Cell strain A375-H for constructing melanoma liver metastasis model as well as construction method and application of cell strain A375-H

Abstract

The invention discloses a cell strain A375-H for constructing a melanoma liver metastasis model as well as a construction method and application of the cell strain A375-H, and relates to the technical field of biology, and the preservation number of the cell strain A375-H is CGMCC No.46344. The cell strain is classified and named as an A375 high metastatic melanoma cell strain. The A375-H cell strain with high metastatic property and liver metastatic potential is efficiently and conveniently obtained by adopting a continuous 10-round Transwell screening combined monoclonal cell selection method. The A375-H cell strain has high liver metastasis invasion potential, and a unique seed cell model is provided for researching the liver metastasis mechanism of melanoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biology, in particular to a cell strain A375-H for constructing a melanoma liver metastasis model and a construction method and application thereof. BACKGROUND

[0002] Melanoma is a highly malignant tumor formed by malignant transformation of skin and other organ melanocytes, and its high metastasis and easy metastasis characteristics are important factors for poor clinical prognosis.

[0003] The liver is one of the most common sites of distant metastasis of melanoma. The incidence of liver metastasis in patients with skin melanoma is about 20%, while the liver metastasis rate of uveal melanoma is as high as 89%; the incidence of liver metastasis in patients with advanced melanoma is as high as 50-80%. Once melanoma patients develop liver metastasis, their prognosis deteriorates dramatically, with a median overall survival of usually less than 6 months, which is 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 landmark event for melanoma to enter the rapid deterioration stage. Therefore, it is of important theoretical value and urgent clinical translation demand to deeply explore the molecular mechanism of melanoma liver metastasis. The existing research method is to construct an animal model of melanoma liver metastasis and then conduct related research.

[0004] At present, the experimental animal model of melanoma liver metastasis is mainly constructed by 1, intrasplenic injection of tumor cells; 2, implantation of orthotopic tumor, but these two experimental methods have high requirements for experimental conditions (sterile environment), experimental operation (surgical suture), and the proportion of experimental animals developing liver metastasis after operation is low, and the success rate is low. Although the A375 cell line is widely used to construct a melanoma liver metastasis model, the cell line lacks stable liver metastasis characteristics.

[0005] Therefore, it is urgent to conduct related research on efficiently inducing melanoma to develop liver metastasis.

[0006] The information disclosed in the background section is only intended to increase the understanding of the general background of the application and should not be considered as admitting or in any form suggesting that the information constitutes prior art that is known to those of ordinary skill in the art. SUMMARY

[0007] The present application provides a cell strain A375-H for constructing a melanoma liver metastasis model and a construction method and application thereof, which can efficiently construct a liver metastasis animal model by tail vein injection into experimental animals, solving the problems of complex operation and difficulty in constructing a melanoma liver metastasis animal model in the prior art.

[0008] The application provides a cell strain A375-H for constructing a melanoma liver metastasis model, which is preserved under the number of CGMCC No. 46344 and classified as A375 high-metastatic melanoma cell strain.

[0009] The application also provides a method for constructing a melanoma liver metastasis model, which comprises injecting a cell suspension containing the cell strain A375-H for constructing a melanoma liver metastasis model as described above into the tail vein of a selected experimental animal.

[0010] Preferably, the concentration of the cell strain A375-H in the cell suspension is 1.5×10 6 cells / 100 μL to 2×10 6 cells / 100 μL.

[0011] Preferably, the preparation of the cell suspension comprises the following steps: taking the cell strain A375-H in the logarithmic growth phase, digesting and centrifuging to collect the cell precipitate, and then resuspending the cell strain A375-H in PBS.

[0012] Preferably, the number of liver metastasis foci of the experimental animal is 6 to 8 after 25 days of tail vein injection, and the melanoma liver metastasis animal model is constructed.

[0013] The application also provides the application of the cell strain A375-H for constructing a melanoma liver metastasis model as described above in constructing a melanoma liver metastasis model.

