A kit for detecting an early marker MED10 of hepatocellular carcinoma and application thereof

Abstract

This invention provides a kit for detecting MED10, an early biomarker for hepatocellular carcinoma, along with its preparation method and applications. The kit includes a nano-dendritic polymer fluorescent probe with an antibody immobilized on it, capable of binding to the MED10 antigen. When detecting MED10 antigen using this kit, the fluorescent probe is added to a serum sample. The MED10 antigen in the sample reacts with the antibody on the nano-fluorescent probe. After washing to remove other substances from the liquid, the fluorescence intensity of the nano-probe is positively correlated with the expression level of MED10 antigen in the sample, thus allowing for the analysis of MED10 antigen expression levels. This kit can be used to detect the diagnostic biomarker MED10 for hepatocellular carcinoma, assisting in the clinical diagnosis of hepatocellular carcinoma, further assessing the patient's condition, and guiding treatment. The kit requires only a blood draw, minimizing patient discomfort and facilitating continuous monitoring of the condition.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a kit for detecting the early marker MED10 of hepatocellular carcinoma, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common type of primary hepatocellular carcinoma, accounting for approximately 85% to 90% of all HCC cases, posing a serious threat to the health and lives of people worldwide. The 5-year overall survival rate for HCC patients is generally less than 20%, primarily because early-stage HCC often presents with no obvious clinical symptoms, leading to diagnosis frequently occurring at an advanced stage. This significantly limits the clinical efficacy of mainstream treatments such as surgical resection, local ablation, and liver transplantation. Therefore, early and timely diagnosis of HCC is crucial for improving patient prognosis and saving lives.

[0003] The clinical diagnosis of HCC mainly relies on imaging techniques such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI). Among these, ultrasound has limited sensitivity in detecting small tumor lesions; CT and MRI require expensive specialized equipment and skilled personnel, and CT examinations carry the risk of radiation exposure, while MRI examinations may cause contrast agent-related adverse reactions.

[0004] Among serum biomarkers, alpha-fetoprotein (AFP) is currently the most widely used indicator in the diagnosis of hepatocellular carcinoma, with a sensitivity of 41% to 65% and a specificity of 80%. However, due to the limited diagnostic efficacy of this indicator, it is prone to causing a large number of missed cases of hepatocellular carcinoma in early screening. At the same time, the high false positive rate can also lead to unnecessary follow-up examinations, increasing the psychological and economic burden on patients.

[0005] Besides AFP, other serum biomarkers such as des-gamma-carboxyprothrombin (DCP) and alpha-fetoprotein-L3 (AFP-L3) are also used in the clinical diagnosis of HCC. Although DCP has high specificity, its sensitivity is limited, making it insufficient for the precise diagnosis of early-stage hepatocellular carcinoma. AFP-L3 also possesses high specificity, but its detection efficacy is limited by the total AFP concentration. When the AFP concentration is below 20 ng / mL, AFP-L3 cannot be effectively detected, significantly limiting its application value in the early diagnosis of hepatocellular carcinoma. Furthermore, the diagnostic efficacy of these biomarkers exhibits significant population heterogeneity; their diagnostic performance varies among patients with underlying liver diseases or hepatocellular carcinoma of different etiologies, thus lacking broad universality. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a kit for detecting MED10, an early biomarker for hepatocellular carcinoma (HCC), along with its preparation method and applications. This invention targets MED10, a superior diagnostic biomarker for HCC compared to the existing biomarker AFP, and develops a detection kit for HCC diagnosis. Detection of this biomarker can assist in the clinical diagnosis of HCC, further assess the patient's condition, and guide treatment. This invention also relates to a nano-dendritic polymer fluorescent probe, which, compared to existing clinical methods for detecting the biomarker AFP, offers advantages such as high sensitivity and specificity, making it suitable for screening and assisting in the diagnosis of early-stage HCC patients and easier to promote. This kit only requires peripheral blood collection from the subject, minimizing invasiveness and facilitating dynamic monitoring of the disease.

[0007] The technical solution of this invention is as follows: A method for preparing a nano-dendritic polymer fluorescent probe includes the following steps: (1) Dissolve the organic fluorescent dye TA (1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride) in dimethylformamide (DMF), and mix by sonication to obtain a TA solution; (2) Add the TA solution to PAMAM (polyamide-amine) aqueous solution and stir the reaction under light-protected conditions; centrifuge the solution after reaction, take the supernatant and dry it to obtain PT nanoparticles; (3) Dissolve the PT nanoparticles obtained in step (2) in water to prepare PT aqueous solution. Take the activated anti-MED10 detection antibody solution and add it to the PT aqueous solution. After reacting in the dark, purify by ultrafiltration to obtain PT nanoparticles with conjugated antibody, which is the nano-dendritic polymer fluorescent probe.

[0008] In step (1), 0.25~1 mg of organic fluorescent dye TA is dissolved in 0.25 mL of DMF to prepare the TA solution.

[0009] In step (2), the PAMAM aqueous solution is a 1 mg / mL PAMAM aqueous solution; the reaction time is 4 h with stirring in the dark. The supernatant was dried by rotary evaporation at 80°C, dissolved in water, centrifuged again, and then freeze-dried. The PT nanoparticles have a particle size of 80~100 nm.

[0010] In step (3), the activation treatment of the anti-MED10 detection antibody solution is as follows: prepare 100 μL of 90~120 μg / mL anti-MED10 detection antibody solution, add 10 μL of NHS solution and 10 μL of EDC solution respectively, activate for 20 min, the concentration of NHS solution and EDC solution is 10 mg / mL; ultrafiltration removes NHS and EDC to terminate activation.

