Construction of a human prostate cancer cell line and its application

Abstract

This invention relates to the fields of tumor biology and cell engineering, specifically disclosing the construction and application of a human prostate cancer cell line. The human prostate cancer cell line is derived from primary prostate cancer tissue, expresses prostate-specific antigen and α-methylacyl-CoA racemic enzyme, and has a 46,XY karyotype. The construction method employs an innovative strategy of co-culturing fully digested cell suspensions with incompletely digested tissue blocks, significantly improving the success rate of establishing the cell line from the primary lesion. This cell line can stably proliferate in conventional culture media supplemented with testosterone and has been successfully applied to subcutaneous tumorigenesis models in immunodeficient mice and in vivo migration models in zebrafish. The primary lesion-derived prostate cancer cell line provided by this invention offers a highly relevant, stable, and reliable new experimental tool for studying the biological characteristics of primary prostate cancer, drug screening, and personalized treatment.

Description

Technical Field

[0001] This invention relates to the fields of tumor biology and cell engineering, specifically to the construction and application of a human prostate cancer cell line. Background Technology

[0002] Prostate cancer is a malignant tumor with a high incidence and mortality rate among men worldwide. Its pathogenesis is complex and exhibits significant heterogeneity, posing a huge challenge to clinical diagnosis and basic research. In the basic research and drug development of prostate cancer, stable, reliable cancer cell lines that can truly reflect the clinical characteristics of the disease are indispensable core experimental tools, widely used in key areas such as tumor biological behavior analysis, signaling pathway mechanism research, drug sensitivity testing, and drug resistance mechanism exploration.

[0003] Currently, the representative prostate adenocarcinoma cell lines widely used internationally mainly include DU145, PC-3, LNCaP, and 22Rv1. While these cell lines have played a crucial role in the history of prostate cancer research, their inherent limitations have become increasingly apparent with a deeper understanding of the disease, severely restricting the accuracy and clinical translational value of research. They exhibit the following significant drawbacks: ① Lack of primary tumor representativeness: Metastatic cancer cells undergo intense clonal selection and microenvironment adaptation, resulting in significant deviations from the primary tumor in terms of genomic, epigenetic, and functional characteristics, making it difficult to accurately reflect the biological behavior of tumors in newly diagnosed patients; ② Inability to simulate high-risk primary subtype characteristics: Existing cell lines were mostly established decades ago, and their genetic background differs from contemporary high-risk primary prostate cancers with high Gleason scores (e.g., 4+4=8), limiting mechanistic research and the development of targeted strategies for this highly aggressive and poor-prognostic subtype; ③ Limitation of "tumor-microenvironment" interaction research: Due to the non-orthotopic prostate tissue, existing cell lines have lost the ability to interact with prostate-specific matrix (e.g., fibroblasts, immune cells), making it difficult to construct physiologically relevant co-culture or organoid models. Based on the above, this invention provides a method for constructing and applying a human prostate cancer cell line. Summary of the Invention

[0004] To address key technical bottlenecks in existing prostate cancer cell lines, such as their origin from metastatic lesions, lack of representativeness of primary tumors, difficulty in simulating the biological behavior of high-risk primary subtypes, and limitations on tumor-microenvironment interaction research, this invention provides a method for constructing and applying a human prostate cancer cell line.

[0005] In a first aspect, the present invention provides a method for constructing a human prostate cancer cell line, employing the following technical solution: A method for constructing a human prostate cancer cell line, comprising the following steps: S1. Obtain a tissue sample from the primary prostate cancer lesion, and perform cutting and washing to obtain pre-treated tissue; S2. The pretreated tissues were divided into a fully digested group and an incompletely digested group. The fully digested group was digested with tissue digestion solution to obtain a cell suspension. The incompletely digested group was digested with tissue digestion solution, digestion was terminated early, and the undigested tissue blocks were collected. S3. Inoculate the cell suspension and the incompletely digested tissue block together into a culture container, add complete culture medium, and carry out primary culture to obtain primary cells; S4. Passage the primary cells to obtain a stable cell line, namely the human prostate cancer cell line.

[0006] Preferably, in step S1, the cleaning is performed using Hank's balanced salt solution, and the material is shredded to 2-4 mm³.

[0007] Preferably, the tissue digestion solution in step S2 contains collagenase IV, penicillin, streptomycin, and gentamicin; the concentration of collagenase IV is 900-1100 U / mL, the concentration of penicillin is 150-250 U / mL, the concentration of streptomycin is 150-250 μg / mL, and the concentration of gentamicin is 90-110 μg / mL.

[0008] Preferably, the complete culture medium in step S3 comprises McCoy's 5A basal medium and fetal bovine serum in a volume ratio of 16-24:3.

[0009] Preferably, the complete culture medium also contains antibiotics and testosterone, the antibiotics including penicillin, streptomycin and gentamicin, and the concentration of testosterone is 8-12 ng / mL.

[0010] Preferably, the complete culture medium contains penicillin at a concentration of 95-105 U / mL, streptomycin at a concentration of 90-110 μg / mL, and gentamicin at a concentration of 40-60 μg / mL.

[0011] Preferably, the primary culture temperature in step S3 is 35-40℃ and the CO2 concentration is 4-6%.

