Construction method and application of asbestos-caused lung carcinoma in situ animal model

Abstract

The invention discloses a construction method and application of an asbestos-caused lung carcinoma-in-situ animal model, and belongs to the technical field of biomedicine. The construction method comprises the following steps: firstly, carrying out multiple poisoning treatments on embryo fibroblasts by chrysotile, and then carrying out malignant transformation to obtain malignant transformed cells; a luciferase gene is introduced by utilizing lentivirus transfection; and injecting the successfully transfected malignant transformed cells into the lung of an experimental animal by adopting an intrapulmonary injection method, and feeding to obtain the lung carcinoma-in-situ animal model. The method creatively uses asbestos to induce malignant transformed embryo fibroblasts with cancerous characteristics to establish the lung in-situ cancer animal model, and has the advantages of short modeling period, high tumor formation rate, visualized cell transfer condition, capability of better reflecting the cancerous characteristics of asbestos and the like. The constructed model can be applied to clinical diagnosis and treatment research such as pathogenesis research and drug screening evaluation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for constructing and applying an animal model of asbestos-induced lung carcinoma in situ. Background Technology

[0002] Asbestos is a natural fibrous silicate mineral with excellent fire-resistant, heat-insulating, and electrical properties, and is widely used in the construction, chemical, and automotive industries. As early as 1987, asbestos was classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). In March 2009, the IARC reassessed the carcinogenicity of asbestos and concluded in a 2012 thematic study that asbestos induces genotoxicity, aneuploidy, polyploidy, and epigenetic alterations. Epidemiological studies have confirmed that lung cancer remains the leading cause of cancer death, and there is a close relationship between asbestos exposure and lung cancer. Studies have shown that occupational exposure and environmental exposure to airborne asbestos have been identified as contributing factors to lung cancer in non-smokers. Asbestos-induced lung cancer is characterized by a long latency period, high malignancy, and poor prognosis. Therefore, in-depth research into the carcinogenic mechanisms of asbestos is crucial for developing more effective treatment strategies.

[0003] In studies of asbestos carcinogenic mechanisms, cell models and animal models are commonly used. For example, patent document CN110423725A discloses that human pleural mesothelial cells (MeT-5A) undergo malignant transformation after multiple stages of exposure to chrysotile asbestos, resulting in a malignant transformed strain of human pleural mesothelial cells, which is used as a cell model for studying carcinogenic mechanisms.

[0004] Constructing animal models that accurately simulate pathological processes can better reconstruct pathogenesis and is of great significance in studying tumor evolution, drug development, and comparing treatment efficacy. Mouse lung tissue is quite similar to human lung tissue in morphology and molecular characteristics, and almost all human genes have homologous genes in mice. Therefore, most existing animal models use mice for research.

[0005] The most common method for constructing an asbestos-induced lung cancer model in mice is the direct asbestos induction method, which induces lung cancer in mice through inhalation, bronchial perfusion, etc. (Research Progress on Early Lung Cancer Animal Models. Radiology Practice, 2013, 28(12):1291-1293). However, this method requires regular intubation and asbestos inoculation of mice, and fibrotic changes in the lungs of the model animals can only be seen after about 6 months of inoculation. The inoculation period is long, the feeding conditions are strict, the reproducibility of the model is poor, and the lung lesions of mice must be observed after dissection. Therefore, it has considerable limitations in its use.

[0006] Therefore, constructing an experimental animal model that has a short modeling cycle, stable tumor formation, visualizes cell metastasis, and accurately reflects the carcinogenic characteristics of asbestos is a technical problem that researchers in the field of asbestos carcinogenic mechanism research need to solve. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing an animal model of lung carcinoma in situ caused by asbestos with a short modeling cycle and stable tumor formation, overcoming the problems of long cycle, low success rate and unclear tumor progression in traditional methods of establishing mouse lung cancer by direct asbestos exposure.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for constructing an animal model of lung carcinoma in situ caused by asbestos, comprising the following steps: (1) Embryonic fibroblasts undergo malignant transformation after being repeatedly exposed to chrysotile asbestos to obtain malignant transformed cells; (2) Malignant transformed cells were transfected with lentiviruses carrying luciferase and resistance genes, and successfully transfected malignant transformed cells were obtained through resistance selection. (3) The successfully transfected malignant transformed cells were injected into the lungs of experimental animals by intrapulmonary injection, and the animal model of lung carcinoma in situ was obtained by feeding them.