[0014] The application also provides a screening method for the cell strain A375-H for constructing a melanoma liver metastasis model as described above, which comprises the following steps: Taking human melanoma A375 as the parent cell, the secondary cell which completes migration and invasion is obtained through Transwell migration and invasion screening of the parent cell, the secondary cell is subjected to Transwell migration and invasion screening again, and after at least 10 times of Transwell migration and invasion screening, a single cell suspension is obtained; After dilution, the single cell suspension is cultured until the number of cells in an independent cell clone reaches 30-50 under microscopic observation, and then the culture is stopped; the target clone which has a regular shape, no contact, a distance of more than 5 mm and a cell number of 30-50 in the cell clone is selected for single clone picking method and cell subculture to obtain the A375-H cell strain.

[0015] Preferably, the Transwell migration and invasion screening comprises the following steps: (1) taking out the upper chamber Transwell chamber from the 6-hole culture plate with a sterile forceps, and adding 2 mL of cell culture medium II preheated to 37 DEG C into each lower chamber of the 6-hole culture plate; (2) Take out the upper chamber Transwell chamber and put it back into the corresponding 6-hole culture plate hole, make sure that the bottom membrane of the chamber is in contact with the lower chamber cell culture medium II but no bubble blockage; (3) Select A375 melanoma parent cells in logarithmic growth phase and good morphology, rinse twice with preheated phosphate buffer solution at 37°C, add 1 mL of 0.25% trypsin to digest the cells, and observe the cells under a microscope when the cells are rounded and the gap is enlarged. Immediately add an equal volume of cell culture medium I to stop digestion, and use a pipette to repeatedly and gently blow the tube wall to prepare a single cell suspension; (4) Use a hemocytometer to count the cells, and dilute the cell suspension to a density of 6.0 x 10 6 cells / mL with cell culture medium I to obtain a cell suspension. Take 1 mL of the cell suspension and add it to the upper chamber Transwell chamber. (5) Place the 6-hole culture plate containing the cell and medium loaded above into a 37°C, 5% CO2 cell incubator and continue to culture for 48 hours. (6) After the culture is completed, take the 6-hole plate out of the incubator and use sterile forceps to remove the Transwell upper chamber from the 6-hole plate hole and discard it. Remove the culture medium in the lower chamber of the 6-hole plate and gently rinse the cells twice with preheated PBS at 37°C. (7) Add 1 mL of preheated 0.25% trypsin at 37°C to each well of the 6-hole plate, and observe under a microscope to confirm that the cells at the bottom of the well are completely detached. Add 1 mL of cell culture medium I to stop digestion, and gently blow the cells repeatedly to prepare a single cell suspension.

[0016] Preferably, the monoclonal selection includes the following steps: (1) Use sterile forceps to take the cloning ring and immerse it in 45°C 0.5% low-melting-point agarose. Place the cloning ring vertically around the target clone. (2) Add 50 μL of preheated 0.25% trypsin to the ring, making sure that the liquid completely covers the cloning digestion cells. Observe under a microscope until the cells are rounded and the edge refraction is enhanced. Add 100 μL of cell culture medium III to stop digestion. (3) Gently blow the cells in the ring area with a pipette to make them completely fall off. Transfer the cell suspension to a well of a 24-hole plate pre-added with 500 μL of cell culture medium III. After the addition is complete, place the 24-hole plate back in a 37°C, 5% CO2 constant-temperature cell incubator for further culture.

[0017] Among them, cell culture medium I: DMEM high-sugar medium without fetal bovine serum; cell culture medium II: DMEM high-sugar medium containing 25% fetal bovine serum; cell culture medium III: DMEM high-sugar medium containing 10% fetal bovine serum.

