Construction method and application of in-situ liver cancer animal model

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a construction method and application of an in-situ liver cancer animal model. The construction method comprises the following steps: inoculating human hepatoma carcinoma cells into a culture medium for culture, digesting with pancreatin, centrifuging, and discarding supernatant to obtain a cell suspension; and injecting the cell suspension into the animal liver in a targeted manner to obtain the in-situ liver cancer animal model. The culture medium comprises the following components: a basic culture medium, fetal calf serum, hepatocyte growth factors, rat tail collagen, beta-mercaptoethanol, dextran 40, a ROCK inhibitor, insulin, non-essential amino acids and antibiotics. Compared with a method for constructing the in-situ liver cancer animal model in the prior art, the method has the advantages that the required tumor cell inoculation amount is small, the modeling period is obviously shortened, the modeling success rate is improved, and scientific guidance is provided for exploring the pathogenesis of liver cancer and formulating the standardized production process of liver cancer model animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for constructing an orthotopic liver cancer animal model and its application. Background Art

[0002] The development process of liver cancer is the result of the interaction of multiple factors. At present, the pathogenic mechanism of liver cancer has not been clearly studied, and in-depth exploration of the pathogenesis is an important basis for the treatment of liver cancer. Experimental animal cancer models are models established in human cancer research that can simulate the manifestations of human cancer. Using mouse models to study the causes and pathogenic mechanisms of liver cancer is an important means for liver cancer research.

[0003] The methods for establishing orthotopic liver cancer animal models are roughly divided into four categories, namely spontaneous liver cancer models, induced liver cancer models, transplanted liver cancer models, and transgenic animal liver cancer models. The most commonly used model animals are mice. Spontaneous liver cancer models are similar to humans in terms of genetics, but the incidence rate is low and unstable, and the occurrence time is difficult to predict; the production technology of transgenic animal liver cancer models requires high technical level, is expensive, and the operation is complex, so there are few studies; the operation of orthotopic transplanted liver cancer models is cumbersome, and the success rate depends on the maturity of technology, so it is not easy to obtain; the induced liver cancer model has simple operation, low cost, strong stability, and is simple and easy to obtain, so it is the most commonly used model establishment method in biomedical research.

[0004] For example, Chinese Patent Publication No. CN104352484A discloses a method for establishing a primary liver cancer mouse model. The steps of the method are as follows: First, select 4-week-old ICR male mice and observe and raise them for 2 weeks; Second, after raising for 2 weeks, measure the weight and daily water intake of each mouse; Third, disinfect the mouse abdomen with alcohol cotton with a volume concentration of 75%, intraperitoneally inject DEN aqueous solution, and start to let the mouse drink water containing 0.15% DEN by volume percentage 12 hours later, and feed for 4 weeks; Fourth, after 4 weeks, intraperitoneally inject DEN aqueous solution again, and then raise, and freely drink water during the raising period, and the water contains DEN; Fifth, starting from the first injection of DEN aqueous solution, raise until the 10th week, and then the primary liver cancer mouse model is obtained. However, this invention has a long modeling time and high breeding cost, and is not suitable for the need of simple and rapid model preparation.

[0005] Another Chinese Patent Publication No. CN101343621A discloses a method for establishing a mouse S180 liver cancer animal model. S180 cells are inoculated into the abdominal cavity or subcutaneous of Kunming (KM) mice to establish a KM mouse ascites cancer animal model. Then, the ascites of the ascites cancer mice is extracted, the supernatant is centrifuged and discarded, washed 2 times with normal saline, and then made into 1.0×10 6 cells / mL - 5.0×10 7A cell suspension of [X] cells / mL was implanted into the livers of mice. After 3 to 4 days, liver cancer masses with blurred boundaries with the liver tissue could be seen. Alternatively, the cells were inoculated subcutaneously in KM mice to establish a subcutaneous sarcoma model in mice. The liver lobes and sarcomas of the above mice were aseptically removed, cut into small pieces, and implanted into the livers of the test mice. After 1 week, liver masses the size of soybean grains with clear boundaries from the normal liver tissue appeared, but the success rate of this model establishment was generally low.

