogen to remove oxygen, the temperature was raised to 70°C, and stirring was continued for 30 min, 0.45 g of acrylonitrile (compound 6) with a Raman characteristic peak of 2241 cm -1 was added, and the reaction was carried out under constant pressure nitrogen protection for 2 h. After the reaction was completed, the unreacted compound 6 and emulsifier were removed by dialysis (the pore size of the dialysis membrane was 8-14 kD) to obtain a pure water dispersion of compound 6 Raman spheres with a Raman characteristic peak of 2241 cm -1 .

[0136] 5. Preparation of compound 7 Raman spheres: 0.05 g of compound 7 with a Raman characteristic peak of 2260 cm -1 was added, 0.03 g of styrene, 0.02 g of acrylic acid, 0.003 g of sodium dodecyl benzene sulfonate, 5 mL of deionized water were added to a reaction vessel, after bubbling nitrogen to remove oxygen, the temperature was raised to 70°C, and stirring was continued for 30 min, 0.01 g of potassium persulfate was added as an initiator, and the reaction was carried out under constant pressure nitrogen protection for 5 h. After the reaction was completed, the unreacted compound 7 and emulsifier were removed by dialysis (the pore size of the dialysis membrane was 8-14 kD) to obtain a pure water dispersion of compound 7 Raman spheres with a Raman characteristic peak of 2260 cm -1 .

[0137] Table 2 Raman spheres obtained in Example 2 and their preparation parameters

[0138]

[0139] Example 3: Synthesis of Raman probes

[0140] In this example, the targeting molecules (AFP antibody, HBX antibody, GPC3 antibody, GS antibody, CD34 antibody) were connected to the surface of the Raman spheres by chemical coupling, and specific recognition ability was given.

[0141] From the five kinds of Raman sphere water dispersions prepared in Example 2, 1 mL was taken separately for treatment: 5 μL of 50 mg / mL EDC aqueous solution was added, and stirring was continued for 30 min to activate the carboxyl groups on the surface of the Raman spheres; 5 μL of 50 mg / mL NHS aqueous solution was added as a coupling agent, and stirring was continued for 30 min; 5 μL of the targeting molecule was added, and the reaction was continued at room temperature for 2 h, so that the targeting molecule was covalently combined with the carboxyl groups on the surface of the Raman spheres through an amide bond, and the Raman probe was obtained.

[0142] In this example, the surface of the compound 2 Raman spheres was modified with AFP antibody, and the characteristic peak was 2160 cm-1 Compound 3 Raman sphere surface modified GPC3 antibody, characteristic peak is 2186 cm -1 Compound 5 Raman sphere surface modified HBX antibody, characteristic peak is 2227 cm -1 Compound 6 Raman sphere surface modified GS antibody, characteristic peak is 2241 cm -1 Compound 7 Raman sphere surface modified CD34 antibody, characteristic peak is 2260 cm -1 .

[0143] Example 4: Preparation of immune Raman multicolor imaging reagent

[0144] The five Raman probes with different Raman characteristic peaks prepared in Example 3 were mixed in a Raman intensity ratio of 1:1:1:1:1 to obtain an immune Raman multicolor imaging reagent. Note: The concentration of each Raman probe is approximately 1 mg / mL, and there will be differences in actual operation. The experiment is mainly to prepare the Raman intensity to be approximately the same.

[0145] Example 5: Application in liver cancer and hepatitis B liver cancer prediction

[0146] The following experiment uses the immune Raman multicolor imaging reagent prepared in Example 4 for labeling and imaging of five liver cancer markers in liver tissue. The specific steps are as follows:

[0147] 1. Baking: The embedded wax block is cut into slices using a Leica pathology slicing machine. The cut tissue slices are placed in a 40°C water bath for slicing, and the glass slide is inserted into the water surface at an angle to retrieve the slice, so that the slice is attached to the appropriate position of the glass slide. It can be baked in a 60°C oven for 3 hours.

[0148] 2. Slice deparaffinization: Place the paraffin sections in xylene I (20 min) - xylene II (20 min) - xylene III (20 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% alcohol (5 min) - 90% alcohol (5 min) - 80% alcohol (5 min) - 70% alcohol (5 min), and then immerse in distilled water for 5 min.