[0018] Preferably, the cell subculture includes the following steps: (1) After observing that the cell confluence meets the requirements, discard the culture medium, rinse the cells with PBS for 1-2 times, add 200 μL trypsin to digest the cells, and when the cells are observed to be shortened and rounded, add 200 μL cell culture medium III to terminate the digestion, resuspend and collect the cells, transfer them to a sterile EP tube, centrifuge, discard the supernatant, resuspend the cells with 1 mL of new cell culture medium III, and transfer them to a new 6-well plate, and return to a 37°C, 5% CO2 constant temperature cell incubator for continuous culture; (2) When the cell confluence reaches 80-90% in the well, repeat the trypsin digestion, centrifugation, resuspension, hole distribution, and bottle inoculation, and subculture at a ratio of 1:2 to 1:4. Through continuous and stable in vitro subculture for more than 15 generations, the cell strain A375-H is obtained.

[0019] The beneficial effects that can be produced by the present application include: 1) The cell strain A375-H for constructing a melanoma liver metastasis model and its application provided by the present application, using 10 rounds of continuous Transwell screening combined with single cell cloning method, efficiently and conveniently obtains A375-H cell strain with high metastasis and liver metastasis potential. The A375-H cell strain has a high liver metastasis potential, providing a unique "seed cell" model for studying the liver metastasis mechanism of melanoma.

[0020] 2) The method for constructing a melanoma liver metastasis model provided by the present application, the A375-H cell strain only needs to be injected into the tail vein of an experimental animal, and a melanoma liver metastasis animal model can be constructed, simplifying the construction process of the liver metastasis animal model. Through cell scratch, Transwell invasion and migration experiment, and tail vein animal model verification, it is found that the A375-H cell strain obtained has strong liver metastasis and invasion ability to experimental animals, and a liver model of experimental animals can be constructed only 25 days after injecting the cell suspension into the tail vein of the experimental animals, effectively shortening the time required for constructing the animal model.

[0021] The A375-H cell was preserved in the China General Microbiological Culture Collection Center on April 16, 2025, at address No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, with a postal code of 100101; the preservation number is CGMCC No. 46344; and the classification and naming is A375 high metastatic melanoma cell strain. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Figure 3 is the result of the cell scratch experiment in Example 3 of the present application, showing the migration ability of A375 and A375-H cells; wherein a is the result of A375 cell culture for 0 h; b is the result of A375-H cell culture for 0 h; c is the result of A375 cell culture for 24 h; d is the result of A375-H cell culture for 24 h; e is the statistical result of the cell scratch method for comparing the migration ability of A375 and A375-H cells; Figure 2 Figure 4 is the result of the Transwell experiment in Example 4 of the present application, showing the invasion and migration ability of A375 and A375-H cells; wherein a is the migration result of A375 cells; b is the migration result of A375-H cells; c is the statistical result of the Transwell experiment for comparing the migration ability of A375 and A375-H cells; d is the invasion result of A375 cells; e is the invasion result of A375-H cells; f is the result of the Transwell experiment for comparing the invasion ability of A375 and A375-H cells; Figure 3 Figure 5 is the result of the qPCR and Western Blot method for detecting the mRNA and protein expression level of EMT-related molecules in A375-H cells in Example 5 of the present application; wherein A is the result of the qPCR method for detecting the mRNA level of EMT-related molecules MMP2 and MMP9 in A375-H cells; B is the result of the Western Blot method for detecting the protein expression level of EMT-related molecules MMP2 and MMP9 in A375-H cells (the internal reference is Tubulin), and C is the statistical result of the gray value of the Western Blot detection of MMP2 and MMP9 expression.

[0023] Figure 4 Figure 6 is the result of the animal model for comparing the liver metastasis of A375 and A375-H cells in Example 6 of the present application; wherein A is the result of the small animal imaging instrument imaging of the nude mice tail vein injection of A375 and A375-H cells for 15 days and 25 days, respectively; B is the columnar graph of the normalized fluorescence intensity of A375 and A375-H cells; C is the real object comparison photograph of the liver and lung of the nude mice tail vein injection of A375 and A375-H cells for 25 days, respectively; D is the photograph of the liver tumor cell infiltration of the nude mice tail vein injection of A375 and A375-H cells for 25 days, respectively; E is the columnar graph of the average number of liver metastasis foci of the nude mice tail vein injection of A375 and A375-H cells for 25 days, respectively. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below in combination with the drawings and examples, but the present application is not limited in any way by the following description. Any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0025] The materials and instruments used in the following examples are obtained from commercial channels unless otherwise specified; the detection methods used are existing methods unless otherwise specified.

