A method for constructing a lung cancer xenograft model

Abstract

The application discloses a method for constructing a lung cancer xenograft tumor model, which comprises the following steps: S1, constructing an asthma model by using a sensitizing agent causing asthma on a rodent; S2, establishing the lung cancer xenograft tumor model by transplanting lung cancer cells from the rodent into the asthma model. The lung cancer xenograft tumor model constructed by the application can simulate the findings of clinical research in basic research, and improve the scientific value and application value of the lung cancer xenograft tumor model.

Description

Technical Field

[0001] This invention relates to the field of animal models, and in particular to a method for constructing a lung cancer xenograft model. Background Technology

[0002] Lung cancer is currently the leading cause of death and the third most common malignant tumor worldwide, and it is also the leading cause of death and morbidity among malignant tumors in my country. The incidence and mortality rates of lung cancer in my country are far higher than the world average, and both are showing an increasing trend year by year. Therefore, the treatment of lung cancer urgently requires the development of new drugs and the formulation of new treatment plans. Elucidating the mechanism of action of drugs in live animal models is undoubtedly the best approach. Thus, suitable animal models of lung cancer are important tools for studying the pathogenesis of lung cancer and developing new drugs for its treatment.

[0003] Lung cancer animal models are mainly classified into spontaneous, induced, transplanted, and genetically modified animal models. Transplanted models refer to tumor animal models formed by transplanting tumor tissue or cells into experimental animals. Based on the origin of the tumor cells or cancerous tissue, they are further divided into allogeneic transplantation models and xenotransplantation models. Allogeneic transplantation means the tumor cell type is consistent with the host animal species, while xenotransplantation means the tumor cell type is different from the host animal species. Furthermore, according to the different sites of lung cancer metastasis, lung cancer animal models are classified into lung metastasis, bone metastasis, brain metastasis, and lymph node metastasis models, etc.

[0004] Transplantable models have become the main modeling method for lung cancer research due to their advantages such as rapid tumor formation, high modeling success rate, relatively convenient operation, and low experimental costs. However, the main drawback of transplantable models is that they cannot simulate clinical pathogenesis characteristics well. This makes the results generated by using the model unable to reflect the real-world lung cancer development process well, thus weakening the translation and application value of experimental results.

[0005] It should be noted that the information disclosed in the background section above is only for understanding the background of this application, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for constructing a lung cancer xenograft model.

[0007] The present invention adopts the following technical solution:

[0008] A method for constructing a lung cancer xenograft model includes the following steps: S1, constructing an asthma model in rodents using an allergen that causes asthma; S2, establishing the lung cancer xenograft model by transplanting rodent-derived lung cancer cells into the asthma model.

[0009] The present invention has the following advantages:

[0010] This invention is the first to experimentally verify (animal experiments) the phenomenon that asthma promotes lung cancer development, and the first to confirm through animal experiments that asthma can indeed promote lung cancer. Based on this, an animal model (asthma-lung cancer model) promoting lung cancer development through allergic inflammation was established for the first time. This invention provides a new approach to the establishment of small animal lung cancer models, which can effectively simulate the findings of clinical research in basic research, thereby improving the scientific and application value of lung cancer xenograft models. Attached Figure Description

[0011] Figure 1 This is a morphological comparison image of tumor-bearing mice obtained from the control group and the asthma group after the experiment of this embodiment of the invention;

[0012] Figure 2 This is a comparison of tumor tissue volume between the control group and the asthma group mice after the experiment of this embodiment of the invention.

[0013] Figure 3 This is a schematic diagram of the pathological changes in mouse lung tissue (Figure A) and the detection of airway hyperresponsiveness (penh) (Figure B) in an embodiment of the present invention;

[0014] Figure 4 This is a graph showing the changes in tumor tissue volume in the control group and the asthma group mice in an experiment according to an embodiment of the present invention.

