Application of reagent for detecting AHSP protein in preparation of product for diagnosing prostatic cancer
Through reagents for detecting AHSP protein, the difficulty of early diagnosis of prostate cancer is solved, and the detection of highly expressed AHSP protein in urine and tissues is achieved, which improves the diagnostic accuracy and treatment guidance of prostate cancer.
Patent Information
- Application Number
- CN202510447391.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-07-11
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The lack of effective markers in the prior art is used for the early diagnosis of prostate cancer, which leads to difficulty in diagnosis and affects the therapeutic effect.
Reagents that detect AHSP proteins, including specific binding agents, oligonucleotide probes or primer pairs, are used to detect AHSP protein expression in urine, prostate tissue or cells, and are tested using protein chips or detection kits.
By detecting the high expression of AHSP protein, the accuracy of early diagnosis of prostate cancer can be significantly improved, the treatment guidance can be provided, and there is good application prospect.
Smart Images
Figure CN120294330A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of a reagent for detecting AHSP protein in the preparation of a product for diagnosing prostate cancer. Background Art
[0002] Prostate cancer (PCa) is one of the common malignant tumors in men, and its incidence has been gradually increasing in recent years. The current early diagnosis of prostate cancer relies on puncture biopsy, combined with prostate-specific antigen (PSA). For some patients, the diagnosis is difficult and there is a lack of effective markers. The complexity of the clinical detection of prostate cancer and the concealment of its clinical manifestations make the early diagnosis and treatment effect of prostate cancer not optimistic, which poses a greater challenge to the treatment of prostate cancer. The importance of early screening, treatment and prognosis of prostate cancer in clinical practice is self-evident.
[0003] Alpha-hemoglobin-stabilizing protein (AHSP). AHSP is a protein that plays an important role in the process of red blood cell development. The protein amount is 12 kDa. It can bind to immature hemoglobin, help it fold and assemble correctly, and as a molecular chaperone, prevent the harmful aggregation of alpha hemoglobin during the normal development of erythroid cells. It especially protects free alpha hemoglobin from precipitation. It is expected to regulate the pathological state of excessive alpha hemoglobin, such as beta-thalassemia. In addition, according to the literature, there is also a certain association between AHSP and the occurrence and development of diseases such as liver cancer and breast cancer, and it may participate in the immune response process of some cancers. At present, there is no report on the relationship between AHSP and the occurrence and development of prostate cancer. Through the research on the relationship between AHSP and prostate cancer, it can provide a theoretical basis for the screening, diagnosis and prognosis prediction of prostate cancer. Summary of the Invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of a reagent for detecting AHSP protein in the preparation of a product for diagnosing prostate cancer.
[0005] The present invention realizes the above purpose through the following technical solutions:
[0006] As the first aspect of the present invention, the application of a reagent for detecting AHSP protein in the preparation of a product for diagnosing prostate cancer.
[0007] Further improvement lies in that the amino acid sequence of the AHSP protein is as shown in SEQ ID No.1, and the GeneBank accession number is Q9NZD4.
[0008] Further improvement lies in that the detection sample of the reagent is urine, prostate tissue or cells.
[0009] A further improvement lies in that the reagent is a binder that specifically binds to the AHSP protein, an oligonucleotide probe that specifically recognizes the gene encoding the AHSP protein, or a primer pair that specifically amplifies the gene encoding the AHSP protein.
[0010] A further improvement lies in that the product includes a protein chip or a protein detection kit.
[0011] A further improvement lies in that the protein chip includes a specific binder for the AHSP protein, and the protein detection kit includes reagents, chips, probes, colloidal gold or test strips for detecting the expression level of the AHSP protein.
[0012] The present invention has the following beneficial effects:
[0013] Through the research of the present invention, it is found that the AHSP protein is highly expressed in prostate cancer tissues, lowly expressed in prostate hyperplasia and normal tissues, and its content in urine is also higher than that in the normal group. AHSP is related to the occurrence and development of prostate cancer, can be used to assist in the diagnosis of prostate cancer, and has guiding significance for the treatment of prostate cancer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This shows the expression of the AHSP protein provided by the present invention in prostate cancer tissues and normal tissues. In the figure, A - B shows that AHSP is highly expressed in prostate cancer tissues (black arrow), and lowly expressed or not expressed in normal tissues (red arrow). A - B are from the same patient (Gleason score 4 + 3 = 7).
[0015] Figure 2 This shows the expression of the AHSP protein provided by the present invention under a 400 - fold high - power microscope in prostate cancer tissues, showing brown - yellow color in the cytoplasm. In the figure, A - B shows that AHSP is highly expressed in prostate cancer tissues. In the figure, Figure A has a Gleason score of 4 + 3 = 7, and Figure B has a Gleason score of 4 + 5 = 9.
[0016] Figure 3 This shows the expression of the AHSP protein provided by the present invention in prostate hyperplasia tissues. In the figure, it is lowly expressed in the hyperplastic prostate glands, and part of the cytoplasm shows light yellow color. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0017] The following further describes the present application in detail with reference to the drawings. It is necessary to point out here that the following specific embodiments are only used to further illustrate the present application and cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make some non - essential improvements and adjustments to the present application based on the above application content.
[0018] For the experimental methods in the following examples, unless otherwise specified, they are all conventional biochemical methods. For the test materials used in the following examples, unless otherwise specified, they are all obtained from regular biochemical reagent stores.
