Cell strain a375-h for constructing melanoma liver metastasis model and construction method and application thereof

Abstract

The application discloses a cell strain A375-H for constructing a melanoma liver metastasis model and a construction method and application thereof, relates to the technical field of biology, and has a preservation number of CGMCC No.46344; and a classification name of A375 high-metastasis melanoma cell strain. A375-H cell strain with high metastasis and liver metastasis potential is efficiently and conveniently obtained by using a continuous 10-round Transwell screening combined with a single clone cell picking method. The A375-H cell strain has high liver metastasis potential, and provides a unique "seed cell" model for studying a liver metastasis mechanism of melanoma.

Description

Technical Field

[0001] This application relates to the field of biological technology, and in particular to a cell line A375-H for constructing a melanoma liver metastasis model, its construction method, and its application. Background Technology

[0002] Melanoma is a highly malignant tumor formed by the malignant transformation of melanocytes in the skin and other organs. Its high metastatic rate and ease of metastasis are important factors contributing to poor clinical prognosis.

[0003] The liver is one of the most common sites of distant metastasis in melanoma. The incidence of liver metastasis in cutaneous melanoma patients is approximately 20%, while the rate is as high as 89% in uveal melanoma; the incidence of liver metastasis in patients with advanced melanoma is as high as 50-80%. Once liver metastasis occurs, the prognosis of melanoma patients deteriorates rapidly, with a median overall survival of less than 6 months, significantly lower than that of patients with lung metastasis (median OS 12-15 months) and subcutaneous metastasis (median OS 18-24 months). Liver metastasis is a hallmark event of melanoma entering a rapid deterioration phase. Therefore, in-depth research into the molecular mechanisms of melanoma liver metastasis has significant theoretical value and an urgent clinical translational need. Current research methods involve constructing animal models of melanoma liver metastasis and then conducting related studies.

[0004] Currently, the main methods for constructing melanoma liver metastasis models in experimental animals are: 1. injection of tumor cells into the spleen; 2. implantation of orthotopic tumors. However, both of these methods have high requirements for experimental conditions (sterile environment) and experimental procedures (surgical suturing), and the proportion of experimental animals developing liver metastasis after these procedures is low, resulting in a low success rate. Although the A375 cell line is widely used to construct melanoma liver metastasis models, it lacks stable liver metastasis characteristics.

[0005] Therefore, there is an urgent need to conduct relevant research on methods that can efficiently induce liver metastasis in melanoma.

[0006] The information disclosed in the background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] This application addresses the aforementioned technical problems by providing a cell line A375-H for constructing a melanoma liver metastasis model, along with its construction method and application. Cell line A375-H can efficiently construct a liver metastasis animal model by intravenous injection into experimental animals via the tail vein, thus solving the problems of complex operation and difficulty in constructing melanoma liver metastasis animal models in existing technologies.

[0008] This application provides a cell line A375-H for constructing a melanoma liver metastasis model, with accession number CGMCC No. 46344; classified and named as: A375 highly metastatic melanoma cell line.

[0009] Another aspect of this application provides a method for constructing a melanoma liver metastasis model, wherein a cell suspension containing the cell line A375-H used to construct the melanoma liver metastasis model is injected into the tail vein of a selected experimental animal.

[0010] Preferably, the concentration of cell line A375-H in the cell suspension is 1.5 × 10⁻⁶. 6 100μL ~ 2×10 6 100μL / piece.

[0011] Preferably, the preparation of the cell suspension includes: taking the logarithmic growth phase cell line A375-H, digesting and centrifuging to collect the cell pellet, and then resuspending the cell line A375-H in PBS.

[0012] Preferably, the number of liver metastases in experimental animals is 6-8 25 days after tail vein injection, thus establishing a melanoma liver metastasis animal model.

[0013] Another aspect of this application provides the application of the A375-H cell line, as described above, in the construction of a melanoma liver metastasis model.

[0014] Another aspect of this application also provides a method for screening the cell line A375-H for constructing a melanoma liver metastasis model as described above, comprising the following steps:

[0015] Using human melanoma A375 as parental cells, the parental cells were screened for migration and invasion using Transwell to obtain secondary cells that had completed migration and invasion. The secondary cells were then screened for migration and invasion again using Transwell. After at least 10 rounds of Transwell migration and invasion screening, a single-cell suspension was obtained.

