Lung adenocarcinoma animal model with high bone metastasis, cell line and construction method and application of lung adenocarcinoma animal model

Abstract

The invention belongs to the technical field of tumor model construction, and discloses a lung adenocarcinoma animal model with high bone metastasis, a cell line and a construction method and application thereof. The lung adenocarcinoma animal model with high bone metastasis is provided through multi-generation in-vivo directional screening, and the construction method is efficient, controllable, high in repeatability, capable of remarkably improving the tumor formation efficiency and rate, capable of being applied to immune healthy mice, low in cost and high in stability. And further extracting tumor cells from the bone metastasis lesion of the animal model to obtain the mouse lung adenocarcinoma cell line with high bone metastasis. The cell line has the advantages of being excellent in bone metastasis performance, stable in phenotype, heritable and the like, can be connected with an existing research system, has universality and economical efficiency, and can efficiently meet the requirement of bone metastasis mechanism research on a high-specificity model. The method can be applied to the fields of construction of tumor animal models or cell lines of bone metastasis, screening or development of lung adenocarcinoma drugs and the like.

Description

Technical Field

[0001] This invention belongs to the field of tumor model construction technology, specifically relating to an animal model of lung adenocarcinoma with high bone metastasis, a cell line, its construction method, and its application. Background Technology

[0002] Lung cancer ranks among the leading causes of cancer-related morbidity and mortality worldwide. Lung adenocarcinoma, as one of the major subtypes of lung cancer, presents significant challenges to clinical diagnosis and treatment due to its insidious early symptoms and rapid progression. According to Globocan data, lung adenocarcinoma has long been among the leading causes of cancer-related morbidity and mortality globally, becoming a major disease that seriously threatens human life and health.

[0003] In the progression of lung adenocarcinoma, distant metastasis is one of the main causes of treatment failure, poor prognosis, and even death, and bone tissue is one of the most common sites of distant metastasis in lung adenocarcinoma. Studies have shown that approximately 36% of lung adenocarcinoma patients will develop bone metastasis during the disease process. Once bone metastasis occurs, patients often experience a series of serious bone-related events, which not only greatly reduce their quality of life but also significantly shorten their lifespan, placing a heavy burden on patients' families and society.

[0004] Animal models or cell lines with high bone metastasis are crucial tools for studying the mechanisms of tumor bone metastasis, screening anti-bone metastasis drugs, and exploring related immune regulatory mechanisms. They can stably and efficiently simulate the entire process of cancer cell migration, invasion, and colonization into the bone. However, in the field of lung adenocarcinoma, the development and application of relevant animal models or cell lines with high bone metastasis still have significant shortcomings. For example, although the existing human lung cancer cell line with high bone metastasis, SPC-A-1BM, can be applied to immunodeficient mouse models, these models cannot simulate the complete immune microenvironment of the body, resulting in low application value of this cell line in the study of the tumor immune microenvironment related to lung adenocarcinoma bone metastasis.

[0005] To overcome the limitations of immunodeficient mouse models, humanized mouse models have been gradually applied. However, these models have problems such as high difficulty in experimental operation and high costs of breeding and experimentation, making it difficult to carry out them widely in routine clinical laboratories. This has seriously limited the popularization and advancement of research on immune-related aspects of lung adenocarcinoma bone metastasis. Summary of the Invention

[0006] In view of the problems existing in the prior art, this invention provides an animal model and cell line of lung adenocarcinoma with high bone metastasis, as well as its construction method and applications. This invention uses a mouse lung adenocarcinoma cell line with a well-established source and stable biological characteristics as parental tumor cells to construct an animal model and cell line of lung adenocarcinoma with high bone metastasis applicable to immunocompetent individuals. This meets the needs of tumor immunology-related research on lung adenocarcinoma bone metastasis and provides a powerful experimental tool for further in-depth exploration of the pathogenesis of lung adenocarcinoma bone metastasis, development of targeted anti-bone metastasis drugs, and optimization of immunotherapy strategies.

