Method for constructing in-situ liver cancer animal model

Abstract

The invention discloses a method for constructing an in-situ liver cancer animal model, and belongs to the technical field of animal models.The method comprises the steps that experimental animals are selected, rodents are selected as the experimental animals, and liver cancer cell strains are prepared, specifically, the liver cancer cell strains are prepared, specifically, cell selection and culture, cell suspension preparation, surgical operation and cell inoculation, according to the method, an experimental animal is anesthetized, then hair in an operation area is shaved off, disinfection treatment is carried out, small incision is carried out on the abdomen of the animal, and liver tissue is exposed, the step needs to be carefully operated, so that other organs are prevented from being damaged, and in the method, culture of liver cancer cells is a complex and fine process; according to the present invention, the sterile operation specification needs to be strictly obeyed, the culture condition needs to be controlled, the cell state needs to be observed and timely adjusted, the liver cancer cells with good growth state are successfully cultured by following the correct culture method and the matters needing attention, the liver cancer cell line with drug resistance is screened, and the powerful support is provided for the subsequent scientific research experiment.

Description

Technical Field

[0001] The invention belongs to the technical field of animal models, and in particular relates to a method for constructing an in situ liver cancer animal model. Background Art

[0002] The biopharmaceutical industry has always been a strategic emerging industry that the country has focused on developing. The development of the biopharmaceutical industry is inseparable from the innovative and cutting-edge basic scientific research in medicine and pharmacy. Animal disease models, as substitute disease models for humans, are important experimental tools for studying the biological characteristics, pathogenesis, drug screening and treatment of diseases. It can be said that without experimental animals, there would be no development and progress in biomedicine today.

[0003] In the study of liver cancer, the current experimental liver cancer animal models still have many shortcomings: (1) The chemical drug-induced model has defects such as a modeling time of more than one year and a high mortality rate; (2) The subcutaneous cell transplant tumor model cannot simulate the environment in which clinical tumors grow; (3) The implantation process of orthotopic tumor models is cumbersome and technically demanding. In addition, most implantation models use immunodeficient mice, which are not suitable for immune system research. Therefore, exploring and establishing more types of tumor experimental animal models that can simulate the occurrence and development of clinical tumors and are easy to obtain and cost-effective is the future development direction.

[0004] The prior art discloses some invention patents in the field of animal model technology, among which the invention patent with publication number CN114540353B discloses a method for constructing an in situ liver cancer animal model, which belongs to the field of animal models. The technical solution obtains sgRNAs targeting AXIN1 gene, PTEN gene and TP53 gene and having good editing ability through screening, wherein the nucleotide sequence of the sgRNA specifically targeting the AXIN1 gene is shown in SEQ ID NO: 1-3, the nucleotide sequence of the sgRNA specifically targeting the PTEN gene is shown in SEQ ID NO: 4-6, and the nucleotide sequence of the sgRNA specifically targeting the TP53 gene is shown in SEQ ID NO, as shown in 7-9, sgRNA was injected into the animal liver through tandem gene editing technology to construct an in situ liver cancer animal model. Through experimental verification, it was found that the knockout model constructed by combining 2-3 of the above tumor suppressor genes had obvious tumor formation at 3-4 months, and the tumor formation rate could be as high as 100%. This technical solution still has some shortcomings in the process of application. Liver cancer treatment includes surgical treatment, interventional treatment, ablation treatment, targeted treatment and immunotherapy, but patients face multiple problems such as recurrence and metastasis, drug ineffectiveness and resistance, and serious adverse reactions. This has also led to slow progress in the development of liver cancer treatment, and it is difficult to meet the needs of different researchers to explore different gene editing backgrounds to induce human tumor research.