[0016] After dilution, culture the single-cell suspension until a single cell clone with 30-50 cells is clearly observed under a microscope. Stop the culture and select target clones with regular morphology, no contact, spacing >5mm, and 30-50 cells per clone for single-clone selection and cell passage culture to obtain the A375-H cell line.

[0017] Preferably, the Transwell migration invasion screening includes the following steps:

[0018] (1) Use sterile forceps to remove the upper Transwell chamber from the 6-well culture plate and add 2 mL of cell culture medium II preheated to 37°C to each lower chamber of the 6-well culture plate;

[0019] (2) Remove the upper Transwell chamber and place it back into the corresponding 6-well culture plate well, ensuring that the membrane at the bottom of the chamber is in contact with the cell culture medium II in the lower chamber but without any air bubbles blocking it;

[0020] (3) Select A375 melanoma parental cells in the logarithmic growth phase with good morphology, rinse twice with phosphate buffer preheated to 37°C, add 1 mL of 0.25% trypsin to digest the cells, observe under a microscope when the cells become round and the gaps increase, immediately add an equal volume of cell culture medium I to stop digestion, and repeatedly and gently blow the tube wall with a pipette to prepare a single cell suspension;

[0021] (4) Use a hemocytometer to count cells, and dilute the cell suspension with cell culture medium I to a density of 6.0 × 10⁻⁶. 6 To obtain a cell suspension, take 1 mL of the cell suspension and add it to the upper Transwell chamber.

[0022] (5) Transfer the 6-well culture plate containing the cells and culture medium, which contains the Transwell chamber system, to a cell culture incubator set at 37°C and 5% CO2, and culture for 48 hours.

[0023] (6) After the culture is completed, remove the 6-well plate from the incubator, use sterile forceps to remove the upper chamber of the Transwell from the well of the 6-well plate and discard it, aspirate the culture medium in the lower chamber of the 6-well plate, and gently wash the cells twice with PBS preheated to 37°C.

[0024] (7) Add 1 mL of 0.25% trypsin preheated to 37°C to each well in the lower chamber of a 6-well plate. After confirming that the cells at the bottom of the well have completely detached under a microscope, add 1 mL of cell culture medium I to stop digestion. Gently and repeatedly pipette the cells to make a single-cell suspension.

[0025] Preferably, monoclonal selection includes the following steps:

[0026] (1) Use sterile forceps to pick up the cloning loop, immerse it in 0.5% low melting point agarose at 45°C, and vertically place the cloning loop over the target clone;

[0027] (2) Add 50 μL of preheated 0.25% trypsin to the ring. The liquid surface should completely cover the clone digested cells. Observe under a microscope until the cells become round and the refractive index of the edges increases. Then add 100 μL of cell culture medium III to stop digestion.

[0028] (3) Gently pipette the area inside the loop to completely detach the cells, and transfer the cell suspension to the wells of a 24-well plate pre-filled with 500 μL of cell culture medium III. After the addition is complete, place the 24-well plate back into a 37°C, 5% CO2 constant temperature cell culture incubator for continued culture.

[0029] Among them, cell culture medium I: DMEM high glucose medium without fetal bovine serum; cell culture medium II: DMEM high glucose medium containing 25% fetal bovine serum; cell culture medium III: DMEM high glucose medium containing 10% fetal bovine serum.

[0030] Preferably, cell passaging includes the following steps:

[0031] (1) After observing that the cell fusion meets the requirements, discard the culture medium, wash the cells with PBS 1-2 times, add 200μL of trypsin to digest the cells, and when the cells shorten and become round, add 200μL of cell culture medium III to stop the digestion, resuspend and collect the cells, transfer them to a sterile EP tube, centrifuge, discard the supernatant, resuspend the cells with 1mL of fresh cell culture medium III, and transfer them to a new 6-well plate, and put them back into a 37℃, 5% CO2 constant temperature cell culture incubator for continued culture;

[0032] (2) When the cells reach 80-90% confluence in the well, repeat trypsin digestion, centrifugation, resuspension, well separation, and bottle inoculation. Passage is performed at a ratio of 1:2 to 1:4. Through continuous and stable in vitro passage culture for more than 15 generations, cell line A375-H is obtained.

