Construction method and application of pulmonary fibrosis and lung cancer combined animal model

Abstract

The invention specifically discloses a construction method and application of a pulmonary fibrosis and lung cancer combined animal model, and relates to the technical field of medical experimental animal modeling. The invention provides a pulmonary fibrosis and lung cancer combined animal model construction method which is characterized by comprising the following steps: S1, inducing a pulmonary fibrosis animal model by using an inducer; s2, using the pulmonary fibrosis related fibroblasts to pretreat lung cancer cells; and S3, inducing the pulmonary fibrosis animal model in the step S1 by using the lung cancer cells in the step S2 to become the pulmonary fibrosis combined lung cancer animal model. The construction method provided by the invention can more effectively simulate the disease process of pulmonary fibrosis-microenvironment change-lung cancer occurrence on the basis of high efficiency, stability, economy and feasibility, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of medical experimental animal modeling, and particularly relates to a method for constructing and applying an animal model of pulmonary fibrosis complicated with lung cancer. Background Art

[0002] Pulmonary fibrosis (IPF) is closely related to lung cancer (LC), and the two often have common etiologies and pathogenesis. However, due to the complex pathogenesis of IPF complicated with LC, there are currently few animal models of IPF complicated with LC, which can be summarized into two categories:

[0003] 1. Induce a pulmonary fibrosis model by intratracheal injection of bleomycin (BLM), and on this basis, induce a model of pulmonary fibrosis complicated with lung cancer by intravenous injection of the tail vein or intralung injection of Lewis lung cancer cells, etc. Such methods may have the following defects: ① By directly injecting ordinary cultured lung cancer cells externally, it is impossible to better simulate the disease process of "pulmonary fibrosis → microenvironment change → lung cancer occurrence" in the human body, and the success rate of intravenous injection of the tail vein for modeling is relatively low, and the intralung injection has a large operation difficulty and is invasive. ② The number of lung cancer cells used for injection is mostly of the order of 10 6 and above, and the number of cells used is relatively large. However, the process of malignant transformation of IPF to IPF-LC often originates from a small number of tumor-initiating cells with a high degree of malignancy. Therefore, this method cannot better simulate the tumor initiation process under real pathological conditions. ③ All the tumor cells used are murine. Due to the differences in genetic and physiological characteristics between ordinary C57 mice and humans, this model cannot be used to explore the in vivo functions of human genes, nor can it be used for the development of human anti-IPF-LC antibody drugs, etc.

[0004] 2. Construct a spontaneous pulmonary fibrosis complicated with lung cancer model by breeding transgenic mice with key genes of IPF and / or LC knocked out. Such methods may have the following defects: ① High experimental cost, long cycle, and large operation difficulty. ② Low model success rate and unstable phenotype. ③ The diseases induced by transgenic engineering technology are not exactly the same as the disease processes that occur naturally clinically. Problems such as the differences in the genetic background between transgenic mice and humans and the need to rely on external factors (such as chemical substances or viruses) to induce diseases may all affect the disease manifestations and the translatability of research results. Summary of the Invention

[0005] (I) Technical Problems to be Solved

[0006] In view of this, one of the main objects of the present invention is to provide a method for constructing an animal model of pulmonary fibrosis complicated with lung cancer, characterized in that the construction method comprises: S1: inducing an animal model of pulmonary fibrosis using an inducer; S2: pretreating lung cancer cells with lung fibrosis-related fibroblasts; S3: using the lung cancer cells in S2 to induce the animal model of pulmonary fibrosis in S1 to become an animal model of pulmonary fibrosis complicated with lung cancer.

[0007] The construction method provided by the present invention more effectively simulates the disease process of "pulmonary fibrosis → microenvironment change → lung cancer occurrence" on the basis of being efficient, stable, economical and easy to implement, and is suitable for popularization and application.

[0008] (II) Technical solution

[0009] To achieve the above object, the present invention provides a method for constructing an animal model of pulmonary fibrosis complicated with lung cancer, characterized in that the construction method comprises:

[0010] S1: inducing an animal model of pulmonary fibrosis using an inducer;

[0011] S2: pretreating lung cancer cells with a culture of lung fibrosis-related fibroblasts;

[0012] S3: using the lung cancer cells in S2 to induce the animal model of pulmonary fibrosis in S1 to become an animal model of pulmonary fibrosis complicated with lung cancer.

[0013] In one embodiment, the inducer is one or a combination of bleomycin, amiodarone, methotrexate.

[0014] In one embodiment, the inducer is bleomycin.

[0015] In one embodiment, the administration method of bleomycin includes one or a combination of intratracheal injection, aerosol inhalation, intratracheal instillation, aerosol inhalation, intraperitoneal injection, oral gavage, intralung injection, subcutaneous injection and intravenous injection.