[0026] Example 1 Obtaining A375-H cell strain I. Experimental materials 1. Screening of A375-H cell strain: A375 cell line was purchased from the Cell Resource Center of Institute of Basic Medicine, Chinese Academy of Medical Sciences. The cell line was isolated from the skin tissue of a 54-year-old female patient with malignant melanoma and showed epithelioid morphological characteristics.

[0027] 2. Cell culture medium (1) Cell culture medium I: DMEM high glucose medium without fetal bovine serum; (2) Cell culture medium II: DMEM high glucose medium containing 25% fetal bovine serum; (3) Cell culture medium III: DMEM high glucose medium containing 10% fetal bovine serum.

[0028] 3. Other experimental materials (1) Transwell 6-well plate: (2) Experimental animals: BALB / c nude mice; gender: male; age: 6-8 weeks; body weight: 18-25 g.

[0029] II. Screening method of A375-H cell strain Using human melanoma A375 as the parent cell, a highly invasive melanoma A375 subline, A375-H cell strain, was obtained by 10 rounds of Transwell combined with single clone selection. The specific method is as follows: 1. Transwell migration / invasion screening: (1) Use sterile forceps to remove the Transwell chamber (Corning, PC membrane, 24 mm, 8 μm) from the 6-well culture plate. Serum-free medium: Matrigel (BD Biosciences, USA) = 8:1 dilution, take 1 mL and evenly spread on the upper chamber of the 6-well plate, and stand overnight in a 37°C incubator.

[0030] (2) The next day, take out the 6-well plate in step (1), and add 2 mL of preheated cell culture medium II to each lower chamber (basal cavity) of the 6-well culture plate.

[0031] (3) Carefully and smoothly put the aforementioned removed Transwell chamber (upper chamber) back into the corresponding 6-well culture plate hole, ensuring that the bottom membrane of the chamber is in contact with the cell culture medium II in the lower chamber without bubble obstruction.

[0032] (4) Select the logarithmic growth phase, good A375 melanoma cells. Discard the original culture medium, with preheated to 37 °C phosphate buffer (PBS, pH 7.4) gently rinse the cells 2 times to remove residual serum. Add 1 mL 0.25% trypsin, digest the cells, observed under a microscope cell round, gap increases, immediately add an equal volume of cell culture medium I to stop digestion. With a pipette repeatedly gently blow the wall of the tube, the preparation of single cell suspension.

[0033] (5) Using a hemocytometer for cell counting. With cell culture medium I medium to dilute the cell suspension to a density of 6.0 x 10 6 The cells / mL. Take 1 mL of cell suspension, carefully added to the upper chamber of the Transwell chamber (ie, inserted into the cavity) in.

[0034] (6) The above loaded cells and medium 6 hole culture plate (containing Transwell chamber system), transfer to set at 37 °C, 5% CO2 cell incubator, continue to culture for 48 hours.

[0035] (7) After the completion of the culture, the 6 hole plate from the incubator. Using sterile forceps Transwell upper chamber from the 6 hole plate hole and discarded. Absorb the culture medium in the 6 hole plate lower chamber (base cavity), with preheated to 37 °C PBS gently rinse the cells 2 times to remove residual serum.

[0036] (8) To 6 hole plate lower chamber each hole 1 mL preheated to 37 °C 0.25% trypsin, observed under a microscope to confirm the hole bottom cell completely detached, add 1 mL cell culture medium I to stop digestion, gently repeatedly blow the cell, the preparation of single cell suspension.