[0033] The beneficial effects that this application can produce include:

[0034] 1) The A375-H cell line for constructing a melanoma liver metastasis model provided in this application and its application: A 10-round Transwell screening combined with monoclonal cell selection method efficiently and conveniently obtained the A375-H cell line with high metastatic potential and liver metastasis potential. The A375-H cell line has a high potential for invasive liver metastasis, providing a unique "seed cell" model for studying the liver metastasis mechanism of melanoma.

[0035] 2) The method for constructing a melanoma liver metastasis model provided in this application allows for the construction of a melanoma liver metastasis animal model simply by injecting the A375-H cell line into the tail vein of experimental animals, thus simplifying the process. Cell scratch assays, Transwell invasion and migration experiments, and tail vein animal model validation revealed that the obtained A375-H cell line exhibits strong metastasis and invasion capabilities in the liver of experimental animals. A liver model can be constructed in experimental animals only 25 days after tail vein injection of the cell suspension, effectively shortening the time required for animal model construction.

[0036] A375-H cells were deposited on April 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 46344; and classified as: A375 highly metastatic melanoma cell line. Attached Figure Description

[0037] Figure 1 The results of the cell scratch assay for A375 and A375-H cells in Example 3 of this application show the migration ability of A375 and A375-H cells; where a represents A375 cells cultured for 0 h; b represents A375-H cells cultured for 0 h; c represents A375 cells cultured for 24 h; d represents A375-H cells cultured for 24 h; and e represents the statistical results of comparing the migration ability of A375 and A375-H cells using the cell scratch assay.

[0038] Figure 2 This image shows the results of the Transwell assay comparing the invasion and migration abilities of A375 and A375-H cells in Example 4 of this application. In the image, a represents the migration results of A375 cells; b represents the migration results of A375-H cells; c represents the statistical results of the Transwell assay comparing the migration abilities of A375 and A375-H cells; d represents the invasion results of A375 cells; e represents the invasion results of A375-H cells; and f represents the results of the Transwell assay comparing the invasion abilities of A375 and A375-H cells.

[0039] Figure 3 The results of qPCR and Western Blot methods for detecting the mRNA and protein expression levels of EMT-related molecules in A375-H cells in Example 5 of this application are shown. Among them, A is the mRNA level result of qPCR method for detecting EMT-related molecules MMP2 and MMP9 in A375-H cells; B is the protein expression level of EMT-related molecules MMP2 and MMP9 in A375-H cells detected by Western Blot method (with Tubulin as the internal control); and C is the statistical result of gray value of MMP2 and MMP9 expression detected by Western Blot.

[0040] Figure 4This document presents a comparison of liver metastasis results using A375 and A375-H cells in an animal model, as described in Example 6 of this application. A shows imaging results from a small animal imaging system on nude mice after 15 and 25 days of feeding following intravenous injection of A375 and A375-H cells, respectively. B is a bar chart showing the normalized fluorescence intensity of A375 and A375-H cells. C is a comparative photograph of the liver and lungs of nude mice after 25 days of feeding following intravenous injection of A375 and A375-H cells, respectively. D is a photograph of liver tumor cell infiltration obtained from dissection of nude mice after 25 days of feeding following intravenous injection of A375 and A375-H cells, respectively. E is a bar chart showing the average number of liver metastases in six nude mice after 25 days of feeding following intravenous injection of A375 and A375-H cells, respectively. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0042] Unless otherwise specified, all materials and instruments used in the following embodiments were obtained through commercial channels; and all detection methods used are existing methods unless otherwise specified.

[0043] Example 1: Obtaining the A375-H cell line

[0044] I. Experimental Materials

[0045] 1. Source of A375-H cell line selection: The A375 cell line was purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. This cell line was isolated from the skin tissue of a 54-year-old female patient with malignant melanoma and exhibited epithelial-like morphological characteristics.

[0046] 2. Cell culture medium

[0047] (1) Cell culture medium I: DMEM high glucose medium without fetal bovine serum;

[0048] (2) Cell culture medium II: DMEM high glucose medium containing 25% fetal bovine serum;

[0049] (3) Cell culture medium III: DMEM high glucose medium containing 10% fetal bovine serum.