[0011] The concentration of the PT aqueous solution is 0.1~0.5 mg / mL, preferably 0.25 mg / mL, and the volume is 100 μL; the concentration of the anti-MED10 detection antibody solution is 90~120 μg / mL, and the volume is 100 μL; in the nano-dendritic polymer fluorescent probe, the reaction time is 2~4 h in the dark.

[0012] The antibody-coupled ratio in the nanodendritic polymer fluorescent probe is 30 μg / mg nanoparticles.

[0013] The method yields a nanodendritic polymer fluorescent probe.

[0014] A kit for detecting serum MED10, a biomarker for hepatocellular carcinoma, comprising the aforementioned nanodendritic polymer fluorescent probe.

[0015] Further preferred embodiments of the kit include phosphate buffer, human MED10 standard, OVA, PT modified with anti-MED10 capture antibody, Tween-20, and well plate.

[0016] The perforated plate is any one of an 8-well plate, a 12-well plate, a 24-well plate, a 96-well plate, and a 384-well plate.

[0017] The kit described herein is used in the preparation of products for the early diagnosis of hepatocellular carcinoma.

[0018] Intermediate complex subunit 10 ( Mediator of RNA polymerase II transcription subunit 10 , MED10MED10 is a protein-coding gene in the human genome, located on the short arm of chromosome 5 (5p15.31). The protein it encodes is a key component of the transcriptional mediator complex. The mediator complex is a core coactivator of the RNA polymerase II transcription system, primarily functioning to mediate the interaction between transcription factors and RNA polymerase II, thereby precisely regulating gene transcription and expression. Studies have confirmed that in hepatocellular carcinoma, MED10 enhances tumor cell resistance to cisplatin by promoting PTEN protein ubiquitination; bioinformatics analysis further indicates that… MED10 Gene expression levels can serve as an important biological indicator for assessing the prognosis of patients with hepatocellular carcinoma.

[0019] The amino acid sequence of MED10 (as shown in SEQ ID No:1) is as follows: MAEKFDHLEEHLEKFVENIRQLGIIVSDFQPSSQAGLNQKLNFIVTGLQDIDKCRQQLHDITVPLEVFEYIDQGRNPQLYTKECLERALAKNEQVKGKIDTMKKFKSLLIQELSKVFPEDMAKYRSIRGEDHPPS.

[0020] Given the advantages of serological testing, such as its non-invasiveness, simplicity, and ability to enrich pathological information in serum samples, the inventors further explored the development of a novel human serum testing kit—a kit based on fluorescent nanoprobes. Antibodies capable of binding to specific antigens are immobilized on nano-dendritic polymers to construct fluorescent nanoprobes. After adding the sample, the specific antigen in the sample reacts with the fluorescent nanoprobes, and other substances in the liquid are removed by washing. The fluorescence intensity of the nanoprobes is positively correlated with the expression level of the specific antigen in the sample, thereby enabling the analysis of antigen expression levels.

[0021] The inventors of this application have confirmed through extensive experimental results that there is a significant difference in MED10 expression in the serum of normal individuals and hepatocellular carcinoma patients. The area under the ROC curve (AUC) of serum MED10 detected by this kit reaches 0.9017, which is higher than the AUC of 0.5713 for AFP, demonstrating the good sensitivity and specificity of this biomarker in the early diagnosis of hepatocellular carcinoma.

[0022] The inventors of this application have demonstrated through extensive experimental results that, in the diagnosis of hepatocellular carcinoma (HCC) at different Barcelona clinical staging stages, the detection rate of serum MED10 detected by this kit is significantly higher than that of AFP, especially in the early diagnosis of stage 0 HCC. Specifically, the Barcelona clinical staging system classifies HCC into four stages: 0, A, B, and C / D. The detection rates of AFP in the corresponding stages are 13.6%, 39.6%, 48.6%, and 75.0%, respectively; while the detection rates of serum MED10 are 88.0%, 93.4%, 88.6%, and 100.0%, respectively.

[0023] The inventors of this application have demonstrated through extensive experimental results that, in the diagnosis of hepatocellular carcinoma (HCC) tumors of different sizes, the detection rate of serum MED10 using this kit is significantly higher than that of AFP, especially in the early diagnosis of HCC tumors with a diameter of less than 2 cm. Specifically, HCC patients are divided into four groups based on tumor size: tumor diameter d≤2 cm, 2 cm, and 3 cm. <d≤5cm、5cm<d≤10cm、d> The detection rates of AFP in the four groups were 22.2%, 50.0%, 61.4%, and 68.8%, respectively; the detection rates of serum MED10 were 93.3%, 93.5%, 93.2%, and 93.8%, respectively.

[0024] The beneficial effects of this invention are as follows: (1) The present invention provides a method for preparing a nano-dendritic polymer fluorescent probe. The method involves first dissolving an organic fluorescent dye TA (1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride) in dimethylformamide (DMF) to prepare a TA solution, then adding the TA solution to a PAMAM aqueous solution and reacting it in the dark to obtain PT nanoparticles; then reacting the activated anti-MED10 detection antibody solution with the PT nanoparticles in the dark to obtain antibody-conjugated PT nanoparticles (PTG), which is the nano-dendritic polymer fluorescent probe; the antibody conjugation rate of the nano-fluorescent probe is 30 μg / mg nanoparticles. This invention constructs a fluorescent nanoprobe by immobilizing an antibody that can bind to the MED10 antigen on a nano-dendritic polymer. After the fluorescent probe is added to a sample, the MED10 antigen in the sample reacts with the antibody on the fluorescent nanoprobe. Other substances in the liquid are removed by washing. The fluorescence intensity of the nanoprobe is positively correlated with the expression level of the MED10 antigen in the sample, thereby analyzing the expression level of the MED10 antigen. Compared with the existing clinical biomarker AFP detection method, it has the advantages of high sensitivity and high specificity, and is suitable for screening and auxiliary diagnosis of early HCC patients, and is easier to promote.

