Acquisition method and application of derivative strain Hepa1-6 substrain of mouse liver cancer cell

Abstract

The invention discloses an acquisition method and application of a derivative strain Hepa1-6 substrain of mouse hepatoma carcinoma cells, the acquisition method comprises the following steps: culturing Hepa1-6 in the market, then transplanting the Hepa1-6 to a C57BL / 6J mouse for multiple passage, establishing the derivative strain Hepa1-6 substrain of the mouse hepatoma carcinoma cells of P3-P4 generation, obtaining tumor cells with the tumor formation rate of 95% or more, naming the tumor cells as Hepa1-6-PSWX002, and establishing the derivative strain Hepa1-6 substrain of the mouse hepatoma carcinoma cells of P3-P4 generation. And a pharmacodynamic test of an antitumor drug is carried out. The method has the advantages of being capable of reducing experiment cost, saving time, being popular and the like, and overcomes the defects that in the prior art, tumors cannot be formed, the tumor forming rate is low, and more mice need to be used for modeling.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and in particular to a method for obtaining a Hepa1-6 substrain derived from mouse liver cancer cells and an application thereof. Background Art

[0002] Liver cancer is one of the most common cancers, causing serious harm to human life and health, and its morbidity and mortality rates have remained high for a long time. The exploration of the pathogenesis of liver cancer, diagnosis, discovery of new targets and drug development are urgent issues to be addressed. Different types of cells in tumor tissues, different ligands expressed by cells, interactions with corresponding receptors and extracellular matrix interactions form a complex regulatory system, which constitutes the tumor microenvironment. Simple in vitro tumor cell culture systems are difficult to fully reveal the complex mutual regulatory network between different cells in the tumor microenvironment.

[0003] Laboratory animals are one of the most commonly used experimental materials in pharmaceutical research and development, drug safety evaluation, hospitals and life sciences. In the process of researching and developing drugs and therapies for human diseases, laboratory animals are the best research subjects and substitutes. All tumor drug research will conduct a large number of animal experiments before entering the clinic. Mice are the most common experimental animals. The establishment of a mouse liver cancer model has become an important solution to this problem and is also the basis for exploring the mechanism of tumor development and drug development.

[0004] Common mouse models used in liver cancer immunology research include transplant tumor models, chemical carcinogen-induced liver cancer models, and genetically engineered liver cancer models. The chemical carcinogen-induced liver cancer model has a long experimental cycle, requires sufficient technical experience to establish the model, and the onset time is difficult to control. The genetically engineered liver cancer model requires specific genetically engineered mice, and its experimental cycle is long and the cost is the highest compared to chemical carcinogen-induced liver cancer. The subcutaneous transplant tumor model of liver cancer has the advantages of simple and convenient operation, intuitive observation of tumor growth, and convenient detection of important data such as animal weight, tumor growth curve, and tumor weight. Therefore, the subcutaneous tumor model is currently the most commonly used tumor model in tumor research, tumor immunity research, and drug development.

[0005] Mouse liver cancer cell line Hepa1-6 is a derivative of BW7756 mouse liver cancer cells produced in C57 / L mice. Currently, C57BL / 6 mice are the most widely used mouse strain in oncology research and tumor immunity research. Mouse liver cancer cell line Hepa1-6 is in great demand in the research direction of mouse liver cancer, but the existing technology has the following defects: the existing Hepa1-6 cells on the market cannot meet the needs of pharmacological and efficacy experiments on the market, such as Figure 1 As shown, when the tumor reaches a certain size, such as less than 1000mm 3The cells start to shrink and eventually shrink to 0. The drug efficacy cannot be measured. They cannot grow to the euthanasia standard like other cells, with an average size of 2000 mm. 3 Or a single length up to 3000mm 3 .

[0006] Therefore, it is necessary to obtain a cell line that can establish a Hepa1-6 liver cancer subcutaneous model in mice, stably grow the Hepa1-6 substrain to the mouse euthanasia standard, and does not require complex equipment. Summary of the invention

[0007] The problem to be solved by the present invention is that the existing Hepa1-6 cells in the market cannot meet the needs of pharmacological and efficacy experiments in the market.

