Application of prognostic marker H4K5la in prognostic evaluation of neuroblastoma

Abstract

The invention discloses application of a prognostic marker H4K5la in prognostic evaluation of neuroblastoma, and relates to the technical field of biology. The invention provides application of the H4K5la detection reagent in preparation of products for judging prognosis of neuroblastoma, prognosis evaluation accuracy is improved by detecting the expression level of H4K5la, the detection cost is reduced by the detection method, detection sensitivity and specificity are enhanced, a theoretical basis is provided for a treatment intervention strategy of targeted H4K5la, and the application of the H4K5la detection reagent in preparation of products for judging prognosis of neuroblastoma is provided. Important reference can be provided for further treatment development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a prognostic marker H4K5la in prognosis assessment of neuroblastoma. Background Art

[0002] Neuroblastoma is a neuroendocrine tumor that originates from the sympathetic nervous system and mainly affects children. It is the most common extracranial solid tumor. Despite recent advances in multimodal anticancer treatment (including surgery, chemotherapy, radiotherapy, and GD2 monoclonal antibody-based immunotherapy), the survival rate of children with high-risk neuroblastoma remains low, and there is an urgent need to find new therapeutic targets and prognostic markers.

[0003] In the study of neuroblastoma, the detection of molecular markers and their role in disease diagnosis and prognosis assessment have received increasing attention. Histone modification, especially histone lactylation, as a new type of epigenetic modification, has gradually shown its important role in tumor progression. Histone lactylation refers to the modification of histone lysine residues by lactic acid molecules through covalent binding, thereby regulating gene expression and cell phenotype. In cancer, especially tumors with significant metabolic abnormalities such as neuroblastoma, histone lactylation may affect tumor growth and metastasis through metabolic-epigenetic reprogramming pathways.

[0004] In the existing technology, common prognostic markers for neuroblastoma include MYCN amplification, ALK mutation and TERT rearrangement. In the existing technologies for diagnosis and prognosis assessment of neuroblastoma, there are several major technical problems: (1) Insufficient sensitivity and specificity of biomarkers: At present, although the commonly used prognostic markers for neuroblastoma such as MYCN amplification, ALK mutation and TERT rearrangement have important prognostic value in some patients, the frequency of these markers in the overall patient population is low, and their sensitivity and specificity need to be further improved. This limitation leads to the inability to timely and accurately identify some high-risk patients through existing markers, which in turn affects the formulation of treatment decisions and the prognosis of patients. (2) Complex and costly detection technology: Existing prognostic marker detection usually requires high-end gene sequencing technology or fluorescence in situ hybridization (FISH) technology. These methods not only have high equipment costs, but also have high requirements on the operating environment and technical level of personnel, making them difficult to be widely used in medical institutions with limited resources. This has, to a certain extent, limited the popularization and promotion of these markers. (3) Insufficient stability and repeatability of test results: Some existing detection technologies rely on nucleic acid detection, which is easily affected by sample quality, experimental conditions, and operator skill level, resulting in unstable test results and poor repeatability. This instability may cause uncertainty in clinical diagnosis, thereby affecting the formulation and implementation of treatment plans. (4) Lack of low-cost, easy-to-use prognostic assessment methods: In many medical settings, especially primary medical institutions, there is a lack of a low-cost, easy-to-use and widely applicable neuroblastoma prognostic assessment method. This makes it impossible for many patients to obtain effective prognostic information, affecting their treatment plans and health management.

[0005] Currently, there is an urgent need to provide a prognostic marker that can improve the sensitivity and specificity of prognostic assessment, reduce detection costs and operational feasibility, and improve the accuracy and reliability of diagnosis. Summary of the invention

[0006] The purpose of the present invention is to provide a prognostic marker H4K5la and its application in the prognosis assessment of neuroblastoma.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides use of an H4K5la detection reagent in the preparation of a product for determining the prognosis of neuroblastoma.

[0008] Specifically, the products include reagents, test kits, test strips, gene chips, and antibody chips.

[0009] Specifically, the detection reagent is a reagent for detecting the expression level of H4K5la.

[0010] Specifically, detecting the H4K51a expression level comprises the following steps: (1) Obtain test samples from neuroblastoma patients; (2) Detect the expression level of H4K5la in the sample; (3) If H4K5la is highly expressed in the sample, it indicates that the prognosis of neuroblastoma patients is poor; if H4K5la is low expressed in the sample, it indicates that the prognosis of neuroblastoma patients is good.