[0025] (2) This invention provides a kit for detecting the serum biomarker MED10 in hepatocellular carcinoma. The kit includes a nano-dendritic polymer fluorescent probe, on which an antibody capable of binding to the MED10 antigen is immobilized. When detecting the MED10 antigen using the kit, the fluorescent probe is added to a serum sample. The MED10 antigen in the sample reacts with the antibody on the nano-fluorescent probe. After washing to remove other substances from the liquid, the fluorescence intensity of the nano-probe is positively correlated with the expression level of the MED10 antigen in the sample, thus enabling analysis of the MED10 antigen expression level. This kit can be used to detect the diagnostic biomarker MED10 in hepatocellular carcinoma, assisting in the clinical diagnosis of hepatocellular carcinoma, further assessing the patient's condition, and guiding treatment. The kit only requires peripheral blood collection from the subject to complete the detection, minimizing invasiveness and facilitating dynamic monitoring of the disease. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This invention demonstrates the detection of serum MED10 using a PT-based nanofluorescent probe; wherein... Figure 1 A represents the particle size distribution of PT. Figure 1 B represents the transmission electron microscopy image of PT. Figure 1 C represents the fluorescence emission spectrum of PT. Figure 1 D represents the detection curve of serum MED10; Figure 2 A shows a comparison of serum MED10 concentrations in healthy individuals and patients with hepatocellular carcinoma; Figure 2 B shows a comparison of serum AFP concentrations between healthy individuals and patients with hepatocellular carcinoma; Figure 3 A shows a comparison of serum MED10 levels between patients with non-hepatocellular carcinoma liver diseases and patients with hepatocellular carcinoma during the training phase. Figure 3 B shows a comparison of serum MED10 AUC between patients with non-hepatocellular carcinoma liver diseases and patients with hepatocellular carcinoma during the training phase. Figure 4 Figure A shows a comparison of the AUC of serum MED10 and AFP in healthy individuals and hepatocellular carcinoma patients during the training phase. Figure 4B shows a comparison of serum MED10 concentrations in healthy individuals and hepatocellular carcinoma patients during the training phase; Figure 4 C shows a comparison of serum AFP concentrations between healthy individuals and hepatocellular carcinoma patients during the training phase; Figure 5 This graph shows a comparison of serum MED10 concentrations in healthy individuals, hepatocellular carcinoma patients without hepatitis, patients with cirrhosis, patients with hepatitis B, patients with benign liver tumors, and patients with hepatocellular carcinoma during the training phase. Figure 6 A shows the concentration of serum MED10 in patients with hepatocellular carcinoma, AFP-negative hepatocellular carcinoma, and AFP-positive hepatocellular carcinoma during the training phase. Figure 6 B shows the proportion of MED10-positive and AFP-positive serum in hepatocellular carcinoma patients during the training phase; Figure 6 C represents the proportion of MED10-positive patients among AFP-positive and AFP-negative hepatocellular carcinoma patients during the training phase; Figure 7 A shows the serum concentration of MED10 during the training phase in healthy individuals and patients with BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D stage hepatocellular carcinoma. Figure 7 B shows the proportion of MED10 positivity in hepatocellular carcinoma patients in the training phase at BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D stages; Figure 7 C represents the serum AFP concentration during the training phase in healthy individuals and patients with BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D hepatocellular carcinoma. Figure 7 D represents the proportion of patients with positive serum AFP during the training phase in BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D stage hepatocellular carcinoma. Figure 8 A shows healthy individuals in the training phase, with four different tumor sizes (tumor diameter d≤2cm, 2cm, etc.). <d≤5cm、5cm<d≤10cm、d> The concentration of serum MED10 in hepatocellular carcinoma patients (10 cm in diameter); Figure 8 B shows the proportion of MED10 positivity in hepatocellular carcinoma patients with four different tumor sizes during the training phase; Figure 8 C shows the serum AFP concentration in healthy individuals and hepatocellular carcinoma patients with four different tumor sizes during the training phase; Figure 8D shows the proportion of AFP-positive patients in hepatocellular carcinoma patients with four different tumor sizes during the training phase; Figure 9 A shows the serum MED10 concentration in healthy individuals and hepatocellular carcinoma patients during the validation phase; Figure 9 B shows the serum AFP concentration in healthy individuals and hepatocellular carcinoma patients during the validation phase; Figure 9 C represents the AUC of serum MED10 and AFP in healthy individuals and patients with hepatocellular carcinoma during the validation phase; Figure 10 A shows the serum concentration of MED10 in patients with hepatocellular carcinoma, AFP-negative hepatocellular carcinoma, and AFP-positive hepatocellular carcinoma during the validation phase. Figure 10 B shows the proportion of MED10-positive and AFP-positive serum in hepatocellular carcinoma patients during the validation phase. Figure 10 C represents the proportion of MED10-positive patients in hepatocellular carcinoma who were AFP-positive and AFP-negative during the validation phase; Figure 11 A shows the serum concentration of MED10 in the validation phase in healthy individuals and patients with BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D stage hepatocellular carcinoma. Figure 11 B shows the proportion of MED10-positive serum MED10 in patients with hepatocellular carcinoma in BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D stages during the validation phase; Figure 11 C shows the serum AFP concentrations in healthy individuals and patients with BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D hepatocellular carcinoma during the validation phase. Figure 11 D represents the proportion of AFP-positive patients in the validation phase of BCLC-0, BCLC-A, BCLC-B, and BCLC-C / D hepatocellular carcinoma. Figure 12 A shows the serum MED10 concentrations in healthy individuals and hepatocellular carcinoma patients with four different tumor sizes during the validation phase. Figure 12 B shows the proportion of MED10 positivity in hepatocellular carcinoma patients with four different tumor sizes during the validation phase; Figure 12 C shows the serum AFP concentrations in healthy individuals and hepatocellular carcinoma patients with four different tumor sizes during the validation phase. Figure 12 D shows the proportion of AFP-positive patients in four different tumor sizes of hepatocellular carcinoma during the validation phase; Figure 13 A shows the serum MED10 concentration in hepatocellular carcinoma patients before and after surgery; Figure 13 B shows the serum MED10 concentration in hepatocellular carcinoma patients before surgery, after surgery (1-3 days), and after surgery (more than 3 days); Figure 14 The proportion of patients with hepatocellular carcinoma who are MED10 positive before surgery, after surgery (1-3 days), and after surgery (more than 3 days) is shown. Figure 15 The results show the serum MED10 concentrations in hepatocellular carcinoma patients before, after, and during postoperative follow-up. Figure 16 This shows the proportion of patients with MED10 positivity in hepatocellular carcinoma who underwent preoperative, postoperative, and postoperative follow-up examinations. Figure 17 The survival rates of patients with MED10-positive and MED10-negative hepatocellular carcinoma are shown. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0029] The above technical solution will be described in detail below with reference to specific embodiments.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Example 1: Preparation of Nanodendritic Polymer Fluorescent Probes This embodiment provides a method for preparing a nano-dendritic polymer fluorescent probe, comprising the following steps: (1) Weigh 1 mg TA and dissolve it in 0.25 mL dimethylformamide (DMF), mix it by sonication to obtain TA solution; (2) The TA solution was added to a 1 mg / mL PAMAM aqueous solution and stirred in the dark for 4 h. The mixed solution was removed, centrifuged, and the supernatant was dried by rotary evaporation at 80 °C. 1 mL of water was added to dissolve the supernatant, and the mixture was centrifuged again. After freeze-drying, PT nanoparticles were obtained with a particle size of approximately 100 nm. Figure 1 A, B).