[0037] (9) Repeat the above steps (1) to (7) operation 10 cycles. Each cycle is used in the last round of screening after the migration of the cells (single cell suspension) as the starting cell.

[0038] (10) after 10 rounds of screening, the cell suspension obtained in the last cycle. Using a hemocytometer for counting. With preheated to 37 °C cell culture medium III to dilute the cell suspension to 500 cells / mL. Take 1 mL of this dilution after the low density cell suspension, added to a new ordinary 6 hole culture plate in a single hole.

[0039] (11) the step (9) in the ordinary 6 hole culture plate, placed back to 37 °C, 5% CO2 constant temperature cell incubator for further culture.

[0040] (12) during the culture process, microscopic observation of cell growth and clonal formation. When observed under a microscope a single cell clone within the cell number reached 30-50, stop culture.

[0041] 2. Single clone ring picking a single clone cell: (1) Clone picking: Observe the 6-well plate of step (11) under an inverted microscope, and circle the target clone (regular morphology, no contact, spacing > 5 mm, and cell number in the cell clone reaches 30-50) on the outside of the bottom of the culture plate with a marker pen. Discard the culture solution, and gently add preheated PBS for rinsing once.

[0042] (2) Clone ring fixing: Pick up the clone ring with a sterile forceps, immerse it in 45°C, 0.5% low-melting-point agarose, shake off the excess liquid, vertically put the clone ring around the target clone, and gently press to tightly seal the ring bottom with the culture dish. Note: The ring diameter needs to be 1-2 mm larger than the clone edge to avoid contacting the cells.

[0043] (3) Digesting the cell clone: Add 50 μL of preheated 0.25% trypsin into the ring, and the liquid surface needs to completely cover the digested cells. Observe the cells under a microscope until the cells become round and the edge refraction increases, and then add 100 μL of cell culture medium III to terminate the digestion.

[0044] (4) Clone amplification: Gently blow the area inside the ring with a pipette 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 constant-temperature cell incubator for continuous culture.

[0045] 3. Cell subculture: (1) Microscopic observation: When the cell confluence reaches about 90%, discard the culture medium, rinse the cells with PBS for 1-2 times, and add 200 μL of trypsin to digest the cells. Observe the cell state under a microscope, and when the cells become shortened and round, add 200 μL of cell culture medium III to terminate the digestion. Resuspend and collect the cells, transfer them to a sterile EP tube, and centrifuge (1000 rpm / min, 5 min). Discard the supernatant, resuspend the cells with 1 mL of new cell culture medium III, and transfer them to a new 6-well plate, and place it back into a 37°C, 5% CO2 constant-temperature cell incubator for continuous culture.

[0046] (2) Cell line establishment: Regularly subculture the cells in step (1). When the cell confluence reaches 80-90% in the well, repeat the trypsin digestion, centrifugation, resuspension, and subculturing processes (usually at a ratio of 1:2 to 1:4). Through continuous subculture (usually more than 15 generations), a melanoma cell subline that can stably proliferate, maintain the expected morphological characteristics, and has liver metastasis potential is successfully constructed, which is named A375-H. Subsequently, the SRT detection (see Example 2) and in vitro and in vivo metastasis potential verification (see Examples 3-5) of the cell line are performed.

[0047] A375 cells were seeded on the upper chamber of Transwell (containing 5% FBS medium), and 15% FBS medium was added to the lower chamber as a chemoattractant. After 48 hours of culture at 37°C, transmembrane cells were collected, and the process was repeated for 10 consecutive rounds. The screened cells were diluted to 500 cells / mL and seeded in a 6-well plate. Well-grown single clones (30-50 cells) were selected under a microscope, trypsinized, and expanded in DMEM medium containing 10% FBS to obtain a stable cell line A375-H.

[0048] Example 2 STR identification results of A375-H cell line The obtained A375-H cells and the parent 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: Table 1 In Table 1, A375 (KCB) is the parent A375 cell purchased from the Kunming Cell Bank of the Chinese Academy of Sciences; when detected, the detection agency detects A375 cells purchased from ATCC (American Type Culture Collection) or DSMZ (German Collection of Microorganisms and Cell Cultures) for detection.

