Construction method of lung cancer transplantation tumor model

Abstract

The invention discloses a construction method of a lung cancer transplantation tumor model. The construction method comprises the following steps: S1, constructing an asthma model for rodents by adopting allergens causing asthma; s2, the lung cancer transplantation tumor model is established by transplanting rodent-derived lung cancer cells in the asthma model. As a novel lung cancer transplantation tumor model, the lung cancer transplantation tumor model constructed by the invention can well simulate the discovery of clinical research in basic research, and the scientific value and the application value of the lung cancer transplantation tumor model are improved.

Description

Technical Field

[0001] The present invention relates to the field of animal models, and in particular to a method for constructing a lung cancer transplant tumor model. Background Art

[0002] Lung cancer is currently the malignant tumor with the highest mortality rate and the third highest incidence rate worldwide. It also has the highest incidence and mortality rates in my country. Lung cancer incidence and mortality rates in my country are far higher than the global average, and the incidence and mortality rates continue to increase annually. The treatment of lung cancer urgently requires the development of new drugs and treatment plans. Elucidating the mechanism of action of drugs in living animal models is undoubtedly the best approach. Therefore, appropriate animal models of lung cancer are important tools for studying the pathogenesis of lung cancer and developing new therapeutic drugs for the treatment of lung cancer.

[0003] Animal models for lung cancer are primarily categorized as spontaneous, induced, transplantable, and genetically modified models. Transplantable models are tumor models formed by transplanting tumor tissue or cells into experimental animals. Depending on the source of the tumor cells or cancerous tissue, these models are categorized as either homologous or xenograft models. A homologous transplant refers to a tumor cell type that is consistent with the host animal species, while a xenograft refers to a tumor cell type that is inconsistent with the host animal species. Furthermore, depending on the site of lung cancer metastasis, animal models for lung cancer are categorized as lung metastasis, bone metastasis, brain metastasis, and lymph node metastasis models.

[0004] Transplantation 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 cost. However, the main drawback of transplantation models is that they cannot simulate clinical disease characteristics well. As a result, the results produced by this model cannot well reflect the real-world development process of lung cancer, weakening the translation and application value of the experimental results.

[0005] It should be noted that the information disclosed in the above background technology section is only used to understand the background of this application, and therefore may include information that does not constitute prior art known to ordinary technicians in this field. Summary of the Invention

[0006] In order to make up for the deficiencies of the prior art, the present invention provides a method for constructing a lung cancer transplant tumor model.

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

[0008] A method for constructing a lung cancer transplant tumor model comprises the following steps: S1, using an asthma-inducing allergen to construct an asthma model in rodents; S2, establishing the lung cancer transplant tumor model by transplanting rodent-derived lung cancer cells into the asthma model.

[0009] The present invention has the following advantages:

[0010] The present invention is the first to confirm the phenomenon that asthma promotes the development of lung cancer through experimental verification (animal experiments), and the first to confirm the fact that asthma can indeed promote lung cancer through animal experiments. Based on this, an animal model (asthma-lung cancer model) in which allergic inflammation promotes the development of lung cancer is established for the first time. The present invention provides a new idea for the establishment of a small animal lung cancer model, which can well simulate the findings of clinical research in basic research and improve the scientific value and application value of the lung cancer transplant tumor model. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0012] Figure 2 This is a comparison of the tumor tissue volumes of mice in the control group and the asthma group after the experiment of the embodiment of the present invention;

[0013] Figure 3 Schematic diagram of the pathological changes in mouse lung tissue (Figure A) and airway hyperresponsiveness (PENH) detection (Figure B) in an embodiment of the present invention;

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

[0015] Figure 5 This is a comparison chart of tumor tissue weights of mice in the control group and the asthma group after the experiment of the embodiment of the present invention. DETAILED DESCRIPTION

