CN111308074B

CN111308074B - Application of diagnosis marker for detecting hepatocellular carcinoma and screening or auxiliary diagnosis product

Info

Publication number: CN111308074B
Application number: CN201911271894.7A
Authority: CN
Inventors: 杨扬; 覃伟; 王励; 刘炜
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2022-11-01
Anticipated expiration: 2039-12-12
Also published as: CN111308074A

Abstract

The invention discloses application of the expression level of Gremlin-1 in plasma in a product of a diagnostic marker for detecting hepatocellular carcinoma, wherein the expression of Gremlin-1 in the plasma in a tissue specimen of hepatocellular carcinoma HCC is mainly positioned at a mesenchymal-tumor junction, and also discloses application of Gremlin-1 in the plasma in screening or auxiliary diagnosis of hepatocellular carcinoma products. By applying the technical scheme of the invention, products such as a kit for detecting a diagnosis marker of hepatocellular carcinoma can be designed, and products for screening or auxiliary diagnosis of hepatocellular carcinoma can also be designed, so that the diagnosis efficiency and the clinical transformation value are further improved.

Description

Application of diagnosis marker for detecting hepatocellular carcinoma and screening or auxiliary diagnosis product

Technical Field

The invention relates to an application of a product of a diagnostic marker for detecting hepatocellular carcinoma and a technology of an application of the product for screening or auxiliary diagnosis of hepatocellular carcinoma.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies in our country or even worldwide. In recent years, with the continuous progress of surgical techniques, comprehensive treatment methods mainly including "surgical treatment" are widely adopted clinically, and the overall treatment effect of hepatocellular carcinoma is remarkably improved. However, the overall therapeutic effect is still poor and there is a high rate of recurrent metastases after surgery. The reason is mainly that the hepatocellular carcinoma patients cannot be treated timely and effectively, and 60-70% of the hepatocellular carcinoma patients enter the middle and late stages when being treated, so that the best treatment period is missed, which is shown in reference data (1-3). Therefore, efficient and early diagnosis is of great importance for hepatocellular carcinoma patients.

At present, only a few tumor markers such as AFP and the like are widely recognized clinically and applied to clinical diagnosis and treatment of hepatocellular carcinoma. Nevertheless, these tumor biomarkers still have certain limitations. Research reports that 15-58% of patients with chronic liver disease and 11-47% of patients with liver cirrhosis have a significant increase in serum alpha-fetoprotein (AFP) as shown in reference data (4-5). There is no obvious increase in some patients with small hepatocellular carcinoma or early hepatocellular carcinoma, and the sensitivity for diagnosis of early hepatocellular carcinoma is only 20-40% as reference (6). Therefore, the combined detection of AFP and imaging examination is often adopted clinically to diagnose hepatocellular carcinoma, and the defects of poor specificity and sensitivity of AFP to partial hepatocellular carcinoma can be effectively overcome. On the other hand, the method actively searches for a diagnostic index with good sensitivity and specificity for diagnosing hepatocellular carcinoma alone, or can improve the diagnostic efficiency of hepatocellular carcinoma by combining with AFP, and has important clinical value.

It is well known that dynamic interactions between tumor cells and their surrounding stroma contribute to the formation, progression, metastasis, and development of drug resistance and immune escape of solid tumors. One study in zhengshun courtyards showed that tumor stroma abundance was closely related to poor prognosis in hepatocellular carcinoma patients, see reference (7), which probably suggests that tumor stroma-related markers could be used as diagnostic and prognostic markers. It is further noted that tumor-associated fibroblasts (CAFs) are a major non-hematopoietic stromal cell type in the tumor microenvironment, and can directly or indirectly act on tumor cells and other non-tumor cells in the microenvironment, thereby promoting the invasion and metastasis of tumor cells. Chen et al indicate that, with the insight into the genetic and molecular phenotypes of CAFs and the mechanisms by which CAFs promote tumor progression, the discovery of CAFs-specific diagnostic and prognostic markers will be facilitated. Searching for specific CAFs markers, and perhaps providing a new treatment strategy for developing precise treatment taking CAFs as targets. Gremlin-1 is a protein highly conserved among species, belongs to one member of the transforming growth factor superfamily, is a member of the Bone Morphogenetic Protein (BMP) antagonist family, and the gene thereof is positioned on the long arm of chromosome 15 and consists of 184 amino acids, and the size of the encoded protein is 20.7KD as the reference material (8-9). Gremlin-1 exists in both secretory and membrane bound forms and is widely expressed in various tissues of the human body including brain, ovary, large intestine, and tissues of esophagus cancer, colorectal cancer and pancreatic cancer (10-12). In colon cancer tissues, gremlin-1 is expressed predominantly in CAFs and hardly in tumor cells (13). Gremlin-1 can form heterodimer with BMP-2, BMP-4, BMP-7, etc., inhibit it from binding with cell surface receptor and thus produce antagonism, and its biological function has not been elucidated. Gremlin-1 was found to be related to reference data for diabetic nephropathy (14), pulmonary fibrosis (15), angiogenesis (16), stem cell differentiation (17), and epithelial-mesenchymal transition (EMT) of colon cancer (13). In recent years, gremlin-1 has received much attention as a role in the fibrosis process. Gremlin-1 is related to hepatic fibrosis and is an important hepatic fibrosis promoting factor (18).

In addition, as the smart medical industry has grown up, more and more focus has been on the use of biomarkers in smart medicine. It can be easily found that the current research on Gremlin-1 mainly focuses on basic research aspects such as molecular mechanism, and the expression level and related transformation research of the Gremlin-1 in the plasma of hepatocellular carcinoma patients are not reported. Compared with a human-Derived tumor cell model, a human-Derived tumor Xenograft model (PDX) has the characteristic of being closer to the tumor of a human body, provides an important in-vivo model for the biological research of the tumor, the search of diagnostic markers and the drug screening, and is beneficial to the development of the individual diagnosis and treatment of the tumor.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the expression level of Gremlin-1 in plasma in products for detecting diagnostic markers of hepatocellular carcinoma and screening or auxiliary diagnosis of hepatocellular carcinoma products, and the application is effectively applied to the design of a kit.

Further aims to construct a human tumor xenograft model, and provide an in vivo model for biological research of tumors, search of diagnostic markers and drug screening.

Use of the expression level of Gremlin-1 in plasma, the expression of Gremlin-1 in a tissue sample of hepatocellular carcinoma HCC being mainly located at the mesenchymal-tumor junction, in a product for the detection of a diagnostic marker for hepatocellular carcinoma.