[0005] Based on this, the present invention designs a method for constructing an in situ liver cancer animal model to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to propose a method for constructing an in situ liver cancer animal model in order to solve the problem that the existing liver cancer treatments include surgical treatment, interventional treatment, ablation treatment, targeted treatment and immunotherapy, but patients face multiple problems such as recurrence and metastasis, drug ineffectiveness and resistance, and serious adverse reactions, which also leads to slow progress in the research and development of liver cancer treatment, and it is difficult to meet the needs of different researchers to explore the research on human tumors induced by different gene editing backgrounds.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A method for constructing an in situ liver cancer animal model, comprising: Experimental animal selection,Rodents were selected as experimental animals; Preparation of liver cancer cell lines, including cell selection and culture, and preparation of cell suspension; Surgical operation and cell inoculation: anesthetize the experimental animals, shave the hair in the surgical area, and disinfect. Make a small incision on the animal's abdomen to expose the liver tissue. This step requires careful operation to avoid damaging other organs. Use a syringe to inject the prepared liver cancer cell suspension into the liver tissue. The speed and depth of injection should be controlled to ensure that the cells can be evenly distributed in the liver. For small rodents, micro-syringes are usually used for precise injection. After the inoculation is completed, the surgical incision is sutured and the animals are given necessary postoperative care. Postoperative observation and data analysis; The liver cancer cell strains are used to screen out liver cancer cell strains with drug resistance.

[0008] As a further description of the above technical solution: The cell selection and culture comprises selecting a suitable liver cancer cell line, culturing the liver cancer cells to an appropriate number before the experiment begins, and ensuring that the cells are in an active state. The cell selection and culture specifically comprises preparing drug-containing serum, liver cancer cell proliferation and liver cancer cell culture. The cell suspension preparation comprises culturing the liver cancer cell activity to more than 90% and after the number of liver cancer cells reaches the experimental requirements, resuspending the cells in serum-free culture medium to prepare a cell suspension.

[0009] As a further description of the above technical solution: The preparation method of the serum comprises: 120 male rodents were divided into 4 groups, namely control group, high-dose drug group, medium-dose drug group and low-dose drug group, with 30 rodents in each group. The drug dosages of high-dose drug group, medium-dose drug group and low-dose drug group were 146g / kg, 72g / kg and 26g / kg respectively. The control group was intragastrically administered with normal saline. The gavage volume of the four groups of rodents was 0.2 ml / kg, once a day for 7 consecutive days; After the last administration, the rodents were fasted but not watered for 10-12 hours, and then blood was collected after intraperitoneal injection of tribromoethanol to anesthetize the rodents; The serum was separated after centrifugation at 2800 r / min for 12 min, and the sera of rodents in the same group were combined and filtered with a 0.2 μm microporous filter membrane. The sera were divided into 2 ml sterile centrifuge tubes and stored in a -22°C quick-freezing device for later use.

[0010] As a further description of the above technical solution: The dosage of the high-dose group, the medium-dose group and the low-dose group is recorded in terms of raw drug dosage, and the clinical equivalent dose of experimental rodents is 12 times the clinical dose for humans as the low-dose group.

[0011] As a further description of the above technical solution: The liver cancer cell proliferation comprises: Liver cancer cells in the logarithmic growth cycle were inoculated into 96-well plates, and drug treatment was quickly performed when the cell confluence reached 80%; A blank group, a control group, and high-, medium-, and low-dose drug groups were arranged. Except for the blank group, three serum concentrations were set for the other groups, which were 20%, 10%, and 5%, respectively, and three replicate wells were set; After 24 h, 48 h, and 72 h of intervention, 10 μl of CCK8 solution was added to each well, gently shaken to mix, and incubated in the dark for 2 h; The absorbance of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument, and the cell proliferation inhibition rate was calculated.

[0012] As a further description of the above technical solution: The blank group was not treated with any intervention, the control group was treated with control group serum, and the high-, medium-, and low-dose drug groups were treated with corresponding drug-containing serum.

[0013] As a further description of the above technical solution: The calculation formula of the cell proliferation inhibition rate is: .

[0014] As a further description of the above technical solution: The liver cancer cell culture comprises: The liver cancer cell lines were inoculated in the upper chamber of the Transwell, and the culture medium was added to the lower chamber, cultured for 22 h, and washed three times with PBS; The lower chamber was added to the culture medium, the blank group was not added with serum, the control group used the control group serum, and the drug groups were added with high, medium and low doses of drug-containing serum respectively.

[0015] As a further description of the above technical solution: The postoperative observation and data analysis include regular observation, imaging examination, pathological examination and data analysis; The regular observation includes regular observation and testing of the experimental animals at appropriate time points after inoculation, and the observation indicators include the health status of the animals, weight changes and tumor growth; Imaging testing uses CT scanning technology to perform non-invasive tests on animals to confirm the formation and growth of in situ liver cancer; Pathological examination: When necessary, the experimental animals were sacrificed and liver tissues were taken for pathological examination to further confirm the presence and characteristics of liver cancer; Data analysis: Perform detailed data analysis on the experimental results, evaluate the success rate and stability of the liver cancer model, and optimize the experimental design as needed.