[0008] In order to solve the above technical problems, one aspect of the present invention provides a method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative, comprising the following steps: S1: using existing mouse liver cancer cells Hepa1-6 to culture and passage mouse liver cancer cells Hepa1-6, and expanding the main library and experimental library; S2: using the cultured mouse liver cancer cells Hepa1-6 for subcutaneous modeling of the first batch of C57BL / 6J mice. During the process, when the normally growing tumor grows to 800-1000mm 3 When the tumors of the second batch of mice in step S2 are formed normally, they are removed, cut into uniform tumor masses after treatment, and the tumor masses are transplanted to the second batch of C57BL / 6J mice for passage, and the tumor formation of this batch of mice is observed; S3: the tumors of the second batch of mice that have formed normally in step S2 are removed, cut into uniform tumor masses after treatment, and transplanted to the third batch of C57BL / 6J mice for passage. After the tumors of this batch of mice grow to the required size, the tumors are removed and frozen; S4: the tumors obtained in step S3 are frozen for many months and then revived, and inoculated into the fourth batch of C57BL / 6J mice. During this tumor formation process, the observed tumors are inoculated into the fifth batch of C57BL / 6J mice when they grow to 800-1500mm3, and the P4 generation of the Hepa1-6 substrain, a derivative of mouse liver cancer cells, is obtained.

[0009] According to an embodiment of the present application, in step S4, the fourth batch of mice is inoculated to obtain the P3 generation of the Hepa1-6 substrain, a derivative of mouse liver cancer cells, named Hepa1-6-PSWX002.

[0010] According to the embodiments of the present application, the deposit number of Hepa1-6-PSWX002 is CCTCCNO.2023331.

[0011] According to an embodiment of the present application, the treatment of the tumor removed from the mouse includes removing the necrotic part, fascia and blood clot of the tumor.

[0012] According to the embodiment of the present application, in step S3, the mouse tumor is removed after it grows to the desired size, that is, the tumor grows to 1000-2000mm 3 Remove it when necessary.

[0013] According to an embodiment of the present application, the culture of mouse liver cancer cells Hepa1-6 in step S1 specifically includes the recovery of mouse liver cancer cells Hepa1-6, the passaging of mouse liver cancer cells Hepa1-6 and the supply of mouse liver cancer cells Hepa1-6.

[0014] According to the embodiment of the present application, the resuscitation of mouse liver cancer cells Hepa1-6 refers to: taking out a frozen mouse liver cancer cell Hepa1-6 screened in step S1, and rapidly thawing it in a 37°C water bath; aspirating the cell suspension into a 15ml centrifuge tube with 9ml complete culture medium added, centrifuging at 1200rpm for 3min, discarding the supernatant, adding complete culture medium to resuspend the cells, transferring to a T25 culture flask, adding culture medium, and adjusting the cell density to 0.2×10 6 cells / mL were placed in a cell culture incubator at 37°C and 5% CO2; the subculture of mouse hepatoma cell Hepa1-6 refers to: when the cell confluence reaches 80%-90%, the cell is subcultured, the supernatant is discarded, the cell is washed with D-PBS 1-2 times and the liquid is discarded, and the cell is digested with 0.25% Trypsin-EDTA for about 1 minute until the cell becomes round and shiny, and the digestion is terminated after the culture is used up; the cells are gently blown to make them fall off the wall, collected into a centrifuge tube, centrifuged at 1200rpm for 3 minutes, the supernatant is discarded, and new complete culture medium is added to resuspend the cells, and the cells are subcultured into a new culture bottle at a ratio of 1:2-1:4, and placed in a cell culture incubator at 37°C and 5 %CO2 culture; the supply of mouse liver cancer cell Hepa1-6 means: discard the supernatant, wash with D-PBS 1-2 times and then discard the liquid, digest with 0.25% trypsin cell digestion solution for about 1 minute, until the cells become round and shiny, and terminate the digestion with complete medium; gently blow to make the cells fall off the wall, collect them into a centrifuge tube, centrifuge at 1200rpm for 3min, discard the supernatant, add new complete medium to resuspend the cells; mix the cell suspension and trypan blue in a 1:1 ratio, count and calculate the activity rate with a cell counter, centrifuge at 4℃, 1200rpm for 3min, discard the supernatant, resuspend the cells with pre-cooled D-PBS, and adjust the cell density to 1×10 8 cells / ml, add an equal volume of matrix gel, mix gently by pipetting; seal the Eppendorf tube with sealing film, put it into the sample bag for recording information and place it in an ice box for supply.