[0016] In one embodiment, the administration method of bleomycin is intratracheal injection.

[0017] In one embodiment, the administration dose of bleomycin is 2 - 5 μg / g.

[0018] In one embodiment, the administration dose of bleomycin is 3 μg / g.

[0019] In one embodiment, the pretreatment includes: contacting a cell population containing lung cancer cells with a culture medium containing a culture of lung fibrosis-related fibroblasts.

[0020] In one embodiment, the cell population containing lung cancer cells is cultured to passage P6 - 8 before contacting with a culture medium containing a lung fibrosis - associated fibroblast culture.

[0021] In one embodiment, the cell population containing lung cancer cells is cultured to 20 - 60% confluence before contacting with a culture medium containing a lung fibrosis - associated fibroblast culture.

[0022] In one embodiment, the cell population containing lung cancer cells is cultured to 40% confluence before contacting with a culture medium containing a lung fibrosis - associated fibroblast culture.

[0023] In one embodiment, the cell population containing lung cancer cells is cultured to 80 - 90% confluence after contacting with a culture medium containing a lung fibrosis - associated fibroblast culture.

[0024] In one embodiment, the culture conditions are 37°C, 5% CO 2 .

[0025] In one embodiment, the culture medium contains 30 - 70% lung fibrosis - associated fibroblast culture.

[0026] In one embodiment, the culture medium contains 50% lung fibrosis - associated fibroblast culture.

[0027] In one embodiment, the lung fibrosis includes idiopathic pulmonary fibrosis (IPF) and / or secondary pulmonary fibrosis.

[0028] In one embodiment, the lung fibrosis is idiopathic pulmonary fibrosis (IPF).

[0029] In one embodiment, the idiopathic pulmonary fibrosis - associated fibroblasts (IPF - associated fibroblasts) are derived from a mammal.

[0030] In one embodiment, examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non - human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc.

[0031] In one embodiment, the IPF - associated fibroblasts are human IPF - associated fibroblasts.

[0032] In one embodiment, the human IPF - associated fibroblast culture includes human IPF - associated fibroblasts.

[0033] In one embodiment, the human IPF - associated fibroblast culture includes a human IPF - associated fibroblast culture medium.

[0034] In one embodiment, the lung cancer cells are derived from a mammal.

[0035] In one embodiment, examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc.

[0036] In one embodiment, the lung cancer cells are human lung cancer cells.

[0037] In one embodiment, the human lung cancer cells are A549 cells.

[0038] In one embodiment, the A549 cells are fresh.

[0039] In one embodiment, the A549 cells are frozen.

[0040] In one embodiment, the A549 cells are stored in liquid nitrogen.

[0041] In one embodiment, the culture medium for the A549 cells is A549 cell complete medium.

[0042] In one embodiment, the A549 cell complete medium comprises 90% F-12K, 10% FBS (Fetal Bovine Serum), and 1% PS (Penicillin-Streptomycin).

[0043] In one embodiment, the pretreatment specifically comprises: culturing a cell population containing A549 cells in A549 cell complete medium until it reaches 20 - 60% confluence at passage P6 - 8, then changing the medium to a mixture of 30 - 70% human IPF-related fibroblast culture and A549 cell complete medium, and culturing it to 80 - 90% confluence under the conditions of 37°C and 5% CO 2 conditions.

[0044] In one embodiment, the pretreatment specifically comprises: culturing A549 cells in A549 cell complete medium until it reaches 40% confluence at passage P6 - 8, then changing the medium to a mixture of 50% human IPF-related fibroblast culture and A549 cell complete medium, and culturing it to 80 - 90% confluence under the conditions of 37°C and 5% CO 2 conditions.

[0045] In one embodiment, the method for activating and expanding human IPF-related fibroblasts comprises: contacting a cell population comprising fetal lung fibroblasts with a culture medium comprising TGF-β, TNF-α, IL-1β and CCL2 to activate and expand lung fibrosis-related fibroblasts.

[0046] In one embodiment, the cell population comprising MRC5 cells is cultured to passage P5 before contacting with a culture medium comprising TGF-β, TNF-α, IL-1β and CCL2.

[0047] In one embodiment, the cell population comprising MRC5 cells is cultured until adherent before contacting with a culture medium comprising TGF-β, TNF-α, IL-1β and CCL2.

[0048] In one embodiment, after the cell population comprising MRC5 cells is contacted with a culture medium comprising TGF-β, TNF-α, IL-1β and CCL2, it is cultured for 24 - 72 h.

[0049] In one embodiment, after the cell population comprising MRC5 cells is contacted with a culture medium comprising TGF-β, TNF-α, IL-1β and CCL2, it is cultured for 48 h.

[0050] In one embodiment, the culture conditions are 37 °C, 5% CO 2 .