[0007] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a method for constructing an animal model of lung adenocarcinoma with high bone metastasis, the method comprising the following steps: S1. Mouse lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 25-30 days, tumor cells were extracted from bone metastases in mice to obtain first-generation lung adenocarcinoma cells. S2. The first generation of lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 14-21 days, tumor cells were extracted from bone metastases in the mice to obtain the second generation of lung adenocarcinoma cells. S3. The second-generation lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 14-21 days, a lung adenocarcinoma animal model with high bone metastasis was obtained -2.

[0008] The construction method provided by this invention is efficient, stable, and scientifically controllable. Through multiple generations of in vivo targeted screening, a lung adenocarcinoma animal model with high bone metastasis was successfully obtained, providing a high-performance and widely adaptable experimental model for lung adenocarcinoma bone metastasis research. The specific beneficial effects are as follows: 1. The construction method provided by this invention is highly efficient, controllable, and reproducible. This invention employs a multi-generational iterative screening strategy involving left ventricular injection and extraction of cells from bone metastases. The steps are clear, and the screening cycle for each generation is well-defined. The tumor formation time for the first generation is approximately 4 weeks, while for the second generation it is shortened to 2-3 weeks. By gradually enriching high-potency bone metastatic cell subsets, the targeted construction of high-potency bone metastatic cell lines is achieved. The entire process is controllable, and the screening cycle for each generation is predictable, effectively solving the problems of long screening cycles and poor stability in traditional bone metastatic cell line screening, ensuring reproducible construction under different experimental scenarios.

[0009] 2. The method for constructing animal models provided by this invention significantly improves tumor formation efficiency and rate, does not rely on immunodeficient mice, and can cover multiple research scenarios such as tumor immunity, regulation of bone metastasis microenvironment, and screening of anti-bone metastasis drugs. It can reduce the cost of replacing experimental models and provide a convenient and reliable tool for basic research and drug development related to bone metastasis.

[0010] 3. The resulting animal models exhibit strong stability. After targeted screening, the high bone metastasis phenotype in the animal models has been stably inherited, with uniform tumor formation time and regular metastatic lesion formation, which can meet the requirements for long-term and reproducible experimental studies, providing a stable model basis for in-depth exploration of bone metastatic diseases.

[0011] Preferably, after obtaining the lung adenocarcinoma animal model-2 with high bone metastasis, the method further includes the following steps: extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model-2 with high bone metastasis to obtain third-generation lung adenocarcinoma cells; The third-generation lung adenocarcinoma cells were injected into normal mice via the left ventricle, and after 14-18 days, a lung adenocarcinoma animal model with high bone metastasis-3 was obtained.

[0012] More preferably, after obtaining the lung adenocarcinoma animal model-3 with high bone metastasis, the method further includes the following step: extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model-3 with high bone metastasis to obtain fourth-generation lung adenocarcinoma cells; The fourth-generation lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 12-16 days, a lung adenocarcinoma animal model with high bone metastasis was obtained.

[0013] Preferably, the mouse lung adenocarcinoma cells include the mouse lung adenocarcinoma cell line Lewis Lung Cancer (LLC).

[0014] Preferably, the normal mouse is a C57 background mouse that is 6-8 weeks old.

[0015] For example, the C57 background mouse in this invention is illustrated using the C57BL / 6 mouse.

[0016] Preferably, in S1, the dose of the injected mouse lung adenocarcinoma cells is 1.5 × 10⁻⁶. 5 cells / each - 3×10 5 cells / each.

[0017] Preferably, in S2, the dose of the first-generation lung adenocarcinoma cells injected is 1.5 × 10⁻⁶. 5 cells / each - 3×10 5 cells / each.

[0018] Preferably, in S3, the dose of the second-generation lung adenocarcinoma cells injected is 1.5 × 10⁻⁶. 5 cells / each - 3×10 5 cells / each.