The application of the expression level of Gremlin-1 in the plasma in a product for detecting a diagnostic marker of hepatocellular carcinoma also comprises the application of the expression level of alpha-fetoprotein (AFP) in the plasma, and the Gremlin-1 in the plasma is used for improving the detection rate of AFP-negative hepatocellular carcinoma; calculating a Gremlin-1 cutoff value in plasma by a Jordan index method, wherein the cutoff value is 53.94ng/ml, dividing hepatocellular carcinoma patients into a Gremlin-1 positive expression group and a Gremlin-1 negative expression group by taking the Gremlin-1 positive expression group as a boundary, and dividing the hepatocellular carcinoma patients into an AFP positive expression group and an AFP negative expression group by taking the Gremlin-1 negative expression group as a boundary, and dividing the hepatocellular carcinoma patients into the AFP positive expression group and the AFP negative expression group by taking the alpha fetoprotein AFP cutoff value as a boundary; the cutoff value of Gremlin-1 in the plasma is greater than or equal to 53.94ng/ml, which is the result of screening or auxiliary diagnosis of hepatocellular carcinoma.

The product for detecting the diagnostic marker of the hepatocellular carcinoma is a kit, and the kit comprises a reagent for detecting the expression level of Gremlin-1 in plasma and the expression level of alpha-fetoprotein AFP in the plasma.

The application of Gremlin-1 in plasma in screening or auxiliary diagnosis of hepatocellular carcinoma products, the application of Gremlin-1 expression level in plasma in products of diagnostic markers for detecting hepatocellular carcinoma, and the screening or auxiliary diagnosis of hepatocellular carcinoma products take plasma of healthy people, chronic liver disease patients and/or hepatocellular carcinoma patients as detection samples.

Aiming at plasma samples of hepatocellular carcinoma patients matched before and after operation, the expression level of Gremlin-1 in the plasma samples of the hepatocellular carcinoma patients before and after the operation is respectively detected, and the Gremlin-1 expression level in the plasma of the hepatocellular carcinoma patients after the operation is obviously reduced compared with that before the operation, so that the method is used for monitoring the postoperative recurrence condition of the hepatocellular carcinoma patients.

Detecting the expression level of alpha-fetoprotein AFP in plasma, and using Gremlin-1 in the plasma to improve the detection rate of AFP negative hepatocellular carcinoma.

Calculating a Gremlin-1 cutoff value in plasma by a Johnson index method, wherein the cutoff value is 53.94ng/ml, dividing hepatocellular carcinoma patients into a Gremlin-1 positive expression group and a Gremlin-1 negative expression group by taking the Gremlin-1 positive expression group as a boundary, and dividing the hepatocellular carcinoma patients into an AFP positive expression group and an AFP negative expression group by taking the Gremlin-1 negative expression group as a boundary, wherein the cutoff value of Alpha Fetoprotein (AFP) in the plasma is 20.0 ng/ml; the cutoff value of Gremlin-1 in the plasma is greater than or equal to 53.94ng/ml, which is the result of screening or auxiliary diagnosis of hepatocellular carcinoma.

Dividing the hepatocellular carcinoma patient into a plasma Gremlin-1 high expression group and a plasma Gremlin-1 low expression group according to the cutoff value of plasma Gremlin-1 of the hepatocellular carcinoma patient, constructing hepatocellular carcinoma PDX models with different plasma Gremlin-1 expression levels, dynamically monitoring the response condition of tumor tissues to the treatment of the Gremlin-1 antibody, and further evaluating the curative effect of the Gremlin-1 antibody in individualized treatment of hepatocellular carcinoma; the plasma Gremlin-1 cutoff value is a approximate-denudation index calculation of the Gremlin-1 cutoff value in the plasma; the hepatocellular carcinoma PDX model is a human tumor Xenograft model namely Patient-Derived mobilizer xenoraft.

In a PDX model established by a hepatocellular carcinoma patient with high Gremlin-1 expression, tumor tissues have good treatment response to a Gremlin-1 antibody and have no obvious toxic or side effect.

The invention has the advantages that:

by applying the technical scheme of the invention, products such as a kit for detecting a diagnosis marker of hepatocellular carcinoma can be designed, products for screening or auxiliary diagnosis of hepatocellular carcinoma can also be designed, and the expression level of Gremlin-1 in plasma is obviously higher than that of normal people and patients with liver cirrhosis based on application, so that the product has better diagnosis efficiency; high expression levels of Gremlin-1 in the plasma of hepatocellular carcinoma patients are closely associated with poor clinical staging. On one hand, the screening or auxiliary diagnosis result of the hepatocellular carcinoma can be better obtained by detecting the expression condition of Gremlin-1 in the plasma of a hepatocellular carcinoma patient; on the other hand, the change of Gremlin-1 in plasma after the operation of the hepatocellular carcinoma patient is also monitored by detecting the expression level of Gremlin-1 in plasma before and after the operation of the hepatocellular carcinoma patient.

The application of the Gremlin-1 expression level in the plasma in products of diagnostic markers for detecting hepatocellular carcinoma and in products for screening or auxiliary diagnosis of hepatocellular carcinoma, such as a kit, can be effectively used for diagnosis of hepatocellular carcinoma by detecting the Gremlin-1 expression level in the plasma; the combined application of the alpha-fetoprotein and AFP can further improve the diagnostic efficacy of the alpha-fetoprotein, and has certain clinical transformation value.

The invention provides an improved hepatocellular carcinoma PDX modeling method, which can further guide the individualized treatment of a hepatocellular carcinoma patient by constructing a hepatocellular carcinoma PDX model and combining the expression level of a plasma marker Gremlin-1 of the hepatocellular carcinoma patient.

The invention relates to an improved hepatocellular carcinoma PDX modeling method. Is used for evaluating the drug effect and toxic and side effect of the antibody of the Ranvatinib and the Gremlin-1.

In a word, the invention is based on that Gremlin-1 is a CAFs specific tumor marker, and has better diagnosis efficiency on hepatocellular carcinoma; after the AFP combined diagnosis, the diagnosis efficiency can be further improved; the expression level of Gremlin-1 in plasma of hepatocellular carcinoma patients is obviously reduced after operation. In addition, by constructing hepatocellular carcinoma PDX models with different plasma Gremlin-1 expression levels, the response condition of tumor tissues to the treatment of the Gremlin-1 antibody is dynamically monitored, and the best benefited population is screened out to guide the individualized treatment of hepatocellular carcinoma patients.