[0012] Secondly, the present invention provides a human prostate cancer cell line (HPC-2) constructed using the above-described method for constructing human prostate cancer cell lines. The human prostate cancer cell line is human prostate cancer cell line HPC-2, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202649 and deposit date of March 17, 2026.

[0013] Preferably, the human prostate cancer cell line is derived from the primary prostate cancer lesion tissue and named human prostate cancer cell HPC-2. It expresses prostate-specific antigen (PSA) and α-methylacyl-CoA racemic enzyme (AMACR) and has a chromosome karyotype of 46,XY.

[0014] The aforementioned human prostate cancer cell line was derived from ethically approved biopsy tissue from primary prostate cancer lesions (Gleason score 4+4=8). No exogenous genes were introduced, and the cells were neither virally transfected nor immortalized. Their sustained in vitro proliferation capacity stems from the spontaneous transformation characteristics of the primary tumor tissue. This cell line was identified as being of human male origin via short tandem repeat (STR) typing and showed no match for known prostate cancer cell lines (including DU145, PC-3, LNCaP, and 22Rv1) in public cell banks such as ATCC and DSMZ, confirming it as a novel cell line with a unique genetic identity. Chromosomal karyotype analysis showed a 46,XY chromosome with no significant numerical or structural abnormalities. HPC-2 cells exhibited typical epithelial-like adherent growth in vitro, stably expressing prostate cancer-specific markers prostate-specific antigen (PSA) and α-methylacyl-CoA racemic enzyme (AMACR), and possessed a high Ki-67 proliferation index. Both in vitro culture and in vivo tumorigenesis depend on exogenous testosterone, and the cells can be stably passaged long-term in a complete culture medium containing testosterone. When subcutaneously inoculated into NSG immunodeficient mice fed a testosterone-containing diet, the tumorigenesis rate is no less than 80%, and the pathological characteristics of the resulting tumor tissue are consistent with the primary lesion. In addition, fluorescently labeled HPC-2 cells can migrate in vivo in a zebrafish model, confirming their invasive potential.

[0015] Thirdly, the present invention provides an application of a human prostate cancer cell line in the preparation of animal models of prostate cancer.

[0016] Preferably, the prostate cancer animal model is an immunodeficient mouse subcutaneous xenograft tumor model.

[0017] In summary, the present invention has the following beneficial effects: (1) Highly closely resembles the real biological characteristics of high-risk primary prostate cancer in clinical practice: This invention is the first to successfully establish a stable cell line HPC-2 from primary lesion tissue obtained through ethically approved prostate biopsy (Gleason score 4+4=8), fundamentally breaking through the limitation that existing mainstream prostate cancer cell lines (such as DU145, PC-3, LNCaP, etc.) all originate from metastatic lesions or xenografts. HPC-2 retains the typical molecular phenotype and pathological characteristics of high-risk primary tumors, and can more realistically simulate the malignant initiation, local invasion, and androgen-dependent growth behavior of early clinical prostate cancer, providing a highly relevant in vitro model for studying the mechanism of primary tumor development, high-risk subtype characteristics, and individualized treatment strategies.

[0018] (2) Clear genetic identity, high biosafety, and strong experimental reproducibility: The HPC-2 cell line was identified as being of human male origin by short tandem repeat (STR) typing. It did not match any known prostate cancer cell lines in authoritative international cell banks such as ATCC and DSMZ, confirming it as a novel cell line with a clear genetic background. Chromosomal karyotype analysis showed a normal male karyotype (46, XY), with no obvious numerical or structural abnormalities. This characteristic effectively avoids research bias caused by cell cross-contamination, misidentification, or genetic drift, and significantly improves the reliability of experimental data and the comparability of multi-center studies.

[0019] (3) Complete functionality with multi-level in vivo-in vitro validation capabilities: HPC-2 exhibits a typical epithelial-like morphology in vitro, adheres firmly to the cell wall, proliferates actively, stably expresses prostate cancer-specific markers PSA and AMACR, and has a high Ki-67 index; its growth is strictly dependent on exogenous testosterone, and it can be stably passaged for a long time in a complete culture medium containing testosterone. More importantly, the tumorigenesis rate of this cell line after subcutaneous inoculation in NSG immunodeficient mice is no less than 80%, and the pathological characteristics of the formed tumor tissue are highly consistent with the primary lesion; at the same time, after fluorescent labeling in a zebrafish model, it can achieve in vivo proliferation and directional migration, confirming its invasive potential. This three-level functional validation system of "in vitro culture-mouse tumorigenesis-zebrafish migration" makes it an ideal preclinical research tool.

[0020] (4) Innovative construction method, significantly improving the success rate of establishing cell lines in primary lesions: This invention adopts a co-culture strategy of "completely digested cell suspension + incompletely digested tissue block", which cleverly combines the rapid expansion advantage of free cells with the microenvironmental support provided by residual tissue blocks (such as extracellular matrix, paracrine factors, etc.), effectively overcoming the industry's technical bottlenecks such as high degree of fibrosis in prostate primary lesion tissue, difficulty in cell dissociation, and low primary cell survival rate. This method is particularly suitable for small samples obtained from routine clinical puncture biopsies, greatly increasing the probability of successfully establishing stable cell lines from real patient specimens, and opening up a feasible path for precision medicine research based on individualized samples.