[0006] In view of this, there is an urgent need in the art to provide an orthotopic liver cancer animal model with a short modeling period and a high modeling success rate, which plays an important role in tumor biological mechanism research, drug development, and treatment strategy evaluation. Summary of the Invention

[0007] The present invention addresses the problems existing in the prior art and provides a method for constructing an orthotopic liver cancer animal model and its application.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for constructing an orthotopic liver cancer animal model, comprising the following steps: (1) Inoculate human liver cancer cells into a culture medium for culture, digest with trypsin, centrifuge, and discard the supernatant to obtain a cell suspension; (2) Then target-inject the cell suspension into the liver of an animal to obtain an orthotopic liver cancer animal model; Among them, the culture medium in step (1) comprises the following components: basal medium, fetal bovine serum, hepatocyte growth factor, mouse tail collagen, β-mercaptoethanol, dextran 40, ROCK inhibitor, insulin, non-essential amino acids, and antibiotics.

[0009] Preferably, the basal medium is DMEM / F12, DMEM, or RPMI-1640.

[0010] More preferably, the basal medium is DMEM.

[0011] Preferably, the ROCK inhibitor is selected from one or more of Y27632, HA1077, and H1152.

[0012] More preferably, the ROCK inhibitor is Y27632.

[0013] Preferably, the non-essential amino acids are selected from one or more of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine.

[0014] More preferably, the non-essential amino acids are selected from at least two of alanine, aspartic acid, glutamic acid, and serine.

[0015] Preferably, the antibiotic is selected from one or more of streptomycin / penicillin, amphotericin B, and Primocin.

[0016] More preferably, the antibiotic is amphotericin B.

[0017] Preferably, in step (1), the medium contains fetal bovine serum at a volume percentage of 8% - 15%, hepatocyte growth factor at 10 ng / mL - 60 ng / mL, mouse tail collagen at 4 ng / mL - 8 ng / mL, β-mercaptoethanol at 1 mM - 3 mM, dextran 40 at 5 ng / mL - 6 ng / mL, ROCK inhibitor at 10 μM - 30 μM, insulin at 5 ng / mL - 12 ng / mL, non-essential amino acids at 2 μM - 4 μM, and antibiotic at 1 μg / mL - 3 μg / mL.

[0018] More preferably, in step (1), the medium contains fetal bovine serum at a volume percentage of 9% - 12%, hepatocyte growth factor at 20 ng / mL - 40 ng / mL, mouse tail collagen at 5 ng / mL - 7 ng / mL, β-mercaptoethanol at 1.5 mM - 2.5 mM, dextran 40 at 5.5 ng / mL - 6 ng / mL, ROCK inhibitor at 12 μM - 28 μM, insulin at 6 ng / mL - 10 ng / mL, non-essential amino acids at 2.5 μM - 3.5 μM, and antibiotic at 1.5 μg / mL - 2.5 μg / mL.

[0019] Preferably, the human hepatocarcinoma cells are Hep G2, Hepa 1-6, Huh-7, or MHCC97H.

[0020] Preferably, the seeding cell density of the human hepatocarcinoma cells is 1.5×10 5 cells / mL - 5×10 5 cells / mL.

[0021] Preferably, in step (1), the specific conditions for the culture are: culturing in a 37°C, 5% CO2 incubator for 48 h - 96 h. After the culture is completed, the medium needs to be discarded, and the cells are washed 2 - 4 times with PBS.

[0022] Preferably, the rotation speed for centrifugation is 1000 rpm - 1500 rpm, and the centrifugation time is 2 min - 5 min.

[0023] Preferably, in step (2), the cell concentration in the cell suspension is 4×10 6 cells / mL - 5×10 6 cells / mL.

[0024] Preferably, the injection volume of the cell suspension is 0.2 - 0.3 mL per animal.

[0025] Preferably, the animal is a mouse.

[0026] The present invention also provides the application of the above construction method in any of the following aspects: (1) Establishing an orthotopic liver cancer model; (2) Screening for the application in drugs or vaccines for preventing or treating liver cancer; (3) Studying the pathogenesis of liver cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects: Compared with the method of constructing an orthotopic liver cancer animal model in the prior art, the present invention requires a small amount of tumor cell inoculation, significantly shortens the modeling period and improves the modeling success rate, providing scientific guidance for exploring the pathogenesis of liver cancer and formulating a standardized production process for liver cancer model animals. Detailed implementation methods

[0028] It should be noted that the raw materials used in the present invention are all ordinary commercially available products.

[0029] Human liver cancer cell Hep G2, model number CL - 0103, was purchased from Wuhan Procell Life Science & Technology Co., Ltd.