[0011] Furthermore, the test sample in step (1) is one or more of tumor tissue, tumor cells, blood or plasma.

[0012] Furthermore, the test sample in step (1) is tumor tissue.

[0013] Furthermore, methods for detecting the expression level of H4K5la in a sample include immunohistochemistry, microarray analysis, polymerase chain reaction, and reverse transcriptase polymerase chain reaction.

[0014] Furthermore, the method for detecting the expression level of H4K5la in the sample is an immunohistochemical method.

[0015] Furthermore, the immunohistochemical method for detecting the expression level of H4K51a includes the following steps: (1) Preparation of FFPE tissue sample sections; (2) Dewaxing and hydration: After the samples were dewaxed in xylene, they were hydrated through graded ethanol. (3) Antigen retrieval: The samples were subjected to antigen retrieval using citrate buffer; (4) Blocking: Block tissue sections with BSA; (5) Primary antibody incubation: Add rabbit anti-H4K5la antibody and incubate for 12-16 hours; (6) Secondary antibody incubation: Use HRP-labeled goat anti-rabbit secondary antibody for incubation for 1-3 hours; (7) Color development: Add color developer and allow to develop for 2-5 minutes; (8) Restaining and sealing: Add hematoxylin for restaining, dehydrate with graded alcohol, add xylene, and seal the slides.

[0016] According to some embodiments of the present invention, the specific steps of dewaxing and hydrating in step (2) are: (1) Place tissue sections in xylene, soak for 5 minutes each time, repeat 3 times; (2) The slices were placed in ethanol solutions of different concentrations for gradient hydration. The immersion time for each step was 3 minutes. The concentrations were 100% anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol and 50% ethanol.

[0017] Specifically, the concentration of the citric acid buffer solution in step (3) is 0.01 M and the pH is 6.0.

[0018] Specifically, the concentration of BSA in step (4) is 5%, and the time for blocking the tissue with BSA is 1 hour.

[0019] Specifically, the primary antibody incubation in step (5) is as follows: adding rabbit anti-H4K51a antibody solution (diluted 1:200) and incubating for 12 hours.

[0020] Specifically, the secondary antibody incubation in step (6) is as follows: adding HRP-labeled goat anti-rabbit secondary antibody (diluted 1:500) and incubating at 37° C. for 1 hour.

[0021] Specifically, after incubation with primary and secondary antibodies, the sections were washed three times with PBS buffer for 5 minutes each time.

[0022] According to some embodiments of the present invention, the re-staining includes: immersing the tissue section in a 5% concentration of hematoxylin stain solution for 3 minutes; placing the section in a 0.1% hydrochloric acid ethanol solution for differentiation for 15 seconds; placing the section in 70%, 80%, 95% and 100% ethanol step by step, soaking for 2 minutes at each level; placing the section in xylene for transparent treatment and soaking for 5 minutes.

[0023] Specifically, the H4K51a expression level was assessed based on the staining intensity.

[0024] Specifically, the prognosis is the predicted 5-year survival rate of the patient.

[0025] Specifically, after immunohistochemical staining, professional pathologists scored the staining intensity from 0 to 4 points according to the scoring system. A score of 0-1 indicates a good prognosis, that is, a high 5-year survival rate; a score of 2 indicates a moderate prognosis; and a score of 3-4 indicates a poor prognosis, that is, a low 5-year survival rate. This scoring system provides an accurate prognostic criterion by comparing the expression differences of H4K5la in neuroblastoma patients and normal samples.

[0026] In the method of the present invention, the criteria for judging weak staining, moderate staining, strong staining and very strong staining are defined as follows: (1) Weak staining: When observed under a microscope, the staining signal is relatively weak, and only the staining of the target area can be distinguished. There is almost no obvious contrast with the background, and the staining area is small.

[0027] (2) Moderate staining: The staining signal is clearly visible with moderate intensity. The staining of the target area forms a certain contrast with the background. The staining coverage area is relatively uniform, showing a clear staining effect.

[0028] (3) Strong staining: The staining signal is significantly enhanced, the staining of the target area is significantly darker than the background, with high contrast, high staining area coverage, and a dense staining effect.

[0029] (4) Very strong staining: The staining signal is extremely strong, the staining depth of the target area significantly exceeds the background, the contrast is extremely clear, the staining area is dark, and the coverage area is almost 100%.

[0030] In yet another aspect, the present invention provides a product for diagnosing or evaluating the prognosis of neuroblastoma, wherein the product comprises a reagent for detecting the expression level of H4K5la.