Drawings

FIG. 1 is a schematic representation of Gremlin-1 expression in liver cancer cell lines and liver cancer tissues;

FIG. 2 schematic diagram of a standard protein fit curve for plasma Gremlin-1;

FIG. 3 is a graph showing the expression levels of plasma Gremlin-1 in normal human, chronic liver disease, benign liver tumor and other patients with different types of tumors;

FIG. 4 is a graph showing the expression levels of plasma AFP in normal population, patients with chronic liver disease and hepatocellular carcinoma;

FIG. 5 is a graphical representation of the working characteristic (ROC) curve of subjects with plasma Gremlin-1, AFP independent and combined diagnosis of hepatocellular carcinoma;

FIG. 6 is a schematic representation of the percentage of plasma Gremlin-1 positive hepatocellular carcinoma patients in AFP positive and negative expressing hepatocellular carcinoma patients;

FIG. 7 is a graphical representation of the dynamic profile of the Gremlin-1 expression levels in plasma of a hepatocellular carcinoma patient before and after surgery;

FIG. 8A schematic representation of Gremlin-1 expression levels in plasma directing accurate treatment of hepatocellular carcinoma patients.

Detailed Description

The present invention is not to be considered as limited by the particular embodiments provided. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention in its aspects. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

The invention relates to application of the expression level of Gremlin-1 in plasma in a product of a diagnostic marker for detecting hepatocellular carcinoma, wherein the expression of Gremlin-1 in a tissue sample of hepatocellular carcinoma HCC in the plasma is mainly positioned at a mesenchymal-tumor junction. The application of the expression level of Gremlin-1 in the plasma in a product for detecting a diagnostic marker of hepatocellular carcinoma also comprises the application of the expression level of Alpha Fetoprotein (AFP) in the plasma, and the Gremlin-1 in the plasma is used for improving the detection rate of AFP negative hepatocellular carcinoma; calculating a Gremlin-1 cutoff value in plasma by a Jordan index method, wherein the cutoff value is 53.94ng/ml, dividing hepatocellular carcinoma patients into a Gremlin-1 positive expression group and a Gremlin-1 negative expression group by taking the Gremlin-1 positive expression group as a boundary, and dividing the hepatocellular carcinoma patients into an AFP positive expression group and an AFP negative expression group by taking the Gremlin-1 negative expression group as a boundary, and dividing the hepatocellular carcinoma patients into the AFP positive expression group and the AFP negative expression group by taking the alpha fetoprotein AFP cutoff value as a boundary; the cutoff value of Gremlin-1 in the plasma is greater than or equal to 53.94ng/ml, which is the result of screening or auxiliary diagnosis of hepatocellular carcinoma.

The product of the diagnostic marker for detecting hepatocellular carcinoma of the present invention refers to a kit comprising a reagent for detecting the expression level of Gremlin-1 in plasma and the expression level of alpha-fetoprotein AFP in plasma.

The application of Gremlin-1 in plasma in screening or auxiliary diagnosis of hepatocellular carcinoma products, the application of Gremlin-1 expression level in the plasma in products of diagnosis markers for detecting hepatocellular carcinoma, and the screening or auxiliary diagnosis of hepatocellular carcinoma products take plasma of healthy people, patients with chronic liver diseases and/or patients with hepatocellular carcinoma as detection samples.

The following are the application of the expression level of Gremlin-1 in plasma in products for detecting diagnostic markers of hepatocellular carcinoma and the basis of the application of Gremlin-1 in plasma in screening or auxiliary diagnosis of hepatocellular carcinoma products, which are performed based on the following technical contents.

The invention adopts enzyme-linked immunosorbent assay (ELISA) method to detect the expression level of Gremlin-1 in the blood plasma of normal people, chronic liver disease, hepatocellular carcinoma, benign liver tumor and different types of tumor patients. The results suggest that the expression level of Gremlin-1 in the plasma of hepatocellular carcinoma patients is obviously higher than that of normal people and patients with chronic liver diseases and benign liver tumors, and has no obvious difference with other tumor patients. Meanwhile, the invention analyzes the correlation between Gremlin-1 in plasma and clinical pathological characteristics. As a result, it was found that the up-regulation of the expression level of Gremlin-1 in plasma was closely related to the Barcelona clinical Liver Cancer staging (BCLC). In addition, the invention also detects the expression level of Gremlin-1 in plasma of hepatocellular carcinoma patients before and after operation. The results suggest that the expression level of Gremlin-1 in plasma of hepatocellular carcinoma patients is significantly reduced after the operation.

To investigate whether Gremlin-1 has clinical transforming value in plasma, the present invention first evaluated its diagnostic efficacy in hepatocellular carcinoma. The invention adopts ELISA method to detect AFP expression level in plasma, and then draws ROC curve according to Gremlin-1 and AFP expression level in plasma. The results suggest that plasma exosomes are less diagnostic potent than AFP when diagnosed alone; when the AFP is combined for diagnosis, the AUC reaches 0.895, the sensitivity is 73.6 percent, and the specificity is 85.4 percent. It is worth mentioning that Gremlin-1 in plasma also has better diagnostic efficacy for AFP negative hepatocellular carcinoma patients, and the probability of positive diagnosis reaches 52.5% (21/40).

Then, the expression level of Gremlin-1 in the plasma of 5 cases of hepatocellular carcinoma patients before and after the operation is detected by adopting an ELISA method. The results suggest that the expression level of Gremlin-1 in plasma of hepatocellular carcinoma patients is significantly reduced after the operation. This result probably suggests that the dynamic measurement of plasma Gremlin-1 after surgery in hepatocellular carcinoma patients could be used to monitor the recurrence after surgery in hepatocellular carcinoma patients.

According to the cutoff value of the plasma Gremlin-1 of the hepatocellular carcinoma patient, the hepatocellular carcinoma patient is divided into a plasma Gremlin-1 high expression group and a plasma Gremlin-1 low expression group. By constructing hepatocellular carcinoma PDX models with different plasma Gremlin-1 expression levels, the subcutaneous tumor mass is increased to 100-200mm³The response of tumor tissues to treatment with the Gremlin-1 antibody was monitored dynamically by tail vein injection of Gremlin-1 antibody (15 mg/kg). The results suggest that Gremlin-1 is highly expressedIn the PDX model established by the hepatocellular carcinoma patient, tumor tissues have good treatment response to the Gremlin-1 antibody, and no obvious toxic or side effect exists. As shown in FIG. 8, the expression level of Gremlin-1 in plasma directs the precise treatment of hepatocellular carcinoma patients. A hepatocellular carcinoma PDX model with high expression of Gremlin-1 in plasma. Increasing subcutaneous tumor mass to 100-200mm³On day 12 after tail vein injection of Gremlin-1 antibody (15 mg/kg) (top panel), efficacy was evaluated using the efficacy evaluation criteria for solid tumors (RECIST). (lower panel) the change in body weight of mice in the PDX model was monitored dynamically. A model of hepatocellular carcinoma PDX with low plasma expression of Gremlin-1. Increasing subcutaneous tumor mass to 100-200mm³On day 30 after tail vein injection of Gremlin-1 antibody (15 mg/kg) (upper panel), efficacy was evaluated using the efficacy evaluation criteria for solid tumors (RECIST). (lower panel) the change in body weight of mice in the PDX model was monitored dynamically.