[0009] This invention employs low-dose, long-term, intermittent exposure to chrysotile asbestos to obtain mouse embryonic fibroblasts undergoing malignant transformation. These malignant transformed cells are then transferred into mouse lung tissue via intrapulmonary injection to construct a lung carcinoma in situ animal model. Studies have shown that the two-step method provided by this invention for establishing asbestos-induced lung carcinoma in experimental animals results in stable tumor formation, with all animals developing tumors in the experiment. This invention introduces a luciferase gene into the malignant transformed cells, enabling them to express luciferase. This allows for the tracking of the survival, proliferation, and migration of malignant transformed cells in lung tissue, and the monitoring of tumor progression in vivo.

[0010] As a preferred option, mice are used as experimental animals, and NIH / 3T3 cells are used as embryonic fibroblasts. Even more preferred is nude mice, as their skin is thinner than other mouse breeds, allowing the ribs and lung parenchyma to be visible under strong light. After skin disinfection, the injection point in the lungs can be clearly located, eliminating the need to cut the skin to expose the lung parenchyma, saving injection time, reducing the risk of mouse mortality, and increasing the success rate of modeling.

[0011] In this invention, the method for constructing the malignant transformed cells includes: 1) Prepare single-cell suspensions of embryonic fibroblasts in the logarithmic growth phase, mix them with an equal volume of matrix gel, and then seed them into cell culture plates and pre-culture them with culture medium. 2) Discard the culture medium, add chrysotile asbestos solution for treatment, and then replace it with culture medium to continue culturing; 3) Repeat step 2) several times; 4) Collect cells, and transfer the luciferase gene, fluorescent protein gene, and anti-purinemycin gene into the cells. Use puromycin to screen for cells with transferred genes.

[0012] 5) Prepare a single-cell suspension and administer it via intrapulmonary injection.

[0013] Preferably, in step 1), the density of the single-cell suspension is 1~2×10⁻⁶. 6 Cells / mL; Under ice bath conditions, mix equal volumes of single-cell suspension and Matrigel, spread into cell culture plates, incubate at 37°C for 30-45 minutes, then add culture medium for pre-culture at 37°C for 20-24 hours.

[0014] Preferably, in step 2), the concentration of the chrysotile asbestos solution is 1.25 μg / mL, and the chrysotile asbestos is dissolved and diluted using phosphate buffer; the treatment time is 48 h; after treatment, the culture medium is replaced with fresh medium and cultured for another 7 days; in step 3), the operation is repeated 1-2 times.

[0015] Through repeated exposure to low doses of the toxin, embryonic fibroblasts underwent malignant transformation, specifically manifested as layered cell growth. Soft agar colony formation experiments showed that the transformed cells exhibited anchorage-independent growth characteristics, indicating a successful malignant transformation cell model.

[0016] This invention introduces the luciferase gene into malignant transformed cells, enabling them to express luciferase after being inoculated and colonized in experimental animals, thereby tracking cell proliferation and migration and visualizing cell transfer.

[0017] Preferably, in step (4), the lentivirus is a triple-labeled lentivirus carrying a luciferase gene, a fluorescent protein gene, and an anti-purinemycin gene; during transfection, the lentivirus and polybrene are added to the culture medium, the culture medium is replaced after 12 h of transfection, and puromycin is added for screening after 32 h of culture.

[0018] Preferably, in step (5), the successfully transfected malignant transformed cells are prepared into a single-cell suspension and mixed with an equal volume of matrix gel to obtain an injection solution, wherein the cell concentration in the injection solution is 10. 8 Cells / mL.

[0019] Preferably, the prepared injection solution is placed at room temperature until it reaches a viscous, non-solidified state before intrapulmonary injection.