[0019] More preferably, the dose of the injected third-generation lung adenocarcinoma cells is 1.5 × 10⁻⁶. 5 cells / each - 3×10 5cells / each.

[0020] More preferably, the dose of the fourth-generation lung adenocarcinoma cells injected is 1.5 × 10⁻⁶. 5 cells / each - 3×10 5 cells / each.

[0021] For example, Nth generation lung adenocarcinoma cells (N being a positive integer from 1 to 4) were injected into mice in the form of a cell suspension at a dose of 75 μL / mouse to 120 μL / mouse, and the concentration of the cell suspension was 2 × 10⁻⁶. 6 cells / mL - 4 × 10 6 cells / mL.

[0022] For example, a lung adenocarcinoma cell suspension was prepared by resuspending tumor cells in PBS buffer.

[0023] In a second aspect, the present invention provides a mouse model of lung adenocarcinoma with high bone metastasis, which is constructed by the method for constructing the animal model of lung adenocarcinoma with high bone metastasis described in the first aspect.

[0024] The lung adenocarcinoma animal model with high bone metastasis provided by this invention uses mouse lung adenocarcinoma cells as parent tumor cells. It has the natural advantage of being applicable to immunocompetent mice and does not rely on immunodeficient mice. After multiple generations of iterative screening, the construction time of the 3rd-4th generation model is shortened to 2 weeks. This animal model has relatively low cost and strong stability.

[0025] Thirdly, the present invention provides the application of the above-mentioned lung adenocarcinoma animal model with high bone metastasis in the preparation of mouse lung adenocarcinoma cell lines with high bone metastasis.

[0026] Fourthly, the present invention provides a method for constructing a mouse lung adenocarcinoma cell line with high bone metastasis, the method comprising: extracting tumor cells from bone metastases in the lung adenocarcinoma animal model with high bone metastasis described in the second aspect to obtain a mouse lung adenocarcinoma cell line with high bone metastasis.

[0027] Fifthly, the present invention provides a mouse lung adenocarcinoma cell line with high bone metastasis, which is constructed by the method for constructing a mouse lung adenocarcinoma cell line with high bone metastasis described in the fourth aspect.

[0028] The mouse lung adenocarcinoma cell line with high bone metastasis provided by this invention is constructed based on the widely used LLC cell line. It not only possesses advantages such as excellent bone metastasis performance and stable, heritable phenotype, but also integrates with existing research systems, combining universality and cost-effectiveness. It can efficiently meet the demand for highly specific models in research on bone metastasis mechanisms. Specific analysis is as follows: 1. Outstanding ability to metastasize to bone marrow The bone metastasis specificity is high. Under the same conditions, lung adenocarcinoma cells of the 3rd-4th generation can form obvious bone metastases in the tibia and femur of mice within 2 weeks after left ventricular injection. The tumor formation time is shortened by about 50% compared with the parent LLC cell line (about 4 weeks), and the tumor formation efficiency and rate are significantly improved. With the increase of screening passage, the bone metastasis invasiveness gradually increases, the tumor formation time is further stabilized, and the bone metastasis efficiency and stability are far superior to the parent cell line, which can meet the needs of high specificity models for bone metastasis mechanism research. 2. Phenotypic stability and inheritance, avoiding traditional defects. After targeted screening, the high bone metastasis phenotype can be stably inherited, effectively solving the phenotypic drift problem that is prone to occur in traditional bone metastasis cell lines, and ensuring the reliability and reproducibility of experimental results; 3. It has strong universality and relatively low cost. The LLC cell line of this invention can be applied to immune-healthy mice, possessing universality for basic tumor research; at the same time, it can directly connect with existing LLC cell line-related research systems, significantly reducing the cost of replacing experimental models.