[0032] (3) PT nanoparticles were dissolved in water to prepare a PT aqueous solution with a concentration of 0.25 mg / mL and a volume of 100 μL. This solution exhibited strong fluorescence emission at 495 nm under an excitation wavelength of 447 nm. Figure 1 C).

[0033] Prepare 100 μL of 100 μg / mL anti-MED10 antibody solution, add 10 μL of 10 mg / mL NHS solution and 10 μL of 10 mg / mL EDC solution, and activate for 20 min. Activation is terminated by ultrafiltration to remove NHS and EDC. The activated antibody is then added to 100 μL of 0.25 mg / mL PT aqueous solution and reacted for 2 h, protected from light throughout the reaction. Afterwards, the antibody-conjugated PT nanoparticles (PTG) are purified by ultrafiltration.

[0034] Example 2: Preparation of Nanodendritic Polymer Fluorescent Probes This embodiment provides a method for preparing a nano-dendritic polymer fluorescent probe, which differs from Example 1 in that: in step (1), 0.25 mg of TA is weighed and dissolved in 0.25 mL of dimethylformamide (DMF) to obtain the TA solution.

[0035] In step (2), the particle size of the PT nanoparticles is approximately 80 nm; In step (3), the concentration of the anti-MED10 antibody solution is 90 μg / mL, the concentration of the PT aqueous solution is 0.1 mg / mL, and the reaction time in the dark is 3 h.

[0036] Example 3: Preparation of Nanodendritic Polymer Fluorescent Probes This embodiment provides a method for preparing a nano-dendritic polymer fluorescent probe, which differs from Example 1 in that: in step (1), 0.5 mg of TA is weighed and dissolved in 0.25 mL of dimethylformamide (DMF) to obtain the TA solution.

[0037] In step (3), the concentration of the anti-MED10 antibody solution is 120 μg / mL, the concentration of the PT aqueous solution is 0.5 mg / mL, and the reaction time in the dark is 4 h.

[0038] Example 4: Establishment of a method for detecting MED10 expression levels This embodiment provides a kit comprising 1 mg / mL of the nano-dendritic polymer fluorescent probe, 0.01 M phosphate buffer, 0.1~1000 ng / mL human MED10 standard, 5 mg / mL ovalbumin (OVA), 0.1% Tween-20, and a well plate; The method for detecting the expression level of MED10 in serum using the aforementioned kit is as follows: Add 100 μL of anti-MED10 capture antibody (Ab1, 30 μg / mL) to the wells of a 96-well plate and incubate overnight. After washing to remove any remaining liquid, add 200 μL of 1 mg / mL ovalbumin to block non-specific binding sites and incubate at 37 °C for 1 hour, then remove all remaining liquid. Prepare MED10 antigen solutions of different concentrations by adding human MED10 standard to phosphate buffer. After washing, add 100 μL of different concentrations (1~10 μg / mL) to each well. 6 MED10 antigen (pg / mL) was incubated at 37°C for 1 hour, followed by washing to remove all liquid from the wells. Each washing step required two washes with 0.05% Tween-20, followed by one wash with 0.03 M phosphate buffer (PB). Finally, 100 μL of 0.1 mg / mL PTG fluorescent probe was added to each well and incubated at 37°C for 1 hour, followed by washing with ultrapure water to remove all liquid from the wells. The fluorescence emission intensity of the fluorescent probe at 495 nm in each well was measured using a microplate reader to establish a linear relationship between fluorescence intensity and MED10 concentration, obtaining a standard curve, as shown below. Figure 1 As shown in Figure D, the detection range of MED10 is 1 pg / mL to 1 ng / mL, indicating that the PT-based nanofluorescent probe can sensitively detect serum MED10.