[0021] (5) Simple operation, low cost, and easy to promote: The HPC-2 cell line does not require viral transfection, gene editing, or feeder layer support, and can proliferate stably under conventional culture conditions. Its complete culture medium has a clear composition and is easy to prepare. The core consists of commercially available McCoy's 5A basal medium and fetal bovine serum, supplemented with conventional antibiotics (penicillin, streptomycin, gentamicin). Only a fixed concentration of testosterone needs to be added before use, avoiding the use of expensive growth factor combinations or complex additives. In addition, the cells adhere firmly, have a stable passage cycle, and are tolerant to conventional trypsin digestion and cryopreservation and thawing operations, which facilitates large-scale expansion and long-term preservation in ordinary laboratories, significantly reducing the technical threshold and operating costs, and has good scalability and industrial application prospects. Attached Figure Description

[0022] Figure 1 An inverted microscope image of epithelial-like cells surrounding a tissue block after primary culture of human prostate cancer cell line HPC-2. Figure 2 An inverted microscope image of the unstained human prostate cancer cell line HPC-2; Figure 3 Inverted microscope image for morphological identification of human prostate cancer cell line HPC-2 after crystal violet staining; Figure 4 Staining results for PSA, AMACR, and Ki-67 immunophenotypic identification of human prostate cancer cell line HPC-2; Figure 5 A short tandem repeat (STR) typing map of the human prostate cancer cell line HPC-2; Figure 6 Chromosomal G-banding karyotype analysis diagram of human prostate cancer cell line HPC-2; Figure 7 Growth curves for in vitro proliferation of the human prostate cancer cell line HPC-2; Figure 8 A photograph of subcutaneous tumor formation of the human prostate cancer cell line HPC-2 in NSG immunodeficient mice; Figure 9 Growth curve of subcutaneous xenograft tumors of the human prostate cancer cell line HPC-2 in NSG immunodeficient mice; Figure 10 Fluorescence observation of the proliferation of human prostate cancer cell line HPC-2 in the yolk sac region of zebrafish; Figure 11 A fluorescence image of the migration of the human prostate cancer cell line HPC-2 in the space between the yolk sac and tail of zebrafish. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0024] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0025] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0026] Example 1 Preparation of experimental reagents: This embodiment provides specific preparation methods for all reagents required for constructing and identifying the human prostate cancer cell line HPC-2. All operations must adhere to aseptic principles to ensure reagent purity and effectiveness. Hank's equilibrium salt solution: Accurately weigh MgSO4 100mg of 7H2O and MgCl2 100 mg of 6H2O, 140 mg of anhydrous CaCl2, 10 mg of phenol red, 350 mg of NaHCO3, and 1000 mg of D-glucose were added sequentially to pure water and dissolved thoroughly. The volume was adjusted to 1 L, the pH was adjusted to 7, and the mixture was filtered through a 0.1 μm filter membrane for sterilization. It was then sealed and stored at room temperature for later use.

[0027] Penicillin-streptomycin-gentamicin mixed solution: Dissolve penicillin, streptomycin, and gentamicin in a 0.9% (w / w) sterile sodium chloride solution, then filter through a 0.1 μm filter membrane to obtain a penicillin-streptomycin-gentamicin mixed solution containing 10 kU / mL penicillin, 10 mg / mL streptomycin, and 5 mg / mL gentamicin. Aliquot the solution into sterile cryovials and store at -20°C. Thaw as needed before use.

[0028] Tissue digestion solution: Take collagenase IV dry powder, dissolve it in Hank's balanced salt solution, then add penicillin-streptomycin-gentamicin mixed solution, and make up to volume with Hank's balanced salt solution to make the final concentration of collagenase IV 1000 U / mL, penicillin 200 U / mL, streptomycin 200 μg / mL, and gentamicin 100 μg / mL; filter through a 0.1 μm filter membrane for sterilization, aliquot, and store at -20℃. Thaw as needed before use.

[0029] Testosterone solution: Dissolve testosterone powder (purity ≥98%) in Hank's balanced salt solution to obtain a testosterone solution with a concentration of 100 ng / mL. Prepare fresh before use.

[0030] Complete culture medium: Take 7.5 mL of fetal bovine serum, 41.5 mL of McCoy's 5A basal medium, and 0.5 mL of penicillin-streptomycin-gentamicin mixed solution, mix well, freeze at -20℃, and thaw as needed; before each use, add 0.5 mL of testosterone solution to make the final testosterone concentration in the culture medium 10 ng / mL.

[0031] Trypsin digestion solution: This solution contains trypsin and EDTA. 2Na and phenol red were dissolved sequentially in Hank's balanced salt solution to make the trypsin concentration 2.5 g / L and EDTA... The 2Na concentration is 0.2 g / L and the phenol red concentration is 10 mg / L. After sterilization by filtration through a 0.1 μm filter membrane, it is aliquoted and stored at -20°C. Thaw as needed before use.

[0032] Cell cryopreservation solution: Mix complete culture medium, fetal bovine serum and dimethyl sulfoxide in a volume ratio of 7:2:1. Prepare fresh before use.