[0029] In a sixth aspect, the present invention provides the use of the lung adenocarcinoma animal model with high bone metastasis as described in the second aspect or the mouse lung adenocarcinoma cell line with high bone metastasis as described in the fifth aspect in screening or developing drugs for lung adenocarcinoma with bone metastasis.

[0030] Given that the lung adenocarcinoma animal model or cell line with high bone metastasis provided by this invention has advantages such as outstanding bone metastasis ability, stable performance, strong universality and relatively low cost, it can be used in the fields of screening or development of lung adenocarcinoma drugs with bone metastasis. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 These are comparative images of the proximal tibia and distal femur of two groups of model mice in Example 1 and Comparative Example 1, illustrating the effects of this invention. Figure 1 A is a representative image of Comparative Example 1. Figure 1 B is a representative image of Example 1; Figure 2 The images show H&E staining of bone metastases in two groups of model mice, Example 1 and Comparative Example 1, in the efficacy examples of this invention; wherein, Figure 2 A is a representative image of Comparative Example 1. Figure 2 B is a representative image of Example 1; Figure 3 These are micrographs of the mouse lung adenocarcinoma cell line LCC and cell line LCC-Bone at different magnifications in the example of the effectiveness of this invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] In this invention, the mouse lung adenocarcinoma cell line LLC was purchased from Shanghai Aoyin Biotechnology Co., Ltd., catalog number Sac0226LG.

[0035] Example 1 This invention provides a method for constructing an animal model of lung adenocarcinoma with high bone metastasis, the method comprising the following steps: S1. Mouse lung adenocarcinoma cell line (LCC) in good logarithmic growth phase was seeded into 10 cm diameter culture dishes. 10 mL of DMEM complete medium containing 10% fetal bovine serum was added, and the dishes were incubated at 37°C with 5% CO2. When cell confluence reached 80%-90%, cells were collected, washed twice with PBS, and then digested with 1 mL of trypsin for 2 min. Digestion was terminated with twice the volume of DMEM containing 15% serum. The cells were centrifuged at 1000 rpm for 5 min at room temperature, counted, and resuspended in PBS. The cell concentration was adjusted to 2 × 10⁶ cells / mL. 6 The concentration of cells / mL was used to obtain a suspension of mouse lung adenocarcinoma cells, which was then placed on ice for later use.

[0036] Six-week-old normal C57BL / 6 mice were shaved and disinfected on the chest to expose the injection area. A vertical injection was made at the 3rd-4th intercostal space, about 1-2 mm from the left sternal border. When ventricular blood pumping was observed, 100 μL of mouse lung adenocarcinoma cell suspension was slowly injected. After 28 days of tumor bearing, the mice were euthanized by cervical dislocation. After disinfection with 75% ethanol, the cells were transferred to the cell manipulation room. Tumor cells were extracted from bone metastases in the femur and tibia of the mice to obtain first-generation lung adenocarcinoma cells.

[0037] The method for obtaining first-generation lung adenocarcinoma cells by extracting tumor cells from bone metastases is as follows: under aseptic conditions, the bone metastases in the femur and tibia are dissected and cut into 1mm pieces. 3 Large and small tissue blocks (which may contain bone fragments) were plated into 10 cm diameter culture dishes and placed in DMEM complete medium containing 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were cultured in a 37°C, 5% CO2 incubator. After one week, the tumor cell density reached 90%. Cells were collected by centrifugation to obtain first-generation lung adenocarcinoma cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 2 × 10⁶ cells / mL. 6cells / mL, to obtain a suspension of first-generation lung adenocarcinoma cells; S2. 100 μL of the first-generation lung adenocarcinoma cell suspension was injected into the left ventricle of 6-week-old normal C57BL / 6 mice. Twenty-one days later, tumor cells were extracted from bone metastases in the mice and cultured in DMEM complete medium containing 15% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured at 37°C and 5% CO2 until the tumor cell density reached 90%. The cells were then collected by centrifugation to obtain second-generation lung adenocarcinoma cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6 cells / mL, to obtain a suspension of second-generation lung adenocarcinoma cells; S3. 100 μL of second-generation lung adenocarcinoma cell suspension was injected into the left ventricle of 6-week-old normal C57BL / 6 mice. Tumor cells were extracted from bone metastases 14 days later, and the cell concentration was obtained at 2 × 10⁻⁶ cells / mL following step S2. 6 A suspension of third-generation lung adenocarcinoma cells / mL; Eight 100 μL suspensions of third-generation lung adenocarcinoma cells were injected into the left ventricle of 6-week-old normal C57BL / 6 mice to obtain a lung adenocarcinoma animal model with high bone metastasis after 14 days.