[0016] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is merely illustrative and is not intended to limit the scope and application of the present invention. In the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0017] Clinically, it has been found that asthma patients are more susceptible to lung cancer than healthy people, while reports show that smoking and secondhand smoke exposure may not be the main factors causing the increased incidence of lung cancer in asthma patients. However, it is currently unclear how asthma causes lung cancer, and there is no mature animal model to solve this problem. To this end, the present invention first uses the main allergens that cause asthma to construct an asthma model in rodents (such as mice), and then establishes a lung cancer transplant model by transplanting rodent-derived lung cancer cells (such as Lewis lung cancer (LLC, lewis lung cancer) cells). Finally, a rodent-based lung cancer transplant model is constructed for the first time (the lung cancer transplant model can be called an "asthma-lung cancer" transplant model, or an asthma-mediated lung cancer model). The lung cancer transplant model constructed by the present invention is a new lung cancer transplant model that can well simulate the findings of clinical studies in basic research, thereby improving the scientific value and application value of the lung cancer transplant model.

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

[0019] S1. Establish an asthma model in rodents using asthma-inducing allergens;

[0020] S2. Establishing the lung cancer transplant tumor model by transplanting rodent-derived lung cancer cells into the asthma model.

[0021] In a preferred embodiment, step S1 comprises: anesthetizing the rodent and subjecting the rodent to allergen stimulation until the asthma pathological characteristics thereof 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 the rodent-derived lung cancer cell solution into the rodents in the asthma model, and stopping the experiment when the mean tumor volume of the rodents is within a predetermined volume range to obtain the lung cancer transplant tumor model.

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

[0024] In a preferred embodiment, step S2 further comprises: during the establishment of the lung cancer transplant tumor model, the rodents in the asthma model are subjected to allergen stimulation every week until the end of the experiment.

[0025] In a preferred embodiment, when constructing an asthma model and / or establishing a lung cancer transplant tumor model, the asthma-causing allergen used is at least one of house dust mites (HDM), pollen, dust mites, cockroaches and molds, and 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; further 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 isopropyl alcohol atomization, administering 10 μg / 30 μL HDM nasal drops to the mice, and then administering 15 μg / 30 μL HDM once a day for 5 consecutive days after 3 days, thereby completing the establishment of the asthma model.

[0030] In a preferred embodiment, step S2 comprises: obtaining Lewis lung cancer cells in the logarithmic growth phase, digesting them with trypsin, and rinsing them twice with pre-cooled phosphate buffered saline (PBS). Then, the Lewis lung cancer cell solution is mixed with Matrigel at a volume ratio of 1:1 (measured by a vernier caliper) to obtain a Lewis lung cancer cell solution. The Lewis lung cancer cell solution is subcutaneously inoculated into mice in the asthma model at a number of 1 million cells / mouse. After one week, the length L and width W of the tumor are measured. The tumor volume V = 1 / 2 × L × W is calculated. 2 The formula was used to calculate the tumor volume of tumor-bearing mice. When the mean tumor volume of mice in the asthma model was 1300 mm 3 -1800mm 3 When the concentration of HDM was within the range, the experiment was stopped and samples were collected; during the establishment of the lung cancer transplant tumor model, the mice in the asthma model also received 15 μg / 30 μL / time HDM stimulation twice a week until the end of the experiment.

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

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

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

[0034] Experimental reagents: house dust mite (Stallergenes Greer), PBS (Thermo Fisher Scientific), isopropyl alcohol (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 House of the Chinese University of Hong Kong)