[0015] Figure 5 This is a comparison chart of tumor tissue weight between the control group and the asthma group mice after the experiment of this embodiment of the invention. Detailed Implementation

[0016] The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary and not intended to limit the scope and application of the present invention. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0017] Clinical findings indicate that asthma patients are more susceptible to lung cancer than healthy individuals, while reports suggest that smoking and secondhand smoke exposure may not be the primary factors contributing to the increased incidence of lung cancer in asthma patients. However, the specific mechanisms by which asthma leads to lung cancer remain unclear, and there are no mature animal models to address this issue. Therefore, this invention first constructs an asthma model in rodents (e.g., mice) using major asthma sensitizers. Then, it establishes a lung cancer xenograft model by transplanting rodent-derived lung cancer cells (such as Lewis lung cancer (LLC) cells). Finally, it is the first to construct a rodent-based lung cancer xenograft model (this lung cancer xenograft model can be called an "asthma-lung cancer" xenograft model, or an asthma-mediated lung cancer model). This lung cancer xenograft model, as a novel lung cancer xenograft model, can effectively simulate clinical findings in basic research, enhancing the scientific and application value of lung cancer xenograft models.

[0018] In a specific embodiment, the present invention provides a method for constructing a lung cancer xenograft model, which includes the following steps:

[0019] S1. Asthma models were constructed in rodents using allergens that induce asthma.

[0020] S2. Establish the lung cancer xenograft model by transplanting rodent-derived lung cancer cells into the asthma model.

[0021] In a preferred embodiment, step S1 includes: anesthetizing rodents, stimulating rodents with allergens until their asthma pathological characteristics meet predetermined requirements, thereby completing the establishment of the asthma model;

[0022] Step S2 includes: taking rodent-derived lung cancer cells in the logarithmic growth phase, inoculating rodent-derived lung cancer cell solution into rodents in the asthma model, stopping the experiment when the average tumor volume of the rodents is within a predetermined volume range, and obtaining the lung cancer xenograft model.

[0023] In a preferred embodiment, the predetermined volume range mentioned in step S2 is 1300 mm. 3 -1800mm 3 .

[0024] In a preferred embodiment, step S2 further includes: during the establishment of the lung cancer xenograft model, the rodents in the asthma model are also subjected to allergen stimulation weekly until the end of the experiment.

[0025] In a preferred embodiment, when constructing an asthma model and / or establishing a lung cancer xenograft model, the allergen causing asthma is at least one of house dust mite (HDM), pollen, dust mites, cockroaches, and mold. More preferably, the allergen is HDM.

[0026] In a preferred embodiment, the rodent is a mouse, and the rodent-derived lung cancer cells are mouse-derived lung cancer cells.

[0027] In a preferred embodiment, the mouse is a male mouse, more preferably a 6-8 week old male mouse; and more preferably, the mouse strain is one of C57BL / 6 mouse and Balc / c mouse.

[0028] In a preferred embodiment, the mouse-derived lung cancer cells are Lewis lung cancer cells.

[0029] In a preferred embodiment, step S1 includes: anesthetizing mice with isopropanol nebulization, stimulating the mice with 10 μg / 30 μL HDM nasal drops, and then stimulating them with 15 μg / 30 μL HDM once a day for 5 consecutive days, thereby completing the establishment of the asthma model.

[0030] In a preferred embodiment, step S2 includes: taking logarithmically growing Lewis lung cancer cells, digesting them with trypsin, rinsing them twice with pre-cooled phosphate-buffered saline (PBS), then mixing the Lewis lung cancer cell solution with matrix gel at a 1:1 volume ratio (measured using calipers) to obtain a Lewis lung cancer cell solution. The Lewis lung cancer cell solution is then subcutaneously inoculated into mice in the asthma model at a quantity of 1 million cells / mouse. One week later, the tumor length L and width W are measured, and the tumor volume V is calculated as V = 1 / 2 × L × W. 2 The formula was used to calculate the tumor volume in tumor-bearing mice. When the average tumor volume in the asthma model mice was 1300 mm, 3 -1800mm 3 When the sample is within the specified range, the experiment is stopped and samples are taken. During the establishment of the lung cancer xenograft model, the mice in the asthma model also receive 15 μg / 30 μL / time of HDM stimulation twice a week until the end of the experiment.

[0031] In a preferred embodiment, the cell fusion rate of the logarithmic growth phase Lewis lung cancer cells is 70%-80%.

[0032] In a preferred embodiment, the mouse is a 6-8 week old male C57BL / 6 mouse.

[0033] The invention is further described below through a more specific embodiment.