FIG. 1 is a schematic diagram showing the expression of Gremlin-1 in liver cancer cell lines and liver cancer tissues. Gremlin-1 is mainly expressed in CAFs, and hardly expressed in normal hepatocytes, liver cancer cells and human hepatic stellate cells LX 2. And D, in the liver cancer tissue, gremlin-1 is intensively expressed in a tumor invasion front area.

FIG. 2 is a schematic diagram of a standard protein fit curve for plasma Gremlin-1. And (3) taking the concentration of the standard substance as a vertical coordinate and the OD value as a horizontal coordinate, drawing a standard fitting curve (figure 2), calculating the corresponding sample concentration according to a formula, and multiplying the corresponding sample concentration by the dilution factor 100 to obtain the actual concentration of the sample.

FIG. 3 is a graph showing the expression levels of plasma Gremlin-1 in normal human, chronic liver disease, benign liver tumor and other patients with different types of tumors. The expression of Gremlin-1 in the plasma of 140 hepatocellular carcinoma patients, 32 chronic hepatitis B patients, 16 healthy blood donors, 9 benign liver disease patients, 3 breast cancer patients, 5 metastatic liver cancer patients, 1 rectal cancer patient and 9 cholangiocarcinoma patients was tested by ELISA. The results suggest that the mean level of expression of Gremlin-1 in plasma of patients with hepatocellular carcinoma is significantly higher than that of normal persons and patients with chronic liver disease ([ P ] 0.001); the expression level of Gremlin-1 in the plasma of hepatocellular carcinoma patients was not significantly increased compared to other tumor types.

FIG. 4 is a graph showing the expression levels of plasma AFP in normal human, chronic liver disease and hepatocellular carcinoma patients. The expression of AFP in plasma of 140 hepatocellular carcinoma patients, 32 chronic hepatitis B patients and 16 healthy blood donors was examined by ELISA. The results suggest that the level of AFP expression in plasma from hepatocellular carcinoma patients was significantly higher than that from normal and chronic liver disease patients (. P.0.01,. P.0.001).

FIG. 5 is a graph showing the working characteristic (ROC) curve of subjects in independent and combined diagnosis of hepatocellular carcinoma by plasma Gremlin-1 and AFP. Gremlin-1 was combined with AFP in plasma for diagnosis. The results show that when the AFP is combined for diagnosis, the sensitivity is 73.6 percent, the specificity is 85.4 percent, and the AUC can reach 0.895.

FIG. 6 is a graph showing the percentage of plasma Gremlin-1 positive hepatocellular carcinoma patients in AFP positive and negative hepatocellular carcinoma-expressing patients. As a result, gremlin-1 in plasma was found to be 72.0% positive in hepatocellular carcinoma patients diagnosed with AFP-positivity. More importantly, gremlin-1 in plasma has a positive rate of 52.5% for diagnosing AFP negative hepatocellular carcinoma patients.

FIG. 7 is a graph showing the dynamic change of the expression level of Gremlin-1 in plasma of a hepatocellular carcinoma patient before and after surgery. And respectively detecting the expression level of Gremlin-1 in plasma specimens of hepatocellular carcinoma patients before and after the operation by adopting an ELISA method. The results suggest that the expression level of Grmelin-1 in plasma of the liver cell patients after the operation is obviously reduced compared with that before the operation.

FIG. 8 is a schematic representation of the expression level of Gremlin-1 in plasma as a guide for accurate treatment of hepatocellular carcinoma patients. According to the cutoff value of plasma Gremlin-1 of hepatocellular carcinoma patients, the inventor divides the hepatocellular carcinoma patients into a plasma Gremlin-1 high expression group and a plasma Gremlin-1 low expression group. Constructing hepatocellular carcinoma PDX models with different plasma Gremlin-1 expression levels, and dynamically monitoring the response condition of tumor tissues to the Gremlin-1 antibody treatment. The results suggest that in the PDX model established by the Gremlin-1 high-expression hepatocellular carcinoma patient, the tumor tissue has good treatment response to the Gremlin-1 antibody and has no obvious toxic or side effect.

The above findings of the present invention are based on the following experimental methods, and the samples and experimental methods used in the present invention are described below, and the following biological means or reagents, which are not provided in detail, are all achieved by the conventional techniques in the art.

Experimental Material

Paraffin hepatocellular carcinoma tissue specimen

The invention also collects fresh tissue specimens which are surgically excised from 9 months 2014 to 9 months 2015 of the third hospital affiliated to the Zhongshan university and are proved to be hepatocellular carcinoma through pathological examination. Meanwhile, fresh normal liver tissue specimens (taken from normal liver tissue beside hepatic hemangioma) were collected as controls. Samples were taken immediately after the isolation of the specimen, while tumor-bleeding necrotic tissue was avoided during the sampling, immediately fixed in 10% formalin and subsequently paraffin-embedded. All patients did not receive other treatment such as Transcatheter Arterial Chemoembolization (TACE) and Radio Frequency Ablation (RFA) before surgery.