[0030] DMEM high - glucose medium was purchased from Wuhan Procell Life Science & Technology Co., Ltd.

[0031] BALB / c male nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0032] In vitro culture of human liver cancer cells: (1) Take out the cryopreserved human liver cancer cell Hep G2 cells, quickly place them in a 37°C water bath for thawing, then transfer the cell suspension to a centrifuge tube, add an appropriate amount of DMEM high - glucose medium, gently mix, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of DMEM high - glucose medium again, gently pipette to form a suspension, transfer it into a culture flask, and culture it in a 37°C, 5% CO2 incubator.

[0033] (2)When the confluence of tumor cells in the culture flask is above 80%, subculture is carried out. Use a pipette to aspirate the old culture medium in the culture flask, add PBS to cover the bottom of the flask, and shake to wash the cells. Aspirate the PBS, add an appropriate amount of trypsin digestion solution to the culture flask so that all the cells at the bottom of the flask are immersed in the trypsin solution. Observe the digested cells under a microscope and perform digestion treatment for 1 min - 2 min (the cells become round). Use a sterile dropper to aspirate the digestion solution, add high-glucose DMEM medium, pipette to make a cell suspension, transfer the suspension into a centrifuge tube and centrifuge (1000 rpm, 5 min), discard the supernatant, add high-glucose DMEM medium to resuspend the cells, and perform subculture inoculation at a ratio of 1:3, with subculture once every 3 - 4 days; the cells used in the experiment are the 3rd generation cells.

[0034] Example 1 A method for constructing an orthotopic liver cancer animal model is as follows: (1)Inoculate human liver cancer cells Hep G2 at a cell density of 3×10 5 cells / mL into a 12-well plate containing high-glucose DMEM medium with 10% (v / v) fetal bovine serum, 30 ng / mL hepatocyte growth factor, 6 ng / mL rat tail collagen, 2 mM β-mercaptoethanol, 6 ng / mL dextran 40, 20 μM ROCK inhibitor Y27632, 8 ng / mL insulin, 2 μM aspartic acid, 2 μM alanine, and 2 μg / mL amphotericin B, and place it in a 5% CO2, 37 °C cell culture incubator for 48 h. Add an appropriate amount of trypsin for digestion. After the cells become round and detach from the wall, add high-glucose DMEM medium to terminate the digestion. Then centrifuge at 1500 rpm for 4 min, discard the supernatant, and obtain a cell suspension with a cell concentration of 5×10 6 cells / mL.

[0035] (2)Use 4-week-old male BALB / c nude mice, raise them in a sterile environment, and let them adapt to the environment for 1 week. Fast the mice for 12 h before the operation, perform routine intraperitoneal anesthesia, fix them in the supine position, and disinfect the skin. Open the abdominal cavity along the midline of the abdomen, expose the liver, and pull the liver lobe out of the incision with a cotton swab; insert the injection needle obliquely, penetrate about 3 mm into the liver, and slowly push in 0.2 mL of the cell suspension. After inoculation, gently press the injection site to avoid overflow of the cell suspension. After observing no active bleeding, gently return the liver to the abdominal cavity, close the abdomen layer by layer, continue to raise the mice after the operation, allow them to eat freely, and observe regularly.

[0036] Example 2 A method for constructing an orthotopic liver cancer animal model is as follows: (1)Inoculate human liver cancer cells Hep G2 at a cell density of 1×10 5Cells were inoculated into a 12-well plate containing high-glucose DMEM medium with 8% fetal bovine serum by volume, 10 ng / mL hepatocyte growth factor, 8 ng / mL rat tail collagen, 3 mM β-mercaptoethanol, 5 ng / mL dextran 40, 10 μM ROCK inhibitor H1152, 12 ng / mL insulin, 3 μM glutamic acid, and 1 μg / mL amphotericin B at a cell density of 5×10 6 cells / mL, and cultured in a 5% CO2, 37°C cell incubator for 48 h. An appropriate amount of trypsin was added for digestion. After the cells became round and detached from the wall, high-glucose DMEM medium was added to terminate the digestion. Then, the cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded to obtain a cell suspension with a cell concentration of 4×10

[0037] (2) Four-week-old male BALB / c nude mice were used and raised in a sterile environment for 1 week to adapt to the environment. The mice were fasted for 12 h before the operation, anesthetized by conventional intraperitoneal injection, fixed in the supine position, and the skin was disinfected. The abdomen was opened along the midline to expose the liver, and the liver lobe was pulled out of the incision with a cotton swab; the injection needle was inserted obliquely into the liver about 3 mm, and 0.3 mL of the cell suspension was slowly injected. After inoculation, the injection site was gently pressed to avoid overflow of the cell suspension. After observing no active bleeding, the liver was gently returned to the abdominal cavity, and the abdomen was closed layer by layer. After the operation, the mice were continued to be raised, allowed to eat freely, and observed regularly.