[0034] Experimental reagents: House dust mites (Stallergenes Greer), PBS (Thermo Fisher Scientific), isopropanol (MCE), LLC cells (ATCC), Matrigel (Corning), trypsin (Thermo Fisher Scientific), methacholine (MCE)

[0035] Animals: 6-8 week old male C57BL / 6 mice (provided by the Animal Facility of the Chinese University of Hong Kong)

[0036] Step 1: Establishing an asthma model. Mice were anesthetized using isopropanol nebulization. Mice were divided into a control group (represented by PBS in the attached diagram) and an asthma group (represented by HDM in the attached diagram). The control group received one 30 μL PBS nasal drop, while the asthma group received one 10 μg / 30 μL HDM nasal drop (HDM was prepared using PBS, the same below). After 3 days, both groups of mice received one 30 μL PBS nasal drop and one 15 μg / 30 μL HDM nasal drop daily for 5 consecutive days. At this point, the asthma pathological characteristics in the asthma group were significantly higher than those in the control group, thus completing the establishment of the asthma model (e.g., ...). Figure 3 (As shown). Step 2: Establishing an "asthma-lung cancer" xenograft model. LLC cells in the logarithmic growth phase (observed as having a cell fusion rate of 70%-80% under a 100x microscope (10x eyepiece, 10x objective)) were digested with trypsin and washed twice with pre-cooled PBS. The LLC cells were then mixed with matrix gel at a 1:1 ratio to obtain an LLC cell solution, which was subcutaneously inoculated into two groups of mice at a density of 1 million cells / mouse. One week later, the tumor length L and width W were measured, and the tumor volume V was calculated as V = 1 / 2 × L × W. 2 The formula was used to calculate the tumor volume in tumor-bearing mice. When the volume in the control group was greater than 100 mm, 3 And less than 2000mm 3 The model was considered successfully established when the mean tumor volume in the asthma group mice reached 1300 mm. 3 -1800mm 3When the tumor reaches the target range, the experiment is stopped (in this example, on day 35), and tissue sampling (i.e., sampling mouse tumor tissue) is performed. During the establishment of the "asthma-lung cancer" model, mice in the control and asthma groups received nasal stimulation of 30 μL / time PBS twice a week and nasal stimulation of 15 μg / 30 μL / time HDM twice a week, respectively (in this example, for the control group, the nasal stimulation of 30 μL / time PBS twice a week could be 30 μL PBS nasal stimulation on Monday and then 30 μL PBS nasal stimulation on Friday; similarly, for the asthma group, the nasal stimulation of 15 μg / 30 μL / time HDM once a week could be 15 μg / 30 μL HDM nasal stimulation on Monday and then 15 μg / 30 μL HDM nasal stimulation on Friday), until the end of the experiment.

[0037] Through the above two-step animal experimental method, the "asthma-lung cancer" xenograft model was established. Figure 1 The image shows a morphological comparison of tumor-bearing mice obtained from the control group (PBS) and the asthma group (HDM) after the experiment, reflecting the overall tumor growth of the mice. The comparison image shows that the tumor volume in the asthma group was larger than that in the control group. Figure 2 The image shows a comparison of tumor tissue volume between the control group (PBS) and the asthma group (HDM) mice after the experiment. By comparing tumor tissue samples taken from the mice, it was found that the tumor tissue in the control group was significantly smaller than that in the asthma group, indicating that asthma can promote lung cancer development, thus proving the success of the asthma-lung cancer model. Figure 3 As shown, Figure A illustrates the pathological changes in mouse lung tissue and the detection of airway hyperresponsiveness (penh) (Figure B shows the airway hyperresponsiveness of mice stimulated with different concentrations of methacholine, with penh values ​​representing the hyperresponsiveness under different stimulation conditions). Figure A shows that the lungs of mice in the asthma group exhibited significant asthma-like pathological changes, while the lungs of mice in the control group showed no pathological changes. Figure B shows that the airway hyperresponsiveness (represented by penh) of mice in the asthma group was significantly higher than that of mice in the control group (p < 0.05), indicating that HDM successfully established an asthma model. Figure 4 The figure shows the tumor tissue volume changes in the control and asthma groups during the experiment. It can be seen that the tumor tissue volume in the asthma group was significantly higher than that in the control group after 28 days of the experiment, and remained higher until the end of the experiment. Figure 5 The figure shows a comparison of tumor weight between the control group and the asthma group after the experiment. It can be seen that the total tumor weight of the asthma group was significantly greater than that of the control group.