[0038] Example 2 This invention provides a method for constructing an animal model of lung adenocarcinoma with high bone metastasis, the method comprising the following steps: S1. Mouse lung adenocarcinoma cell line (LCC) in good logarithmic growth phase was seeded into 10 cm diameter culture dishes. 10 mL of DMEM complete medium containing 10% fetal bovine serum was added, and the dishes were incubated at 37°C with 5% CO2. When cell confluence reached 80%-90%, cells were collected, washed twice with PBS, and then digested with 1 mL of trypsin for 2 min. Digestion was terminated with twice the volume of DMEM containing 15% serum. The cells were centrifuged at 1000 rpm for 5 min at room temperature, counted, and resuspended in PBS. The cell concentration was adjusted to 2 × 10⁶ cells / mL. 6 The concentration of cells / mL was used to obtain a suspension of mouse lung adenocarcinoma cells, which was then placed on ice for later use.

[0039] Eight-week-old normal C57BL / 6 mice were shaved and disinfected on the chest to expose the injection area. A vertical injection was made at the 3rd-4th intercostal space, about 1-2 mm from the left sternal border. When ventricular blood pumping was observed, 75 μL of mouse lung adenocarcinoma cell suspension was slowly injected. The mice were euthanized by cervical dislocation 30 days after tumor bearing. After disinfection with 75% ethanol, the mice were transferred to the cell manipulation room. Tumor cells were extracted from bone metastases in the femur and tibia of the mice to obtain first-generation lung adenocarcinoma cells.

[0040] The method for obtaining first-generation lung adenocarcinoma cells by extracting tumor cells from bone metastases is as follows: under aseptic conditions, the bone metastases in the femur and tibia are dissected and cut into 1mm pieces. 3 Large and small tissue blocks (which may contain bone fragments) were plated into 10 cm diameter culture dishes and placed in DMEM complete medium containing 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were cultured in a 37°C, 5% CO2 incubator. After one week, the tumor cell density reached 90%. Cells were collected by centrifugation to obtain first-generation lung adenocarcinoma cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 1.5 × 10⁻⁶ cells / mL. 6 cells / mL, to obtain a suspension of first-generation lung adenocarcinoma cells; S2. 120 μL of the first-generation lung adenocarcinoma cell suspension was injected into 8-week-old normal C57BL / 6 mice via left ventricle. Twenty-one days later, tumor cells were extracted from bone metastases in the mice and cultured in DMEM complete medium containing 15% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured at 37°C and 5% CO2 until the tumor cell density reached 90%. The cells were then collected by centrifugation to obtain second-generation lung adenocarcinoma cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 1.5 × 10⁻⁶ cells / mL. 6 cells / mL, to obtain a suspension of second-generation lung adenocarcinoma cells; S3. 120 μL of second-generation lung adenocarcinoma cell suspension was injected into 8-week-old normal C57BL / 6 mice via left ventricle. Tumor cells were extracted from bone metastases 18 days later, and the cell concentration was 1.5 × 10⁻⁶ cells / mL following step S2. 6 A suspension of third-generation lung adenocarcinoma cells / mL; 120 μL of a suspension of third-generation lung adenocarcinoma cells was injected into the left ventricle of 8-week-old normal C57BL / 6 mice. Twenty-one days later, tumor cells were extracted from bone metastases in the mice, and the cell concentration was 1.5 × 10⁻⁶ cells / mL according to step S2. 6 A suspension of fourth-generation lung adenocarcinoma cells / mL; Eight 120 μL suspensions of fourth-generation lung adenocarcinoma cells were taken and injected into eight normal C57BL / 6 mice at 8 weeks of age via the left ventricle. After 12 days, a lung adenocarcinoma animal model with high bone metastasis-4 was obtained.