[0033] 0.1M Sodium Phosphate Buffer: Weigh 1.56g of sodium dihydrogen phosphate and 8.96g of disodium hydrogen phosphate, add 800mL of pure water, and stir magnetically until completely dissolved; adjust the pH of the solution to 7.4 using a pH meter, bring the volume to 1L with pure water, filter through a 0.1μm filter membrane for sterilization, and store in a sealed container at room temperature for use in washing steps of experiments such as immunostaining.

[0034] PBS-Tx solution: Add 5 μL of Triton X-100 to 5 mL of 0.1 M sodium phosphate buffer, mix well by pipetting, and prepare fresh for use in cell permeation treatment.

[0035] PBS-T solution: Add 5 μL of Tween-20 to 5 mL of 0.1 M sodium phosphate buffer, mix well by pipetting, and prepare fresh for use in the washing step of the immunostaining process.

[0036] Blocking solution: Weigh 500 mg bovine serum albumin, dissolve it in PBS-T solution and bring the volume to 10 mL. Mix thoroughly and store at 4°C for later use. It is used for non-specific binding blocking in immunostaining.

[0037] E3 culture medium (1× working solution): Accurately weigh 292.2 mg NaCl, 12.7 mg KCl, and 2 mg CaCl2. 2H₂O 48.5mg, MgSO₄ 81.3 mg of 7H2O was added to pure water and dissolved completely. The volume was adjusted to 1 L, and the pH was adjusted to 7.4. After sterilization by filtration through a 0.1 μm filter membrane, the solution was sealed and stored at room temperature for later use.

[0038] Carno fixative: Mix methanol and glacial acetic acid in a volume ratio of 3:1.

[0039] Example 2 Construction of the human prostate cancer cell line HPC-2: A method for constructing a human prostate cancer cell line, comprising the following steps: S1. Obtain a tissue sample from the primary prostate cancer lesion. In a sterile laminar flow hood, use sterile scissors to cut the tissue into 2 mm³ pieces and place them in a centrifuge tube. Add 2 mL of Hank's balanced salt solution to the centrifuge tube and gently pipette five times to suspend the tissue fragments. Let it stand for 5 minutes until the tissue fragments settle naturally. Carefully aspirate the supernatant. Repeat this step twice to thoroughly wash away any residual red blood cells and impurities in the sample, obtaining the pretreated tissue. S2. Divide the pretreated tissue into two equal portions, designated as the complete digestion group and the incomplete digestion group, respectively. Transfer each portion to a sterile 15mL centrifuge tube, add 10mL of tissue digestion solution preheated to 37℃ to each tube, mix gently, and place both tubes in a shaker at 37℃ and 100rpm for digestion. The complete digestion group is digested for 15 minutes, with gentle pipetting 10 times every 5 minutes. After digestion, immediately stop the reaction by placing the tube on ice, centrifuge at 1000rpm for 4 minutes, discard the supernatant, and resuspend the precipitate in 1mL of complete culture medium to obtain a cell suspension. The incomplete digestion group is digested for only 8 minutes, then stopped by placing the tube on ice, centrifuge at 1000rpm for 2 minutes, discard the supernatant, and collect the undigested tissue fragments at the bottom. S3. Combine the cell suspension with the incompletely digested tissue blocks and inoculate them together in a 6cm culture dish. Add complete culture medium to a total volume of 5mL, gently shake in a crosswise direction, and incubate at 37℃ with 5% CO2 for 48 hours for primary culture. When a large number of epithelial-like cells are observed crawling out from around the tissue blocks under a microscope, and the cell confluence reaches 80%, primary cells are obtained (e.g., ...). Figure 1 (as shown) S4. Discard the old culture medium of the primary cells and gently wash once with 1 mL of Hank's balanced salt solution; add 1 mL of trypsin digestion solution and incubate at 37°C, 5% CO2 for 3 min. Observe under a microscope until the cells become rounded and the gaps are obvious, then immediately add 1 mL of complete culture medium to stop digestion; gently pipette 8 times to detach the cells, collect the suspension in a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 4 min; discard the supernatant, resuspend in 1 mL of complete culture medium, seed into a new 6 cm culture dish, add complete culture medium to 5 mL, and culture at 37°C, 5% CO2. Change the medium every 2 days thereafter. When the cell confluence reaches 80%, passage in the same way; after 10 passages, the cells are morphologically uniform, firmly adhered, and proliferate stably, without obvious senescence or floating, thus obtaining a human prostate cancer cell line that can be expanded for a long time.

[0040] like Figure 2 As shown, in the unstained state, HPC-2 cells exhibit a typical epithelial-like morphology with tight intercellular junctions, clear cytoplasm containing a small number of granules, and round or oval nuclei with a high nucleocytoplasmic ratio, consistent with the morphological characteristics of prostate cancer cells.