[0015] According to an embodiment of the present application, the complete culture medium includes 90% DMEM, 10% fetal bovine serum and 100 units per milliliter of penicillin plus streptomycin.

[0016] According to an embodiment of the present application, mouse liver cancer cell Hepa1-6 is an adherent cell growing in an epithelial-like monolayer.

[0017] Another aspect of the present invention provides the use of the above-mentioned mouse liver cancer cell derivative strain Hepa1-6 substrain in the study of drug efficacy of mouse liver cancer.

[0018] The beneficial effects of the technical solution of the present invention compared with the prior art are:

[0019] 1. The present invention obtains the Hepa1-6 substrain, a derivative strain of mouse liver cancer cells, by using a method of multiple transplantation and subculture in mice to continuously screen mouse liver cancer cells with a high tumor formation rate and that will not shrink, and ultimately obtains primary cells with a tumor formation rate of up to 100%, thereby solving the problem of low tumor formation rate of Hepa1-6 cells cultured in the prior art.

[0020] 2. The present invention has the advantages of reducing experimental costs, saving time, and being popular, and overcomes the defects of the prior art that tumors cannot be formed, the tumor formation rate is low, and more mice need to be used for modeling.

[0021] 3. Provide a liver cancer model Hepa1-6 for preclinical pharmacology and efficacy studies: The liver cancer model established by the present invention does not require induction by carcinogenic agents and other reagents, and has high safety throughout the experimental process. The establishment of carcinoma in situ only requires C57BL / 6 background mice, and there is no need to construct and breed specific transgenic mice. Only one subcutaneous injection of mice is required to complete the model construction. Therefore, the model establishment operation is simple, the entire experimental cycle is short, and the research cycle is effectively shortened and the research cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a data diagram of tumor formation of the existing Hepa1-6 cell line in the first batch of C57BL / 6J mice in the examples of the present invention;

[0023] Figure 2 This is a growth data diagram of the P1 generation tumor in the example of the present invention;

[0024] Figure 3 This is a growth data diagram of the P2 generation tumor in the example of the present invention;

[0025] Figure 4 This is a growth data diagram of the P3 generation tumor in the example of the present invention;

[0026] Figure 5 This is a growth data diagram of the P4 generation tumor in the example of the present invention;

[0027] Figure 6 This is a growth curve of liver cancer tumors in P4 mice that were given the drug once a day in the examples of the present invention;

[0028] Figure 7 This is a growth curve of liver cancer tumors in P4 mice that were given the drug twice a day in the example of the present invention; The Hepa1-6 cell substrain Hepa1-6-PSWX002 is classified and named as mouse hepatocarcinoma cell Hepa1-6-PSWX002. The preservation unit is China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, Postal Code: 430072; the preservation date is November 7, 2023, and the preservation number is CCTCC NO: C2023331. Specific implementation method:

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0030] Hepa1-6 is a mouse liver cancer cell, a derivative of BW7756 mouse liver cancer cells produced in C57 / L mice, and is negative for ectromelia virus (ECTV). It is often used to establish an animal model of mouse liver cancer. The cell was screened for several liver-specific expression factors (such as albumin, α1 antitrypsin, α-fetoprotein, and amylase), and confirmed to be able to grow in a serum-free medium supplemented with dexamethasone and selenium. Hepa1-6 is an adherent cell that grows in an epithelial-like monolayer. High concentrations can cause tumors in animals, and it is recommended to be injected with matrix gel. DMEM medium (1.5g / LNaHCO3) is recommended for Hepa1-6 cell culture, containing 10% fetal bovine serum, and 1% sodium pyruvate is recommended. It is recommended to replace fresh culture medium 2-3 times a week, and subculture at a ratio of 1:3 to 1:4. Trypsin digestion at room temperature for 2-3 minutes, and the subculture cycle is usually 2-3 days.

[0031] In order to overcome the insufficient tumor formation rate of mouse liver cancer cell Hepa1-6 and achieve a higher tumor formation rate of mouse liver cancer cell Hepa1-6, the inventors of the present invention have long been committed to the study of mouse liver cancer cell Hepa1-6 passage, and finally obtained the P3 generation substrain of mouse liver cancer cell Hepa1-6 with a higher tumor formation rate.