[0041] Example 3 This invention provides a method for constructing an animal model of lung adenocarcinoma with high bone metastasis, the method comprising the following steps: S1. Mouse lung adenocarcinoma cell line (LCC) in good logarithmic growth phase was seeded into 10 cm diameter culture dishes. 10 mL of DMEM complete medium containing 10% fetal bovine serum was added, and the dishes were incubated at 37°C with 5% CO2. When cell confluence reached 80%-90%, cells were collected, washed twice with PBS, and then digested with 1 mL of trypsin for 2 min. Digestion was terminated with twice the volume of DMEM containing 15% serum. The cells were centrifuged at 1000 rpm for 5 min at room temperature, counted, and resuspended in PBS. The cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 The concentration of cells / mL was used to obtain a suspension of mouse lung adenocarcinoma cells, which was then placed on ice for later use.

[0042] Seven-week-old normal C57BL / 6 mice were shaved and disinfected on the chest to expose the injection area. A vertical injection was made at the 3rd-4th intercostal space, about 1-2 mm from the left sternal border. When ventricular blood pumping was observed, 80 μL of mouse lung adenocarcinoma cell suspension was slowly injected. The mice were euthanized by cervical dislocation 25 days after tumor bearing. After disinfection with 75% ethanol, the mice were transferred to the cell manipulation room. Tumor cells were extracted from bone metastases in the femur and tibia of the mice to obtain first-generation lung adenocarcinoma cells.

[0043] The method for obtaining first-generation lung adenocarcinoma cells by extracting tumor cells from bone metastases is as follows: under aseptic conditions, the bone metastases in the femur and tibia are dissected and cut into 1mm pieces. 3 Large and small tissue blocks (which may contain bone fragments) were plated into 10 cm diameter culture dishes and placed in DMEM complete medium containing 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics. The cells were cultured in a 37°C, 5% CO2 incubator. After one week, the tumor cell density reached 90%. Cells were collected by centrifugation to obtain first-generation lung adenocarcinoma cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 cells / mL, to obtain a suspension of first-generation lung adenocarcinoma cells; S2. 80 μL of the first-generation lung adenocarcinoma cell suspension was injected into the left ventricle of 7-week-old normal C57BL / 6 mice. After 14 days, tumor cells were extracted from bone metastases in the mice and cultured in DMEM complete medium containing 15% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured in a 37°C, 5% CO2 incubator until the tumor cell density reached 90%. The cells were then collected by centrifugation to obtain second-generation lung adenocarcinoma cells. The cells were resuspended in PBS buffer and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 cells / mL, to obtain a suspension of second-generation lung adenocarcinoma cells; S3. 80 μL of second-generation lung adenocarcinoma cell suspension was injected into the left ventricle of 7-week-old normal C57BL / 6 mice. Tumor cells were extracted from bone metastases 14 days later, and the cell concentration was obtained at 3 × 10⁻⁶ cells / mL following step S2. 6A suspension of third-generation lung adenocarcinoma cells / mL; Eight 120 μL suspensions of third-generation lung adenocarcinoma cells were taken and injected into eight normal C57BL / 6 mice at 7 weeks of age via the left ventricle. After 14 days, a lung adenocarcinoma animal model with high bone metastasis-3 was obtained.