[0031] Specifically, the products include reagents, test kits, test strips, gene chips, and antibody chips.

[0032] Furthermore, the product is a test kit.

[0033] Specifically, the product includes a reagent for detecting the expression level of H4K5la.

[0034] Furthermore, methods for detecting the expression level of H4K5la include immunohistochemistry, microarray analysis, polymerase chain reaction, and reverse transcriptase polymerase chain reaction.

[0035] Furthermore, the method for detecting the expression level of H4K5la is an immunohistochemical method.

[0036] Specifically, detecting the H4K51a expression level comprises the following steps: (1) Obtain test samples from neuroblastoma patients; (2) Detect the expression level of H4K5la in the sample; (3) If H4K5la is highly expressed in the sample, it indicates that the prognosis of neuroblastoma patients is poor; if H4K5la is low expressed in the sample, it indicates that the prognosis of neuroblastoma patients is good.

[0037] Furthermore, the test sample is one or more of tumor tissue, tumor cells, blood or plasma.

[0038] Furthermore, the test sample is tumor tissue.

[0039] Principle explanation: Application of IHC technology: Immunohistochemistry (IHC) combines specific antibodies with target proteins in tissue sections and uses color development to display the location and abundance of proteins. The optimized IHC technology can accurately detect the expression of H4K5la in FFPE (formalin-fixed paraffin-embedded) tissue samples, providing quantitative or semi-quantitative test results and providing a basis for prognostic evaluation.

[0040] The beneficial effects of the present invention are: The present invention has the following beneficial effects by detecting the expression level of histone H4 lysine 5 lactylation (H4K5la) in tumor tissues of neuroblastoma (NB) patients: (1) Improved the accuracy of prognostic assessment: High expression of H4K5la is significantly associated with poor prognosis in patients with neuroblastoma. Compared with traditional prognostic markers (such as MYCN amplification), H4K5la shows significant expression differences in more patients, so its detection can provide more extensive prognostic assessment information, thereby improving the accuracy of prognostic assessment.

[0041] (2) Simplified detection method and reduced detection cost: The present invention adopts immunohistochemistry (IHC) technology and uses specific antibodies to detect the expression of H4K5la, which can be completed in a conventional pathology laboratory, significantly reducing the cost and technical threshold of detection and improving the popularization and convenience of the technology.

[0042] (3) Enhanced detection sensitivity and specificity: By optimizing the immunohistochemical staining conditions and color development reaction, the present invention can accurately and sensitively detect the expression level of H4K5la, improve the detection sensitivity of the target protein, and reduce background noise, thereby enhancing the specificity of the detection and ensuring the reliability of the detection results.

[0043] (4) Provides a new direction for therapeutic intervention: This invention not only provides a new prognostic assessment marker, but also reveals the biological function of H4K5la in neuroblastoma, indicating the important role of H4K5la in tumor metabolism and invasion. This provides a theoretical basis for the therapeutic intervention strategy targeting H4K5la and can provide an important reference for further treatment development. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The H4K5la staining expression in tumor tissues of children with neuroblastoma (survival group and death group).

[0045] Figure 2 Kaplan-Meier survival curve analysis of patients with high and low H4K5la expression.

[0046] Figure 3 This is the ROC curve in Experimental Example 1. DETAILED DESCRIPTION

[0047] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0048] Example 1 Detection of H4K5la in patients with neuroblastoma Experimental Materials: Neuroblastoma FFPE tissue samples were obtained from Shanghai Children's Medical Center; Rabbit anti-H4K5la polyclonal antibody (Jingjie Biotechnology, catalog number PTM-1407RM); HRP-labeled goat anti-rabbit secondary antibody (Signal Transduction Company, catalog number 7074S); DAB colorimetric kit (Dako, Denmark, catalog number K3468); Conventional IHC staining reagents (formalin, methanol, citrate buffer, etc.).

[0049] Method steps: Tissue section preparation: FFPE tissue samples were sliced ​​to 4 μm thickness, placed on glass slides, and baked in an oven at 60°C for 1 hour.

[0050] Deparaffinization and hydration: (1) Place the tissue sections in 50 mL of xylene and soak for 5 minutes each time. Repeat 3 times to fully remove the paraffin. After each soaking, gently absorb the excess xylene with clean filter paper.

[0051] (2) Place the slices in ethanol solutions of different concentrations for gradient hydration. The immersion time for each step is 3 minutes. The concentrations are 100% anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol and 50% ethanol, respectively. After each step, use filter paper to gently absorb the excess ethanol.