[0149] 3. Antigen retrieval: use electric stove to carry out antigen retrieval, place the dewaxing and hydrated tissue section on the high-temperature-resistant plastic section holder in the beaker (or repair box), add appropriate amount of repair solution (0.01 M Tris-EDTA repair solution (pH 9.0)) to the beaker, the liquid surface should be immersed in the section tissue to a certain height, the electric stove can be first heated to boiling, and when it is boiled, it is adjusted to medium, at this time, the timer starts, and the repair time is 15 min, and the tissue should not be dried during the process (the repair solution should be sufficient). After the time, take the beaker out of the microwave oven, and cool it naturally. When the repair solution reaches room temperature, take out the glass slide and wash it with TBST (pH 7.4) for 3 times, 3 min each time (do not wash the tissue during the washing process to avoid damaging the tissue).

[0150] 4. Garaman probe: drop the diluted immune raman multicolor imaging reagent, and incubate it in a 4°C wet box for 12 h.

[0151] 5. Sealing: wash the glass slide with PBS for three times, wipe the liquid on the section with a water-absorbing paper, and seal it with a cover glass.

[0152] 6. Image the liver tissue section using a commercially available ordinary raman imaging system: use the microscope bright field to capture the position of the tissue section, select the imaging area, and then set the following experimental conditions: laser 532 nm, laser power 50 mW, objective 50X, exposure time 1 s, integration number 1, and step length 50 um.

[0153] 7. Image processing: after the imaging test is completed, select 2160 cm -1 , 2186 cm -1 , 2227 cm -1 , 2241 cm -1 , and 2260 cm -1 Raman characteristic peaks to give different colors to the tissue imaging area, and the characteristic peak position corresponds to the positive expression position of the marker.

[0154] Results and analysis:

[0155] As shown in Figures 6a to 6j : AFP (2160 cm -1 ) and HBX (2227 cm -1 ) have obvious positive expression, which indicates that the patient carries hepatitis B virus and is preliminarily diagnosed with liver cancer; GPC3 (2186 cm -1 ), GS (2241 cm -1 ), and CD34 (2260 cm -1 ) have low positive expression, which confirms that the cancer has not deteriorated and the current deterioration degree is low, which is consistent with the early diagnosis of liver cancer.

[0156] Example 6: Application in predicting the progression of hepatitis B-related liver cancer

[0157] Experimental procedure: Liver tissue sections were processed according to the procedure in Example 5, and Raman imaging was performed after incubation with immunoRaman multicolor imaging reagent for 12 h.

[0158] Results and Analysis:

[0159] like Figures 7a to 7j As shown, AFP, HBX, and GPC3 were clearly positive, confirming that the patient had hepatitis B-related liver cancer and was at risk of worsening (GPC3 grows synchronously with liver cancer cells, and positive expression confirms that liver cancer cells grow rapidly and have the risk of proliferation and spread, thus indicating that the patient's liver cancer may further worsen); GS and CD34 were not highly positive but were expressed to some extent, suggesting that the cancer had not yet progressed to an advanced stage, which is consistent with the clinical assessment of the risk of worsening of "tumor size exceeding 10 cm".

[0160] Example 7: Application in assessing the progression of hepatitis B-related liver cancer

[0161] Experimental procedure: Liver tissue sections were processed according to the procedure in Example 5, and Raman imaging was performed after incubation with immunoRaman multicolor imaging reagent for 12 h.

[0162] Results and Analysis:

[0163] like Figures 8a to 8j As shown: five liver cancer markers, AFP, HBX, GPC3, GS, and CD34, were clearly expressed positively; positive expression of AFP and HBX can clearly identify the patient as a hepatitis B-related liver cancer patient; positive expression of GPC3 can reflect the proliferation of malignant tumors, and its positive expression confirms that the liver cancer is in the growth and evolution stage; GS and CD34 reflect that the entire liver detection area is in a state of high metabolic activity and abnormal blood and energy supply, which can confirm that this sample has hepatitis B tumor metastasis and that the hepatitis B-related liver cancer has seriously deteriorated, consistent with the clinical result of "bone metastasis".