[0044] Example 4 This invention provides a method for constructing a lung adenocarcinoma cell line with high bone metastasis. The method includes extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model-3 with high bone metastasis provided in Example 1, i.e., the fourth generation of lung adenocarcinoma cells, which is a mouse lung adenocarcinoma cell line with high bone metastasis.

[0045] Example 5 This invention provides a method for constructing a lung adenocarcinoma cell line with high bone metastasis. The method includes extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model-4 with high bone metastasis provided in Example 2, i.e., the fifth generation of lung adenocarcinoma cells, to obtain a mouse lung adenocarcinoma cell line with high bone metastasis.

[0046] Example 6 This invention provides a method for constructing a lung adenocarcinoma cell line with high bone metastasis. The method includes extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model-3 with high bone metastasis provided in Example 3, i.e., fourth-generation lung adenocarcinoma cells, to obtain a mouse lung adenocarcinoma cell line with high bone metastasis.

[0047] Comparative Example 1 This invention provides a comparative example of a method for constructing an animal model of bone metastasis in lung adenocarcinoma, the method comprising the following steps: Following the method described in Example 1, a cell concentration of 2 × 10⁻⁶ was obtained. 6 A suspension of mouse lung adenocarcinoma cells / mL was placed on ice for later use.

[0048] Eleven normal C57BL / 6 mice aged 6 weeks were used. The chest was shaved and disinfected to expose the injection area. The injection was performed vertically at the 3rd-4th intercostal space, about 1-2 mm from the left sternal border. When ventricular blood was observed to be pumped out, 100 μL of mouse lung adenocarcinoma cell suspension was slowly injected. After 28 days of tumor bearing, a lung adenocarcinoma animal model with bone metastasis was obtained.

[0049] Example of effect This invention evaluated the femoral and tibial bone metastases in the mouse models constructed in Example 1 and Comparative Example 1. Statistical analysis showed that the osteoblastogenesis rate in the animal model provided in Example 1 was 100%, while the osteoblastogenesis rate in the animal model-3 provided in Comparative Example 1 was 45.5%.

[0050] The morphological comparison of the proximal tibia and distal femur of the two groups of model mice in Example 1 and Comparative Example 1 is shown in the figure below. Figure 1 As shown; where, Figure 1 A is a representative image of Comparative Example 1. Figure 1 B is a representative image from Example 1. Histological H&E staining images of bone metastases in the two groups of model mice, Example 1 and Comparative Example 1, are shown below. Figure 2 As shown; where, Figure 2 A is a representative image of Comparative Example 1. Figure 2 B is a representative image of Example 1.

[0051] Depend on Figure 1-2 It can be visually observed that there are significant differences in the tissue morphology of the proximal tibia and distal femur of the two groups of model mice. Specifically, the bone metastases in the Example 1 model mice exhibited significant oncological characteristics, with clear tumorigenesis; while the Comparative Example 1 model mice did not show obvious tumorigenesis. Analysis of H&E staining results revealed no obvious tumor formation in the tibial tissue of the Comparative Example 1 model mice, while obvious bone metastases were observed in the tibial tissue of the Example 1 model mice, confirming its significant tumorigenesis.

[0052] The mouse lung adenocarcinoma cell line with high bone metastasis provided in Example 4 is designated as cell line LCC-Bone.

[0053] Morphological images of mouse lung adenocarcinoma cell lines LCC and LCC-Bone observed under an optical microscope. Microscopic images of mouse lung adenocarcinoma cell lines LCC and LCC-Bone at different magnifications are shown below. Figure 3 As shown.

[0054] Depend on Figure 3 It can be seen that the fourth-generation lung adenocarcinoma LLC-Bone constructed in Example 4 is a tumor cell with a consistent morphology that grows adherently, and it is homologous to LLC.

[0055] The above research results show that LLC-Bone has a stronger bone metastasis potential than existing LLC cell lines and can be used to efficiently and stably construct a mouse lung adenocarcinoma bone metastasis model.