[0039] Collect the blood sample to be tested into a centrifuge tube and let it stand at room temperature for 30 minutes until the blood separates into layers. Centrifuge the tube at 5000 rpm for 10 minutes and collect the supernatant for use. Dilute the collected serum 100-fold and add the serum dilution to wells pre-fixed with capture antibody and OVA. Incubate at 37 °C for 1 hour, then wash to remove all liquid from the wells. Add 100 μL of 0.1 mg / mL PTG fluorescent probe and incubate at 37 °C for 1 hour, then wash to remove all liquid from the wells. Measure the fluorescence emission intensity of the fluorescent probe at 495 nm using a microplate reader. The concentration of MED10 antigen in the sample can be obtained from the fluorescence intensity using a standard curve.

[0040] Detection and Expansion Validation of Serum MED10 in Experimental Examples 1. Detection of serum MED10 expression levels in healthy individuals and hepatocellular carcinoma patients during the detection phase. To preliminarily evaluate the diagnostic potential of MED10 as a serum biomarker for hepatocellular carcinoma, a total of 108 serum samples were included, from 68 hepatocellular carcinoma patients and 40 healthy controls. The serum concentration of MED10 in all samples was measured using the nanofluorescent probe detection method established in Example 2, and the results were statistically analyzed. For comparison, the expression level of alpha-fetoprotein (AFP) in serum samples was also measured simultaneously to evaluate the difference in diagnostic efficacy between MED10 and the traditional biomarker AFP.

[0041] According to the formula, the mean serum MED10 concentration in healthy individuals is 1.078 ng / mL, and the mean serum MED10 concentration in patients with hepatocellular carcinoma is 2.015 ng / mL. Figure 2 A), AUC = 0.9020, p < 0.001, mean AFP concentration in healthy individuals = 15.11 ng / mL, mean AFP concentration in hepatocellular carcinoma patients = 552.39 ng / mL (A), AUC = 0.9020, p < 0.001, mean AFP concentration in healthy individuals = 15.11 ng / mL, mean AFP concentration in hepatocellular carcinoma patients = 552. Figure 2 B), AUC = 0.5188, p=0.75. The AUC of MED10 was significantly greater than that of AFP, meaning that MED10 was significantly better than AFP in the detection stage, suggesting that MED10 may be a novel serum biomarker for hepatocellular carcinoma with high clinical application potential.

[0042] 2. Detection of serum MED10 expression levels in patients with non-hepatocellular carcinoma liver diseases and hepatocellular carcinoma during the training phase. The nano-fluorescent probe detection method established in Example 2 was used to detect serum samples from 200 patients with hepatocellular carcinoma and 294 patients with liver diseases other than hepatocellular carcinoma. The results were statistically analyzed to systematically evaluate the ability of this biomarker to distinguish between patients with hepatocellular carcinoma and patients with liver diseases other than hepatocellular carcinoma in a larger sample size.

[0043] According to the formula, the mean MED10 concentration in patients with non-hepatocellular carcinoma liver diseases was 0.9819 ng / mL, and the mean MED10 concentration in patients with hepatocellular carcinoma was 2.251 ng / mL. Figure 3 A), AUC = 0.9380 (A) Figure 3 B). Statistical analysis showed that the average concentration of MED10 in the serum of patients with hepatocellular carcinoma was significantly higher than that in patients with other liver diseases (p < 0.0001), indicating that it has high sensitivity and specificity in differentiating hepatocellular carcinoma from other liver diseases (such as hepatitis, cirrhosis, benign tumors, etc.).

[0044] To directly compare with the traditional hepatocellular carcinoma marker AFP, MED10 and AFP were detected in serum samples from 200 hepatocellular carcinoma patients and 100 healthy individuals using the nanofluorescent probe detection method established in Example 2, and the detection results were statistically analyzed.

[0045] According to the formula, the mean serum MED10 concentration in healthy individuals is 1.184 ng / mL, and the mean MED10 concentration in hepatocellular carcinoma patients is 2.251 ng / mL. Figure 4 B), p<0.0001, AUC = 0.8889 ( Figure 4 A). The mean serum AFP concentration in healthy individuals was 38.09 ng / mL, while the mean serum AFP concentration in patients with hepatocellular carcinoma was 381.7 g / mL. Figure 4 C), p<0.0001, AUC =0.5428 ( Figure 4 A). The results showed that the mean concentration of MED10 in hepatocellular carcinoma patients was significantly higher than that in healthy individuals, with an AUC of 0.8889, which was significantly better than that of AFP (0.5428).

[0046] The 310 patients with non-hepatocellular carcinoma liver diseases were further subdivided into five subgroups: no hepatitis (n=41), cirrhosis (n=60), chronic hepatitis B (n=60), benign liver tumors (n=45), and healthy individuals (n=100).

[0047] According to the formula, the mean serum MED10 concentration was 1.0219 ng / mL in patients without hepatitis, 0.8013 ng / mL in patients with cirrhosis, 0.6633 ng / mL in patients with chronic hepatitis B, 0.2162 ng / mL in patients with benign liver tumors, 1.1839 ng / mL in healthy individuals, and 2.2500 ng / mL in patients with hepatocellular carcinoma. The results indicate that the mean concentration of MED10 in patients with hepatocellular carcinoma was significantly higher than that in healthy individuals and patients without hepatocellular carcinoma (p < 0.001). Figure 5 ).

[0048] Further analysis revealed no difference in serum MED10 concentrations among all hepatocellular carcinoma patients and between AFP-negative and AFP-positive hepatocellular carcinoma patients. Figure 6 A). Statistical analysis using Youden's J determined the cutoff value for MED10 to be 1.317 ng / mL, and the cutoff value for AFP to be 20 ng / mL. Under these conditions, the sensitivity of MED10 in assisting the diagnosis of hepatocellular carcinoma was 92.9%, while the sensitivity of AFP was only 41.4% (…). Figure 6 B). More importantly, regardless of whether AFP is positive or negative, the sensitivity of MED10 in assisting the diagnosis of hepatocellular carcinoma exceeds 90%. Figure 6 C), indicating that MED10 is an ideal biomarker for the diagnosis of hepatocellular carcinoma.