[0032] The inventors successfully obtained Hepa1-6 cells with a high tumor formation rate by performing multiple in vivo passages on adherent mouse liver cancer cells Hepa1-6. The specific steps are as follows:

[0033] S1: Use the existing mouse liver cancer cell Hepa1-6 to culture and transmit the mouse liver cancer cell Hepa1-6 to expand the main library and experimental library. In actual operation, this step will purchase commercially available mouse liver cancer cell Hepa1-6 for culture to expand the inventory.

[0034] S2: The cultured mouse liver cancer cells Hepa1-6 were used for subcutaneous modeling of the first batch of C57BL / 6J mice. During the process, when the normally grown tumor grew to 800-1000mm3, it was removed, cut into uniform tumor masses after treatment, and the tumor masses were transplanted to the second batch of C57BL / 6J mice for passage to observe the tumor formation of this batch of mice. The tumor formation rate during this step is extremely low, and the tumor begins to shrink after growing to a certain size, so the key to this step is to screen the tumors that meet the conditions in time for transplantation and passage. The purpose of this step is to expand the amount of tumors for easy screening.

[0035] S3: The tumors of the second batch of mice in step S2 are removed and cut into uniform tumor pieces after treatment, and then transplanted into the third batch of C57BL / 6J mice for passage. After the tumors of this batch of mice grow to the required size, the tumors are removed and frozen. The key to this step is to count the tumor formation rate and observe whether the tumor has shrunk.

[0036] S4: The tumor obtained in step S3 was frozen for several months and then revived, and inoculated into the fourth batch of C57BL / 6J mice to obtain the mouse liver cancer cell derivative Hepa1-6 substrain Hepa1-6-PSWX002. Figure 4 As shown, the experiment shows that the P3 generation tumors did not shrink, and the tumor formation rate reached 60%. The key to this step is to verify whether the cultured P2 generation tumors can survive and grow normally after being revived after low temperature freezing. The experiment shows that the revived Hepa1-6 substrain tumor cells can survive and grow normally.

[0037] Furthermore, the present invention freezes part of the mouse liver cancer tumor obtained in the P3 generation, provides part for in vitro isolation of primary cell culture expansion, and continues to pass the part in vivo for the P4 generation experiment. As shown in the figure, the tumor formation rate of the P4 generation is as high as 100%.

[0038] Example 1 Cultivation of mouse liver cancer cells Hepa1-6

[0039] 1) Resuscitation of mouse hepatocellular carcinoma cell line Hepa1-6

[0040] Take out a frozen commercially purchased mouse liver cancer cell Hepa1-6 from liquid nitrogen and thaw it quickly in a 37°C water bath. Pipette the cell suspension into a 15ml centrifuge tube with 9ml complete medium (90% DMEM + 10% FBS + 100U / ml PS), centrifuge at 1200rpm for 3min, discard the supernatant, add complete medium to resuspend the cells, transfer to a T25 culture flask, add culture medium, adjust the cell density to 0.2×106cells / mL, and place in a cell culture incubator at 37°C and 5% CO2 for culture;

[0041] 2) Passaging of mouse hepatocellular carcinoma cell line Hepa1-6

[0042] When the cells reach a confluence of 80% to 90%, the cells are passaged. Discard the supernatant, wash with D-PBS 1 to 2 times and discard the liquid, use 0.25% Trypsin-EDTA, digest for about 1 minute, until the cells become round and shiny, and terminate the digestion when the culture medium is used up. Gently blow the cells to make them fall off the wall, collect them into a centrifuge tube, centrifuge at 1200rpm for 3 minutes, discard the supernatant, and add new complete culture medium to resuspend the cells. Passage into new culture bottles at a ratio of 1:2 to 1:4, and culture in a cell culture incubator at 37°C and 5% CO2;