[0056] Given that the lung adenocarcinoma animal model or cell line with high bone metastasis provided by this invention has advantages such as outstanding bone metastasis ability, stable performance, strong universality and relatively low cost, it can be used in the fields of screening or development of lung adenocarcinoma drugs with bone metastasis.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of lung adenocarcinoma with high bone metastasis, characterized in that, The construction method includes the following steps: S1. Mouse lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 25-30 days, tumor cells were extracted from bone metastases in mice to obtain first-generation lung adenocarcinoma cells. S2. The first generation of lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 14-21 days, tumor cells were extracted from bone metastases in the mice to obtain the second generation of lung adenocarcinoma cells. S3. The second-generation lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 14-21 days, a lung adenocarcinoma animal model with high bone metastasis was obtained-2.

2. The method for constructing an animal model of lung adenocarcinoma with high bone metastasis as described in claim 1, characterized in that, After obtaining the lung adenocarcinoma animal model with high bone metastasis-2, the following steps are also included: extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model with high bone metastasis-2 to obtain third-generation lung adenocarcinoma cells; The third-generation lung adenocarcinoma cells were injected into normal mice via the left ventricle, and after 14-18 days, a lung adenocarcinoma animal model with high bone metastasis-3 was obtained.

3. The method for constructing an animal model of lung adenocarcinoma with high bone metastasis as described in claim 2, characterized in that, After obtaining the lung adenocarcinoma animal model-3 with high bone metastasis, the following steps are also included: extracting tumor cells from the bone metastases of the lung adenocarcinoma animal model-3 with high bone metastasis to obtain fourth-generation lung adenocarcinoma cells; The fourth-generation lung adenocarcinoma cells were injected into normal mice via the left ventricle. After 12-16 days, a lung adenocarcinoma animal model with high bone metastasis was obtained.

4. The method for constructing an animal model of lung adenocarcinoma with high bone metastasis as described in any one of claims 1-3, characterized in that, The mouse lung adenocarcinoma cells include the mouse lung adenocarcinoma cell line LLC; and / or The normal mice were C57 background mice aged 6-8 weeks.

5. The method for constructing an animal model of lung adenocarcinoma with high bone metastasis as described in claim 1, characterized in that, In S1, the dose of the mouse lung adenocarcinoma cells injected was 0.8 × 10⁻⁶. 5 cells / each - 5×10 5 cells; and / or In S2, the dose of the first-generation lung adenocarcinoma cells injected is 0.8 × 10⁻⁶. 5 cells / each - 5×10 5 cells; and / or In S3, the dose of the second-generation lung adenocarcinoma cells injected is 0.8 × 10⁻⁶. 5 cells / each - 5×10 5 cells / each.

6. An animal model of lung adenocarcinoma with high bone metastasis, characterized in that, The lung adenocarcinoma animal model with high bone metastasis as described in any one of claims 1-5 was constructed.

7. The application of the lung adenocarcinoma animal model with high bone metastasis as described in claim 6 in the preparation of mouse lung adenocarcinoma cell lines with high bone metastasis.

8. A method for constructing a mouse lung adenocarcinoma cell line with high bone metastasis, characterized in that, The construction method includes: extracting tumor cells from bone metastases in the lung adenocarcinoma animal model with high bone metastasis as described in claim 6 to obtain a mouse lung adenocarcinoma cell line with high bone metastasis.

9. A mouse lung adenocarcinoma cell line with high bone metastasis, characterized in that, It was constructed using the method for constructing a mouse lung adenocarcinoma cell line with high bone metastasis as described in claim 8.

10. The use of the lung adenocarcinoma animal model with high bone metastasis as described in claim 6 or the mouse lung adenocarcinoma cell line with high bone metastasis as described in claim 9 in screening or developing drugs for lung adenocarcinoma with bone metastasis.

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