[0038] Example 3 A method for constructing an orthotopic liver cancer animal model is as follows: (1) Human liver cancer cells Hep G2 were inoculated into a 12-well plate containing high-glucose DMEM medium with 15% fetal bovine serum by volume, 60 ng / mL hepatocyte growth factor, 4 ng / mL rat tail collagen, 1 mM β-mercaptoethanol, 6 ng / mL dextran 40, 30 μM ROCK inhibitor HA1077, 5 ng / mL insulin, 4 μM glycine, and 3 μg / mL Primocin at a cell density of 5×10 5 cells / mL, and cultured in a 5% CO2, 37°C cell incubator for 96 h. An appropriate amount of trypsin was added for digestion. After the cells became round and detached from the wall, high-glucose DMEM medium was added to terminate the digestion. Then, the cells were centrifuged at 1500 rpm for 2 min, and the supernatant was discarded to obtain a cell suspension with a cell concentration of 5×10 6 cells / mL.

[0039] (2) Four-week-old male BALB / c nude mice were used and raised in a sterile environment for 1 week to adapt to the environment. The mice were fasted for 12 h before surgery, then anesthetized intraperitoneally routinely, fixed in the supine position, and the skin was disinfected. The abdominal cavity was opened along the midline of the abdomen to expose the liver, and the liver lobe was pulled out of the incision with a cotton swab; the injection needle was inserted obliquely into the liver about 3 mm, and 0.2 mL of the cell suspension was slowly injected. After inoculation, the injection site was gently pressed to avoid the overflow of the cell suspension. After observing no active bleeding, the liver was gently returned to the abdominal cavity, and the abdomen was closed layer by layer. After the operation, the mice were continuously raised, allowed to eat freely, and observed regularly.

[0040] Example 4 A method for constructing an orthotopic liver cancer animal model is as follows: (1) Human liver cancer cells Hep G2 were inoculated at a cell density of 2×10 5 cells / mL into a 12-well plate containing DMEM high-glucose medium with 9% fetal bovine serum by volume percentage, 20 ng / mL hepatocyte growth factor, 5 ng / mL rat tail collagen, 2.5 mM β-mercaptoethanol, 6 ng / mL dextran 40, 12 μM ROCK inhibitor H1152, 10 ng / mL insulin, 2.5 μM glutamic acid, 1 μM alanine, and 1.5 μg / mL streptomycin / penicillin, and cultured in a 5% CO2, 37 °C cell culture incubator for 72 h. An appropriate amount of trypsin was added for digestion. After the cells became round and detached from the wall, DMEM / F12 medium was added to terminate the digestion. Then, centrifugation was performed at 1400 rpm for 2.5 min, and the supernatant was discarded to obtain a cell suspension with a cell concentration of 4.5×10 6 cells / mL.

[0041] (2) Four-week-old male BALB / c nude mice were used and raised in a sterile environment for 1 week to adapt to the environment. The mice were fasted for 12 h before surgery, then anesthetized intraperitoneally routinely, fixed in the supine position, and the skin was disinfected. The abdominal cavity was opened along the midline of the abdomen to expose the liver, and the liver lobe was pulled out of the incision with a cotton swab; the injection needle was inserted obliquely into the liver about 3 mm, and 0.2 mL of the cell suspension was slowly injected. After inoculation, the injection site was gently pressed to avoid the overflow of the cell suspension. After observing no active bleeding, the liver was gently returned to the abdominal cavity, and the abdomen was closed layer by layer. After the operation, the mice were continuously raised, allowed to eat freely, and observed regularly.