[0050] 3. Other experimental materials

[0051] (1) Transwell 6-well plate:

[0052] (2) Experimental animals: BALB / c nude mice; sex: male; age: 6-8 weeks; weight: 18-25g.

[0053] II. Screening Methods for A375-H Cell Lines

[0054] Using human melanoma A375 as the parental cell line, a highly aggressive melanoma subline, A375-H, was obtained through 10 consecutive rounds of Transwell assays combined with monoclonal selection. The specific methods are as follows:

[0055] 1. Transwell migration / invasion screening:

[0056] (1) Use sterile forceps to remove the Transwell chamber (Corning, PC membrane, 24 mm, 8 μm) from the 6-well culture plate. Dilute serum-free medium: matrix gel (BD Biosciences, USA) = 8:1, take 1 mL and spread it evenly on the 6-well plate chamber, and incubate overnight at 37°C.

[0057] (2) On the second day, take out the 6-well plate from operation (1) and add 2 mL of cell culture medium II preheated to 37°C to each lower chamber (basal cavity) of the 6-well culture plate.

[0058] (3) Carefully and steadily place the previously removed Transwell chamber (upper chamber) back into the corresponding 6-well culture plate well, ensuring that the bottom membrane of the chamber is in contact with the cell culture medium II of the lower chamber but without any air bubbles blocking it.

[0059] (4) Select A375 melanoma cells in the logarithmic growth phase with good morphology. Discard the original culture medium and gently wash the cells twice with phosphate-buffered saline (PBS, pH 7.4) preheated to 37°C to remove residual serum. Add 1 mL of 0.25% trypsin to digest the cells. When the cells become rounded and the gaps between them increase under a microscope, immediately add an equal volume of cell culture medium I to stop the digestion. Gently pipette the cells repeatedly to prepare a single-cell suspension.

[0060] (5) Cell counting was performed using a hemocytometer. The cell suspension was diluted with Cell Culture Medium I to a density of 6.0 × 10⁻⁶. 6 Cells / mL. Take 1 mL of cell suspension and carefully add it to the upper chamber (i.e., insertion chamber) of the Transwell chamber.

[0061] (6) Transfer the 6-well culture plate (containing the Transwell chamber system) loaded with cells and culture medium to a cell culture incubator set at 37°C and 5% CO2 and culture for 48 hours.

[0062] (7) After culture is complete, remove the 6-well plate from the incubator. Use sterile forceps to remove the upper Transwell chamber from the well of the 6-well plate and discard it. Aspirate the culture medium from the lower chamber (basal cavity) of the 6-well plate and gently wash the cells twice with PBS preheated to 37°C to remove residual serum.

[0063] (8) Add 1 mL of 0.25% trypsin preheated to 37°C to each well in the lower chamber of a 6-well plate. After confirming that the cells at the bottom of the well have completely detached under a microscope, add 1 mL of cell culture medium I to stop digestion. Gently and repeatedly pipette the cells to make a single-cell suspension.

[0064] (9) Repeat steps (1) to (7) above for 10 cycles. Each cycle uses cells (single-cell suspension) that migrated from the previous round of screening as the starting cells.

[0065] (10) After completing 10 rounds of screening, collect the cell suspension obtained from the last cycle. Count the cells using a hemocytometer. Dilute the cell suspension to 500 cells / mL with cell culture medium III preheated to 37°C. Take 1 mL of this diluted low-density cell suspension and add it to a single well of a new standard 6-well culture plate.

[0066] (11) Place the ordinary 6-well culture plate from step (9) back into a 37°C, 5% CO2 constant temperature cell culture incubator for continued culture.

[0067] (12) During the culture process, observe the cell growth and clone formation under a microscope. Stop the culture when the number of cells in an independent cell clone reaches 30-50 and can be clearly observed under a microscope.

[0068] 2. Monoclonal loop selection of monoclonal cells:

[0069] (1) Selecting cell clones: Observe the 6-well plate of step (11) above under an inverted microscope, and use a marker to circle the target clone (regular shape, no contact, spacing > 5 mm, cell number in the cell clone reaches 30-50) on the outside of the bottom of the culture plate. Aspirate the culture medium and gently add preheated PBS to rinse once.