[0041] Example 3 Passaging and cryopreservation of human prostate cancer cell line HPC-2 (1) Cell passage A1. Remove the HPC-2 cell culture dish to be passaged and remove the old culture medium in a sterile laminar flow hood. A2. Add 1 mL of Hank's balanced salt solution, gently shake the culture dish to cover the entire cell layer with the solution, wash once to remove residual culture medium and serum components. A3. Add 1 mL of trypsin digestion solution, shake the culture dish to evenly cover the cells with the digestion solution, and incubate in a 37°C, 5% CO2 incubator for 3 min. During this time, observe the cells through an inverted microscope. When the cells become round, the gaps between them increase and they begin to detach, immediately add 1 mL of complete culture medium to stop the digestion. A4. Gently blow on the bottom and edge of the culture dish with a pipette to completely detach all adherent cells and form a single-cell suspension. A5. Transfer the cell suspension to a sterile centrifuge tube, centrifuge at 1000 rpm for 4 min, discard the supernatant, and obtain the cell pellet. A6. Add 1 mL of fresh complete culture medium to the cell pellet and gently pipette 8 times to resuspend the cell pellet into a uniform cell suspension. A7. Inoculate the cell suspension into a new 6cm culture dish, add complete culture medium to 5mL, and continue culturing in a 37℃, 5%CO2 incubator. Subsequent passages are performed using the same method, with a stable passage period of 2 days.

[0042] (2) Cell cryopreservation B1. Follow steps A1-A5 in the passage procedure above to obtain the cell pellet after centrifugation; B2. Add freshly prepared cell freezing solution to the cell pellet, gently pipette to resuspend the cells, and adjust the cell concentration to 1×10⁻⁶. 7 Cells / mL were used to obtain a cell suspension; B3. Aliquot the cell suspension into sterile cryovials, 1 mL per tube. After tightening the cap, label the tube with the cell name, cryopreservation date and operator information. B4. Place the cryovials in the programmed cooling box and put them in an ultra-low temperature freezer at -80℃ overnight to achieve gradient cooling (cooling rate 1℃ / min). B5. The next day, remove the cryovials from the -80°C freezer and quickly transfer them to a liquid nitrogen tank for long-term storage. When thawing, immediately place the cryovials in a 37°C water bath to thaw quickly. After centrifuging to remove the cryopreservation solution, resuspend the cells in complete culture medium and inoculate them for culture.

[0043] Example 4 Identification of the human prostate cancer cell line HPC-2 (1) Morphological identification C1. Take HPC-2 cells in the logarithmic growth phase and administer at a dose of 1×10⁻⁶. 4 Seeds were seeded at a density of cells / well into 96-well cell culture plates (each well had 250 μL of complete culture medium added) and incubated at 37°C in a 5% CO2 incubator for 24 h. C2. Remove the culture medium, add 50 μL of 4 wt% paraformaldehyde solution to each well, fix at room temperature for 20 min, remove the paraformaldehyde solution, add 100 μL of distilled water to each well, and wash twice, 1 min each time. C3. Add 50 μL of crystal violet staining solution to each well, soak at room temperature for 20 min, remove the staining solution, add 100 μL of distilled water to each well, and wash 3 times for 1 minute each time. C4. Add 100 μL of distilled water to each well and observe using an inverted microscope.

[0044] like Figure 3 As shown, HPC-2 cells exhibit typical epithelial-like morphology, adherent growth, uniform shape, clear cell boundaries, and are arranged in a polygonal or cobblestone pattern with close interlocking structures; the nuclei are large, round or oval, with a high nucleocytoplasmic ratio, clear nuclear membrane, and 1-2 distinct nucleoli are visible, consistent with the morphological characteristics of prostate cancer cells.

[0045] (2) Immunophenotyping identification D1. Cell seeding and fixation: HPC-2 cells were seeded at a rate of 1×10⁶ cells / year. 4 Seeds were seeded into 96-well cell culture plates (each well had 250 μL of complete culture medium added), and cultured in a 37°C, 5% CO2 incubator for 24 h. After the culture medium was removed, 50 μL of 4 wt% paraformaldehyde solution was added to each well and fixed at room temperature for 20 min. D2. Washing and permeation: Remove the paraformaldehyde solution, add 100 μL of PBS-T solution to each well, wash twice for 5 min each time; remove the PBS-T solution, add 50 μL of PBS-Tx solution to each well, permeate at room temperature for 5 min, remove the PBS-Tx solution, add 100 μL of PBS-T solution to each well, wash twice for 2 min each time, and finally remove the PBS-T solution. D3. Sealing: Add 50 μL of sealing solution to each well and place on a shaker at room temperature for 1 hour (shaking speed 200 rpm). D4. Primary antibody incubation: Remove the blocking buffer, add 50 μL of primary antibody diluted with blocking buffer (PSA antibody, AMACR antibody, Ki-67 antibody, dilution ratio recommended according to the antibody instructions) to each well, and incubate overnight at 4°C. D5. Secondary antibody incubation: Recover the primary antibody, add 100 μL of PBS-T solution to each well, wash 3 times, 5 min each time; remove the PBS-T solution, add 50 μL of fluorescently labeled secondary antibody diluted with blocking buffer to each well, and incubate at room temperature in the dark for 1 h. D6. Color Development and Counterstaining: Recover the secondary antibody, add 100 μL of PBS-T solution to each well, wash 3 times for 5 min each time in the dark; follow the instructions of the commercial DAB horseradish peroxidase colorimetric kit, add colorimetric solution and develop color until a clear brown precipitate appears; remove the colorimetric solution, add 50 μL of hematoxylin staining solution to each well, counterstain at room temperature for 20 min, then add 100 μL of PBS-T solution to each well and wash once, remove the PBS-T solution, add 100 μL of distilled water to each well, and observe under an inverted microscope.