[0042] Example 5 A method for constructing an orthotopic liver cancer animal model is as follows: (1) Human liver cancer cells Hep G2 were inoculated at a cell density of 4×10 5Cells were inoculated at a density of 6 cells / mL into a 12-well plate containing high-glucose DMEM medium supplemented with 12% (v / v) fetal bovine serum, 40 ng / mL hepatocyte growth factor, 7 ng / mL murine tail collagen, 1.5 mM β-mercaptoethanol, 5.5 ng / mL dextran 40, 28 μM ROCK inhibitor HA1077, 6 ng / mL insulin, 1 μM serine, 2.5 μM glutamic acid, and 2.5 μg / mL Primocin, and cultured in a 5% CO2, 37 °C cell incubator for 60 h. An appropriate amount of trypsin was added for digestion. After the cells became round and detached from the wall, RPMI-1640 medium was added to terminate the digestion. The cells were then centrifuged at 1200 rpm for 3 min, and the supernatant was discarded to obtain a cell suspension with a cell concentration of 5×10

[0043] (2) Four-week-old male BALB / c nude mice were used and raised in a sterile environment for 1 week to adapt to the environment. The mice were fasted for 12 h before the operation, anesthetized by conventional intraperitoneal injection, fixed in the supine position, and the skin was disinfected. The abdomen was opened along the midline to expose the liver, and the liver lobe was pulled out of the incision with a cotton swab; the injection needle was inserted obliquely into the liver about 3 mm, and 0.3 mL of the cell suspension was slowly injected. After inoculation, the injection site was gently pressed to avoid overflow of the cell suspension. After observing no active bleeding, the liver was gently returned to the abdominal cavity, and the abdomen was closed layer by layer. After the operation, the mice were continuously raised, allowed to eat freely, and observed regularly.

[0044] Comparative Example 1 The difference from Example 1 was only that the high-glucose DMEM medium did not contain murine tail collagen, and the specific components of this medium were as follows: high-glucose DMEM medium containing 10% (v / v) fetal bovine serum, 30 ng / mL hepatocyte growth factor, 2 mM β-mercaptoethanol, 6 ng / mL dextran 40, 20 μM ROCK inhibitor Y27632, 8 ng / mL insulin, 2 μM aspartic acid, 2 μM alanine, and 2 μg / mL amphotericin B.

[0045] Comparative Example 2 The difference from Example 1 was only that the high-glucose DMEM medium did not contain β-mercaptoethanol and dextran 40, and the specific components of this medium were as follows: high-glucose DMEM medium containing 10% (v / v) fetal bovine serum, 30 ng / mL hepatocyte growth factor, 6 ng / mL murine tail collagen, 20 μM ROCK inhibitor Y27632, 8 ng / mL insulin, 2 μM aspartic acid, 2 μM alanine, and 2 μg / mL amphotericin B.

[0046] Comparative Example 3 The difference from Example 1 is only that the high-glucose DMEM medium does not contain β-mercaptoethanol, and the specific components of this medium are as follows: DMEM high-glucose medium containing 10% fetal bovine serum by volume percentage, 30 ng / mL hepatocyte growth factor, 6 ng / mL mouse tail collagen, 6 ng / mL dextran 40, 20 μM ROCK inhibitor Y27632, 8 ng / mL insulin, 2 μM aspartic acid, 2 μM alanine, and 2 μg / mL amphotericin B.

[0047] Comparative Example 4 The difference from Example 1 is only that the high-glucose DMEM medium does not contain dextran 40, and the specific components of this medium are as follows: DMEM high-glucose medium containing 10% fetal bovine serum by volume percentage, 30 ng / mL hepatocyte growth factor, 6 ng / mL mouse tail collagen, 2 mM β-mercaptoethanol, 20 μM ROCK inhibitor Y27632, 8 ng / mL insulin, 2 μM aspartic acid, 2 μM alanine, and 2 μg / mL amphotericin B.

[0048] Comparative Example 5 The difference from Example 1 is only that the content of the components in the high-glucose DMEM medium is different, and the specific components of this medium are as follows: DMEM high-glucose medium containing 10% fetal bovine serum by volume percentage, 80 ng / mL hepatocyte growth factor, 10 ng / mL mouse tail collagen, 6 mM β-mercaptoethanol, 1 ng / mL dextran 40, 20 μM ROCK inhibitor Y27632, 20 ng / mL insulin, 6 μM aspartic acid, 2 μM alanine, and 6 μg / mL amphotericin B.