[0051] The culture medium is a complete culture medium for MRC5 cells.

[0052] In one embodiment, the concentration of TGF-β is 500 - 5000 pg / ml; and / or the concentration of TNF-α is 50 - 500 pg / ml; and / or the concentration of IL-1β is 5 - 100 pg / ml; and / or the concentration of CCL2 is 100 - 2000 pg / ml.

[0053] In one embodiment, the concentration of TGF-β is 1000 pg / ml; and / or the concentration of TNF-α is 100 pg / ml; and / or the concentration of IL-1β is 20 pg / ml; and / or the concentration of CCL2 is 700 pg / ml.

[0054] In one embodiment, the concentration of TGF-β is 1000 pg / ml, the concentration of TNF-α is 100 pg / ml, the concentration of IL-1β is 20 pg / ml, and the concentration of CCL2 is 700 pg / ml.

[0055] In one embodiment, the fetal lung fibroblasts are derived from a mammal.

[0056] In one embodiment, examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc.

[0057] In one embodiment, the embryonic lung fibroblasts are human embryonic lung fibroblasts.

[0058] In one embodiment, the human embryonic lung fibroblasts are MRC5 cells.

[0059] In one embodiment, the MRC5 cells are fresh.

[0060] In one embodiment, the MRC5 cells are frozen.

[0061] In one embodiment, the MRC5 cells are stored in liquid nitrogen.

[0062] In one embodiment, the MRC5 cell culture medium is MRC5 cell complete medium.

[0063] In one embodiment, the MRC5 cell complete medium is 90% MEM (Minimum Essential Medium), 10% FBS (Fetal Bovine Serum), and 1% PS (Penicillin - Streptomycin).

[0064] In one embodiment, the method for activating and amplifying human IPF - related fibroblasts specifically includes: culturing a cell population containing MRC5 cells in MRC5 cell complete medium until passage P5, and when the cells adhere, replacing the MRC5 cell complete medium with MRC5 cell complete medium containing 500 - 5000 pg / ml of TGF - β; and / or 50 - 500 pg / ml of TNF - α; and / or 5 - 100 pg / ml of IL - 1β; and / or 100 - 2000 pg / ml of CCL2, and culturing at 37°C, 5% CO 2 for 24 - 72 h.

[0065] In one embodiment, the method for activating and amplifying human IPF - related fibroblasts specifically includes: culturing a cell population containing MRC5 cells in MRC5 cell complete medium until passage P5, and when the cells adhere, replacing the MRC5 cell complete medium with MRC5 cell complete medium containing 1000 pg / ml of TGF - β; and / or 100 pg / ml of TNF - α; and / or 20 pg / ml of IL - 1β; and / or 700 pg / ml of CCL2, and culturing at 37°C, 5% CO2 Cultivate for 24 h below.

[0066] In one embodiment, the method for activating and amplifying human IPF-related fibroblasts specifically includes: culturing a cell population containing MRC5 cells in complete MRC5 cell medium until passage P5, and when the cells adhere to the wall, replacing the complete MRC5 cell medium with complete MRC5 cell medium containing 1000 pg / ml of TGF-β, 100 pg / ml of TNF-α, 20 pg / ml of IL-1β, and 700 pg / ml of CCL2, and culturing at 37 °C, 5% CO 2 Cultivate for 24 h below.

[0067] In one embodiment, the human IPF-related fibroblast culture is obtained by purifying the activation and amplification culture solution of human IPF-related fibroblasts.

[0068] In one embodiment, the purification includes centrifuging the culture supernatant at 1000 rpm for 5 min at room temperature, and taking the supernatant and filtering it through a 0.22 μm filter.

[0069] In one embodiment, the lung cancer cells are administered on the 14th day of the induced pulmonary fibrosis animal model.

[0070] In one embodiment, the method for administering the lung cancer cells includes one or a combination of intratracheal injection, aerosol inhalation, intratracheal instillation, aerosol inhalation, intraperitoneal injection, oral gavage, intralung injection, subcutaneous injection, and intravenous injection.

[0071] In one embodiment, the method for administering the lung cancer cells is intratracheal injection.

[0072] In one embodiment, the dosage of the lung cancer cells administered is 8×10 4 -4×10 5 cells / animal.

[0073] In one embodiment, the animals include mammals and non-mammals.

[0074] Examples of mammals include, but are not limited to, any member of the class Mammalia: non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals, etc.

[0075] In one embodiment, the animal is a mouse.

[0076] In one embodiment, the mouse is an immunodeficient mouse.

[0077] In one embodiment, the immunodeficient mice are BALB / c Nude Mice.