[0036] Step 1: Construct an asthma model. Mice were anesthetized with isopropyl alcohol nebulization. The mice were divided into a control group (indicated by PBS in the attached figure) and an asthma group (indicated by HDM in the attached figure). The control group received 30 μL PBS nasal drops once, and the asthma group received 10 μg / 30 μL HDM nasal drops once (wherein, HDM was prepared with PBS, the same below). After 3 days, the two groups of mice received 30 μL PBS nasal drops and 15 μg / 30 μL HDM nasal drops once a day for 5 consecutive days. At this point, the asthma pathological characteristics of the asthma group were significantly higher than those of the control group, thus completing the establishment of the asthma model (as shown in Figure 1). Figure 3 As shown). Step 2: Establish an "asthma-lung cancer" transplant tumor model. Take LLC cells in the logarithmic growth phase (cell fusion rate is 70%-80% under a 100x (eyepiece 10x, objective lens 10x) microscope), digest them with trypsin, and rinse them twice with pre-cooled PBS. Then, mix LLC cells with matrix gel at a ratio of 1:1 to obtain LLC cell solution, and subcutaneously inoculate LLC cell solution into two groups of mice, respectively, at a number of 1 million cells / mouse. After 1 week, measure the length L and width W of the tumor, and calculate the tumor volume V = 1 / 2 × L × W 2 The formula was used to calculate the tumor volume of tumor-bearing mice. When the volume of the control group was greater than 100 mm 3 And less than 2000mm 3 The model was considered successful when the mean tumor volume of the asthma group mice was 1300 mm 3 -1800mm 3When the level of HDM in the control group was within the range of 400 μg / mL, the experiment was stopped (in this case, the experiment was stopped on the 35th day) and samples were collected (i.e., mouse tumor tissue was collected). During the establishment of the "asthma-lung cancer" model, mice in the control group and the asthma group received 30 μL / time PBS nasal drops and 15 μg / 30 μL / time HDM nasal drops twice a week, respectively (in this case, for example, for the control group, the 30 μL / time PBS nasal drops received twice a week can be 30 μL PBS nasal drops on Monday and then 30 μL PBS nasal drops on Friday; similarly, for the asthma group, the 15 μg / 30 μL / time HDM nasal drops received weekly can be 15 μg / 30 μL HDM nasal drops on Monday and then 15 μg / 30 μL HDM nasal drops on Friday) until the end of the experiment.

[0037] Through the above two-step animal experiment method, the establishment of the "asthma-lung cancer" transplant tumor model was completed. Figure 1 As shown in the figure, the morphological comparison of tumor-bearing mice obtained from the control group (PBS) and the asthma group (HDM) after the experiment, which reflects the overall tumor growth of the mice. From the comparison figure, it can be seen that the tumor volume of the mice in the asthma group is larger than that in the control group. Figure 2 The figure below shows the volume comparison of tumor tissues in mice in the control group (PBS) and the asthma group (HDM) after the experiment. By comparing the tumor tissues of mice, it was found that the tumor tissues in the control group were significantly smaller than those in the asthma group, indicating that asthma can promote the development of lung cancer and proving the success of the asthma-lung cancer model. Figure 3 The following are the pathological changes in the lung tissue of mice (Figure A) and the detection of airway hyperresponsiveness (penh) (Figure B, using different concentrations of methacholine to stimulate airway hyperresponsiveness in mice, and observing the airway hyperresponsiveness of mice under different stimulation conditions, represented by penh values). As shown in Figure A, the lungs of mice in the asthma group showed obvious asthma-like pathological changes, while the lungs of mice in the control group did not show pathological changes. As shown in Figure B, 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 curve of tumor tissue volume changes in the control group and the asthma group during the experiment. It can be seen that the tumor tissue volume of the asthma group mice was significantly higher than that of the control group after 28 days of the experiment until the end of the experiment. Figure 5 The figure shows the comparison of tumor weights of mice in the control group and the asthma group after the experiment. It can be seen that the total amount of tumors in the mice in the asthma group was significantly greater than that in the mice in the control group.

[0038] In the above examples, HDM was used as an allergen to stimulate mice to establish an asthma model, and then Lewis lung cancer cells were inoculated to establish the lung cancer xenograft model. In other examples, the allergens may be pollen, dust mites, cockroaches, mold, or other allergens with similar carcinogenic effects to HDM. Furthermore, the rodents may be other experimental rodents besides mice.