[0046] like Figure 4 As shown, HPC-2 cells highly express PSA and AMACR, confirming that they are prostate cancer cells; the Ki-67 positivity rate is over 75%, indicating that this cell line has active malignant proliferation ability, consistent with the characteristics of the high-risk prostate cancer tissue from which it originated.

[0047] (3) STR typing identification E1. Genomic DNA Extraction: Take HPC-2 cells in the logarithmic growth phase, use a commercial genomic DNA extraction kit, and strictly follow the instructions to extract genomic DNA. Detect the DNA concentration and purity using a nucleic acid quantification instrument. If the A260 / A280 ratio is between 1.8, it meets the requirements for PCR amplification, and HPC-2 genomic DNA is obtained. E2, STR site amplification: Using the PowerPlex® 16 HS System kit (Promega Biotechnology), 50 ng of HPC-2 genomic DNA was used as a template and mixed with the primer mixture and PCR master mixture in the kit according to the instructions. Multiplex PCR amplification was performed in a thermal cycler. The amplification program was set as follows: 95℃ pre-denaturation for 10 min; 94℃ denaturation for 1 min; 60℃ annealing for 1 min; 72℃ extension for 1 min, for a total of 30 cycles; and finally, 72℃ extension for 10 min. The PCR amplification products were stored at 4℃. E3. Capillary electrophoresis and data analysis: 1 μL of PCR amplification product was mixed with 0.5 μL of Size Standard internal standard and separated by capillary electrophoresis using a genetic analyzer; the size of the fragments was determined based on their migration location using the instrument's software and compared with allele typing standards to obtain the allele call value for each STR locus. E4. Result Comparison: The STR typing data of HPC-2 cells were compared with all known human cell lines (including prostate cancer cell lines such as DU145, PC-3, LNCaP, and 22Rv1) in the ATCC and DSMZ databases.

[0048] like Figure 5 As shown, all detection peaks were human-specific, and no interference signals from other species were detected; the AMEL loci were X and Y types, confirming that they originated from males; the matching degree with existing known cell lines was less than 80%, confirming that HPC-2 is a novel cell line with unique genetic identity and no cross-contamination.

[0049] (4) Chromosome karyotype identification F1. Cell cycle arrest: HPC-2 cells in the logarithmic growth phase were seeded in T25 culture flasks. When the cell confluence reached 70%, colchicine solution was added to the culture medium to make the final colchicine concentration 0.1 μg / mL. The flasks were then placed in a 37℃, 5% CO2 incubator and cultured for another 4 h to inhibit tubulin polymerization and arrest cell division in metaphase of mitosis. F2. Cell Collection and Hypotonic Treatment: Aspirate the culture medium from the T25 culture flask, add 1 mL of trypsin digestion solution to digest the cells, and after the cells detach from the cell wall, add complete culture medium containing 10% fetal bovine serum to stop the digestion. Pipe and form a single-cell suspension. Transfer the single-cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 5 mL of 0.075 M potassium chloride solution preheated to 37°C to the cell pellet, gently pipette and mix, and place in a 37°C water bath for hypotonic treatment for 25 min to allow the cells to swell and the chromosomes to disperse. F3. Fixation: After the hypotonic treatment, add 1 mL of pre-cooled Carno fixative to the centrifuge tube, gently pipette to mix and pre-fix; then centrifuge at 1000 rpm for 5 min, discard the supernatant; add 5 mL of Carno fixative, resuspend the cells and fix at room temperature for 20 min, repeat the fixation steps 3 times to ensure that the cells are fully fixed. F4. Cell suspension preparation and aging: After the final fixation, centrifuge and discard the supernatant. Add 5 mL of fresh Carnot fixative and resuspend the cells to obtain a cell suspension. Use a pipette to draw up the cell suspension and drop it from a height of 30 cm onto a pre-cooled, clean glass slide. The impact force and rapid evaporation of the liquid will cause the chromosomes to disperse fully. Place the glass slide in a 75°C oven for aging for 2 hours to enhance the binding force between the chromosomes and the slide. F5, G banding and staining: After aging, the slides were placed in a 0.25% trypsin solution for 60 seconds, removed, and immediately rinsed with physiological saline to stop the reaction; then the slides were placed in a 5% Giemsa stain solution (Beyotime Biotechnology) for 10 minutes, gently rinsed with deionized water, and air-dried. F6. Karyotype analysis: Under a high-powered microscope, 100 well-dispersed metaphase cells with clear bands were randomly selected for chromosome counting; the 5 best-morphologically selected cells were photographed, spliced, and arranged using an automated chromosome karyotype analysis system.

[0050] like Figure 6 As shown, the HPC-2 cell line has 46 chromosomes and a karyotype of 46,XY. No obvious abnormalities in chromosome number or structural aberrations were observed.