[0070] (2) Cloning loop fixation: Use sterile forceps to pick up the cloning loop, immerse it in 45°C, 0.5% low melting point agarose, shake off the excess liquid, and vertically place the cloning loop over the target clone, pressing gently to ensure a tight seal between the bottom of the loop and the culture dish. Note: The diameter of the loop should be 1-2 mm larger than the edge of the clone to avoid contact with the cells.

[0071] (3) Digestion of cell clones: Add 50 μL of preheated 0.25% trypsin to the loop, ensuring the liquid completely covers the digested cells. Observe under a microscope until the cells become round and the refractive index of the edges increases, then add 100 μL of cell culture medium III to terminate the digestion.

[0072] (4) Cloning and expansion: Gently pipette the area within the loop to completely detach the cells, and transfer the cell suspension to a 24-well plate (pre-added with 500 μL of cell culture medium III). Place the 24-well plate back into a 37°C, 5% CO2 incubator for further culture.

[0073] 3. Cell passage culture:

[0074] (1) Microscopic observation: When the cell confluence reaches about 90%, discard the culture medium, wash the cells with PBS 1-2 times, and add 200 μL of trypsin to digest the cells. Observe the cell state under a microscope. When the cells shorten and become rounded, add 200 μL of cell culture medium III to stop the digestion. Resuspend and collect the cells, transfer them to sterile EP tubes, and centrifuge (1000 rpm / min, 5 min). Discard the supernatant, resuspend the cells with 1 mL of fresh cell culture medium III, and transfer them to a new 6-well plate. Place the plate back into a 37℃, 5% CO2 incubator for further culture.

[0075] (2) Cell line establishment: The cells from step (1) were passaged regularly. When the cells reached 80-90% confluence in the wells, the trypsin digestion, centrifugation, resuspension, and seeding process in separate wells / bottles were repeated (usually passaged at a ratio of 1:2 to 1:4). Through continuous passage culture (usually more than 15 generations), a melanoma cell subline that could proliferate stably, maintain the expected morphological characteristics, and had liver metastasis potential was successfully constructed and named A375-H. Subsequently, the cell line was subjected to SRT detection (see Example 2) and in vivo and in vitro metastasis potential verification (see Examples 3-5).

[0076] A375 cells were seeded in the upper chamber of a Transwell plate (containing 5% FBS medium), while the lower chamber was supplemented with 15% FBS medium as a chemotactic source. After incubation at 37°C for 48 hours, cells that had transmembrane were collected. This process was repeated for 10 consecutive rounds. The selected cells were diluted to 500 cells / mL and seeded into 6-well plates. Well-grown single clones (30-50 cells) were selected under a microscope, digested with trypsin, and then expanded in DMEM medium containing 10% FBS to obtain the stable cell line A375-H.

[0077] Example 2: STR identification results of A375-H cell line

[0078] The obtained A375-H cells and parental A375 cells were sent to the Kunming Cell Bank of the Kunming Institute of Zoology, Chinese Academy of Sciences for STR cell identification. The results are shown in Table 1.

[0079] Table 1

[0080]

[0081] In Table 1, A375 (KCB) refers to parental A375 cells purchased from the Kunming Cell Bank of the Chinese Academy of Sciences; for testing, the testing institution may choose to purchase A375 cells from ATCC (American Center for Type Culture Collection) or DSMZ (German Center for Microbiology and Cell Culture Collection) for testing.

[0082] The identification results show that: ① STR analysis revealed no third allele in the A375-H cell line, ruling out the possibility of cross-contamination from human cell lines. ② The STR typing data of the A375-H cell line at all loci were completely consistent with the A375 cell line data recorded in the ATCC, KCB, and DSMZ databases. Therefore, the A375-H cells are indeed A375 cells.

[0083] The obtained A375-H cells were deposited on April 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China; accession number: CGMCC No. 46344; and classified as: A375 highly metastatic melanoma cell line.

[0084] Example 3: Cell scratch assay comparing the migration ability of A375 and A375-H cells.