[0016] As a further description of the above technical solution: The rodent is preferably a mouse or a rat as the experimental animal, and the liver cancer cell line is Hepa1-6 or HepG2.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: In the present invention, the cultivation of liver cancer cells is a complex and delicate process, which requires strict compliance with aseptic operation specifications, control of culture conditions, observation of cell status and timely adjustment. By following the correct culture methods and precautions, liver cancer cells with good growth status are successfully cultivated, and liver cancer cell lines with drug resistance are screened, providing strong support for subsequent scientific research experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of a process for constructing an in situ liver cancer animal model proposed by the present invention; Figure 2 This is a schematic diagram of the process of serum preparation in the method for constructing an in situ liver cancer animal model proposed by the present invention; Figure 3 A schematic diagram of the process of liver cancer cell proliferation in a method for constructing an in situ liver cancer animal model proposed by the present invention; Figure 4 This is a schematic diagram of the process of postoperative observation and data analysis in the method for constructing an in situ liver cancer animal model proposed in the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Please refer to the attached Figure 1 -Attached Figure 4 The present invention provides a technical solution: a method for constructing an in situ liver cancer animal model, comprising: Experimental animal selection,Rodents were selected as experimental animals; Preparation of liver cancer cell lines, including cell selection and culture, and preparation of cell suspension; Surgical operation and cell inoculation: anesthetize the experimental animals, shave the hair in the surgical area, and disinfect. Make a small incision on the animal's abdomen to expose the liver tissue. This step requires careful operation to avoid damaging other organs. Use a syringe to inject the prepared liver cancer cell suspension into the liver tissue. The speed and depth of injection should be controlled to ensure that the cells can be evenly distributed in the liver. For small rodents, micro-syringes are usually used for precise injection. After the inoculation is completed, the surgical incision is sutured and the animals are given necessary postoperative care. Postoperative observation and data analysis; The liver cancer cell strains are used to screen out liver cancer cell strains with drug resistance.

[0021] Specifically, the cell selection and culture includes selecting a suitable liver cancer cell line, culturing the liver cancer cells to an appropriate number before the experiment begins, and ensuring that the cells are in an active state. The cell selection and culture specifically include preparing drug-containing serum, liver cancer cell proliferation and liver cancer cell culture. The cell suspension preparation includes culturing the liver cancer cell activity to more than 90% and the number of liver cancer cells reaches the experimental requirements, resuspending the cells in serum-free culture medium to prepare a cell suspension.

[0022] Specifically, the method for preparing the serum comprises: 120 male rodents were divided into 4 groups, namely control group, high-dose drug group, medium-dose drug group and low-dose drug group, with 30 rodents in each group. The drug dosages of high-dose drug group, medium-dose drug group and low-dose drug group were 146g / kg, 72g / kg and 26g / kg respectively. The control group was intragastrically administered with normal saline. The gavage volume of the four groups of rodents was 0.2 ml / kg, once a day for 7 consecutive days; After the last administration, the rodents were fasted but not watered for 10-12 hours, and then blood was collected after intraperitoneal injection of tribromoethanol to anesthetize the rodents; The serum was separated after centrifugation at 2800 r / min for 12 min, and the sera of rodents in the same group were combined and filtered with a 0.2 μm microporous filter membrane. The sera were divided into 2 ml sterile centrifuge tubes and stored in a -22°C quick-freezing device for later use.

[0023] Specifically, the dosage of the high-dose drug group, the medium-dose drug group and the low-dose drug group is recorded in terms of raw drug dosage, and the clinical equivalent dose of experimental rodents is 12 times the clinical dose for humans as the low-dose drug group.

[0024] Specifically, the proliferation of liver cancer cells includes: Liver cancer cells in the logarithmic growth cycle were inoculated into 96-well plates, and drug treatment was quickly performed when the cell confluence reached 80%; A blank group, a control group, and high-, medium-, and low-dose drug groups were arranged. Except for the blank group, three serum concentrations were set for the other groups, which were 20%, 10%, and 5%, respectively, and three replicate wells were set; After 24 h, 48 h, and 72 h of intervention, 10 μl of CCK8 solution was added to each well, gently shaken to mix, and incubated in the dark for 2 h; The absorbance of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument, and the cell proliferation inhibition rate was calculated.