[0051] Example 5 In vitro proliferation assay of human prostate cancer cell line HPC-2 G1. Take HPC-2 cells in the logarithmic growth phase, digest, centrifuge, and resuspend them to prepare a single-cell suspension. Count the cells using a cell counting chamber and adjust the cell concentration to 1×10⁻⁶. 4 cells / mL; G2. In a 96-well cell culture plate, add 100 μL of cell suspension (containing 1 × 10³ cells) to each well, set up 6 replicates at each time point, and set up blank control wells without cells (add only 100 μL of complete culture medium). G3. Place the 96-well cell culture plate in a 37℃, 5% CO2 incubator for 4 hours. After the cells have completely adhered to the plate, add 10μL of CCK-8 reagent (Beyotime Biotechnology) to each well and continue culturing for 1.5 hours. Use an ELISA reader to detect the optical density (OD) value at a wavelength of 450nm as the baseline value at 0h. G4. After 24h, 48h, 72h and 96h of culture, the OD value was measured: the old culture medium in each well was removed, 100μL of fresh complete culture medium and 10μL of CCK-8 reagent were added, avoiding the generation of air bubbles, and the well was incubated in a 37℃, 5% CO2 incubator for 1.5h, and the 450nm OD value was measured. G5. Data Processing: Plot the HPC-2 cell growth curve with culture time as the x-axis and the average OD value (excluding the OD value of the blank control well) at each time point as the y-axis.

[0052] like Figure 7 As shown, HPC-2 cells were in the logarithmic growth phase for 24-48 hours after in vitro culture, and entered the plateau phase after 72 hours, showing a stable proliferation trend overall, which confirms that they have reliable in vitro proliferation ability.

[0053] Example 6 Animal experimental applications of the human prostate cancer cell line HPC-2 (1) Construction of a subcutaneous tumorigenesis model in NSG-immunodeficient mice H1. Preparation of experimental animals: Six 6-week-old male NSG immunodeficient mice were selected and housed in an SPF-grade animal room at an ambient temperature of 25℃ and a humidity of 50%, with a circadian rhythm of 12h / 12h. Starting one week before inoculation, the mice were continuously fed a special diet containing 10mg / kg testosterone to maintain the physiological level of androgens in the body. After one week of acclimatization, the mice were used for the experiment and were numbered No.1-No.6 for subsequent individual data tracking and recording. H2. Cell Preparation: HPC-2 cells in the logarithmic growth phase and in good growth condition were digested with trypsin and washed twice with physiological saline. Finally, the cells were resuspended in a 1:1 mixture of physiological saline and matrix gel pre-cooled to 4°C, kept on ice to maintain viability, and the cell concentration was adjusted to 1.0 × 10⁻⁶. 7 pcs / 100μL; H3. Cell inoculation: Using a 1 mL insulin syringe, draw up 100 μL of cell suspension and aseptically inject it into the subcutaneous region of the right posterior back of mice. The dose per mouse is 1.0 × 10⁻⁶. 7 One cell; H4. Tumor formation observation and monitoring: After inoculation, the mice's mental state, activity level and weight changes were observed twice a week. At the same time, the long axis (a) and short axis (b) of the tumor nodules were measured using calipers. The tumor volume of each mouse (No.1-No.6) was calculated according to the formula V=0.5×a×b² (V is the tumor volume, unit mm³). H5. Experimental endpoint and sample collection: 21 days after inoculation, the average tumor volume of mice No.1, No.2, No.3, No.5, and No.6 reached 800 mm³. The mice were euthanized by cervical dislocation. The subcutaneous tumor tissue was completely dissected, weighed, and photographed. The tumor tissue was divided into two parts. One half was fixed in 4 wt% paraformaldehyde solution for subsequent paraffin sectioning and histological analysis. The other half was quickly frozen in liquid nitrogen for molecular biology research.

[0054] The results showed that 5 out of 6 mice (No. 1, No. 2, No. 3, No. 5, and No. 6) successfully developed tumors, with a tumor formation rate of 83.3%. Figure 8 As shown, the tumor tissue has a regular morphology and uniform texture. Pathological section staining observation showed that its histological characteristics were consistent with those of primary prostate cancer tissue, confirming that the HPC-2 cell line has high tumorigenicity in vivo and retains the malignant biological characteristics of the source tumor.

[0055] The tumor volume of mice No.1, No.2, and No.3 was measured periodically, and growth curves were plotted. Figure 9As shown, from day 7 post-inoculation, the tumor volume in all groups showed a continuous and rapid growth trend, with groups No.1 and No.2 exhibiting more active proliferation. By the experimental endpoint (day 21 post-inoculation), the tumor volume reached approximately 950 mmHg. 3 1000mm 3 Group No. 3 showed slightly slower proliferation, with a final volume of approximately 500 mm. 3 The overall growth curves show that HPC-2 cells have a stable and efficient tumorigenic capacity in immunodeficient mice, and can serve as a reliable model for in vivo proliferation studies and drug sensitivity evaluation of prostate cancer.