[0078] In one embodiment, the construction method specifically includes:

[0079] S1: Inject bleomycin into BALB / c Nude Mice through intratracheal injection at a dose of 2 - 5 μg / g to induce pulmonary fibrosis mice;

[0080] S2: Culture the cell population containing MRC5 cells in MRC5 cell complete medium until passage P5. When the cells adhere, replace the MRC5 cell complete medium with MRC5 cell complete medium containing 500 - 5000 pg / ml of TGF-β; and / or 50 - 500 pg / ml of TNF-α; and / or 5 - 10 pg / ml of IL-1β; and / or 100 - 2000 pg / ml of CCL2, and culture at 37°C, 5% CO 2 2 for 24 - 72 h; When the cell population containing A549 cells is cultured in A549 cell complete medium until passage P6 - 8 and reaches 20 - 60% confluence, replace the medium with 30 - 70% human IPF-related fibroblast culture and A549 cell complete medium, and culture at 37°C, 5% CO 2 until 80 - 90% confluence;

[0081] S3: Inject A549 cells into the pulmonary fibrosis mice through intratracheal injection at a dose of 8×10 4 -4×10 5 cells / mouse.

[0082] In one embodiment, the construction method specifically includes:

[0083] S1: Inject bleomycin into BALB / c Nude Mice through intratracheal injection at a dose of 3 μg / g to induce pulmonary fibrosis mice;

[0084] S2: Culture the cell population containing MRC5 cells in MRC5 cell complete medium until passage P5. When the cells adhere, replace the MRC5 cell complete medium with MRC5 cell complete medium containing 1000 pg / ml of TGF-β; and / or 100 pg / ml of TNF-α; and / or 20 pg / ml of IL-1β; and / or 700 pg / ml of CCL2, and culture at 37°C, 5% CO 2 2 for 24 h; When the A549 cells are cultured in A549 cell complete medium until passage P6 - 8 and reach 40% confluence, replace the medium with 50% human IPF-related fibroblast culture and A549 cell complete medium, and culture at 37°C, 5% CO 2Cultured to 80 - 90% confluence under the conditions;

[0085] S3: Inject A549 cells into the pulmonary fibrosis mice via intratracheal injection at a dose of 8×10 4 - 4×10 5 cells per mouse.

[0086] In one embodiment, the construction method specifically includes:

[0087] S1: Inject bleomycin into BALB / c Nude Mice via intratracheal injection at a dose of 3 μg / g to induce pulmonary fibrosis mice;

[0088] S2: Culture the cell population containing MRC5 cells in the complete MRC5 cell medium until passage P5. When the cells adhere, replace the complete MRC5 cell medium with the complete MRC5 cell medium containing 1000 pg / ml of TGF-β, 100 pg / ml of TNF-α, 20 pg / ml of IL-1β, and 700 pg / ml of CCL2, and culture for 24 h at 37°C and 5% CO 2 . Culture A549 cells in the complete A549 cell medium until passage P6 - 8 and 40% confluence, then replace the medium with 50% human IPF-related fibroblast culture and the complete A549 cell medium, and culture to 80 - 90% confluence under the conditions of 37°C and 5% CO 2 ;

[0089] S3: On the 14th day after inducing pulmonary fibrosis in mice, inject A549 cells into the pulmonary fibrosis mice via intratracheal injection at a dose of 8×10 4 - 4×10 5 cells per mouse.

[0090] On the other hand, the present invention also provides an animal with pulmonary fibrosis complicated with lung cancer, which is obtained by the above construction method.

[0091] On the other hand, the present invention also provides the application of the animal with pulmonary fibrosis complicated with lung cancer, which includes any one of the following: (1) Application in constructing a disease model related to pulmonary fibrosis complicated with lung cancer; (2) Application in studying the pathogenesis of diseases related to pulmonary fibrosis complicated with lung cancer; (3) Application in screening candidate drugs for diseases related to pulmonary fibrosis complicated with lung cancer.

[0092] On the other hand, the present invention also provides a method for screening candidate drugs for diseases related to pulmonary fibrosis complicated with lung cancer. The method includes administering a drug to the animal model. If the drug can improve the symptoms related to pulmonary fibrosis complicated with lung cancer in the animal model, then the drug is a candidate drug for diseases related to pulmonary fibrosis complicated with lung cancer.

[0093] (III) Beneficial effects

[0094] The present invention provides a method for constructing an animal model of pulmonary fibrosis complicated with lung cancer, characterized in that the construction method includes: S1: inducing an animal model of pulmonary fibrosis using an inducer; S2: pretreating lung cancer cells with a fibroblast culture related to pulmonary fibrosis; S3: using the lung cancer cells in S2 to induce the animal model of pulmonary fibrosis in S1 to become an animal model of pulmonary fibrosis complicated with lung cancer. Compared with the prior art, the following beneficial effects are achieved:

[0095] 1. Before transplanting lung cancer cells, Fibrotic cocktail (fibrosis mixed inducer) is used to induce IPF-related lung fibroblasts, and then their culture is used to treat lung cancer cells. Compared with the existing direct transplantation model, it can more effectively simulate the disease process of "pulmonary fibrosis → microenvironment change → lung cancer occurrence".