[0043] 3) Supply of mouse liver cancer cell Hepa1-6

[0044] When the cell confluence is 80% to 90% and the required amount is reached, it can be supplied. Discard the supernatant, wash with D-PBS 1 to 2 times and then discard the liquid, use 0.25% Trypsin-EDTA, digest for about 1 minute, until the cells become round and shiny, and terminate the digestion when the medium is used up. Gently blow to make the cells fall off the wall, collect them into a centrifuge tube, centrifuge at 1200rpm for 3min, discard the supernatant, add new complete culture medium to resuspend the cells. Take the cell suspension: Trypan blue and mix them evenly in a 1:1 ratio, count and calculate the viability with a cell counter. Centrifuge at 4℃, 1200rpm for 3min, discard the supernatant, resuspend the cells with pre-cooled D-PBS, adjust the cell density to 1×108cells / ml, add an equal volume of Matrigel, and gently blow to mix. After sealing the Ep tube with sealing film, put it in the sample bag with recorded information and place it in an ice box for supply.

[0045] Example 2 Obtaining the Hepa1-6 substrain of mouse liver cancer cell derivatives

[0046] The following steps are involved:

[0047] 1) The mouse liver cancer cells Hepa1-6 obtained in Example 1 were used for subcutaneous modeling of the first batch of C57BL / 6J mice. The first batch of C57BL / 6J mice consisted of 5 mice. As shown in Table 1, only the tumor of 1 mouse did not shrink. Figure 2 As shown, the tumor can grow normally. When the only normal growth obtained is the tumor growing to 800-1000mm 3 When the tumor was detected, it was removed, processed and cut into uniform tumor masses, which were then transplanted into the second batch of C57BL / 6J mice for passage. The second batch of C57BL / 6J mice consisted of 10 mice. The tumor formation in this batch of mice was observed. As shown in Table 2, 3 / 10 mice developed tumors and the tumors did not shrink, forming the growth data of the P1 generation tumors.

[0048]

[0049] Table 1

[0050]

[0051] Table 2

[0052] 2) The tumors of the second batch of mice in the above steps were removed, cut into uniform tumor pieces after treatment, and transplanted into the third batch of C57BL / 6J mice for passage. The third batch of C57BL / 6J mice consisted of 24 mice. As shown in Table 3, the results of the P2 generation tumors formed by passage of the third batch of C57BL / 6J mice showed that 15 / 24 mice did not show tumor shrinkage. After the tumors of this batch of mice grew to the desired size, the tumors were removed and frozen. The growth curve of the P2 generation tumors is shown in Figure 3 As shown, relative to Figure 2 The P1 generation of tumors grew faster and larger.

[0053]

[0054] Table 3

[0055] 3) P2 generation tumors or P2 generation tumor cells were frozen for many months and then revived, inoculated into C57BL / 6J mice, and then passaged to obtain P3 generation tumors. As shown in Table 4, P3 generation tumors can still grow successfully, with a high tumor formation rate, and can grow stably until the euthanasia standard. Figure 4 As shown in the figure, the tumor growth curve observed in some mice during the tumor formation process. During the tumor formation process, some mice were observed to grow to 2000mm in a single mouse. 3 Above, some are 800-1500mm 3 At passage P4, a portion of the tumor mass was given to the in vitro group to extract primary cells. After success, the frozen portion was expanded, provided to the body to establish normal CDX subcutaneous inoculation, and provided to a third party for STR identification.

[0056]

[0057] Table 4

[0058] Among them, the treatment of the removed tumor includes removing necrotic parts, fascia, blood clots, etc.

[0059] Specifically, in this embodiment, after the Hepa1-6 cell culture was completed, it was supplied to the subcutaneous modeling experiment process. As shown in Table 1, among the five experiments, only one tumor (1 / 5) grew normally, and the rest of the tumors shrank to 0. When it grew to 1000-1500 mm3 (it did not shrink when it exceeded 1000 mm3, proving that it was different from other tumors), it was removed, the necrotic part, fascia, blood clots, etc. were removed, and it was cut into a uniform tumor mass; the tumor mass was transplanted to a new batch of C57BL / 6J mice for passage, and the tumor formation of this batch of mice was observed, as shown in Table 2. The final result was that 3 / 10 tumors did not shrink. These three tumors were removed, and the calcified parts, fascia, blood clots, etc. were removed, cut into uniform tumor blocks, and transplanted again into a new batch of C57BL / 6J mice for passage. As shown in Table 3, the results of this time were that 15 / 24 tumors did not shrink, and 15 mice did not shrink their tumors. After the tumors of this batch of mice grew to a certain size, they were removed and frozen; after being frozen for many months, they were revived and inoculated into C57BL / 6J mice. As shown in Table 4, the P3 generation can still grow successfully, with a high tumor formation rate, and can grow stably to the euthanasia standard. For the tumors removed from the P3 generation, part of the P3 generation was frozen, part was provided for in vitro isolation of primary cell culture expansion, and part continued in vivo passage experiment P4 generation. As shown in Table 5, the tumor formation rate of the P4 generation was as high as 100%.