[0020] Preferably, the intrapulmonary injection method includes: fixing the experimental animal in a supine position after anesthesia, inserting a needle into the lung at a 10-15° angle at the 3rd-4th intercostal space, and injecting 50 μL of cell injection solution. The injection volume varies depending on the type of cells; the amount of cells can be determined by consulting relevant literature. In this invention, injecting the above-mentioned number of malignant transformed cells ensures tumor formation in all mice.

[0021] One week after intrapulmonary injection, in vivo imaging was performed weekly. Over time, the intensity and extent of fluorescence signals in the chest of the experimental animals increased, indicating that malignant transformed cells survived, proliferated, and migrated within the lungs, thus confirming the successful establishment of the lung carcinoma in situ animal model. The constructed animal model exhibited transparent, fish-flesh-like tumors in the lung tissue, along with lung tissue deformation and adhesion to the chest wall. This pathological manifestation is consistent with the clinical pathological features of asbestos-induced lung cancer.

[0022] Preferably, the in vivo imaging method includes: injecting a luciferase substrate into the peritoneum, allowing it to act for 5 minutes, and then placing it in an in vivo imaging device for in vivo imaging.

[0023] This invention constructs an experimental animal model with stable tumors, short cycle, visualized cell metastasis, and better reflection of the carcinogenic properties of asbestos through the above method.

[0024] This invention provides the application of the aforementioned animal model of lung carcinoma in situ in the study of the mechanism of asbestos-induced lung cancer. The animal model constructed by this invention will be applied to research on the carcinogenicity assessment of carcinogens, discussion of carcinogenic mechanisms, and clinical diagnosis and treatment of carcinogen-induced specific tumors.

[0025] This invention also provides the application of the aforementioned animal model of lung carcinoma in situ in screening or evaluating drugs for treating asbestos-induced lung cancer. The experimental animal model established in this invention is used as a drug delivery vehicle to screen drugs that have therapeutic effects on asbestos-induced lung cancer, or to evaluate the efficacy of drugs in treating asbestos-induced lung cancer.

[0026] The beneficial effects of this invention are as follows: This invention innovatively utilizes asbestos-induced malignant transformed cells to construct a lung carcinoma in situ model, achieving a short modeling cycle and high tumorigenesis rate. By transferring the luciferase gene into asbestos-induced malignant transformed cells, cell metastasis can be visualized, allowing for the tracking of tumor progression. The model constructed using this invention enables early mechanistic studies of asbestos-induced carcinogenesis, offering good specificity, reproducibility, and traceability of cancer cell spread in carcinogen research. This model is of great significance for assessing the carcinogenicity of chemical carcinogens, discussing carcinogenic mechanisms, and the clinical diagnosis and treatment of carcinogen-induced specific tumors. Attached Figure Description

[0027] Figure 1The images shown are fluorescence images of mice in Example 1, where A is a fluorescence image of mice on day 7 after intrapulmonary injection; B is a fluorescence image of mice on day 14 after intrapulmonary injection; and C is a fluorescence image of mice on day 21 after intrapulmonary injection.

[0028] Figure 2 The image shows the changes in fluorescence intensity in mouse in vivo imaging in Example 1.

[0029] Figure 3 The image shows the morphology of mouse lung tissue in Example 1.

[0030] Figure 4 These are four in vivo fluorescence imaging images of the control group mice in Example 2.

[0031] Figure 5 These are four in vivo fluorescence imaging images of mice in the PX-478 group in Example 2.

[0032] Figure 6 The image shows a comparison of the fluorescence intensity of the control group mice and the PX-478 group mice after intrapulmonary injection for 7 and 14 days in Example 2.

[0033] Figure 7 The image shows a comparison of the fluorescence intensity of the control group mice and the PX-478 group mice after intrapulmonary injection for 21 and 28 days in Example 2. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] The NIH / 3T3 cells used in the following examples were purchased from the China Cell Resource Center; trypsin (Gibco), chrysotile asbestos (Japan Mineral Fibers Association), triple-labeled lentivirus (Lingke Biotechnology), Matrigel (Corning), nude mice (Zhejiang Medical Animal Center), DMEM medium (Gibco), fetal bovine serum (Gibco), polyglobulin (Lingke Biotechnology), puromycin (Fushen Biotechnology), and luciferase substrate (Fushen Biotechnology) were all purchased commercially.