Liver cancer-associated fibroblast

Under aseptic conditions, 3-5g of a fresh hepatocellular carcinoma tissue specimen excised by surgery was stored in a high-glucose DMEM (Dulbecco's modified Eagle's medium) medium containing 10% Fetal Bovine Serum (FBS), labeled and stored in an ice box for a short time. Hepatocellular carcinoma tissue specimens were taken from the cancerous limbal region and tissue spared for hemorrhagic necrosis. The sample is washed several times with Phosphate Buffered Saline (PBS) containing double antibody, adipose tissues and blood on the sample are removed as much as possible, and non-liver cancer tissues adjacent to the sample and obvious vascular structures are cut off. Cutting the trimmed specimen into pieces of about 1mm × 1mm³The tissue fragments were washed 2 times with PBS. The minced tissue pieces were placed in 8-10ml high-glucose DMEM (containing 1g/L collagenase type IV, 10% FBS) medium and the tissue was digested for 6h at 37 ℃ on a shaker. The tissue mass appeared floccular after shaking indicating complete digestion. Gently blow the digestive juice until the loose tissue mass dissipates. Washing with PBS for 2 times, resuspending with high-sugar DMEM culture solution containing 10% fetal calf serum, filtering with 200 mesh filter screen, and adjusting cell density to 1.0 × 10⁶Per ml, inoculated at 25cm²Placing in a culture flask at 37 deg.C and 5% CO₂Culturing in a cell culture box. And transferring the cells into a culture bottle for 48h, then carrying out the cell liquid change for the 1 st time, and observing the cell adherence and growth conditions. Thereafter, the cells were completely fused by changing the medium 1 time every 2 to 3 days with a high-glucose DMEM medium containing 10% FBS.

Cell lines

The human normal hepatocyte (L02) and human hepatoma cell lines (HepG 2, SMMC-7721 and Huh 7) and human hepatic stellate cell (LX 2) cell lines used in this experiment were preserved by the liver disease laboratory at the third Hospital affiliated to Zhongshan university; human hepatoma cell lines (MHCC 97-L, MHCC97H and HCCLM 3) were purchased from the liver cancer institute of Dandan university. All cell cultures in this study were carried out at 37 ℃ and 5% CO using high-glucose DMEM containing 10% fetal bovine serum, except as otherwise specified₂Culturing in a cell culture box. Then freezing, recovering and subculturing the cells. All operations were performed in a clean bench.

Plasma sample

In the examples of the present invention, plasma specimens were obtained from 140 hepatocellular carcinoma patients (confirmed by combining AFP, imaging, histopathology, and the like), 32 chronic hepatitis b liver disease patients, 16 healthy blood donors, 9 liver benign disease patients, 9 cholangiocellular carcinoma patients, 5 metastatic liver cancer patients, 1 rectal cancer patient, and 3 breast cancer patients, who were approved and approved by the ethical committee of the third hospital affiliated to zhongshan university and the huashan hospital affiliated to the compound university, and who underwent consent.

Construction of tissue specimens for PDX hepatocellular carcinoma

The study in this section collected fresh tissue specimens surgically excised between 2019 and 2019, 8 months and confirmed to be hepatocellular carcinoma by pathological examination at the third hospital affiliated to zhongshan university. The specimens were isolated, immediately sampled, placed in DMEM medium containing double antibody and 20% FBS, and placed on ice. All patients did not receive any other treatment before surgery, such as TACE and RFA.

Main reagents and test materials

PBS(Cat No.:SH30256.01,HyClone,USA)；

Gremlin-1ELISA Kit(Cat No.:SEC128Hu,Cloud-Clone,USA)；

Transcriptor First Strand cDNA Synthesis Kit(Roche,Switzerland)；

Max DNA Polymerase(Takara,China)；

Gremlin-1Antibody(N-20):(Cat No.:sc-18274,Santa Cruz,USA)；

Gremlin-1Antibody:(Novus Biologicals,USA)；

FBS(GIBCO,USA)；

High glucose DMEM (GIBCO, USA);

Matrigel(Corning,USA)；

serum-free frozen stock (bambaker, japan);

Recombinant Human EGF(PeproTech,USA)；

Recombinant Human FGF-basic(PeproTech,USA)。

main instrument equipment

Refrigerator at 4 deg.C, -20 deg.C and-80 deg.C

Electric constant temperature incubator (Shanghai Jinghong experiment equipment Co., ltd., china)

Enzyme-labeling instrument (TECAN, switzerland)

Experimental methods

Reverse transcription-polymerase chain reaction (RT-PCR)

The extraction of cell total ribonucleic acid (RNA) is carried out by the following steps: adherent cells in logarithmic growth phase are taken, and the cells can be directly washed for 2-3 times by PBS solution. Then 1ml of Trizol solution was added and blown with a sample applicator to a clear liquid without cell clumps. The whole liquid was poured into a 1.5ml EP tube, and after the liquid was mixed by inversion 10 times, it was allowed to stand at room temperature for 5min. Adding 1/5 (0.2 ml) volume of chloroform, covering the centrifugal tube, shaking the centrifugal tube forcefully for 15sec, standing at room temperature for 5min, and centrifuging at 12000rpm for 15min at 4 ℃. Carefully pipette the supernatant into another 1.5ml centrifuge tube, add 0.5ml of isopropanol to the supernatant, reverse the tube and mix the solution well, place at room temperature for 10min, centrifuge at 12000rpm for 10min at 4 ℃. The supernatant was discarded and l ml of pre-cooled 75% ethanol was slowly added along the tube wall, the tube was inverted gently several times and centrifuged at 12000rpm for 5min at 4 ℃. Carefully discard the ethanol, and place the precipitate on a clean bench to dry for about 30min by starting a blower, at which time the RNA precipitate becomes transparent. The RNA pellet was dissolved by adding a suitable amount of diethyl pyrocarbonate (DEPC). Mu.l of the RNA solution was dropped on a micro nucleic acid analyzer to measure the absorbance (OD) and concentration. If the ratio of OD260/OD280 is between 1.8 and 2.1, it indicates that the RNA sample has a higher purity. Another 1. Mu.l of RNA sample was added with 1% agarose gel, sufficient 1 XTAE electrophoresis buffer was added to the electrophoresis tank until the gel surface was completely covered by 5-10mm, electrophoresis was performed at constant pressure of 100V for 20min, and the integrity of bands of 5s ribosomal RNA (ribosomal RNA, rRNA), 18s rRNA and 28s rRNA was observed with a gel imaging system, and the bands of 5s rRNA,18s rRNA and 28s rRNA were considered to be acceptable if they were evident.