[0056] (2) Construction of a model of proliferation and migration in zebrafish I1. Cell Preparation and Labeling: HPC-2 cells in logarithmic growth phase were digested with trypsin, counted using a cell counting chamber, and the cell concentration was adjusted to 1×10⁻⁶. 4 Collect cell suspension at 1 mL / mL, centrifuge at 600 rpm for 5 min, and discard the supernatant; add 1 mL of 0.1 M sodium phosphate buffer, rinse the cells by pipetting, and repeat the centrifugation and rinsing step once; add 1 mL of 5 μM red fluorescent staining reagent for cell membrane (Dil, Beyotime Biotechnology), gently pipet to resuspend the cells, incubate at 37°C for 5 min, then transfer to a 4°C refrigerator for 15 min to complete the fluorescent labeling; centrifuge again at 600 rpm for 5 min, discard the staining solution, add 100 μL of cell-specific PBS (C0221B, Beyotime Biotechnology), mix by pipetting to obtain the labeled HPC-2 cell suspension, and place on ice for later use; I2. Zebrafish embryo preparation: Select healthy AB strain zebrafish for pairing and breeding, collect 30 fertilized eggs, put them into 10mL of E3 culture medium, and culture them in a constant temperature incubator at 28.5℃ until they are 2 days old; select zebrafish juveniles that are developing normally and without deformities, anesthetize them with an appropriate amount of anesthetic, and arrange them neatly in the groove of an agar plate to expose the yolk sac and the gap between the yolk sac and the tail. I3. Cell transplantation: Using a microinjection apparatus, labeled HPC-2 cell suspensions were transplanted into the yolk sac of zebrafish juveniles (for proliferation detection) and the gap between the yolk sac and the tail (for migration detection), with 5 × 10³ cells transplanted into each juvenile. 4. Incubation and In vivo imaging: After transplantation, zebrafish juveniles were transferred to 6-well plates, with 3 juveniles per well and 5 mL of E3 culture medium added to each well. The plates were then incubated at 32°C. At 2 h, 24 h, 48 h, and 72 h post-transplantation, 6 juveniles were selected for observation and imaging of fluorescence signals using a stereomicroscope. For the yolk sac transplantation group, the fluorescence area at different time points was measured to assess cell proliferation capacity. For the interstitial transplantation group, the diffusion range of the fluorescence signal was observed to assess cell migration capacity.

[0057] like Figure 10 As shown, in the yolk sac transplantation group, the fluorescence area gradually increased with prolonged culture time, and the fluorescence area at 72 h increased more than three times compared to 2 h, confirming that HPC-2 cells have significant proliferation capacity in zebrafish; Figure 11 As shown, in the interstitial transplantation group, fluorescence signals were observed to diffuse towards the tail of zebrafish after 24 hours. Between 48 and 72 hours, the diffusion range further expanded, indicating that HPC-2 cells have active migration ability. This model can be used for research on the mechanisms related to the proliferation and migration of prostate cancer cells and for drug screening.

[0058] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for constructing a human prostate cancer cell line, characterized in that, Includes the following steps: S1. Obtain a tissue sample from the primary prostate cancer lesion, and perform cutting and washing to obtain pre-treated tissue; S2. The pretreated tissues were divided into a fully digested group and an incompletely digested group. The fully digested group was digested with tissue digestion solution to obtain a cell suspension. The incompletely digested group was digested with tissue digestion solution, digestion was terminated early, and the undigested tissue blocks were collected. S3. Inoculate the cell suspension and the incompletely digested tissue block together into a culture container, add complete culture medium, and carry out primary culture to obtain primary cells; S4. Passage the primary cells to obtain a stable cell line, namely the human prostate cancer cell line.

2. The method for constructing a human prostate cancer cell line according to claim 1, characterized in that, In step S1, the cleaning process uses Hank's balanced salt solution, and the material is cut into pieces to 2-4 mm³.

3. The method for constructing a human prostate cancer cell line according to claim 1, characterized in that, The tissue digestion solution in step S2 contains collagenase IV, penicillin, streptomycin, and gentamicin; the concentration of collagenase IV is 900-1100 U / mL, the concentration of penicillin is 150-250 U / mL, the concentration of streptomycin is 150-250 μg / mL, and the concentration of gentamicin is 90-110 μg / mL.

4. The method for constructing a human prostate cancer cell line according to claim 1, characterized in that, In step S3, the complete culture medium consists of McCoy's 5A basal medium and fetal bovine serum in a volume ratio of 16-24:

3.

5. The method for constructing a human prostate cancer cell line according to claim 4, characterized in that, The complete culture medium also contains antibiotics and testosterone, including penicillin, streptomycin and gentamicin, and the concentration of testosterone is 8-12 ng / mL.

6. The method for constructing a human prostate cancer cell line according to claim 1, characterized in that, In step S3, the primary culture temperature is 35-40℃ and the CO2 concentration is 4-6%.

7. A human prostate cancer cell line constructed using the method for constructing a human prostate cancer cell line according to any one of claims 1-6, characterized in that, The human prostate cancer cells described are human prostate cancer cells HPC-2, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:C202649 and deposit date of March 17, 2026.

8. The use of the human prostate cancer cell line according to claim 7 in the preparation of animal models of prostate cancer.

9. The application of the human prostate cancer cell line according to claim 8 in the preparation of animal models of prostate cancer, characterized in that, The prostate cancer animal model is a subcutaneous xenograft model in immunodeficient mice.

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