[0037] Example 1 This embodiment provides a method for constructing a nude mouse lung in situ carcinoma model by asbestos-induced malignant transformed cells. In this embodiment, fluorescence in vivo imaging is performed on mice to assess tumor formation and determine the feasibility of the model. The specific details of this embodiment are as follows: 1. Malignant transformation of cells One day before the malignant transformation of NIH / 3T3 cells, the matrix gel was thawed overnight at 4°C. Once cells reached 70%-80% confluence, they were trypsin-digested, resuspended in complete culture medium, and the cell density was adjusted to 1×10⁶ cells / year. 6 Cells / mL. The cell suspension was mixed with matrix gel at a 1:1 ratio and seeded into 24-well plates, 200 μL per well. The plates were incubated at 37°C for 30-45 minutes, and then 500 μL of complete culture medium was added to each well. 24 h after seeding, the old culture medium was discarded. The transformed cell group was treated with 1.25 μg / mL chrysotile asbestos diluted in PBS, while the control group received the same volume of PBS. Three replicates were prepared for each group. After 48 h, the medium was replaced with asbestos-free complete culture medium. The medium was changed after 7 days, and the treatment with chrysotile asbestos was repeated for 48 h, followed by fresh medium. This process was repeated for another 7 days, for a total of 21 days, after which the cells were collected. After malignant transformation of NIH / 3T3 cells using this double-dose treatment method, the cells exhibited cascading growth. The soft agar colony formation experiment showed anchorage-independent growth characteristics, and the malignant transformed cells were harvested.

[0038] 2. Cell transfection NIH / 3T3 cells transformed by asbestos were cultured at 5 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 5 × 10⁶ cells / well in 24-well plates to establish a puromycin killing curve. 4 The cells were seeded at a density of [missing information] into 24-well plates and incubated overnight. When the cells reached 80% confluence, the medium was replaced with a medium containing triple-labeled lentivirus (containing green fluorescent protein, puromycin resistance, luciferase gene, and polybrene). The lentivirus was diluted to 1 × 10⁻⁶. 7 T / mL, Polybrene diluted to 50 µg / mL, and complete culture medium, lentivirus dilution, and Polybrene solution were prepared in a 7:2:1 ratio to transfect cells. After 12 hours, the culture medium containing lentivirus was replaced with lentivirus-free culture medium. After 32 hours, puromycin was added to screen for successfully transfected cells.

[0039] 3. Preparation of cell suspension 5 × 10⁵ cells were successfully transfected. 6The cells were mixed in a 1:1 ratio with 25 µL of Matrigel. After reviewing relevant literature, the number of cells to be injected into the lungs was determined. This number ensured that tumor formation occurred in the lungs of mice injected with chrysotile-transformed cells under 3D conditions.

[0040] 4. Intrapulmonary injection in nude mice Five nude mice were anesthetized by intraperitoneal injection of 0.1 mL / 20 g of 1% sodium pentobarbital. Cell suspension was aspirated into an insulin needle, 50 µL per mouse, and left at room temperature until it reached a viscous, non-coagulated state. The mouse was positioned approximately 1.5 cm above the costal arch on the left anterior axillary line, at the 3rd-4th intercostal space. Using an insulin needle, 50 μL of the cell mixture was slowly injected into the left lung at a 10-15° angle, 3-5 mm in diameter. After injection, the needle was withdrawn after 5-10 seconds of intubation and rotated out. The mice were then placed in cages after regaining consciousness.

[0041] 5. Nude mouse in vivo imaging One week after intrapulmonary injection, in vivo imaging was performed weekly. Nude mice were injected intraperitoneally with luciferase substrate at a concentration of 15 mg / mL, at a dose of 200 µL per 20 g of mouse body weight. After 5 minutes of action, the mice were anesthetized with isoflurane for 5 minutes and then placed in a small animal in vivo imaging system for image capture.

[0042] See results Figure 1 and Figure 2 The mice showed obvious fluorescent signals in their chests, and the intensity and range of the fluorescent signals increased over time, suggesting that asbestos-induced malignant transformed cells survived, proliferated and migrated in the mouse lungs.