[0049] From the identification results, it can be seen that: ① STR analysis shows that the A375-H cell line does not detect the third allele, which excludes the possibility of cross contamination of human cell lines. ② The STR typing data of A375-H cell line at all sites are completely consistent with the data of A375 cell line recorded in the ATCC, KCB and DSMZ databases. Therefore, A375-H cells are A375 cells.

[0050] The obtained A375-H cells were preserved in the China General Microbiological Culture Collection Center on April 16, 2025, at address: No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, with a postal code of 100101; the preservation number is CGMCC No. 46344; the classification name is A375 high-metastatic melanoma cell line.

[0051] Example 3 Cell scratch experiment to compare the migration ability of A375 and A375-H cells A375 and A375-H cells were seeded in a 6-well plate at a density of 5x10 5 cells / well, and cultured in DMEM high glucose medium containing 10% FBS until 100% confluence. A 200 μL sterile gun head was used to vertically scrape off the cell layer to form a scratch about 0.5 mm wide. After washing with PBS for 3 times to remove the detached cells, the medium was replaced with a maintenance medium containing 1% FBS. At 0h and 24h, photographs were taken under an inverted microscope (a-e). Figure 1 ​

[0052] ImageJ software to calculate the relative wound healing area 。

[0053] From Figure 1 As shown in a-e, the migration ability of A375-H cells was significantly stronger than that of A375 cells.

[0054] Example 4 Transwell experiment to evaluate the invasion and migration ability of A375 and A375-H cells 24-well Transwell upper chamber (Corning, 8 μm pore size) was pre-coated with 50 μL Matrigel (1:8 = Matrigel: serum-free DMEM) and solidified at 37°C for 1 hour. The lower chamber was added with 600 μL of DMEM containing 15% FBS (chemotactic source). The upper chamber was inoculated with 1 x 10 6 cells (DMEM containing 5% FBS). After 48 hours of incubation at 37°C, the chamber was removed, and the upper chamber was wiped with a cotton swab to remove the non-invasive cells. The cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.3% crystal violet for 20 min. Five fields were randomly selected under a microscope for counting the cells that passed through the membrane. The results of the Transwell migration experiment are shown in Figure 2 a and b, and the results of the invasion experiment are shown in d and e. Figure 2

[0055] Migration experiment: The operation steps were the same as those of the invasion experiment, except that the Matrigel coating step was omitted, and the cells were directly inoculated into the upper chamber.

[0056] From Figure 2 As shown in a-f, the invasion and migration abilities of A375-H cells were significantly stronger than those of A375 cells, and the invasion rate was nearly 2 times that of A375-H cells.

[0057] Example 5 qRCR and Western Blot methods to detect the mRNA and protein expression levels of EMT-related molecules in A375 and A375-H cells Logarithmic growth phase A375 and A375-H cells were inoculated in T25 culture bottles and cultured in DMEM high glucose medium containing 10% FBS to 80% confluence. The culture medium was discarded, and the cells were washed twice with PBS. The cells were digested with 1 mL trypsin, and the digestion was terminated by culturing the cells with 2 mL DMEM high glucose medium containing 10% FBS. 1 mL of the cell suspension was taken into an EP tube for RNA extraction, and another 2 mL of the cell suspension was used for protein extraction to detect the mRNA and protein expression levels of EMT-related molecules, respectively.

[0058] ​RNA extraction: centrifugal precipitation of cells, discard supernatant. Add 1 mL TRIzol to each EP tube, stand on ice for 30 min, then centrifuge (4°C, 12000g, 15 min), carefully pipette the upper colorless aqueous phase to a new centrifuge tube, then add an equal volume of isopropanol, mix gently, stand at 4°C for 30 min or more. Centrifuge (4°C, 12000g, 10 min) to obtain RNA precipitate. Discard the supernatant, add an appropriate amount of 75% ethanol, wash at 4°C (12000g, 5 min). Air dry the precipitate on the clean bench for 3-5 min, dissolve the RNA with 20-50 μL DEPC water, determine the concentration and purity (A260 / A280 ≥ 1.8). The mRNA levels of MMP2 and MMP9 in cells were detected by qRCR as shown in Table 2.