[0052] (3) After completing the ethanol gradient, rinse the sections in distilled water for 5 minutes to remove any ethanol residue and ensure that the samples are fully hydrated.

[0053] Antigen retrieval: Samples were subjected to antigen retrieval in an autoclave with citrate buffer (0.01 M) at pH 6.0 for 10 min.

[0054] Blocking: Block tissue sections with 5% BSA and incubate at room temperature for 1 hour to reduce nonspecific binding.

[0055] Primary and secondary antibody incubation: First, place the dewaxed and hydrated tissue sections in a humidified chamber containing blocking solution and block at room temperature for 30 minutes to reduce nonspecific binding. After blocking, aspirate the blocking solution, add 50 μL of rabbit anti-H4K5la antibody solution (diluted 1:200) to each section, ensuring that the entire section surface is covered, and then place the humidified chamber in a 4°C refrigerator and incubate overnight (about 12 hours). The next day, take out the sections and rinse them with PBS buffer 3 times for 5 minutes each to remove unbound primary antibodies. Subsequently, add 100 μL of HRP-labeled goat anti-rabbit secondary antibody (diluted 1:500) to each section and incubate in a 37°C humidified chamber for 1 hour. After secondary antibody incubation, rinse 3 times with PBS buffer for 5 minutes each.

[0056] Color development: Use DAB color development kit to develop color. Control the time according to the color intensity, usually 3 minutes.

[0057] Counterstaining and sealing: First, immerse the tissue sections in a 5% hematoxylin stain for 3 minutes to color the cell nuclei. After staining, rinse the sections under running water for 3 minutes to remove excess stain. Then, place the sections in a 0.1% hydrochloric acid ethanol solution for 15 seconds to adjust the staining depth, and rinse again with running water for 1 minute to neutralize. Next, place the sections in 70%, 80%, 95% and 100% ethanol step by step, soaking for 2 minutes at each level for dehydration. After dehydration, place the sections in xylene for transparent treatment, soak for 5 minutes, and repeat twice to ensure transparency. Finally, add neutral gum drops to the sections and gently cover them with a coverslip to avoid bubbles, and let them stand to dry.

[0058] The results are as follows Figure 1 Shown: The H4K5la expression level was evaluated by microscopic observation based on the DAB staining intensity of the cell nucleus, and a standard scoring system (0-4 points) was established.

[0059] Example 2 Correlation between H4K51a detection and patient prognosis Experimental materials: IHC stained sections obtained in Example 1.

[0060] A total of 50 children with neuroblastoma were included in this study, with an average age of 4.6±3.1 years, of which 55.6% were male. Analysis of patient survival status showed that 38 children survived for more than 5 years, accounting for 76.0% of the total; another 12 children did not survive for more than 5 years, accounting for 24.0%. These data provide a reliable basis for further analysis of the relationship between H4K5la expression level and survival prognosis of children.

[0061] Diagnostic criteria: According to the 2019 version of the Chinese Guidelines for the Diagnosis and Treatment of Childhood Neuroblastoma, the clinical diagnostic criteria for neuroblastoma include the following key elements: (1) Clinical symptoms and signs: abdominal or chest mass, bone pain, weight loss, fever, fatigue, etc. Some patients also show hypertension and sweating due to catecholamine secretion by the tumor. (2) Imaging examination: Ultrasound, CT or MRI are used to locate and evaluate the mass; MIBG scan (iodine-labeled methyl iodophenylguanidine scan) is specific for the identification of neuroblastoma cells. (3) Laboratory examination: Detection of catecholamine metabolites in urine or serum, especially vanillyl mandelic acid (VMA) and homovanillic acid (HVA), which are often elevated in patients with neuroblastoma. (4) Histopathological analysis: Tumor tissue samples are obtained by biopsy and pathological analysis is performed to confirm neuroblastoma and evaluate the histological grade and cell differentiation of the tumor. (5) Genetic testing: Detection of MYCN gene amplification, ALK gene mutation, etc., to determine the biological characteristics and invasiveness of the tumor and assist in prognosis assessment. (6) Bone marrow biopsy and bone scan: used to check whether there is tumor metastasis in the bone marrow, especially for the diagnosis and staging of advanced neuroblastoma. (7) Clinical staging: Staging according to the International Neuroblastoma Staging System (INSS) or the International Neuroblastoma Risk Stratification System (INRGSS) to guide the selection of treatment options and prognosis judgment.