The procedure for synthesizing complementary deoxyribonic acid (cDNA) is briefly described below. Using the Transcriptor First Strand cDNA Synthesis Kit, cDNA was synthesized using an anchored oligo (dT) 18 primer and hexamer random primers to create a 20. Mu.l reverse transcription reaction: total RNA 1.0. Mu.g, adsorbed-oligo (dT) 18.0. Mu.l, random hexamer primer 2.0. Mu.l, water (PCR-grade) to 13. Mu.l. The template-primer mixture was denatured by heating the nuclease-free PCR tube at 65 ℃ for 10min, a step that ensures denaturation of the RNA secondary structure. Immediately, the PCR tube was cooled on ice. Subsequently, the following RT reaction components were added to the PCR tube containing the template-primer mixture: 4.0. Mu.l of Transcriptor reverse transcription reaction buffer (5X), 0.5. Mu.l of Protector RNase inhibitor, 2.0. Mu.l of Dexynuleotide mix (10 mM), and 0.5. Mu.l of Transcriptor reverse transcription buffer. The 20. Mu.l of reaction solution was mixed well (care: not vortex). RT was performed under the following conditions: 10min at 25 ℃,30min at 65 ℃ and 5min at 85 ℃. The RT product was stored at-20 ℃. According to TaKaRa Co

Max DNA Polymerase instruction, the following reaction system was set up: 2.0. Mu.l of cDNA, 1.0. Mu.l of each of the upstream and downstream primers, 8.5. Mu.l of DEPC water,

max DNA Polymerase 12.5. Mu.l. The above 25. Mu.l of the reaction solution was thoroughly mixed. PCR amplification was performed under the following conditions: preheating at 98 deg.C for 5min; heating at 98 ℃ for 10sec, annealing at 56 ℃ for 15sec, and stretching at 72 ℃ for 30sec for 35 cycles; fully extending for 5min at 72 ℃; maintaining the temperature at 4 ℃. Mu.l of the PCR product was electrophoresed in 1.5% agarose gel at 100V, and the subject band was photographed and analyzed using a gel imaging analysis system.

Enzyme-linked immunosorbent assay (ELISA)

The expression level of Gremlin-1 in the plasma samples was determined by ELISA. Diluting the plasma sample by 100 times, and completing the detection work according to the following steps:

1. sample adding: respectively provided with a standard hole, a sample hole to be detected and a blank hole. Setting 7 standard holes, and adding 100 mul of standard substances with different concentrations in sequence. Adding 100 mul of standard substance diluent into a blank hole, adding 100 mul of sample to be detected into the rest hole, adding a film on an enzyme label plate, and incubating for 1 hour at 37 ℃;

2. discarding the liquid, and spin-drying without washing;

3. adding 100 mul of working solution A (prepared before use) into each hole, covering a film on an enzyme label plate, and incubating for 1 hour at 37 ℃;

4. the well contents were discarded, each well was washed with 350. Mu.l of washing solution, soaked for 1-2 minutes, and the microplate was tapped on absorbent paper to remove all the contents of the wells. And repeating the plate washing for 3 times, sucking or pouring out the residual washing buffer solution after the last washing, reversely buckling the enzyme label plate on the water absorption paper, and sucking and drying all the liquid remained in the holes.

5. Each well was filled with 100. Mu.l of the working solution (prepared immediately before use) of the detection solution B, coated with a microplate, and incubated at 37 ℃ for 30 minutes.

6. Discarding the liquid, spin-drying, and washing the plate for 5 times, wherein the method is the same as the step (4);

7. adding 90 μ l of Tetramethylbenzidine (TMB) substrate solution into each well, coating with an enzyme label plate, and developing in dark at 37 deg.C for 10min (when the first 3-4 wells of the standard wells have obvious gradient blue, and the second 3-4 wells have no obvious gradient, the reaction can be stopped).

8. The reaction was stopped by adding 50. Mu.l of stop solution to each well, whereupon the blue color turned to yellow. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution. If the color is not uniform, the enzyme label plate is slightly shaken to mix the solution evenly.

9. Immediately after ensuring that no water drops are formed at the bottom of the microplate and no air bubbles are formed in the wells, the OD value of each well is measured by a 450nm wavelength of a microplate reader.

10. And (4) plotting the OD value of each standard and sample after subtracting the OD value of the blank hole. And (3) taking the concentration of the standard substance as a vertical coordinate and the OD value as a horizontal coordinate, drawing a standard fitting curve (figure 2), calculating the corresponding sample concentration according to a formula, and multiplying the corresponding sample concentration by the dilution factor 100 to obtain the actual concentration of the sample.

Immunohistochemistry

Tissue sections with a thickness of 3 μm were routinely made. Placing the paraffin section on a copper frame, and baking in a thermostat at 60-70 ℃ for 30-60min. After the paraffin sections were taken out of the oven, they were immediately placed in xylene and soaked for 15min × 2 times. The slices are taken out from xylene and then sequentially soaked in ethanol with different concentrations for dehydration, absolute ethanol (10 min × 2 times) → 95% ethanol (5 min) → 80% (5 min) → 70% ethanol (5 min), and then the slices are washed with tap water for 5min and then the slices are washed with distilled water for one time. The slices are placed in citrate buffer, heated in a microwave oven at high heat mode for 5min, then heated in a microwave oven at low heat mode for 25min (to avoid boiling dry), and the slices are naturally cooled to room temperature to expose the repair antigen. PBS soak, 5min X2 times (wherein the first soak is a new PBS), the section from PBS, distilled water. Incubating with 3% hydrogen peroxide in dark at room temperature for 20min to eliminate endogenous peroxidase, soaking the slices in distilled water for 3-5min, and soaking in PBS for 5min. Adding 10% normal goat serum dropwise for sealing, incubating at 37 deg.C for 30min, removing excess liquid, and wiping the excess liquid with absorbent paper. The diluted primary antibody (PBS dilution) is added dropwise, the temperature is kept overnight at 4 ℃, the section is rewarmed for 30min at 37 ℃ the next day, and the section is soaked for 5min multiplied by 3 times in PBS. Adding secondary antibody dropwise, incubating at 37 deg.C for 20-30min, soaking in PBS for 5min × 3 times. Preparing a color developing agent (taking a clean EP tube containing 1ml of Diaminobenzidine (DAB) substrate liquid, adding 20 mul of concentrated DAB solution (50 x), uniformly mixing, and then (placing in the dark for use within 30 min.) controlling color development under a microscope, dipping with tap water to stop dyeing, dripping a proper amount of hematoxylin (1.