[0043] See results Figure 3 In mice, transparent, fish-flesh-like tumors developed in the lung tissue, causing deformation of the lung tissue and adhesion to the chest wall. The color and morphology of the tumors were consistent with descriptions in literature related to intrapulmonary injection of lung cancer cells.

[0044] Example 2 This embodiment provides the application of an asbestos-induced malignant transformed cell model of nude mouse lung in situ carcinoma in the evaluation of therapeutic drug efficacy. In this embodiment, the drug was administered to mice, and tumor formation was assessed to determine the feasibility of the model. The specific details of this embodiment are as follows: 1. Malignant transformation of cells The transformation process was the same as in Example 1. One day before the malignant transformation of NIH / 3T3 cells, the matrix gel was thawed overnight at 4°C. When cells reached 70%-80% fusion, they were digested with trypsin, resuspended in complete culture medium, and the cell density was adjusted to 1×10⁶ cells / year. 6Cells / mL. The cell suspension was mixed with matrix gel at a 1:1 ratio and seeded into 24-well plates, 200 μL per well. The plates were incubated at 37°C for 30-45 minutes, and then 500 μL of complete culture medium was added to each well. 24 h after seeding, the old culture medium was discarded. The transformed cell group was treated with chrysotile asbestos diluted in PBS, while the control group was treated with the same volume of PBS. Three replicates were set for each group. After 48 h, the medium was replaced with normal medium and cultured for another 7 days. The medium was then changed again, followed by another 48 h of chrysotile asbestos treatment, and then fresh medium was added. This process was repeated for another 7 days, for a total of 21 days, after which cells were collected. NIH / 3T3 cells were malignantly transformed using this double-dose method, and the malignant transformed cells were harvested.

[0045] 2. Cell transfection The transfection process was the same as in Example 1. NIH / 3T3 cells transformed by asbestos were transfected at a rate of 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 5 × 10⁶ cells / well in 24-well plates to establish a puromycin killing curve. 4 The cells were seeded at a density of 100% into 24-well plates and incubated overnight. When the cells reached 80% confluence, the regular medium was replaced with a medium containing triple-labeled lentivirus with green fluorescent protein, anti-purinemycin, luciferase gene, and polybrene. The cells were then transfected. After 12 hours, the medium containing lentivirus was replaced with the regular medium. After 32 hours, puromycin was added to screen for successfully transfected cells.

[0046] 3. Cell suspension preparation: 5 × 10⁵ cells of successfully transfected cells... 6 Mix 1 part with 25 µL of Matrigal matrix gel in a 1:1 ratio.

[0047] 4. Intrapulmonary injection in nude mice The injection procedure was the same as in Example 1. Nude mice were anesthetized by intraperitoneal injection of 0.1 mL / 20 g of 1% sodium pentobarbital. 50 µL of cell suspension was drawn from each mouse using an insulin needle and allowed to stand at room temperature until it reached a viscous, non-solidified state. The nude mouse was positioned approximately 1.5 cm above the costal arch on the left anterior axillary line, at the 3rd-4th intercostal space. Using an insulin injection needle, 50 μL of the cell mixture was slowly injected into the left lung at a 10-15° angle, 3-5 mm into the lung. After injection, the needle was withdrawn after 5-10 seconds of intubation and rotated out. The mice were then placed in cages for rearing after regaining consciousness.

[0048] 5. HIF inhibitor intervention PX-478 solution was prepared, and the inhibitor was dissolved in physiological saline to a final concentration of 2 mg / mL. Each mouse was injected intraperitoneally at a dose of 20 mg / kg, starting the day after cell inoculation. The weight of the nude mice was measured before injection, and the injection dose was calculated based on body weight. Injections were administered continuously for 5 days, with mouse weight recorded every other day. The control group received physiological saline, and there were 12 mice in each of the HIF inhibitor group and the control group.