[0062] Method steps: Establishment of the scoring system: A semi-quantitative scoring system was used based on the H4K5la staining intensity and distribution (0 point: no staining; 1 point: weak staining; 2 points: moderate staining; 3 points: strong staining; 4 points: very strong staining).

[0063] IHC staining intensity: DAB staining intensity was quantitatively analyzed by ImageJ software, and the mean ± standard deviation of H4K5la expression level was calculated.

[0064] In the method of the present invention, the judgment criteria for weak staining, moderate staining, strong staining and very strong staining are defined as follows in order to standardize the evaluation of the staining results: (1) Weak staining: When observed under a microscope, the staining signal is relatively weak, and only the staining of the target area can be distinguished. There is almost no obvious contrast with the background, and the staining area is small.

[0065] (2) Moderate staining: The staining signal is clearly visible with moderate intensity. The staining of the target area forms a certain contrast with the background. The staining coverage area is relatively uniform, showing a clear staining effect.

[0066] (3) Strong staining: The staining signal is significantly enhanced, the staining of the target area is significantly darker than the background, with high contrast, high staining area coverage, and a dense staining effect.

[0067] (4) Very strong staining: The staining signal is extremely strong, the staining depth of the target area significantly exceeds the background, the contrast is extremely clear, the staining area is dark, and the coverage area is almost 100%.

[0068] This standard ensures consistent and reproducible staining results, allowing technicians to accurately judge staining intensity and obtain standardized staining assessments across samples.

[0069] Data collection and analysis: The IHC scores of patients were correlated with their prognostic data (such as disease-free survival and overall survival), and the Kaplan-Meier survival analysis method was used to compare the survival curves of patients with different H4K5la expression levels.

[0070] Statistical analysis: The log-rank test was used to assess the significance of differences in survival curves.

[0071] Experimental results: The survival analysis results are as follows Figure 2 As shown: The Kaplan-Meier survival curve showed that the prognosis of patients with high and low H4K5la expression was significantly different, and the P value of the log-rank test was <0.01.

[0072] Results: The prognosis of patients with high H4K5la expression was significantly worse than that of patients with low H4K5la expression, indicating that H4K5la is an independent marker for the prognosis of neuroblastoma.

[0073] Comparative Example 1 Detection of traditional prognostic markers Experimental Materials: Neuroblastoma FFPE tissue samples; MYCN gene amplification detection kit (purchased from Thermo Fisher Scientific).

[0074] Method steps: DNA extraction: DNA was extracted from FFPE tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen, Catalog No. 56404).

[0075] qPCR detection: Use the MYCN gene amplification detection kit and follow the manufacturer's instructions to perform qPCR detection to evaluate the gene amplification status.

[0076] Results: Although MYCN amplification is a recognized prognostic marker, its amplification rate is low, with only 18% of patients showing MYCN amplification (Table 1), and detection requires complex equipment and technology, which is difficult to promote in resource-limited settings.

[0077] Table 1 MYCN amplification detection results in children with neuroblastoma who survived and died (n=50)

[0078] Experimental Example 1 This study reviewed 50 high-risk neuroblastoma cases that were pathologically examined and confirmed by more than two pathologists. By comparing the immunohistochemical results of H4K5la and MYCN, ROC curves were drawn to evaluate the diagnostic performance of these two markers in high-risk neuroblastoma. Figure 3 As shown in the figure, H4K5la showed a higher diagnostic accuracy through ROC curve analysis. This indicates that H4K5la has a better ability to distinguish neuroblastoma. The analysis results suggest that H4K5la may have more application potential in the diagnosis and prognosis evaluation of high-risk neuroblastoma.

[0079] Comparative Example 2 FISH detection of MYCN amplification to evaluate the prognosis of neuroblastoma Experimental Materials: neuroblastoma FFPE tissue samples (paraffin-embedded tissue from neuroblastoma patients); FISH MYCN amplification detection kit (purchased from Kanglu Biotechnology Co., Ltd., catalog number FP-048); Rabbit anti-H4K5la antibody (dilution ratio 1:200), PTM BIO, catalog number PTM-1407RM; HRP-labeled goat anti-rabbit secondary antibody (Signal Transduction Company, catalog number 7074S); DAB color development kit (Dako Company, Denmark, K3468); DAPI (4',6-diamidino-2-phenylindole) stain (for FISH detection) was purchased from Sigma-Aldrich with the catalog number D9542.

[0080] Method steps: Step 1: Sample preparation Sections of 4 μm thickness were cut from FFPE (formalin-fixed paraffin-embedded) tissue blocks of patients with neuroblastoma and placed on slides.