[0096] 2. Before transplantation, the lung cancer cells are pretreated with a human IPF-related lung fibroblast culture, which causes the lung cancer cells to transform into a tumor-initiating cell phenotype with a stronger malignancy degree and significantly reduces the number of transplanted lung cancer cells. Compared with the existing transplantation models, it can more effectively simulate the characteristics of tumor-initiating cells.

[0097] 3. When transplanting lung cancer cells by intratracheal injection, compared with the tail vein injection method, it has better lung targeting and there is no risk of transplantation leakage in other organs; compared with the intralung injection method, the operation is simple and non-invasive.

[0098] 4. Using immunodeficient mice to transplant human lung cancer cells is more suitable for exploring the in vivo functions of human genes and developing human drugs for treating IPF-LC compared with using ordinary C57 mice.

[0099] 5. Compared with the spontaneous pulmonary fibrosis complicated with lung cancer transgenic mouse model, the construction method of the animal model of pulmonary fibrosis complicated with lung cancer provided by the present invention has low experimental cost, short cycle, small operation difficulty and stable phenotype, and avoids the influence of genetic background differences of transgenic mice and artificial intervention on the research results.

[0100] (IV) Terms and Definitions

[0101] As used herein, the term "isolate" refers to the process of increasing the percentage of a certain substance in a composition. For example, isolating a type of cell from a cell population refers to the process of producing a cell population in which the percentage of this type of cell is increased compared with the percentage of this type of cell in the original cell population. Therefore, when used in the context of a type of cell, the term "isolated" does not mean that the isolated cell population contains 100% of this type of cell, but means that the percentage of this type of cell in the cell population is increased after the isolation process.

[0102] It is known in the art that lung cancer cells can be obtained from a variety of sources. In some embodiments, a cell population comprising lung cancer cells is obtained from a cultured lung cancer cell line. In some embodiments, the cell population comprising lung cancer cells is collected, isolated, purified, or induced from a body fluid, tissue, or organ (including but not limited to peripheral blood, cord blood, bone marrow, lymph nodes, spleen, or other tissues or fluids of a subject). In certain embodiments, the cell population comprising lung cancer cells is a cultured lung cancer cell line. In certain embodiments, the cultured lung cancer cell line is a freshly obtained cultured lung cancer cell line. In certain embodiments, the cultured lung cancer cell line is a cryopreserved cultured lung cancer cell line. Various methods for collecting and preparing cultured lung cancer cell lines are known in the art.

[0103] In some embodiments, the cell population comprising cells of a cultured lung cancer cell line is a mammalian cell. In certain embodiments, the mammalian cell is a human cell. In certain embodiments, the human cell is an engineered cell. In certain embodiments, the human cell is a non-engineered cell. In certain embodiments, the mammalian cell is a non-human cell. In specific embodiments, the non-human cell is an engineered cell or a non-engineered cell. In some embodiments, the cell population comprising lung cancer cells is obtained from a subject. In certain embodiments, the cell population comprising lung cancer cells is obtained from a non-healthy subject. In certain embodiments, the non-healthy subject has a solid tumor cancer. In certain embodiments, the non-healthy subject has lung cancer.

[0104] In some embodiments, the activation and amplification conditions for human IPF-related fibroblasts further include cytokines. Non-limiting examples of cytokines include lectins, hepatocyte growth factor, prostaglandins, fibroblast growth factors, prolactin, placental lactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β, Müllerian inhibitory substance, murine gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factor (NGF), platelet growth factor, TGF-α, TGF-β, insulin-like growth factor-1, insulin-like growth factor-II, erythropoietin (EPO), bone-inducing factor, interferon-α, interferon-β, interferon-λ, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), interleukin-1 (IL-1), IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, L-8, IL-9, IL-10, IL-11, L-12, L-13, IL-14, 1L-15, IL-16, IL-17, IL-18, IL-21, LIF kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor, and LT (lymphotoxin). BRIEF DESCRIPTION OF THE DRAWINGS

[0105] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0106] Figure 1 It is a schematic diagram of model establishment.

[0107] Figure 2 It is the induction of IPF-related lung fibroblast phenotype by Fibrotic cocktail. (A) After treatment with PBS, 1 ng / ml TGF-β, or 1×fibrotic cocktail for 48 h, the expression levels of Fibronectin, CollagenI, and α-SMA in MRC5 cells were detected by Western blot, with GAPDH as an internal reference. (B) Western blot gray-scale scanning statistical chart. (C, D) Collagen contraction experiments were used to detect the collagen deposition ability of A549 cells in each group, and photos were taken (C) and statistics were performed (D).