[0060]

[0061] Table 5

[0062] Among them, the Hepa1-6 cell substrain obtained in the P3 generation was named Hepa1-6-PSWX002 and classified as mouse hepatocarcinoma cell Hepa1-6-PSWX002. The cell line is preserved by the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, Postal Code: 430072; the preservation date is November 7, 2023, and the preservation number is CCTCC NO: C2023331.

[0063] Example 3 Application of a mouse liver cancer cell derivative Hepa1-6 substrain in drug efficacy research after establishing a mouse liver cancer model

[0064] Construction of mouse subcutaneous tumor model

[0065] 1) C57BL / 6J female WT mice, about 8 weeks old, were selected, and the animal breeding conditions were consistent with the above example.

[0066] 2) Prepare a cell suspension of the P4 generation tumor cells obtained in Example 2, adjust the cell suspension concentration to 5.0 x 10^6 / ml, inject 100 ul subcutaneously into each mouse, and monitor the tumor volume.

[0067] 3) Tumor growth curve Figure 6 and 7 The control group showed

[0068] (2) Liver cancer drug efficacy model

[0069] 1) The above C57BL / 6J mice were divided into three groups, one group as a control group and two groups as experimental groups. The mice in one experimental group were injected with 30 mg / kg sorafenib once a day, and the tumor volume was monitored; the mice in the other experimental group were injected with 30 mg / kg sorafenib twice a day, and the tumor volume was monitored.

[0070] 2) The tumor growth curve of the experimental group and the tumor growth curve of the control group are as follows Figure 6 and 7 Shown

[0071] By comparing the tumor growth curves, we can see the impact of anti-tumor drugs on the tumor growth process.

[0072] In summary, the present invention has the following beneficial effects:

[0073] 1. The present invention obtains the Hepa1-6 substrain, a derivative strain of mouse liver cancer cells, and adopts the transplantation and subculture method to continuously screen mouse liver cancer cells with a high tumor formation rate and non-shrinking, and finally obtains primary cells with a tumor formation rate of up to 100%, which solves the problem of low tumor formation rate of Hepa1-6 cells cultured in the prior art.

[0074] 2. The present invention has the advantages of reducing experimental costs, saving time, and being popular, and overcomes the defects of the prior art that tumors cannot be formed, the tumor formation rate is low, and more mice need to be used for modeling.

[0075] 3. Provide a liver cancer model Hepa1-6 for preclinical pharmacology and efficacy studies: The liver cancer model established by the present invention does not require induction by carcinogenic agents and other reagents, and has high safety throughout the experimental process. The establishment of the model only requires C57BL / 6 background mice, and there is no need to construct and breed specific transgenic mice. Only one subcutaneous injection of mice is required to complete the model construction. Therefore, the model establishment operation is simple, the entire experimental cycle is short, and the research cycle is effectively shortened and the research cost is reduced.

[0076] The above are only preferred embodiments of the present invention, and are not intended to limit the implementation methods and protection scope of the present invention. Those skilled in the art should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for obtaining a Hepa1-6 substrain derived from mouse liver cancer cells, characterized in that: The following steps are involved: S1: Use the existing mouse liver cancer cell Hepa1-6 to culture and subculture the mouse liver cancer cell Hepa1-6 to expand the main library and experimental library; S2: The cultured mouse liver cancer cells Hepa1-6 were used for subcutaneous modeling of the first batch of C57BL / 6J mice. During the process, when the normally growing tumor grew to 800-1000mm 3 When the tumor was 600 μg / cm3, it was removed and cut into uniform tumor masses after treatment. The tumor masses were transplanted into the second batch of C57BL / 6J mice for passage, and the tumor formation of this batch of mice was observed. S3: The tumors of the second batch of mice that normally develop tumors in step S2 are removed, cut into uniform tumor pieces after treatment, and transplanted into the third batch of C57BL / 6J mice for passage. After the tumors of this batch of mice grow to the desired size, the tumors are removed and frozen; S4: The tumors obtained in step S3 were frozen for several months and then revived and inoculated into the fourth batch of C57BL / 6J mice. During this tumor formation process, the tumors observed grew to 800-1500 mm 3 The cells were inoculated into the fifth batch of C57BL / 6J mice to obtain the P4 generation of the Hepa1-6 substrain, a derivative of mouse liver cancer cells.

2. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 1, characterized in that: In step S4, the fourth batch of mice is inoculated to obtain the P3 generation of the Hepa1-6 substrain, a derivative of mouse liver cancer cells, which is named Hepa1-6-PSWX002.

3. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 1, characterized in that: The deposit number of Hepa1-6-PSWX002 is CCTCC NO: C2023331.

4. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 1, characterized in that: The treatment of tumors removed from mice included the removal of necrotic tumor parts, fascia, and blood clots.

5. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 1, characterized in that: In step S3, the mouse tumor is removed after it grows to a desired size.

6. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 1, characterized in that: The culture of mouse liver cancer cells Hepa1-6 in step S1 specifically includes the recovery of mouse liver cancer cells Hepa1-6, the passaging of mouse liver cancer cells Hepa1-6 and the supply of mouse liver cancer cells Hepa1-6.

7. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 6, characterized in that: The resuscitation of mouse liver cancer cell Hepa1-6 is as follows: taking out a frozen mouse liver cancer cell Hepa1-6 selected in step S1, and rapidly thawing it in a 37°C water bath; aspirating the cell suspension into a 15ml centrifuge tube with 9ml complete culture medium, centrifuging at 1200rpm for 3min, discarding the supernatant, adding complete culture medium to resuspend the cells, transferring to a T25 culture flask, adding culture medium, and adjusting the cell density to 0.2×10 6 cells / mL were placed in a cell culture incubator at 37°C and 5% CO2; The subculture of mouse liver cancer cell Hepa1-6 refers to: when the cells are cultured to a confluence of 80%-90%, the cells are subcultured, the supernatant is discarded, the cells are washed with D-PBS 1-2 times and the liquid is discarded, and the cells are digested with 0.25% Trypsin-EDTA for about 1 minute until the cells become round and shiny, and the digestion is terminated after the culture is used up; the cells are gently blown to make them fall off the wall, collected into a centrifuge tube, centrifuged at 1200rpm for 3 minutes, the supernatant is discarded, new complete culture medium is added to resuspend the cells, and the cells are subcultured into a new culture bottle at a ratio of 1:2-1:4, and placed in a cell culture incubator at 37°C and 5% CO2 for culture; The supply of mouse liver cancer cell Hepa1-6 is as follows: discarding the supernatant, washing with D-PBS 1-2 times and then discarding the liquid, digesting with 0.25% trypsin cell digestion solution for about 1 minute, until the cells become round and shiny, and then using complete medium to terminate the digestion; gently blowing to make the cells fall off the wall, collecting them into a centrifuge tube, centrifuging at 1200rpm for 3min, discarding the supernatant, adding new complete medium to resuspend the cells; mixing the cell suspension and trypan blue at a ratio of 1:1, counting and calculating the activity rate with a cell counter, centrifuging at 4°C and 1200rpm for 3min, discarding the supernatant, resuspending the cells with pre-cooled D-PBS, and adjusting the cell density to 1×10 8 cells / ml, add an equal volume of matrix gel, mix gently by pipetting; seal the Eppendorf tube with sealing film, put it into the sample bag for recording information and place it in an ice box for supply.

8. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to claim 1, characterized in that: The complete culture medium includes 90% DMEM, 10% fetal bovine serum and 100 units per ml of penicillin plus streptomycin.

9. The method for obtaining a Hepa1-6 substrain of a mouse liver cancer cell derivative according to any one of claims 1 to 8, characterized in that: The mouse liver cancer cell Hepa1-6 is an adherent cell growing in an epithelial-like monolayer.

10. Application of a mouse liver cancer cell derivative strain Hepa1-6 substrain in mouse liver cancer drug efficacy research.

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