[0059] Table 2 MMP2 and MMP9 primer sequences Take 1 μg of the total RNA extracted before to reverse transcription, according to the PrimeScript RT kit instructions. Calculate the relative expression of the gene by the 2-ΔΔCt method.

[0060] Protein extraction: centrifugal precipitation of cells, discard supernatant. Add 200 μL 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), take the supernatant to determine the protein concentration.

[0061] Western Blot detection of EMT-related molecular protein expression level: take 30 μg of protein sample for SDS-PAGE (separation gel concentration: 10%, concentration gel: 5%), constant voltage 80V, 30min→ 120V, 60min. Semi-dry transfer to PVDF membrane, constant current 220mA, 60min, the transfer buffer contains 10% methanol. Membrane with 5% skim milk (TBST preparation) room temperature blocking 1h. 4°C overnight incubation, MMP2 or MMP9 primary antibody (1:2000), TBST membrane washing 3 times, 10min / time, add the corresponding HRP labeled secondary antibody (1:10000) room temperature incubation 1h. Development and quantification: ECL chemiluminescence reagent exposure, ImageLab software analysis of band gray value.

[0062] Relative expression of target protein The results are shown in A~C of Table 3, and it can be seen from A~C that compared with A375 cells, the expression of EMT-related molecules MMP2 and MMP9 in A375-H cells is significantly increased. Figure 3 Figure 3 ​​​

[0063] Example 6: Construction of an animal model of melanoma liver metastasis using A375 and A375-H cells 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Depend on Figure 4As can be seen from A to E, after the A375 and A375-H cells were injected into the tail veins of the nude mice, systemic metastasis occurred; and after the mice were fed for 25 days, the average results obtained from 6 mice were as follows: the number of liver metastasis foci of the nude mice inoculated with the A375 cells was only 1, while the number of liver metastasis foci of the nude mice inoculated with the A375-H cells was 6 to 8, and the liver metastasis trend was more significant. From the above results, it can be known that the rapid construction of the melanoma liver metastasis model is realized by the tail vein injection of the A375-H cells provided in the application.

[0069] Although the present application has been described in detail with reference to the foregoing embodiments, technical solutions recorded in the foregoing embodiments can be modified or some technical features can be replaced by equivalent ones by those skilled in the art, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A cell line A375-H for constructing a melanoma liver metastasis model, characterized in that, The preservation number is CGMCC No. 46344, and the classification name is A375 high-metastatic melanoma cell strain.

2. A method of constructing a melanoma liver metastasis model, characterized by, The selected experimental animals are injected with a cell suspension containing the A375-H cell strain for constructing a melanoma liver metastasis model according to claim 1 in the tail vein.

3. The construction method of claim 2, wherein, The concentration of cell line A375-H in the cell suspension was 1.5 x 10 6 cells / 100 μL to 2 x 10 6 cells / 100 μL.

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

5. The construction method of claim 3, wherein, Twenty-five days after the tail vein injection, the number of liver metastasis foci of the experimental animals is 6-8, and the melanoma liver metastasis animal model is constructed.

6. The use of the A375-H cell strain for constructing a melanoma liver metastasis model according to claim 1 in constructing a melanoma liver metastasis model.

7. A screening method for cell line A375-H for constructing a model of melanoma liver metastasis according to claim 1, characterized in that, The method comprises the following steps: Taking human melanoma A375 as the parent cell, the secondary cell that completes migration and invasion is obtained by Transwell migration and invasion screening of the parent cell, and the secondary cell is subjected to Transwell migration and invasion screening again. After at least 10 times of Transwell migration and invasion screening, a single cell suspension is obtained; After dilution, the single cell suspension is cultured until the number of cells in an independent cell clone reaches 30-50 under a microscope, and the culture is stopped. The target clone with regular shape, no contact, spacing > 5 mm, and cell number in the cell clone reaching 30-50 is selected for single clone selection, and the A375-H cell strain is obtained by cell subculture.