[0108] Figure 3It is the scratch assay of A549 cells. (A) The scratch assay was used to detect the migration ability of A549 cells in the treatment group of IPF-related lung fibroblast cultures and A549 cells in the untreated group cultured in normal medium. Photos were taken (A) and statistics were performed (B).

[0109] Figure 4 It is the comparison of the tumorigenic ability of A549 cells.

[0110] Figure 5 It is the imaging and gross changes after mouse modeling. (A) On Day 34, micro-CT was used to detect the changes in the sagittal lung images of mice in each group. 3D slicer was used to segment the abnormal lung regions and perform three-dimensional reconstruction of the lungs. (B) Gross lung pictures of mice in each group.

[0111] Figure 6 It is the morphological changes after mouse modeling. (A, B) HE and Masson staining were used to detect the changes in the lung structure, fibrosis, and tumor burden of mice in each group. (C) The lung fibrosis of mice in each group was statistically analyzed according to the Masson staining area. (D) Hydroxyproline quantitative detection was used to statistically analyze the collagen deposition in the lungs of mice in each group. (E) The lung tumor burden of mice in each group was statistically analyzed according to HE staining. Detailed implementation manners

[0112] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0113] As used herein, "comprising", "having", or "including" include "containing", "consisting essentially of", "substantially consisting of", and "consisting of"; "consisting essentially of", "substantially consisting of", and "consisting of" are subordinate concepts of "comprising", "having", or "including".

[0114] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The reagents, methods, and equipment used are all conventional reagents, methods, and equipment in the technical field of the present invention unless otherwise specified.

[0115] Example 1

[0116] Construct a pulmonary fibrosis combined with lung cancer model:

[0117] S1: Induce pulmonary fibrosis in mice with bleomycin:

[0118] Seven-week-old male BALB / c Nude Mice (CAnN.Cg-Foxn1nu / Crl) were housed in a SPF environment for 1 week to adapt to the experimental conditions. They were anesthetized by intraperitoneal injection of 1.25% tribromoethanol at a dose of 20 μl / g. After complete anesthesia, they were fixed in the supine position on a 40-degree inclined plane plate. The glottis was exposed using a lighted laryngoscope to locate the trachea, and 3 μg / g bleomycin (BLM) was injected intratracheally using an atomizing syringe. This was day 0 of model establishment. After the injection, the mice were gently rotated left and right and the chest was gently massaged to ensure uniform distribution of bleomycin in the lungs. After the animals woke up, they were reared routinely.

[0119] S2: Pretreatment of A549 cells with human IPF-related fibroblast cultures:

[0120] 1. Cell resuscitation: Human embryonic lung fibroblasts MRC5 and human lung cancer cells A549 cryopreserved in liquid nitrogen were taken out and immediately thawed in a 37°C water bath. The thawed cell suspension was aspirated in a laminar flow hood and transferred to a 15-ml centrifuge tube containing 10 ml of medium. The cells were centrifuged at 800 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was resuspended and inoculated into a culture dish containing complete medium. The dish was placed in an incubator at 37°C and 5% CO 2 . The complete medium for MRC5 cells consisted of 90% MEM (Minimum Essential Medium), 10% FBS (Fetal Bovine Serum), and 1% PS (Penicillin-Streptomycin). The complete medium for A549 cells consisted of 90% F-12K, 10% FBS (Fetal Bovine Serum), and 1% PS (Penicillin-Streptomycin).

[0121] 2. Inducing human IPF-related lung fibroblasts using Fibrotic cocktail: Refer to Table 1

[0122] . The working concentration of Fibrotic cocktail was used to prepare a 1000× Fibrotic cocktail stock solution in PBS containing 0.1% BSA. P5-generation MRC5 cells were inoculated into a 10-cm dish at a density of 4×10 6 cells / dish. After the cells adhered overnight, the complete medium was replaced with complete medium containing 1× Fibrotic cocktail, and the dish was placed in an incubator at 37°C and 5% CO2 for 48 hours. The obtained cell culture supernatant was collected, centrifuged at 1000 rpm for 5 minutes at room temperature, and then the supernatant was separated and filtered using a 0.22-μm filter to obtain the human IPF-related lung fibroblast culture.

[0123] Table 1. Working concentrations of Fibrotic cocktail

[0124] Component Working Concentration (pg / ml) Catalog Number TGF-β 1000 7754-BH, R&D systems TNF-α 100 T6674, Sigma-Aldrich IL-1β 20 IL038, Sigma-Aldrich CCL2 700 300-04, PeproTech

[0125] 3. Pretreat A549 cells with human IPF-related lung fibroblast cultures: When the P6-8 generation of A549 cells cultured normally reach 40% confluence, replace the A549 complete medium with 50% human IPF-related lung fibroblast cultures plus 50% A549 complete medium, and culture in an incubator at 37°C and 5% CO2 for 96 hours, changing the medium every 24 hours.