[0025] Specifically, the blank group was not treated with any intervention, the control group was treated with control group serum, and the high-, medium-, and low-dose drug groups were treated with corresponding drug-containing serum.

[0026] Specifically, the calculation formula of the cell proliferation inhibition rate is: .

[0027] Specifically, the liver cancer cell culture includes: The liver cancer cell lines were inoculated in the upper chamber of the Transwell, and the culture medium was added to the lower chamber, cultured for 22 h, and washed three times with PBS; The lower chamber was added to the culture medium, the blank group was not added with serum, the control group used the control group serum, and the drug groups were added with high, medium and low doses of drug-containing serum respectively.

[0028] Specifically, the postoperative observation and data analysis include regular observation, imaging examination, pathological examination and data analysis; The regular observation includes regular observation and testing of the experimental animals at appropriate time points after inoculation, and the observation indicators include the health status of the animals, weight changes and tumor growth; Imaging testing uses CT scanning technology to perform non-invasive tests on animals to confirm the formation and growth of in situ liver cancer; Pathological examination: When necessary, the experimental animals were sacrificed and liver tissues were taken for pathological examination to further confirm the presence and characteristics of liver cancer; Data analysis: Perform detailed data analysis on the experimental results, evaluate the success rate and stability of the liver cancer model, and optimize the experimental design as needed.

[0029] Specifically, the rodent is preferably a mouse or a rat as the experimental animal, and the liver cancer cell line is Hepa1-6 or HepG2.

[0030] Working principle, when using: Experimental animal selection: Select rodents as experimental animals, preferably mice or rats; Preparation of liver cancer cell lines: Cell selection and culture: select appropriate liver cancer cell lines and culture liver cancer cells to an appropriate number and activity state before the experiment begins; Preparation of cell suspension: When the cell activity is above 90% and the number reaches the experimental requirements, the cells are resuspended in serum-free medium to prepare a cell suspension; Surgical operation and cell inoculation, animal anesthesia and disinfection: anesthetize the experimental animals, shave the hair in the surgical area, and disinfect it; Surgical incision and liver exposure: A small incision is made in the animal's abdomen to expose the liver tissue. This step requires careful operation to avoid damaging other organs.

[0031] Cell inoculation: Use a syringe to inject the prepared liver cancer cell suspension into the liver tissue. The speed and depth of injection should be controlled to ensure that the cells can be evenly distributed in the liver. Usually, a micro-syringe is used for precise injection; Wound suturing and care: After the inoculation is completed, the surgical incision is sutured and the animals are given necessary postoperative care; Postoperative observation and data analysis: Regular observation: At appropriate time points after inoculation, the experimental animals are regularly observed and tested. The observation indicators include the health status of the animals, weight changes, and tumor growth. Imaging testing uses CT scanning technology to perform non-invasive tests on animals to confirm the formation and growth of in situ liver cancer; Pathological examination: When necessary, the experimental animals were sacrificed and liver tissues were taken for pathological examination to further confirm the presence and characteristics of liver cancer; Data analysis: Conduct detailed data analysis on the experimental results, evaluate the success rate and stability of the liver cancer model, and optimize the experimental design as needed; In summary, throughout the entire experimental process, aseptic operation specifications need to be strictly followed to avoid complications such as infection. During the experiment, attention should be paid to the welfare of the animals to ensure that they complete the experiment with minimal pain. After the model is constructed, necessary verification experiments need to be carried out to confirm the validity and reliability of the model.

[0032] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for constructing an in situ liver cancer animal model, characterized in that: include: Experimental animal selection,Rodents were selected as experimental animals; Preparation of liver cancer cell lines, including cell selection and culture, and preparation of cell suspension; Surgical operation and cell inoculation: anesthetize the experimental animals, shave the hair in the surgical area, and disinfect. Make a small incision on the animal's abdomen to expose the liver tissue. This step requires careful operation to avoid damaging other organs. Use a syringe to inject the prepared liver cancer cell suspension into the liver tissue. The speed and depth of injection should be controlled to ensure that the cells can be evenly distributed in the liver. For small rodents, micro-syringes are usually used for precise injection. After the inoculation is completed, the surgical incision is sutured and the animals are given necessary postoperative care. Postoperative observation and data analysis; The liver cancer cell strains are used to screen out liver cancer cell strains with drug resistance.