[0049] Nude mouse in vivo imaging: In vivo imaging was performed weekly one week after intrapulmonary injection. Nude mice were injected intraperitoneally with luciferase substrate at a concentration of 15 mg / mL, with 200 µL injected per 20 g of mouse body weight. After 5 minutes of action, the mice were anesthetized with isoflurane for 5 minutes and then placed in a small animal in vivo imaging system for image capture.

[0050] See results Figures 4-7 In vivo fluorescence imaging was performed on mice at 7, 14, 21, and 28 days after intrapulmonary injection. Significant fluorescence signals appeared in the chest area of ​​the mice, with the intensity and extent of the fluorescence signal increasing over time, indicating that asbestos-induced malignant transformed cells survived, proliferated, and migrated in the mouse lungs. From the second imaging onwards, the total fluorescence intensity in the lungs of mice in the HIF inhibitor group was lower than that in the control group, indicating that the HIF inhibitor could inhibit the growth and reproduction rate of malignant transformed cells in mouse lung tissue.

[0051] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of asbestos-induced lung carcinoma in situ, characterized in that, Includes the following steps: (1) Embryonic fibroblasts undergo malignant transformation after being repeatedly exposed to chrysotile asbestos to obtain malignant transformed cells; (2) Malignant transformed cells were transfected with lentiviruses carrying luciferase and resistance genes, and successfully transfected malignant transformed cells were obtained through resistance selection. (3) The successfully transfected malignant transformed cells were injected into the lungs of experimental animals by intrapulmonary injection, and the animal model of lung carcinoma in situ was obtained by feeding them.

2. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 1, characterized in that, Mice were used as experimental animals, and NIH / 3T3 cells were used as embryonic fibroblasts.

3. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 1, characterized in that, In step (1), the method for constructing the malignant transformed cells includes: 1) Prepare single-cell suspensions of embryonic fibroblasts in the logarithmic growth phase, mix them with an equal volume of matrix gel, and then seed them into cell culture plates and pre-culture them with culture medium. 2) Discard the culture medium, add chrysotile asbestos solution for treatment, and then replace it with culture medium to continue culturing; 3) Repeat step 2) several times; 4) Collect cells and screen to obtain malignant transformed embryonic fibroblasts.

4. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 3, characterized in that, In step 1), the density of the single-cell suspension is 1~2×10⁻⁶. 6 Cells / mL; pre-culture conditions: 37℃ for 20-24 h.

5. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 3, characterized in that, In step 2), the concentration of the chrysotile asbestos solution is 1.25 μg / mL, and the treatment time is 48 h; in step 3), the operation is repeated 1-2 times.

6. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 1, characterized in that, In step (2), the lentivirus is a triple-labeled lentivirus carrying the luciferase gene, the fluorescent protein gene, and the anti-purinemycin gene; during transfection, the lentivirus and polybrene are added to the culture medium, the culture medium is changed after 12 h of transfection, and puromycin is added for screening after 32 h of culture.

7. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 1, characterized in that, In step (3), the successfully transfected malignant transformed cells are prepared into a single-cell suspension and mixed with an equal volume of matrix gel to obtain an injection solution. The cell concentration in the injection solution is 10. 8 Cells / mL; administer intrapulmonary injection when the prepared injection solution has been left at room temperature to a viscous, non-solidified state.

8. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 1 or 7, characterized in that, In step (3), the method of intrapulmonary injection includes: fixing the experimental animal in a supine position after anesthesia, inserting the needle into the lung at a 10-15° angle at the 3rd-4th intercostal space, and injecting cell injection solution in a volume of 50 μL.

9. The method for constructing an animal model of asbestos-induced lung carcinoma as described in claim 1, characterized in that, In step (3), in vivo imaging is performed weekly after intrapulmonary injection one week later. As time goes by, the intensity and range of fluorescence signal in the chest of the experimental animal increase, indicating that the animal model of lung carcinoma in situ has been successfully constructed. The in vivo imaging method includes: injecting luciferase substrate into the peritoneum and performing in vivo imaging after 5 minutes.

10. The application of the lung in situ carcinoma animal model prepared by the construction method according to any one of claims 1-9 in the study of the mechanism of asbestos-induced lung cancer or in the screening or evaluation of drugs for the treatment of asbestos-induced lung cancer.

Images