8. The screening method according to claim 7, characterized in that, The Transwell migration and invasion screening comprises the following steps: (1) The upper chamber Transwell chamber is taken out from the 6-well culture plate with sterile forceps, and 2 mL of cell culture medium II preheated to 37°C is added to each lower chamber of the 6-well culture plate; (2) Take out the upper chamber Transwell chamber and put it back into the corresponding 6-well culture plate hole, ensuring that the bottom membrane of the chamber is in contact with the lower chamber cell culture medium II but no bubble blockage; (3) Select the A375 melanoma parent cell in the logarithmic growth phase and with good morphology, rinse it twice with phosphate buffered saline preheated to 37°C, add 1 mL of 0.25% trypsin to digest the cells, and when the cells are observed to be rounded and the gap is enlarged under a microscope, immediately add an equal volume of cell culture medium I to stop the digestion, and repeatedly and gently blow the tube wall with a pipette to prepare a single cell suspension; (4) Cell counting was performed using a hemocytometer, and the cell suspension was diluted with Cell Culture Medium I to a density of 6.0 x 10 6 cells / mL to obtain a cell suspension, and 1 mL of the cell suspension was added to the upper chamber of the Transwell chamber. (5) The 6-well culture plate loaded with cells and medium is transferred to a cell culture incubator set at 37°C and 5% CO2, and cultured for 48 hours; (6) After the culture is completed, the 6-well plate is taken out of the incubator, the Transwell upper chamber is taken out of the 6-well plate hole with sterile forceps and discarded, and the medium in the lower chamber of the 6-well plate is aspirated, and the cells are gently rinsed twice with PBS preheated to 37°C; (7) Add 1 mL of 0.25% trypsin preheated to 37°C to each well of the 6-well plate lower chamber, and after confirming that the cells at the bottom of the well are completely detached under a microscope, add 1 mL of cell culture medium I to stop the digestion, and gently blow the cells repeatedly to prepare a single cell suspension.

9. The screening method according to claim 7, wherein, The single clone selection comprises the following steps: (1) Cloning ring was taken with sterile forceps, and immersed in 0.5% low-melting-point agarose at 45°C. The cloning ring was vertically fitted on the target clone; (2) 50 μL of preheated 0.25% trypsin was added into the ring, and the liquid surface should completely cover the cloning digestion cells. The cells were observed under a microscope until the cells were rounded and the edge refraction was enhanced. Then, 100 μL of cell culture medium III was added to terminate the digestion; (3) The cells were completely detached by gently blowing the ring area with a pipette. The cell suspension was transferred to a 24-well plate pre-added with 500 μL of cell culture medium III. After the addition was completed, the 24-well plate was placed back into a 37°C, 5% CO2 constant-temperature cell incubator for further culture.

10. The screening method according to claim 7, characterized by, Cell subculture included the following steps: (1) After observing that the cell confluence met the requirements, the culture medium was discarded, and the cells were rinsed with PBS for 1-2 times. Then, 200 μL of trypsin was added to digest the cells. When the cells were shortened and rounded, 200 μL of cell culture medium III was added to terminate the digestion. The cells were resuspended and collected, transferred to a sterile EP tube, centrifuged, and the supernatant was discarded. The cells were resuspended with 1 mL of new cell culture medium III and transferred to a new 6-well plate. The plate was placed back into a 37°C, 5% CO2 constant-temperature cell incubator for further culture; (2) When the cell confluence reached 80-90% in the well, the trypsin digestion, centrifugation, resuspension, hole transfer, and bottle inoculation were repeated. The cell strain A375-H was obtained by continuous and stable in vitro subculture for more than 15 generations.

Images