[0049] 3. Serum MED10 concentration detection in hepatocellular carcinoma patients at different clinical stages in Barcelona during the training phase. To further evaluate the diagnostic efficacy of MED10 for different stages of hepatocellular carcinoma during the training phase, hepatocellular carcinoma patients were divided into four stages (0, A, B, and C / D) according to the Barcelona Clinical Hepatocellular Carcinoma Staging Method, and compared with healthy individuals. Serum samples were tested using the nanofluorescent probe detection method established in Example 2, and the results were statistically analyzed.

[0050] The results showed that the mean serum MED10 concentration in patients with different stages of hepatocellular carcinoma was significantly higher than that in healthy controls (p < 0.001). Figure 7 (A) The detection rates for the four stages were 88.0%, 93.4%, 88.6%, and 100.0%, respectively. Figure 7 B), indicating that it has good diagnostic sensitivity in all stages.

[0051] The mean serum AFP concentration in hepatocellular carcinoma patients was significantly higher than that in healthy controls only in the A and C / D stage groups. Figure 7 C), suggesting that its detection capability is insufficient in early and some intermediate-stage hepatocellular carcinoma patients. Figure 7 C). The detection rates of AFP in the four stages 0, A, B, and C / D are 13.6%, 39.6%, 48.6%, and 75.0%, respectively. Figure 7 D), both were significantly lower than MED10.

[0052] In summary, MED10 demonstrated superior diagnostic sensitivity and detection rate compared to AFP in hepatocellular carcinoma patients at all Barcelona clinical stages, especially in the early stages, and has the potential to become an ideal serum biomarker for hepatocellular carcinoma.

[0053] 4. Detection of serum MED10 expression levels in hepatocellular carcinoma patients with different tumor sizes during the training phase. To further evaluate the diagnostic efficacy of MED10 for hepatocellular carcinoma of different tumor sizes, hepatocellular carcinoma was divided into tumor diameters ≤2 cm and ≤2 cm. <d≤5cm、5cm<d≤10cm、d> Four groups were divided into 10cm groups. All serum samples were tested for MED10 according to the nanofluorescent probe method established in Example 2 and compared with healthy controls.

[0054] According to the formula calculation, the mean serum MED10 concentration in patients with hepatocellular carcinoma of different tumor sizes was significantly higher than that in healthy controls (p < 0.001) ( Figure 8 A). The detection rates for different tumor sizes were 93.3%, 93.5%, 93.2% and 93.8% in sequence ( Figure 8 B), indicating that it has good diagnostic sensitivity for hepatocellular carcinoma of different tumor sizes.

[0055] The average concentration of AFP in the groups with 5 cm < d ≤ 10 cm and d > 10 cm was significantly higher than that in healthy controls ( Figure 8 C), and there was no significant difference in the average concentration between the groups with d ≤ 2 cm and 2 cm < d ≤ 5 cm and healthy controls ( Figure 8 C), indicating its insufficient detection ability in patients with smaller tumors. The detection rates of AFP in the four groups were 22.2%, 50.0%, 61.4% and 68.8% in sequence ( Figure 8 D), all significantly lower than that of MED10.

[0056] In summary, MED10 showed better diagnostic sensitivity and detection rate than AFP in patients with hepatocellular carcinoma of different tumor sizes. Especially in the early stage, it still had a high detection ability and had the potential application value of becoming an ideal serum biomarker for hepatocellular carcinoma.

[0057] 5. Verification stage: Verification of the diagnostic effect of serum MED10 in patients with hepatocellular carcinoma from multi - center case sources To further verify the stability and wide applicability of MED10 as a serum biomarker for hepatocellular carcinoma, a multi - center clinical study was carried out in the verification stage. The case sources covered many places across the country and involved five representative tertiary - level hospitals. A total of 172 patients with hepatocellular carcinoma and 101 healthy control subjects were included. The MED10 was detected according to the nano - fluorescent probe method established in Example 2, the serum samples were detected, and the statistical analysis was carried out on the detection results, and a comparative analysis was made with the traditional biomarker AFP.

[0058] The mean MED10 concentration in patients with hepatocellular carcinoma = 2.462 ng / mL was significantly higher than the MED10 concentration in healthy people = 1.246 ng / mL (p < 0.0001), and the statistical analysis result was significant ( Figure 9 A). The mean AFP concentration in patients with hepatocellular carcinoma = 696.680 ng / mL was significantly higher than the MED10 concentration in healthy people = 12.970 ng / mL (p < 0.0001), and the statistical analysis result was significant ( Figure 9B). MED10 had an AUC of 0.8531, indicating high diagnostic accuracy. In contrast, AFP had an AUC of 0.5329 in the same population, showing significantly lower diagnostic ability than MED10. Figure 9 C).

[0059] There was no difference in serum MED10 concentration among all hepatocellular carcinoma patients and between AFP-negative and AFP-positive hepatocellular carcinoma patients. Figure 10 A). The cutoff value of MED10 was 1.317 ng / mL, and the cutoff value of AFP was 20 ng / mL. Under these conditions, the sensitivity of MED10 in assisting the diagnosis of hepatocellular carcinoma was 92.4%, while the sensitivity of AFP was only 45.9% (…). Figure 10 B). More importantly, regardless of whether AFP is positive or negative, the sensitivity of MED10 in assisting the diagnosis of hepatocellular carcinoma exceeds 90%. Figure 10 C).