[0081] Bake the sections in a 60 °C oven for 1 h to fix the samples.

[0082] Step 2: Dewaxing and hydration The samples on the slides were placed in xylene and dewaxed for 10 minutes, repeated twice.

[0083] The cells were rehydrated step by step using gradient ethanol solutions (100%, 95%, 70%) for 2 min each step, and finally rinsed with distilled water.

[0084] Step 3: Preprocessing Incubate tissue sections in a 98°C water bath for 10 minutes using the pretreatment buffer provided in the FISH detection kit to dissociate tightly bound DNA strands in the tissue.

[0085] The samples were cooled and rinsed with PBS (phosphate buffered saline).

[0086] Step 4: Enzyme digestion Add proteinase K solution and digest the sample for 10 minutes at room temperature to remove proteins and expose the target DNA sequence. Rinse the sample again with PBS.

[0087] Step 5: DNA probe hybridization Add MYCN DNA probe labeled with fluorescent dye, cover the sample and gently flatten with a coverslip.

[0088] The samples were placed in the mixed solution and denatured at 80°C for 5 minutes to unwind the DNA double helices.

[0089] Incubate samples overnight (12-16 hours) at 37°C in a humidified chamber to allow complete hybridization of the probe to the target sequence.

[0090] Step 6: Cleaning Wash samples with FISH wash buffer at 45°C for 5 min to remove unbound probe.

[0091] The samples were rinsed again with PBS.

[0092] Step 7: Nuclear staining The samples were stained with DAPI stain and incubated for 5 minutes to contrast stain the cell nuclei.

[0093] Step 8: Observe the results MYCN amplification was observed using a fluorescence microscope, and the distribution of positive signals was recorded.

[0094] The amplification status of MYCN is assessed based on the degree of signal amplification. Positive amplification is usually manifested as multiple green fluorescent signal spots.

[0095] Positive detection rate: MYCN amplification positive signals only appear in about 20% of the samples, and the amplification signals appear as multiple green fluorescent spots.

[0096] Operational complexity: FISH detection requires a long experimental cycle (including an overnight hybridization step), high equipment requirements, and high cost.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of H4K5la detection reagent in the preparation of products for determining the prognosis of neuroblastoma.

2. The use according to claim 1, characterized in that: The products include reagents, test kits, test strips, gene chips, and antibody chips.

3. The use according to claim 1, characterized in that: The detection reagent is a reagent for detecting the expression level of H4K5la.

4. The use according to claim 1, characterized in that: Detection of H4K5la expression levels includes the following steps: (1) Obtain test samples from neuroblastoma patients; (2) Detect the expression level of H4K5la in the sample; (3) If H4K5la is highly expressed in the sample, it indicates that the prognosis of neuroblastoma patients is poor; if H4K5la is low expressed in the sample, it indicates that the prognosis of neuroblastoma patients is good.

5. The use according to claim 4, characterized in that: The test sample in step (1) is one or more of tumor tissue, tumor cells, blood or plasma.

6. The use according to claim 4, characterized in that: Methods for detecting the expression level of H4K5la include immunohistochemistry, microarray analysis, polymerase chain reaction, and reverse transcriptase polymerase chain reaction.

7. The use according to claim 6, characterized in that: The immunohistochemical method for detecting the expression level of H4K5la includes the following steps: (1) Preparation of FFPE tissue sample sections; (2) Dewaxing and hydration: After the samples were dewaxed in xylene, they were hydrated through graded ethanol. (3) Antigen retrieval: The samples were subjected to antigen retrieval using citrate buffer; (4) Blocking: Block tissue sections with BSA; (5) Primary antibody incubation: Add rabbit anti-H4K5la antibody and incubate for 12-16 hours; (6) Secondary antibody incubation: Use HRP-labeled goat anti-rabbit secondary antibody for incubation for 1-3 hours; (7) Color development: Add color developer and allow to develop for 2-5 minutes; (8) Restaining and sealing: Add hematoxylin for restaining, dehydrate with graded alcohol, add xylene, and seal the slides.

8. A product for diagnosing or evaluating prognosis of neuroblastoma, characterized in that: The product includes a reagent for detecting the expression level of H4K5la.

9. The product according to claim 8, characterized in that The product described is a test kit.

10. The product according to claim 8, characterized in that The product performs diagnosis or prognosis assessment by detecting the H4K5la expression level in the sample tissue of the subject.

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