[0019] 1. Urinary proteome analysis
[0020] In October 2024, 20 ml of preoperative morning urine was collected from 10 patients clinically diagnosed with prostate cancer. At the same time, 20 ml of urine from 5 normal males was collected as a control group for analysis. Proteomics was analyzed using the T-test. The analysis process is as follows:
[0021] (1) Protein extraction
[0022] The urine samples were taken out from -80 °C. pH 8.0 Tris-HCl was added to all samples to a final concentration of 50 mM, and centrifuged at 1000 g for 5 min. The supernatant was taken, and then centrifuged at 17000 g for 10 min. The supernatant was taken and an equal volume of methanol and 1 / 4 volume of chloroform were added. After shaking for 15 s, it was left at room temperature for 5 min. After centrifuging at 12000 g at room temperature for 15 min, the supernatant was discarded. Then, an equal volume of methanol was added, shaken for 15 sec, and centrifuged at 12000 g at room temperature for 15 min. The supernatant was discarded. 80 μl of lysis buffer (containing 1% SDC) was added for reconstitution, and the protein concentration was measured using a BCA kit.
[0023] (2) Trypsin digestion
[0024] Equal amounts of protein from each sample were digested. The volume was adjusted to be consistent with the lysis buffer, and then dithiothreitol (DTT) was added to a final concentration of 5 mM, and reduced at 56 °C for 30 min. Then, iodoacetamide (IAA) was added to a final concentration of 11 mM, and incubated in the dark at room temperature for 15 min. TEAB was added to dilute urea to ensure the concentration was lower than 2 M. Trypsin was added at a ratio of 1:50 (protease:protein, m / m), and digested overnight. Then, trypsin was added at a ratio of 1:100 (protease:protein, m / m), and digestion continued for 4 h.
[0025] (3) Liquid chromatography-mass spectrometry (LC-MS) analysis
[0026] The peptides obtained by trypsin digestion were dissolved in liquid chromatography mobile phase A and separated using the NanoElute ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an acetonitrile-water solution containing 0.1% formic acid. The liquid phase gradient was set as follows: 0-14min, 6%-24% B; 14-16min, 24%-35% B; 16-18min, 35%-80% B; 18-20min, 80% B, and the flow rate was maintained at 500nl / min. After separation by the ultra-high performance liquid phase system, the peptides were injected into the Capillary ion source for ionization and then into the timsTOF Pro 2 mass spectrometer for data acquisition. The ion source voltage was set to 1.75kV, and the peptide parent ions and their secondary fragments were detected and analyzed using TOF. The data acquisition mode used was data-independent parallel accumulation serial fragmentation (dia-PASEF) mode. The primary mass spectrometry scanning range was set to 300-1500 m / z. After one primary mass spectrum was acquired, 20 PASEF mode acquisitions were performed. The secondary mass spectrometry scanning range was 400-850, with every 7 m / z as a window.
[0027] Using urine proteome analysis, through a series of analyses such as protein extraction, trypsin hydrolysis, and liquid chromatography-mass spectrometry analysis, it was found that the concentration of AHSP protein in the urine of prostate cancer patients was significantly higher than that in the normal male group (P=0.008). The results are shown in Table 1.
[0028] Table 1. Comparison of AHSP protein content in urine of prostate cancer group and normal group urine
[0029]
[0030]
[0031] Note: C1 to C5 are normal male groups; P1 to P10 are prostate cancer groups.
[0032] 2. Immunohistochemical Analysis
[0033] Archival wax blocks of 20 prostate cancer patients and 10 normal prostate tissues were collected from July 2021 to November 2024, and 10 cases of prostate hyperplasia were collected. The monoclonal antibody (clone number: EPR12319) purchased from abcam was diluted to 1:30, and the immunohistochemistry EnVision one-step method was used. 4um thick sections were dewaxed to water, and heat antigen retrieval was performed with EDTA, and DAB was used for color development. SPSS25.0 statistical software was used, and Fisher ， s exact probability test method was used for analysis.
[0034] It was found by immunohistochemical staining results that the AHSP protein was expressed in all prostate cancer patients. The expression intensity in prostate cancer patients was higher than that in benign prostatic hyperplasia (P<0.05) and also higher than that in normal prostate tissue (P<0.05). AHSP was weakly expressed in benign prostatic hyperplasia and normal tissues. The AHSP protein was stained brown-yellow in the cytoplasm of prostate cancer cells ( Figures 1-3 ), which can be used for the diagnosis of prostate cancer.
[0035] The above-described embodiments merely represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed. However, it should not be construed as a limitation to the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and all of these fall within the protection scope of the present invention.
Claims
1. Use of a reagent for detecting AHSP protein in the preparation of a product for diagnosing prostate cancer.
2. The application according to claim 1, wherein The amino acid sequence of the AHSP protein is as shown in SEQ ID No.
1.
3. The application according to claim 1, characterized in that, The detection sample of the reagent is urine, prostate tissue or cells.
4. The application according to claim 1, characterized in that The reagent is a binder that specifically binds to the AHSP protein, an oligonucleotide probe that specifically recognizes the gene encoding the AHSP protein, or a primer pair that specifically amplifies the gene encoding the AHSP protein.
5. The application according to claim 1, characterized in that, The product includes a protein chip or a protein detection kit.
6. The application according to claim 5, characterized in that, The protein chip includes a specific binder for the AHSP protein, and the protein detection kit includes a reagent, a chip, a probe, colloidal gold or a test strip for detecting the expression level of the AHSP protein.