[0038] The above embodiments establish an asthma model in mice by stimulating them with HDM as an allergen, and then establish the lung cancer xenograft model by inoculating Lewis lung cancer cells. In other embodiments, the allergens can also be pollen, dust mites, cockroaches, and mold, which have similar carcinogenic effects to HDM; the rodents can also be other rodents besides mice that can be used for animal experiments.

[0039] The above description provides a further detailed explanation of the present invention in conjunction with specific / preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the concept of the present invention, and all such substitutions or modifications should be considered within the scope of protection of the present invention. In the description of this specification, the reference to terms such as "an embodiment," "some embodiments," "preferred embodiment," "example," "specific example," or "some examples," etc., indicates that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A method for constructing a lung cancer xenograft model, characterized in that, Includes the following steps: S1. Constructing an asthma model in rodents using allergens that cause asthma, including: anesthetizing rodents, stimulating rodents with allergens until their asthma pathological characteristics meet predetermined requirements, thereby completing the establishment of the asthma model; S2. Establish the lung cancer xenograft model by transplanting rodent-derived lung cancer cells into the asthma model; The rodent is a mouse, and the rodent-derived lung cancer cells are mouse-derived lung cancer cells.

2. The method for constructing a lung cancer xenograft model as described in claim 1, characterized in that: Step S2 includes: taking rodent-derived lung cancer cells in the logarithmic growth phase, inoculating rodent-derived lung cancer cell solution into rodents in the asthma model, stopping the experiment when the average tumor volume of the rodents is within a predetermined volume range, and obtaining the lung cancer xenograft model.

3. The method for constructing a lung cancer xenograft model as described in claim 2, characterized in that: Step S2 further includes: during the establishment of the lung cancer xenograft model, the rodents in the asthma model are also subjected to allergen stimulation weekly until the end of the experiment.

4. The method for constructing a lung cancer xenograft model as described in any one of claims 1-3, characterized in that: The allergens that cause asthma are at least one of house dust mites, pollen, dry dust mites, cockroaches, and mold.

5. The method for constructing a lung cancer xenograft model as described in claim 1, characterized in that: The mice are male; the mouse strain is either C57BL / 6 or Balc / c; the mouse-derived lung cancer cells are Lewis lung cancer cells.

6. The method for constructing a lung cancer xenograft model as described in any one of claims 1-3, characterized in that, Step S1 includes: anesthetizing mice with isopropanol nebulization, stimulating the mice with 10μg / 30μL HDM nasal drops, and then stimulating them with 15μg / 30μL HDM once a day for 5 consecutive days, thus completing the establishment of the asthma model.

7. The method for constructing a lung cancer xenograft model as described in claim 6, characterized in that, Step S2 includes: taking logarithmically growing Lewis lung cancer cells, digesting them with trypsin, washing them twice with pre-cooled PBS, then mixing the Lewis lung cancer cell solution with matrix gel at a 1:1 ratio to obtain a Lewis lung cancer cell solution, and subcutaneously inoculating the Lewis lung cancer cell solution into mice in the asthma model at a quantity of 1 million cells / mouse. One week later, the tumor length L and width W are measured, and the tumor volume V is calculated as V = 1 / 2 × L × W. 2 The formula was used to calculate the tumor volume in tumor-bearing mice. When the mean tumor volume in the asthma model mice was 1300 mm, 3 -1800mm 3 When the sample is within the acceptable range, stop the experiment and take samples. During the establishment of the lung cancer xenograft model, the mice in the asthma model were also stimulated with 15 μg / 30 μL / time of HDM twice a week until the end of the experiment.

8. The method for constructing a lung cancer xenograft model as described in claim 7, characterized in that, The cell fusion rate of the logarithmic growth phase Lewis lung cancer cells was 70%-80%.

9. The method for constructing a lung cancer xenograft model as described in claim 7, characterized in that, The mice were 6-8 week old male C57BL / 6 mice.

10. The method for constructing a lung cancer xenograft model as described in claim 2, characterized in that, The predetermined volume range is 1300 mm. 3 -1800mm 3 .

11. The method for constructing a lung cancer xenograft model as described in claim 5, characterized in that, The mice were 6-8 week old male mice.

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