[0126] S3: Establish a model of pulmonary fibrosis complicated with lung cancer by intratracheal injection of A549 cells

[0127] 1. Prepare an A549 cell suspension: When the cells reach 80-90% confluence, aspirate the medium, add 4 ml of PBS to each 10 cm dish to wash the cells 2 times, then add 3 ml of 0.25% trypsin to each dish, place at 37°C and digest until the cells become round and slightly detached, add the previously aspirated medium to terminate digestion, centrifuge at 800 rpm at room temperature for 5 minutes, discard the supernatant, add an appropriate amount of PBS to resuspend the cells, count and adjust the cell density to 8×106 / ml, which is the A549 cell suspension. Place the obtained A549 cell suspension on ice for standby and complete the in vivo injection within 2 hours.

[0128] 2. Establish an IPF-LC model by intratracheal injection of A549 cells: On the 14th day of bleomycin-induced pulmonary fibrosis mouse model establishment, anesthetize the mice by intraperitoneal injection of 1.25% tribromoethanol at a dose of 20 μl / g. After complete anesthesia, fix the mice in the supine position on a 40-degree inclined plane plate, use a lighted laryngoscope to expose the glottis to locate the trachea, and use an atomizing syringe to intratracheally inject 8×10 4 or 4×10 5 A549 cells. After injection, gently rotate the mice left and right moderately and gently massage the chest of the mice to ensure that the cell suspension is evenly distributed in the lungs. After the animals wake up, raise them routinely.

[0129] Example 2

[0130] Evaluation of modeling effect:

[0131] 1. Observe the activities, respiratory rate, food intake, hair color changes, and survival of the mice daily, and measure the body weight of the mice every 3 days and make good records.

[0132] On the 34th day of model establishment, high-resolution micro-CT scanning was performed on the lungs of mice, and the parameter settings were as follows: the scanning accuracy was 5 μm, the X-ray energy was 60 kV / 134 mA / 50 ms, and 3D Slicer software was used to perform three-dimensional reconstruction of the scanned images and analyze the ROI (Region of interest) area of pulmonary fibrosis.

[0133] On the 35th day of model establishment, the mice were euthanized, the whole lungs were taken for photography, and then lung tissues were taken for histomorphological detection (HE staining, Masson staining) and scoring; the lung tissues of the mice were taken to detect collagen deposition (hydroxyproline content detection).

[0134] The results were as Figure 5 shown. Compared with the untreated group, the area of lung lesions increased after modeling with A549 cells in the treatment group ( Figure 5 A). In addition, compared with the untreated group, the pulmonary fibrosis and tumor burden of the mice increased after modeling with A549 cells in the treatment group ( Figure 5 B). Compared with the untreated group, both the pulmonary fibrosis and tumor burden of the mice increased significantly after modeling with A549 cells in the treatment group. The quantitative results of hydroxyproline showed that the collagen deposition in the lungs of the mice increased after modeling with A549 cells in the treatment group ( Figure 6 A-E). The above results indicate that the method of treating A549 with the selected IPF-related lung fibroblast culture in the present invention can significantly improve the modeling effect of pulmonary fibrosis complicated with lung cancer, manifested as the aggravation of pulmonary fibrosis and lung cancer phenotypes.

[0135] Example 3

[0136] Effect of inducer on the phenotype of IPF-related lung fibroblasts:

[0137] Currently, the widely used method for in vitro induction of IPF-related lung fibroblasts is to add exogenous TGF-β for stimulation. However, IPF has a complex pathological process, and it is difficult to simulate the changes in the pulmonary microenvironment of IPF patients under real clinical conditions with a single exogenous TGF-β stimulation. According to existing reports, TGF-β, TNF-α, IL-1β, and CCL2 are key profibrotic cytokines that induce the transformation of lung fibroblasts into IPF-related lung fibroblasts. However, the currently commonly used method for inducing IPF-related lung fibroblasts in the field is still single TGF-β treatment. Therefore, the present invention creatively uses the Fibroticcocktail composed of the above factors to replace the single TGF-β stimulation, and compares the induction effects of the two on IPF-related lung fibroblasts. Western blot was used to detect the expression of Fibronectin, Collagen I, and α-SMA in MRC5 cells, and GAPDH was used as an internal reference. The collagen contraction experiment was used to detect the collagen deposition ability of A549 cells in each group.

[0138] The results are as Figure 2 shown. Compared with single TGF-β stimulation, Fibrotic cocktail stimulation can better induce the transformation of lung fibroblasts into IPF-related lung fibroblasts, manifested by upregulation of the expression of myofibroblast markers Fibronectin, Collagen I, and α-SMA ( Figure 2 A, B), and the cell collagen deposition ability is significantly improved ( Figure 2 C, D).