2. The method for constructing an in situ liver cancer animal model according to claim 1, characterized in that: The cell selection and culture comprises selecting a suitable liver cancer cell line, culturing the liver cancer cells to an appropriate number before the experiment begins, and ensuring that the cells are in an active state. The cell selection and culture specifically comprises preparing drug-containing serum, liver cancer cell proliferation and liver cancer cell culture. The cell suspension preparation comprises culturing the liver cancer cell activity to more than 90% and after the number of liver cancer cells reaches the experimental requirements, resuspending the cells in serum-free culture medium to prepare a cell suspension.

3. The method for constructing an in situ liver cancer animal model according to claim 2, characterized in that: The preparation method of the serum comprises: 120 male rodents were divided into 4 groups, namely control group, high-dose drug group, medium-dose drug group and low-dose drug group, with 30 rodents in each group. The drug dosages of high-dose drug group, medium-dose drug group and low-dose drug group were 146g / kg, 72g / kg and 26g / kg respectively. The control group was intragastrically administered with normal saline. The gavage volume of the four groups of rodents was 0.2 ml / kg, once a day for 7 consecutive days; After the last administration, the rodents were fasted but not watered for 10-12 hours, and then blood was collected after intraperitoneal injection of tribromoethanol to anesthetize the rodents; The serum was separated after centrifugation at 2800 r / min for 12 min, and the sera of rodents in the same group were combined and filtered with a 0.2 μm microporous filter membrane. The sera were divided into 2 ml sterile centrifuge tubes and stored in a -22°C quick-freezing device for later use.

4. The method for constructing an in situ liver cancer animal model according to claim 3, characterized in that: The dosage of the high-dose group, the medium-dose group and the low-dose group is recorded in terms of raw drug dosage, and the clinical equivalent dose of experimental rodents is 12 times the clinical dose for humans as the low-dose group.

5. The method for constructing an in situ liver cancer animal model according to claim 4, characterized in that: The liver cancer cell proliferation comprises: Liver cancer cells in the logarithmic growth cycle were inoculated into 96-well plates, and drug treatment was quickly performed when the cell confluence reached 80%; A blank group, a control group, and high-, medium-, and low-dose drug groups were arranged. Except for the blank group, three serum concentrations were set for the other groups, which were 20%, 10%, and 5%, respectively, and three replicate wells were set; After 24 h, 48 h, and 72 h of intervention, 10 μl of CCK8 solution was added to each well, gently shaken to mix, and incubated in the dark for 2 h; The absorbance of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument, and the cell proliferation inhibition rate was calculated.

6. The method for constructing an in situ liver cancer animal model according to claim 5, characterized in that: The blank group was not treated with any intervention, the control group was treated with control group serum, and the high-, medium-, and low-dose drug groups were treated with corresponding drug-containing serum.

7. The method for constructing an in situ liver cancer animal model according to claim 6, characterized in that: The calculation formula of the cell proliferation inhibition rate is:

8. The method for constructing an in situ liver cancer animal model according to claim 7, characterized in that: The liver cancer cell culture comprises: The liver cancer cell lines were inoculated in the upper chamber of the Transwell, and the culture medium was added to the lower chamber, cultured for 22 h, and washed three times with PBS; The lower chamber was added to the culture medium, the blank group was not added with serum, the control group used the control group serum, and the drug groups were added with high, medium and low doses of drug-containing serum respectively.

9. The method for constructing an in situ liver cancer animal model according to claim 8, characterized in that: The postoperative observation and data analysis include regular observation, imaging examination, pathological examination and data analysis; The regular observation includes regular observation and testing of the experimental animals at appropriate time points after inoculation, and the observation indicators include the health status of the animals, weight changes and tumor growth; Imaging testing uses CT scanning technology to perform non-invasive tests on animals to confirm the formation and growth of in situ liver cancer; Pathological examination: When necessary, the experimental animals were sacrificed and liver tissues were taken for pathological examination to further confirm the presence and characteristics of liver cancer; Data analysis: Perform detailed data analysis on the experimental results, evaluate the success rate and stability of the liver cancer model, and optimize the experimental design as needed.

10. The method for constructing an in situ liver cancer animal model according to claim 9, characterized in that: The rodent is preferably a mouse or a rat as the experimental animal, and the liver cancer cell line is Hepa1-6 or HepG2.

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