[0060] Extensive validation across multiple centers and clinical samples systematically confirmed the consistent diagnostic performance of MED10 in populations from different regions and hospitals. Compared to the traditional biomarker AFP, MED10 demonstrated significant advantages in diagnostic sensitivity, specificity, and AUC, further establishing its clinical application potential as a serum biomarker for hepatocellular carcinoma.

[0061] 6. Validation Phase: Validating the diagnostic efficacy of serum MED10 in hepatocellular carcinoma patients at different Barcelona clinical stages. To further validate the excellent stability and broad applicability of serum MED10 in hepatocellular carcinoma patients at different Barcelona Clinical Hepatocellular Carcinoma (BCLC) stages, a multicenter clinical study was conducted during the validation phase. Cases were collected from multiple locations across China, involving five representative tertiary hospitals. To systematically evaluate the performance of MED10 in different BCLC stages, cases were divided into four groups according to the BCLC criteria: healthy individuals, groups 0-A, B, and C / D. MED10 concentration was detected using the nanofluorescent probe method established in Example 2, serum samples were analyzed, and the results were statistically analyzed.

[0062] The results showed that the mean serum MED10 concentration in patients with different stages of hepatocellular carcinoma was significantly higher than that in healthy controls (p < 0.001). Figure 11 The detection rates for A in the 0-A and BC / D stages were 94.9% and 86.7%, respectively. Figure 11 B), indicating that it has good diagnostic sensitivity in all stages.

[0063] The mean serum AFP concentration in hepatocellular carcinoma patients was significantly higher than that in healthy controls only in the 0-A stage group. Figure 11C). The detection rates for stages 0-A and BC / D were 41.4% and 66.7%, respectively. Figure 11 D), both were significantly lower than MED10.

[0064] In summary, MED10 not only maintains stable detection performance in multicenter, multi-site clinical samples, but also has excellent recognition ability in patients with different BCLC stages of hepatocellular carcinoma, especially maintaining high sensitivity in the early stage (0-A stage), and has good application prospects in early screening of hepatocellular carcinoma.

[0065] 7. Validation Phase: Validate the diagnostic efficacy of serum MED10 in hepatocellular carcinoma patients with tumors of different sizes. To further validate the excellent stability and broad applicability of serum MED10 in hepatocellular carcinoma patients with tumors of different sizes, a multicenter clinical study was conducted during the validation phase. Cases were collected from multiple locations across China, involving five representative tertiary hospitals.

[0066] To further evaluate the diagnostic efficacy of MED10 for hepatocellular carcinomas of different sizes, hepatocellular carcinomas were divided into tumor diameters ≤2 cm and ≤2 cm. <d≤5cm、5cm<d≤10cm、d> Four groups were divided into 10cm groups. All serum samples were tested for MED10 using the nanofluorescent probe method established in Example 2, and compared with healthy controls. AFP concentration was simultaneously measured in all serum samples, and all data were statistically analyzed.

[0067] According to the formula, the mean serum MED10 concentration in hepatocellular carcinoma patients with different tumor sizes was significantly higher than that in healthy controls (p < 0.001). Figure 12 A). The detection rates for different tumor sizes were 95.5%, 92.3%, 88.2%, and 98.5%, respectively. Figure 12 (B) indicates that it has good diagnostic sensitivity for hepatocellular carcinoma of different tumor sizes.

[0068] AFP concentrations were significantly higher in the >10 cm group than in healthy controls. Figure 12 C), indicating that its detection capability is insufficient in patients with smaller tumors. The detection rates of AFP in the four groups were 31.8%, 61.5%, 64.7% and 50.7%, respectively. Figure 12 D), both were significantly lower than MED10.

[0069] In summary, serum MED10 not only maintains good consistency and detection stability in multicenter, multi-site clinical samples, but also demonstrates excellent detection rate and sensitivity in hepatocellular carcinoma patients with different tumor sizes. It is particularly suitable for early hepatocellular carcinoma screening and has significant potential as a broad-spectrum, stable serum biomarker for hepatocellular carcinoma.

[0070] 8. Independent Validation Phase I: Preoperative and postoperative serum MED10 levels in hepatocellular carcinoma patients. In the independent validation phase I, to further evaluate the feasibility of MED10 as a serum diagnostic biomarker for hepatocellular carcinoma (HCC) patients and its application value in dynamic disease monitoring, a systematic study of preoperative and postoperative serum MED10 concentrations was conducted. The study included 76 pathologically diagnosed HCC patients. Serum samples were collected preoperatively, 1-3 days postoperatively, and more than 3 days postoperatively. Serum MED10 concentrations were detected using the nanofluorescent probe method established in Example 2. By comparing MED10 concentrations at different time points, the expression trend over time postoperatively was explored.

[0071] Compared to preoperative levels, serum MED10 concentrations decreased significantly at all postoperative time points. Specifically, the preoperative MED10 concentration was 2.400 ng / mL, while the postoperative MED10 concentration was 1.127 ng / mL. Figure 13 A). Another set of results showed that the preoperative MED10 concentration was 2.547 ng / mL, and the MED10 concentration in the group 1-3 days postoperatively was significantly lower than the preoperative concentration (1.257 ng / mL, p < 0.0001). In the group more than 3 days postoperatively, the MED10 concentration further decreased to 1.048 ng / mL (p < 0.0001), showing a trend of gradual decrease with the extension of postoperative time. Figure 13 B).

[0072] Furthermore, MED10 also showed a significant trend in positive detection rates at different time points. The preoperative MED10 positive detection rate was 87.18%, while it decreased to 25.00% in the 1-3 day postoperative group, and further decreased to 23.53% in the group more than 3 days postoperatively, all significantly lower than the preoperative rate. Figure 14 ).