[0085] A375 and A375-H cells were divided into groups of 5 × 10 5 Cells were seeded at a density of cells / well in 6-well plates and cultured to 100% confluence in DMEM high-glucose medium containing 10% FBS. The cell layer was vertically scraped off using a 200 μL sterile pipette tip, creating a scratch approximately 0.5 mm wide. Cells were washed three times with PBS to remove detached cells, and the medium was replaced with maintenance medium containing 1% FBS. Images were taken at 0 h and 24 h under an inverted microscope in a fixed position. Figure 1 in a~e).

[0086] ImageJ software calculates the relative healing area of ​​scratches. .

[0087] Depend on Figure 1 As can be seen from a to e, the migration ability of A375-H cells is significantly stronger than that of A375 cells.

[0088] Example 4: Transwell assay to evaluate the invasion and migration capabilities of A375 and A375-H cells.

[0089] Pre-coat the upper chamber (Corning, 8μm wells) of a 24-well Transwell plate with 50μL of Matrigel (1:8 = Matrigel: Serum-free DMEM) and cure at 37°C for 1 hour. Add 600μL of DMEM (chemokine) containing 15% FBS to the lower chamber. Seed 1×10⁶ cells / well in the upper chamber.6 Cells (5% FBS in DMEM). After incubation at 37°C for 48 hours, the chamber was removed, and uninvaded cells in the upper chamber were wiped off with a cotton swab. The cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.3% crystal violet for 20 min. Five fields of view were randomly selected under a microscope to count the cells that had penetrated the membrane. Transwell migration assay results are shown below. Figure 2 The results of the invasion experiments for a and b are as follows: Figure 2 d and e.

[0090] Migration experiment procedure: The steps are the same as the invasion experiment, except that the matrix gel plating step is omitted, and cells are directly seeded into the upper chamber.

[0091] Depend on Figure 2 As can be seen from a to f, the invasion and migration abilities of A375-H cells are significantly stronger than those of A375 cells, with an invasion rate nearly twice that of A375-H cells.

[0092] Example 5: Detection of mRNA and protein expression levels of EMT-related molecules in A375 and A375-H cells using qRCR and Western Blot methods.

[0093] Logarithmic growth phase A375 and A375-H cells were seeded in T25 culture flasks and cultured in DMEM high-glucose medium containing 10% FBS until 80% confluence. The medium was discarded, and the cells were washed twice with PBS. The cells were digested with 1 mL of trypsin until they became round and bright. Digestion was terminated by culturing in 2 mL of DMEM high-glucose medium containing 10% FBS. 1 mL of the cell suspension was placed in an EP tube to extract RNA, and another 2 mL of the cell suspension was used to extract protein. The mRNA and protein expression levels of EMT-related molecules were then detected.

[0094] RNA extraction: Centrifuge to precipitate cells and discard the supernatant. Add 1 mL of TRIzol to each EP tube, incubate on ice for 30 min, then centrifuge (4℃, 12000g, 15 min). Carefully transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, mix gently, and incubate at 4℃ for at least 30 min. Centrifuge (4℃, 12000g, 10 min) to obtain RNA precipitate. Discard the supernatant, add an appropriate amount of 75% ethanol, wash, and centrifuge at 4℃ (12000g, 5 min). Air-dry the precipitate in a clean bench for 3-5 min, dissolve the RNA in 20-50 μL of DEPC water, and determine the concentration and purity (A260 / A280 ≥1.8). The mRNA levels of MMP2 and MMP9 in cells were detected by qRI, as shown in Table 2.

[0095] Table 2 Primer sequences for MMP2 and MMP9

[0096]

[0097] Take 1 μg of the extracted total RNA and perform reverse transcription according to the PrimeScript RT kit instructions. The method calculates the relative expression level of genes.

[0098] Protein extraction: Centrifuge to precipitate cells and discard the supernatant. Add 200 μL of RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) to each EP tube, lyse on ice for 30 min, centrifuge (12,000 rpm, 10 min, 4 °C), and collect the supernatant to determine the protein concentration.

[0099] Western blotting detection of EMT-related protein expression levels: 30 μg protein sample was subjected to SDS-PAGE (separating gel concentration: 10%, stacking gel: 5%), constant voltage 80V, 30 min → 120V, 60 min. The sample was then transferred to a PVDF membrane using a semi-dry transfer method, constant current 220mA, 60 min, transfer buffer containing 10% methanol. The membrane was blocked with 5% skim milk (prepared with TBST) at room temperature for 1 h. Incubation was performed overnight at 4℃. The membrane was washed three times with MMP2 or MMP9 primary antibody (1:2000), 10 min each time, with the corresponding HRP-labeled secondary antibody (1:10000), and incubated at room temperature for 1 h. Development and quantification: Exposure with ECL chemiluminescence reagent, and analysis of band grayscale values ​​using ImageLab software.