Western blot

The extraction of the total cell protein is briefly described as follows: the culture medium in the cell culture flask was discarded, the cells were rinsed twice with ice PBS, the PBS in the flask was aspirated off, and 0.5ml of 0.5M ethylenediaminetetraacetic acid (EDTA) was added to promote cell shedding. Then, the cells were scraped with a cell spatula, transferred into a 1.5ml centrifuge tube together with EDTA, centrifuged at 200g for 3-5min at room temperature, and the supernatant was removed as much as possible. Adding appropriate amount of RIPA lysate (containing phosphatase inhibitors such as PMSF) into the cell precipitate, and incubating on ice for 30min (during which the cell is vortexed once every 4-5 min) to fully lyse the cells. Protein sample quantification is carried out according to BCA (Bicinchoninic acid) protein concentration determination kit instructions (Kaiky, china), the total cell protein is deformed for 10min at 100 ℃, the sample is added into a hole, and 3-5 mu l of protein Marker is added as a Marker according to actual needs. During electrophoresis, a constant current of 70mA × 10min was used to compress each well sample into a straight line, and then a constant voltage of 150V was used until bromophenol blue ran to the edge of the lower edge green line. During film transfer, a sponge pad, filter paper, gel, a 0.45 mu M polyvinylidene fluoride (PVDF) film, the filter paper and the sponge pad are sequentially placed in the direction from the negative electrode to the positive electrode, 1 x film transfer liquid is poured into a tray, then a clip is placed into the film transfer liquid, the clip is opened, the sponge pad is firstly placed on the black surface, the filter paper and separation glue are sequentially placed on the sponge pad, then the PVDF film (the upper right corner of the film is cut off in advance to be used as a mark, and the PVDF film is soaked in methanol for 5 min) to be covered on the separation glue (the bubbles are removed), then the filter paper and the sponge pad are sequentially covered on the PVDF film, and the clip is closed, so that a sandwich structure of a sandwich is formed. The clips are placed into a film transferring groove (black frame to black frame, white frame to red frame), 1 x film transferring liquid is poured until the film transferring clips are immersed, ice blocks are placed around the electrophoresis groove, the pressure is constant at 100V, and the film transferring time is 90min. After washing, the PVDF membrane was placed in a blocking solution containing 5% skimmed milk powder and incubated on a slow shaker at room temperature for 1h. The membrane was cut according to the molecular weight of the protein, and the cut PVDF membrane was placed in the diluted primary antibody solution and incubated overnight in a shaker at 4 ℃. Wash membrane 5min 3 times. The PVDF membrane is put into a secondary antibody of the corresponding species, and incubated for 1h at room temperature in a slow shaker. The membrane was washed 5min × 3 times. Absorbing water with filter paper, soaking the luminescent liquid for 1min, and exposing with automatic exposure machine.

Construction of a model for PDX of hepatocellular carcinoma

Immediately preserving a part of the obtained fresh hepatocellular carcinoma tissues in liquid nitrogen or at the temperature of minus 80 ℃ for extracting DNA/RNA and protein; meanwhile, a portion of the hepatocellular carcinoma tissue was formalin-fixed, paraffin-embedded, and used for HE staining and tissue immunohistochemical examination. Rapidly placing hepatocellular carcinoma tissue for constructing PDX model in DMEM culture solution containing double antibody and 20% FBS, and cutting tumor tissue into 2-10mm under aseptic condition³Tissue mass of size. Subsequently, the tissue mass was transferred into 100% matrigel containing fibroblast growth factor (bFGF) and Epidermal Growth Factor (EGF), and after 30 minutes of the placement, the right abdominal wall and right inguinal region of immunodeficient mice (NOD/SCID) were rapidly implanted. When the tumor volume reaches 500-1000mm³On the left and right, they were passaged in vivo. After the model is successfully established, obtaining a part of fresh tissues after passage, and quickly shearing the fresh tissues into 50-100mm³And (3) placing the tumor mass in a freezing tube pre-filled with Bambanker serum-free freezing solution, and then transferring the freezing tube into liquid nitrogen for freezing and storing for later use.

Statistical analysis method

The invention adopts IBM SPSS Statistics Version 20.0 and GraphPad Prism Version 6.0 statistical software to carry out statistical analysis. The measured data are expressed by mean + -standard deviation and tested by independent sample t(ii) a Acquisition of counting data by X²And (6) checking. Analyzing the correlation between the Gremlin-1 expression level and clinical pathological characteristics by adopting a chi 2 test; ROC was plotted and AUC was calculated and diagnostic efficacy evaluations were performed. All tests are bilateral, P<0.05 was considered statistically significant.

By adopting the experimental method, the invention obtains the following results:

gremlin-1mRNA and protein are significantly highly expressed in liver cancer tissues and CAFs, and significantly lowly expressed in liver cancer cell lines.

In order to further understand the expression conditions of Gremlin-1mRNA and protein in different liver cancer cell lines and CAFs, the expression levels of Gremlin-1 messenger RNA (messenger RNA, mRNA) and protein in six liver cancer cell lines with different invasive metastatic potentials, LX2 and CAFs of a normal liver cell line L02, hepG2, SMMC-7721, huh7, MHCC97-L, MHCC97-H and HCCLM3 are detected by adopting an RT-PCR and Western blot method. Interestingly, the expression level of Gremlin-1 in CAFs was significantly higher than that of a series of liver cancer cell lines, L02 and LX2, with almost no Gremlin-1 expression in the liver cancer cell lines, L02 and LX2 as shown in FIGS. 1A-C.

Next, immunohistochemical detection is performed on a paraffin hepatocellular carcinoma specimen, so that the expression condition of Gremlin-1 is determined. The results suggest that Gremlin-1 expression in HCC tissue specimens is predominantly located at the stroma-tumor junction, i.e. the pre-tumor invasion line is shown in figure 1D.

2. Gremlin-1 is significantly highly expressed in plasma in hepatocellular carcinoma patients.

The expression of Gremlin-1 in plasma of 140 hepatocellular carcinoma patients (confirmed by AFP, imaging, histopathology, etc.), 32 chronic hepatitis B patients, 16 healthy blood donors, 9 patients with liver benign disease, 3 breast cancer patients, 5 metastatic liver cancer patients, 1 rectal cancer patient, and 9 cholangiocarcinoma patients was first examined by ELISA. The results suggest that the average expression level of Gremlin-1 in the plasma of hepatocellular carcinoma patients is obviously higher than that of normal people and chronic liver disease patients (P < 0.01); the results of the lack of significant elevation of Gremlin-1 expression in the plasma of hepatocellular carcinoma patients compared to other tumor types are shown in FIG. 3, which suggests that Gremlin-1 expression in the plasma of hepatocellular carcinoma patients is not significantly tumor tissue specific.

The invention further analyzes the correlation between the expression level of Gremlin-1 in plasma and the clinical pathological characteristics of liver cancer. The levels of Gremlin-expression in the plasma of hepatocellular carcinoma patients were found to be closely related to the clinical pathology of the BCLC staging, but not to the presence or absence of cirrhosis, tumor size, number of tumor nodules, presence or absence of envelopes or pseudoenvelopes, and Edmendson-Steiner staging (Table 1).