[0073] The above results indicate that serum MED10 not only has good diagnostic sensitivity in patients with hepatocellular carcinoma, but also decreases rapidly over time after tumor resection, suggesting its potential value in dynamic disease monitoring and prognostic assessment. As a serum biomarker reflecting changes in tumor burden, MED10 has broad application prospects as an indicator for evaluating postoperative treatment efficacy and monitoring recurrence risk in hepatocellular carcinoma.

[0074] 9. Independent validation phase II: Detection of serum MED10 in hepatocellular carcinoma patients after surgery. To further validate the diagnostic value of serum MED10 in hepatocellular carcinoma (HCC) patients and its potential application in postoperative disease monitoring, a study on postoperative and follow-up serum MED10 concentrations was conducted during the validation phase. A total of 59 HCC patients were included in the study, and their serum samples were divided into preoperative, postoperative, and postoperative follow-up groups based on the timeline of surgical treatment. Serum MED10 concentrations were detected using the nanofluorescent probe method described in Example 2, and the relevant detection data were statistically analyzed.

[0075] The test results showed that the preoperative serum MED10 concentration was 2.184 ng / mL, and the postoperative serum MED10 concentration was 1.119 ng / mL, which was significantly lower than the preoperative concentration (p < 0.001). During the follow-up period, the MED10 concentration further decreased to 1.028 ng / mL (p < 0.001), suggesting that MED10 concentration can dynamically reflect changes in tumor burden. Figure 15 ).

[0076] Meanwhile, the analysis of the positive detection rate of MED10 at different time points also showed significant differences. The positive detection rate of MED10 in the preoperative group was 96.61%, which decreased significantly to 19.35% in the postoperative group, and further decreased to 14.03% in the postoperative follow-up group. Figure 16 The above changes indicate that MED10 expression is significantly reduced in HCC patients during the postoperative period, exhibiting good dynamic response characteristics.

[0077] A 10-year follow-up cohort survival analysis of hepatocellular carcinoma patients (n=225) showed that the overall survival of MED10-positive patients was significantly shorter than that of MED10-negative patients (p<0.001). Figure 17 ).

[0078] In summary, serum detection of MED10, an early marker of hepatocellular carcinoma, has advantages such as high sensitivity and strong specificity. It is suitable for screening and auxiliary diagnosis of patients with early hepatocellular carcinoma, as well as for postoperative efficacy evaluation and prognostic monitoring, further demonstrating its application value as a tool for the diagnosis and dynamic monitoring of hepatocellular carcinoma.

[0079] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a nano-dendritic polymer fluorescent probe, characterized in that, Includes the following steps: (1) Dissolve the organic fluorescent dye TA in DMF, and mix it by sonication to obtain a TA solution; (2) Add the TA solution to the PAMAM aqueous solution and stir the reaction under light-protected conditions; centrifuge the solution after the reaction, take the supernatant and dry it to obtain PT nanoparticles; (3) Dissolve the PT nanoparticles obtained in step (2) in water to prepare a PT aqueous solution. Take the activated anti-MED10 detection antibody solution and add it to the PT aqueous solution. After the reaction is carried out in the dark, the solution is purified by ultrafiltration to obtain the PT nanoparticles with conjugated antibody, which is the nano-dendritic polymer fluorescent probe.

2. The method for preparing the nano-dendritic polymer fluorescent probe according to claim 1, characterized in that, In step (1), 0.25~1 mg of organic fluorescent dye TA is dissolved in 0.25 mL of DMF to prepare the TA solution.

3. The method for preparing the nano-dendritic polymer fluorescent probe according to claim 1, characterized in that, In step (2), the PAMAM aqueous solution is a 1 mg / mL PAMAM aqueous solution; the reaction time is 4 h with stirring in the dark. The supernatant was dried by rotary evaporation at 80℃, dissolved in water, centrifuged again, and then freeze-dried. The PT nanoparticles have a particle size of 80~100 nm.

4. The method for preparing the nano-dendritic polymer fluorescent probe according to claim 1, characterized in that, In step (3), the activation treatment of the anti-MED10 antibody solution is as follows: prepare 100 μL of 90~120 μg / mL anti-MED10 antibody solution, add 10 μL of NHS solution and 10 μL of EDC solution respectively, activate for 20 min, the concentration of NHS solution and EDC solution is 10 mg / mL; ultrafiltration removes NHS and EDC to terminate activation.

5. The method for preparing the nano-dendritic polymer fluorescent probe according to claim 1, characterized in that, The concentration of the PT aqueous solution is 0.1~0.5 mg / mL; the concentration of the anti-MED10 antibody solution is 90~120 μg / mL; and the reaction time in the dark is 2~4 h. In the nanodendritic polymer fluorescent probe, the antibody coupling rate is 30 μg / mg nanoparticles.

6. The nanodendritic polymer fluorescent probe prepared by the method according to any one of claims 1-5.

7. The application of the nanodendritic polymer fluorescent probe prepared by the method according to any one of claims 1-5 or the nanodendritic polymer fluorescent probe according to claim 6 in the detection of the hepatocellular carcinoma biomarker MED10.

8. A kit for detecting the hepatocellular carcinoma biomarker MED10, characterized in that, The kit includes the aforementioned nanodendritic polymer fluorescent probe.

9. The reagent kit according to claim 5, characterized in that, Includes 0.01 M phosphate buffer, 0.1–1000 ng / mL human MED10 standard, 5 mg / mL OVA, 1 mg / mL nanodendritic polymer fluorescent probe, 0.1% Tween-20, and well plates; The perforated plate is any one of an 8-well plate, a 12-well plate, a 24-well plate, a 96-well plate, and a 384-well plate.

10. The use of the kit according to any one of claims 8-9 in the preparation of a diagnostic product for early hepatocellular carcinoma.

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