[0100] relative expression level of target protein

[0101] The results are as follows Figure 3 As shown in A~C, by Figure 3 As seen in cells A through C, compared to A375 cells, A375-H cells showed significantly increased expression of EMT-related molecules MMP2 and MMP9.

[0102] Example 6: Construction of an animal model of melanoma liver metastasis using A375 and A375-H cells

[0103] Nude mouse tail vein injection model (liver metastasis potential verification): Sixteen male BALB / c nude mice (6-8 weeks old, weighing 18-25g) were randomly divided into two groups, n=8 in each group. Logarithmically growing A375 and A375-H cells were collected, digested, and centrifuged to collect the cell pellet. Cells were counted, and an appropriate amount of cells was resuspended in PBS to a concentration of 2×10⁻⁶. 6 Cells / 100μL. Nude mice were fixed using a tail vein syringe, their tails were disinfected with 75% alcohol, and the cell suspension was slowly injected.

[0104] The nude mice were observed daily after injection. From the third day onwards, tumor metastasis was monitored every three days using a small animal imaging system. On day 25, the mice were euthanized, and the livers were completely dissected. Tissue samples were fixed in 4% paraformaldehyde. The tissues were embedded in paraffin, prepared into paraffin sections, and H&E staining was performed to confirm tumor cell infiltration. Simultaneously, the number of liver metastases on the livers of nude mice after 25 days of feeding following tail vein injections of A375 and A375-H cells was statistically analyzed and averaged. The results are shown in [Figure number missing]. Figure 4 Middle A~E, Figure 4 The results showed that after 25 days of feeding, mice inoculated with A375-H cells had a greater number and area of ​​metastatic lesions than mice inoculated with A375 cells.

[0105] Figure 4 The results showed that mice inoculated with A375-H cells had significantly higher photon flux compared to mice inoculated with A375 cells, especially after 25 days of feeding. This indicates that the number of metastatic lesions formed in mice inoculated with A375-H cells was higher than that in mice inoculated with A375 cells.

[0106] Figure 4 The results showed that mice inoculated with A375-H cells had a significantly higher number of liver metastases compared to mice inoculated with A375 cells, and also had a higher number of lung metastases.

[0107] Figure 4 The results of the D~E experiment showed that mice inoculated with A375-H cells had significantly larger and more numerous liver metastases compared to mice inoculated with A375 cells.

[0108] Depend on Figure 4 As shown in Figures A through E, both A375 and A375-H cells injected into the tail vein of nude mice resulted in systemic metastasis. After 25 days of feeding, the average number of liver metastases in six mice was only one in the nude mice inoculated with A375 cells, while the number of liver metastases in the nude mice inoculated with A375-H cells reached 6-8, indicating a more significant trend towards liver metastasis. These results demonstrate that the tail vein injection of A375-H cells provided in this application enables the rapid construction of a melanoma liver metastasis model.

[0109] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cell line A375-H for constructing a melanoma liver metastasis model, characterized in that, The cell line A375-H has the accession number CGMCC No. 46344 and is classified as: A375 highly metastatic melanoma cell line.

2. A method for constructing a melanoma liver metastasis model, characterized in that, The selected experimental animals were injected with a cell suspension containing the cell line A375-H as described in claim 1 for constructing a melanoma liver metastasis model via the tail vein.

3. The construction method according to claim 2, characterized in that, The concentration of cell line A375-H in the cell suspension was 1.5 × 10⁻⁶. 6 100μL ~ 2×10 6 100μL / piece.

4. The construction method according to claim 3, characterized in that, The preparation of the cell suspension includes: taking the logarithmic growth phase cell line A375-H, digesting and centrifuging to collect the cell pellet, and then resuspending the cell line A375-H in PBS.

5. The application of the cell line A375-H as described in claim 1 for constructing a melanoma liver metastasis model in the construction of a melanoma liver metastasis model.

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