[0049] Test Example After inoculating the human hepatocellular carcinoma cell suspension, the growth of tumors in nude mice was observed using SPECT / CT on the 7th day, 10th day, and 14th day respectively. The long diameter (a) and short diameter (b) of the tumors were measured with an electronic vernier caliper, and the average tumor volume was calculated and recorded (V = 1 / 2 × a × b 2 )

[0050] Usually when the tumor volume reaches 100 mm 3 -200 mm 3 or so, it is considered that the model establishment is initially successful, and the experiment starts after the tumor reaches 100 mm 3 . The measurement results are shown in Table 1. As can be seen from Table 1, compared with the groups of Comparative Example 1 - Comparative Example 5, tumors appeared in the livers of mice in the groups of Example 1 - Example 5 after the 7th day, and the liver tumor volumes of the mice in Example 1 were significantly higher than those of Comparative Example 1 - Comparative Example 5 ( P <0.05 or P(<0.01). It shows that by using the orthotopic liver cancer animal models provided in Examples 1 - 5 for modeling, tumor-bearing mice with larger tumor volumes can be obtained.

[0051] Table 1 Tumor Volume Table of Orthotopic Liver Cancer Animal Models (n = 8)

[0052] Note: Compared with the 7th day of Example 1, * Indicates P <0.05, ** Indicates P <0.01; compared with the 10th day of Example 1, # Indicates P <0.05, ## Indicates P <0.01; compared with the 14th day of Example 1, & Indicates P <0.05, && Indicates P <0.01.

[0053] In addition, according to the method for constructing the orthotopic liver cancer animal model of Example 1, the success rate of modeling BALB / c nude mice in different batches (3 batches, 10 mice in each batch) was tested. After 7 days of inoculating the human liver cancer cell suspension, the tumor volumes of all 30 mice reached 100 mm 3 -200 mm 3 , and all 30 mice developed tumors, with a tumorigenesis rate of 100%.

[0054] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing an in-situ liver cancer animal model, characterized in that, It includes the following steps: (1) Inoculate human liver cancer cells into a culture medium for culturing, digest with trypsin, centrifuge, discard the supernatant, and obtain a cell suspension; (2) Then targetedly inject the cell suspension into the animal liver to obtain an orthotopic liver cancer animal model; Among them, the culture medium described in step (1) includes the following components: basal medium, fetal bovine serum, hepatocyte growth factor, rat tail collagen, β-mercaptoethanol, dextran 40, ROCK inhibitor, insulin, non-essential amino acids, and antibiotics.

2. The construction method according to claim 1, wherein The basal medium is DMEM / F12, DMEM or RPMI-1640.

3. The construction method according to claim 1, characterized in that, The ROCK inhibitor is selected from one or more of Y27632, HA1077, and H1152.

4. The construction method according to claim 1, wherein The non-essential amino acids are selected from one or more of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine.

5. The construction method according to claim 1, characterized in that The antibiotics are selected from one or more of streptomycin / penicillin, amphotericin B, and Primocin.

6. The construction method according to claim 5, wherein In the culture medium described in step (1), it contains 8%-15% (v / v) fetal bovine serum, 10 ng / mL - 60 ng / mL hepatocyte growth factor, 4 ng / mL - 8 ng / mL rat tail collagen, 1 mM - 3 mM β-mercaptoethanol, 5 ng / mL - 6 ng / mL dextran 40, 10 μM - 30 μM ROCK inhibitor, 5 ng / mL - 12 ng / mL insulin, 2 mM - 4 μM non-essential amino acids, and 1 μg / mL - 3 μg / mL antibiotics.

7. The construction method according to claim 1, characterized in that, The human hepatoma cells are Hep G2, Hepa 1-6, Huh-7 or MHCC97H, and the seeding cell density of the human hepatoma cells is 1.5×10 5 cells / mL - 5×10 5 cells / mL.

8. The construction method according to claim 1, wherein In step (1), the specific conditions for culturing are: culture in a 37°C, 5% CO2 incubator for 48 h - 96 h; after the culturing is completed, the culture medium needs to be discarded, and the cells are washed 2 - 4 times with PBS. The rotation speed for centrifugation is 1000 rpm - 1500 rpm, and the centrifugation time is 2 min - 5 min.

9. The construction method according to claim 1, characterized in that In step (2), the cell concentration in the cell suspension is 4×10 6 cells / mL - 5×10 6 cells / mL, the injection volume of the cell suspension is 0.2 - 0.3 mL / animal, and the animal is a mouse.

10. The application of the construction method according to any one of claims 1 - 9 in any of the following aspects: (1) Establishing an orthotopic liver cancer model; (2) Screening drugs or vaccines for preventing or treating liver cancer; (3) Studying the pathogenesis of liver cancer.

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