[0039] The above description further details the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be construed as being limited to these descriptions. Persons skilled in the art will appreciate that, without departing from the spirit of the present invention, they may make various substitutions or modifications to the described embodiments, and these substitutions or modifications should be considered to fall within the scope of protection of the present invention. Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples" indicates that the specific features, structures, materials, or characteristics described in conjunction with such embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Persons skilled in the art may combine and assemble the different embodiments or examples described in this specification, as well as features of different embodiments or examples, without conflicting opinions. 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 may be made herein without departing from the scope of protection of the patent application.

Claims

1. A method for constructing a lung cancer transplant tumor model, characterized in that: The steps include: S1. Establish an asthma model in rodents using asthma-inducing allergens; S2. Establishing the lung cancer transplant tumor model by transplanting rodent-derived lung cancer cells into the asthma model.

2. The method for constructing a lung cancer transplant tumor model according to claim 1, wherein: Step S1 comprises: anesthetizing the rodent and subjecting the rodent to allergen stimulation until the asthma pathological characteristics thereof meet predetermined requirements, thereby completing the establishment of the asthma model; Step S2 comprises: taking rodent-derived lung cancer cells in the logarithmic growth phase, inoculating the rodent-derived lung cancer cell solution into the rodents in the asthma model, and stopping the experiment when the mean tumor volume of the rodents is within a predetermined volume range to obtain the lung cancer transplant tumor model; preferably, the predetermined volume range is 1300 mm 3 -1800mm 3 .

3. The method for constructing a lung cancer transplant tumor model according to claim 2, wherein: Step S2 also includes: during the establishment of the lung cancer transplant tumor model, the rodents in the asthma model are further subjected to allergen stimulation every week until the end of the experiment.

4. The method for constructing a lung cancer transplant tumor model according to any one of claims 1 to 3, wherein: The allergen causing asthma is at least one of house dust mites, pollen, dust mites, cockroaches and mold.

5. The method for constructing a lung cancer transplant tumor model according to any one of claims 1 to 3, wherein: The rodent is a mouse, and the rodent-derived lung cancer cells are mouse-derived lung cancer cells.

6. The method for constructing a lung cancer transplant tumor model according to claim 5, wherein: The mice are male mice, preferably 6-8 week old male mice; the mouse strain is one of C57BL / 6 mice and Balc / c mice; the mouse-derived lung cancer cells are Lewis lung cancer cells.

7. The method for constructing a lung cancer transplant tumor model according to any one of claims 1 to 3, wherein: Step S1 includes: using isopropyl alcohol atomization to anesthetize mice, subjecting the mice to nasal stimulation with 10 μg / 30 μL HDM, and after 3 days, subjecting the mice to nasal stimulation with 15 μg / 30 μL HDM once a day for 5 consecutive days, thereby completing the establishment of the asthma model.

8. The method for constructing a lung cancer transplant tumor model according to claim 7, wherein: Step S2 comprises: taking Lewis lung cancer cells in the logarithmic growth phase, digesting them with trypsin, and rinsing them twice with pre-cooled PBS, then mixing the Lewis lung cancer cell solution with matrigel at a ratio of 1:1 to obtain a Lewis lung cancer cell solution, and inoculating the Lewis lung cancer cell solution subcutaneously into mice in the asthma model at a number of 1 million cells / mouse. After one week, the length L and width W of the tumor were measured, and the tumor volume V = 1 / 2 × L × W was calculated. 2 The formula was used to calculate the tumor volume of tumor-bearing mice. When the mean tumor volume of mice in the asthma model was 1300 mm 3 -1800mm 3 When it is within the range, stop the experiment and collect the sample; During the establishment of the lung cancer transplant tumor 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.

9. The method for constructing a lung cancer transplant tumor model according to claim 8, wherein: The cell fusion rate of the Lewis lung cancer cells in the logarithmic growth phase is 70%-80%.

10. The method for constructing a lung cancer transplant tumor model according to claim 8, wherein: The mice were 6-8 week old male C57BL / 6 mice.

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