[0139] The above results indicate that the method of inducing IPF-related lung fibroblasts with Fibrotic cocktail selected in the present invention has a more obvious effect compared with the existing single TGF-β stimulation method, and theoretically can better simulate the changes in the pulmonary microenvironment of IPF patients.

[0140] Example 4

[0141] Effect of treatment method on the migration ability of A549:

[0142] The migration ability of A549 cells in the treatment group of IPF-related lung fibroblast cultures and untreated A549 cells cultured in normal medium was detected by scratch assay.

[0143] The results are as Figure 3 shown. Compared with A549 cells cultured in normal complete medium, A549 cells after the above-mentioned culture treatment showed stronger in vitro migration ability ( Figure 3 A, B).

[0144] The above results indicate that the method of treating A549 with IPF-related lung fibroblast cultures selected in the present invention can significantly improve the migration ability of tumor cells.

[0145] Example 5

[0146] Effect of treatment method on the tumorigenic ability of A549 cells:

[0147] The tumorigenic ability of A549 cells in different treatment groups with a certain number of transplanted cells was statistically analyzed.

[0148] The results are as Figure 4 shown. Compared with A549 cells cultured in normal complete medium, A549 cells in the treatment group of IPF-related lung fibroblast cultures showed stronger in vivo tumorigenic ability with a small number of cells. The tumorigenesis rates of untreated and treated A549 cells at a transplantation dose of 8×10 4 were 2 / 8 and 7 / 8 respectively, and the tumorigenesis rates of untreated and treated A549 cells at a transplantation dose of 4×10 5 were 6 / 8 and 8 / 8 respectively, and there were significant statistical differences between the two.Figure 4 )。

[0149] The above results indicate that tumor-initiating cells possess characteristics of tumor stem cells such as high migratory ability and high tumorigenic ability of a small number of cells in vivo. The method of treating A549 with the IPF-related lung fibroblast culture selected in this invention can significantly improve the migratory ability of tumor cells and the tumorigenic ability of a small number of cells in vivo. Compared with untreated A549 cells, it can better mimic the characteristics of tumor-initiating cells.

[0150] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the process, method, article or device comprising the element.

[0151] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for constructing an animal model of pulmonary fibrosis combined with lung cancer, characterized in that: The construction method comprises: S1: Inducing animal model of pulmonary fibrosis using induction agents; S2: Pretreatment of lung cancer cells with lung fibrosis-associated fibroblast cultures; S3: Use the lung cancer cells in S2 to induce the pulmonary fibrosis animal model in S1 to become an animal model of pulmonary fibrosis combined with lung cancer.

2. The construction method according to claim 1, characterized in that: The inducing agent is one of bleomycin, amiodarone, methotrexate or a combination thereof.

3. The construction method according to claim 1, characterized in that: The pretreatment comprises: contacting a cell population containing lung cancer cells with a culture medium containing pulmonary fibrosis-associated fibroblast culture.

4. The construction method according to claim 3, characterized in that: The cell population comprising lung cancer cells is cultured to a confluence of 20-60% before contacting with a culture medium containing pulmonary fibrosis-associated fibroblast culture.

5. The construction method according to claim 4, characterized in that: The culture medium contains 30-70% lung fibrosis-associated fibroblast cultures.

6. The construction method according to claim 5, characterized in that: The method for activating and expanding pulmonary fibrosis-related fibroblasts comprises: contacting a cell population containing embryonic lung fibroblasts with a culture medium containing TGF-β, TNF-α, IL-1β and CCL2 to activate and expand pulmonary fibrosis-related fibroblasts.

7. The construction method according to claim 6, characterized in that: The cell population comprising human embryonic lung fibroblasts is cultured until adherence before contacting with a culture medium comprising TGF-β, TNF-α, IL-1β and CCL2.

8. The construction method according to claim 1, characterized in that: The administration method of the lung cancer cells in S3 includes one or a combination of intratracheal injection, nebulization inhalation, intratracheal instillation, aerosol inhalation, intraperitoneal injection, oral gavage, intrapulmonary injection, subcutaneous injection and intravenous injection.

9. The construction method according to claim 8, characterized in that: The method of administering lung cancer cells in S3 is intratracheal injection.

10. Use of the animal model obtained by the construction method according to any one of claims 1 to 9, characterized in that: The application includes any of the following: (1) Application in constructing disease models related to pulmonary fibrosis and lung cancer; (2) Application in the study of the pathogenesis of pulmonary fibrosis combined with lung cancer-related diseases; (3) Application in screening candidate drugs for pulmonary fibrosis and lung cancer-related diseases.

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