[0164] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. An immunoraman multicolor imaging reagent for liver cancer prediction and evaluation, characterized in that, The five Raman probes are composed of Raman spheres and antibodies connected to the surface of the Raman spheres; wherein: The Raman spheres are polymerized from a three-key monomer and (i) acrylic acid or (ii) styrene and acrylic acid, with a particle size of 10-100 nm and a Raman shift of 1800-2800 cm -1 ; the three-key monomers of the five Raman probes are selected from compounds 1 to 7, wherein compounds 1 and 2 are not selected simultaneously, compounds 3 and 4 are not selected simultaneously, and the structural formulas of compounds 1-7 are shown as follows: ； The antibodies of the five Raman probes are respectively taken from one of AFP antibodies, HBX antibodies, GPC3 antibodies, GS antibodies and CD34 antibodies.

2. The immunoraman multichromatic imaging reagent according to claim 1, characterized in that: The three-key monomers in the five Raman probes are respectively compound 2, compound 3, compound 5, compound 6 and compound 7. The characteristic peak of the triple bond monomer for the Raman probe of compound 2 is 2160 cm -1 ; The characteristic peak of the triple bond monomer is 2186 cm -1 ; The characteristic peak of the triple bond monomer for the Raman probe of compound 5 is 2227 cm -1 ; The characteristic peak of the triple bond monomer is 2241 cm -1 ; The characteristic peak of the triple bond monomer is 2260 cm -1 .

3. The immunoraman multicolor imaging reagent according to claim 2, wherein: The antibody on the Raman probe with the three-key monomer of compound 2 is AFP antibody; The antibody on the Raman probe with the three-key monomer of compound 3 is GPC3 antibody; The antibody on the Raman probe with the three-key monomer of compound 5 is HBX antibody; The antibody on the Raman probe with the three-key monomer of compound 6 is GS antibody; The antibody on the Raman probe with the three-key monomer of compound 7 is CD34 antibody.

4. The immunoraman multicolor imaging reagent according to claim 1, wherein: The Raman spheres are connected to the antibodies by chemical bonds; and / or, The particle size of the Raman spheres is 30-70 nm.

5. An immunoraman multicolor imaging kit for liver cancer prediction and evaluation, characterized in that, The immunoraman multicolor imaging reagent according to any one of claims 1-4; in use, a liver tissue slice is co-incubated with the immunoraman multicolor imaging reagent in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL. 6.The immunoraman multicolor imaging kit for liver cancer prediction and evaluation according to claim 5, characterized in that, The immunoraman multicolor imaging kit further comprises PBS, wherein the total volume of the PBS is configured so that after mixing with all the immunoraman multicolor imaging reagents, the concentration of each Raman probe is not less than 0.5 mg / mL.

7. A liver cancer prediction and evaluation system, characterized by, The system comprises a sample processing module, a collection module and an acquisition module, which are physically connected or communicatively connected; wherein: The sample processing module is configured to co-incubate one liver tissue slice of a subject with the immunoraman multicolor imaging reagent according to any one of claims 1-4 in PBS, and the concentration of each Raman probe in the incubation solution is not less than 0.5 mg / mL; The collection module is configured to collect Raman imaging information of the liver tissue slice; The acquisition module is configured to analyze the expression levels of alpha-fetoprotein, liver susceptible virus protein, glypican 3, glutamine synthetase and highly glycosylated type I transmembrane glycoprotein according to the Raman imaging information.

8. The liver cancer prediction and evaluation system according to claim 7, characterized by: The liver cancer prediction and evaluation system further comprises an evaluation module: The evaluation module is configured to evaluate the risk of liver cancer, the cause of liver cancer, the degree of malignancy and the metastasis of the subject according to the expression levels.

9. The liver cancer prediction and evaluation system according to claim 8, wherein The evaluation module is specifically configured to take the average level of healthy liver tissue as a reference, and realize multidimensional evaluation based on the expression level difference of each marker, including: According to the expression level of alpha-fetoprotein, the risk of liver cancer of the subject is evaluated; According to the expression level of liver susceptible virus protein, it is determined whether the liver cancer is caused by hepatitis B virus; According to the expression level of glypican 3, the degree of malignancy and the risk of deterioration of liver cancer are evaluated; According to the combined expression level of glutamine synthetase and highly glycosylated type I transmembrane glycoprotein, the metastasis of liver cancer is evaluated.

10. A liver cancer prediction and assessment instrument, characterized in that: The liver cancer prediction and evaluation system according to any one of claims 7 to 9.

Images