3. Gremlin-1 in plasma is helpful to improve the ability of clinical diagnosis of hepatocellular carcinoma.

The present invention calculates the expression of Gremlin-1 and AFP in the plasma of healthy population, chronic liver disease patient and hepatocellular carcinoma patient as shown in FIG. 4, and evaluates the efficiency of diagnosing hepatocellular carcinoma. Plotting ROC curves for the individual and combined diagnosis of primary hepatocellular carcinoma with Gremlin-1 in plasma and AFP, and calculating the area under the curves as shown in FIG. 5, it can be seen that the diagnosis efficacy of Gremlin-1 in plasma is slightly inferior to AFP when diagnosed alone; on the basis, the invention carries out combined diagnosis on Gremlin-1 and AFP in plasma. It was found that AUC reached 0.895 in combination with AFP diagnosis.

In addition, the cutoff value (cutoff value) of Gremlin-1 was calculated by the Jordan index method and was determined to be 53.94ng/ml. Dividing the hepatocellular carcinoma patients into a Gremlin-1 positive expression group and a Gremlin-1 negative expression group by taking the value as a boundary; the cutoff value was also determined to be 20.0ng/ml based on the range of normal values for clinical AFP, and hepatocellular carcinoma patients were also classified into AFP-positive expression group and AFP-negative expression group based on this value. According to the invention, when the AFP diagnosis hepatocellular carcinoma patient is negative, the probability that the Gremlin-1 in the plasma is positive to the diagnosis is 52.5%, which shows that the Gremlin-1 in the plasma can obviously improve the detection rate of the AFP negative hepatocellular carcinoma, and is shown in figure 6.

TABLE 1.106 relationship between Gremlin-1 expression level in plasma of hepatocellular carcinoma patients and clinical pathological features

4. The expression level of Gremlin-1 in the blood plasma of hepatocellular carcinoma patients after operation is in a descending trend.

The invention collects 5 plasma samples of hepatocellular carcinoma patients matched before and after operation, and respectively detects the expression level of Gremlin-1 in the plasma samples of the hepatocellular carcinoma patients before and after operation by adopting an ELISA method. The results suggest that the expression level of Grmelin-1 in plasma of the hepatocyte patients after surgery is significantly reduced compared to before surgery as shown in FIG. 7.

5. The expression level of Gremlin-1 in plasma directs the use of PDX models in the precision treatment of hepatocellular carcinoma patients.

According to the cutoff value of plasma Gremlin-1 of the hepatocellular carcinoma patients, the inventor divides the hepatocellular carcinoma patients into a plasma Gremlin-1 high expression group and a plasma Gremlin-1 low expression group. Constructing hepatocellular carcinoma PDX models with different plasma Gremlin-1 expression levels, and dynamically monitoring the response condition of tumor tissues to the Gremlin-1 antibody treatment. The results suggest that in the PDX model established for the Gremlin-1 high-expression hepatocellular carcinoma patient, the tumor tissue has good treatment response to the Gremlin-1 antibody, and no obvious toxic or side effect is shown in figures 8A-B.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Reference to the literature

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Claims

1. The application of a reagent for detecting the expression levels of Gremlin-1 protein and AFP protein in blood plasma in preparing a product of a diagnostic marker for detecting hepatocellular carcinoma is disclosed, wherein Gremlin-1 in the blood plasma is used for improving the detection rate of AFP negative hepatocellular carcinoma; and the expression level of the Gremlin-1 protein in the plasma of the hepatocellular carcinoma patient is obviously higher than that of normal people, chronic liver disease patients and benign tumor patients.

2. Use of a reagent for detecting the expression levels of the Gremlin-1 protein and the AFP protein in plasma for the preparation of a product for the detection of diagnostic markers for hepatocellular carcinoma according to claim 1, characterized in that the cutoff value for Gremlin-1 in plasma is calculated by the john index method, said cutoff value being 53.94ng/ml and the hepatocellular carcinoma patients are classified into a Gremlin-1 positive expression group and a Gremlin-1 negative expression group by using this value as a boundary, the cutoff value for the alpha fetoprotein AFP in plasma is 20.0ng/ml and the hepatocellular carcinoma patients are classified into an AFP positive expression group and an AFP negative expression group by using this value as a boundary; the cutoff value of Gremlin-1 in the plasma is greater than or equal to 53.94ng/ml, which is the result of screening or auxiliary diagnosis of hepatocellular carcinoma.

3. The use of the reagent for detecting the expression levels of Gremlin-1 protein and AFP protein in plasma for the preparation of a product for the detection of a diagnostic marker for hepatocellular carcinoma according to claim 1, characterized in that the product for the detection of a diagnostic marker for hepatocellular carcinoma is a kit comprising the reagent for detecting the expression levels of Gremlin-1 protein in plasma and the expression level of alpha-fetoprotein AFP in plasma.

4. The application of a reagent for detecting the expression level of Gremlin-1 protein in blood plasma in preparing a PDX model for guiding accurate treatment of hepatocellular carcinoma patients,

dividing the hepatocellular carcinoma patient into a plasma Gremlin-1 high expression group and a plasma Gremlin-1 low expression group according to the cutoff value of plasma Gremlin-1 of the hepatocellular carcinoma patient, constructing hepatocellular carcinoma PDX models with different plasma Gremlin-1 expression levels, dynamically monitoring the response condition of tumor tissues to the treatment of the Gremlin-1 antibody, and evaluating the curative effect of the Gremlin-1 antibody in individualized treatment of the hepatocellular carcinoma; the plasma Gremlin-1 cutoff value is a approximate-denudation index calculation of the Gremlin-1 cutoff value in the plasma; in a PDX model established by a Gremlin-1 high-expression hepatocellular carcinoma patient, tumor tissues have good treatment response to a Gremlin-1 antibody, and no obvious toxic or side effect exists.

5. The application of the reagent for detecting the expression level of the Gremlin-1 protein in the plasma in the preparation of a product for monitoring postoperative recurrence of a hepatocellular carcinoma patient is characterized in that the expression level of Gremlin-1 in a plasma sample of a hepatocellular carcinoma patient before and after surgery is respectively detected aiming at the plasma sample of the hepatocellular carcinoma patient matched between the preoperative operation and the postoperative operation, and the Gremlin-1 expression level in the postoperative plasma of the hepatocellular carcinoma patient is obviously reduced compared with that before the surgery, so that the reagent is used for monitoring the postoperative recurrence condition of the hepatocellular carcinoma patient.