A method for constructing a chronic stress gastric cancer metastasis model based on immune-competent 615 inbred mice

Abstract

The application discloses a kind of chronic stress gastric cancer metastasis model construction methods based on immune sound 615 inbred line mouse, belong to the field of biological medicine, the method is by chronic restraint stress pretreatment to immune sound 615 inbred line mouse, and after pretreatment end, inoculate mouse front gastric cancer MFC cell, continue to implement chronic restraint stress treatment after inoculation, to construct chronic stress gastric cancer metastasis animal model;The model constructed by the application can simulate the process that chronic stress promotes gastric cancer metastasis under complete immune background, can stably present the characteristics such as behavior change, neuroendocrine change and transfer load increase higher;The application provides a kind of new chronic stress gastric cancer metastasis model construction method, model construction process is clear, stability is good, can be used to study the mechanism that chronic stress promotes gastric cancer metastasis and the screening and evaluation of related drug or intervention measure.

Description

Technical Field

[0001] This invention relates to the field of tumor metastasis research or animal model construction, and more specifically, to a method for constructing a chronic stress gastric cancer metastasis model based on immune-healthy 615 inbred mice. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors of the digestive system, and metastasis is a significant factor affecting patient prognosis and treatment outcomes. Recent studies suggest that chronic stress can not only cause persistent neuroendocrine disorders but also promote tumor development and metastasis, thereby exacerbating disease progression and impacting prognosis. Therefore, research on how chronic stress promotes gastric cancer metastasis is of great importance, and establishing suitable animal models is the foundation and key to conducting related mechanistic studies and drug screening.

[0003] Currently, animal models of gastric cancer metastasis and chronic stress have a certain research foundation. Animal models of gastric cancer metastasis can be constructed based on different host immune backgrounds, tumor cell sources, and inoculation methods. For example, in immunodeficient hosts, human gastric cancer cells, patient-derived tumor tissue, or organoids can be used to establish relevant models; in immunocompetent hosts, murine gastric cancer cells are often used, such as combinations of MFC cells and 615 inbred mice, or YTN16 cells and C57BL / 6 mice. Chronic stress models include various methods such as chronic restraint stress and chronic unpredictable mild stress. Among them, chronic restraint stress (CRS) has been widely used in stress-related research due to its relatively simple operation and good reproducibility. There are also reports of combining stress treatment with tumor models in studies on chronic stress promoting tumor progression.

[0004] The aforementioned models and methods provide a foundation for related research, but some shortcomings remain in studies on chronic stress promoting gastric cancer metastasis: First, existing models often fail to simultaneously consider chronic stress treatment, gastric cancer metastasis evaluation, and a complete immune background. In particular, while immunodeficient hosts such as nude mice can be used to observe tumor growth and metastasis, their lack of complete T-cell immune function results in a host immune background that differs significantly from the actual immune microenvironment, thus limiting their applicability in studies of chronic stress-related gastric cancer metastasis. Second, reports on constructing a composite model of chronic stress promoting gastric cancer metastasis using 615 inbred mice as an immune-healthy host, combined with chronic restraint stress and MFC cell inoculation, are relatively scarce, especially lacking a systematic technical solution that considers modeling timing, endpoint evaluation, and model stability.

[0005] Therefore, providing a method for constructing an animal model of gastric cancer metastasis under chronic stress based on an immune-intact host is of great significance for evaluating the impact of chronic stress on gastric cancer metastasis in an intact immune background and for providing experimental tools for related mechanism research and drug screening.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for constructing a chronic stress gastric cancer metastasis model based on immune-intact 615 inbred mice, so as to simulate the process of chronic stress promoting gastric cancer metastasis in a complete immune background, and to provide experimental tools for related mechanism research and screening and evaluation of drugs or interventions.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for constructing a chronic stress gastric cancer metastasis model based on immune-healthy 615 inbred mice includes the following steps:

[0010] 1) Select immune-healthy 615 inbred mice and subject them to adaptive rearing;

[0011] 2) The mice were subjected to chronic restraint stress pretreatment;

[0012] 3) After the chronic restraint stress pretreatment was completed, the mice were inoculated with mouse forestomy cancer MFC cells;

[0013] 4) After tumor cell inoculation, the mice were subjected to chronic restraint stress to obtain a chronic stress gastric cancer metastasis animal model.

[0014] The chronic restraint stress treatment is carried out using a restraint device that has the function of restricting movement and has good ventilation, preferably a 50mL centrifuge tube with a total of 9 ventilation holes on the side wall and tip, and is treated for 8 hours a day, with a pretreatment time of 14 days.

[0015] Preferably, the mice are SPF-grade male 615 inbred mice, aged 3-4 weeks, weighing 18-22g, and acclimatized for 5-7 days, preferably 7 days.

[0016] Preferably, the total cycle of the construction method is 5 weeks, of which the first 14 days are a chronic restraint stress pretreatment phase, and after inoculation of MFC cells, a 21-day chronic restraint stress treatment is continued.

[0017] Preferably, the MFC cells are revived and cultured before inoculation, preferably cells that have been passaged up to 5 times after revival, and are inoculated 1 day after passage and when the cell confluence reaches 70% to 80%.

[0018] Preferably, the MFC cells are inoculated using serum-free culture medium or PBS after resuspension, and the inoculation dose is 1.0×10^6 to 1.5×10^6 cells per mouse, preferably 1.25×10^6 cells; the inoculation volume is 0.1 to 0.2 mL per mouse, preferably 0.2 mL per mouse.

[0019] Preferably, the MFC cells are inoculated via either tail vein injection or intraperitoneal injection.

[0020] Preferably, when tail vein injection is used, lung metastasis is used as the primary observation endpoint; the evaluation indicators include lung in vivo imaging signal, number of metastatic nodules in lung tissue and / or the proportion of metastatic area in lung tissue pathological sections.

[0021] Preferably, when intraperitoneal injection is used, peritoneal metastasis is used as the primary observation endpoint; the evaluation indicators include peritoneal in vivo imaging signal, number of peritoneal metastatic nodules and / or peritoneal pathological changes.

[0022] A second aspect of the present invention also provides the application of the animal model obtained by the construction method in studying the mechanism by which chronic stress promotes gastric cancer metastasis.

[0023] A third aspect of the present invention also provides the application of the animal models obtained by the construction method in screening or evaluating drugs or interventions that inhibit chronic stress-related gastric cancer metastasis.

[0024] Preferably, the evaluation criteria for animal models include the following aspects: behavioral test indicators, anti-metastatic pharmacodynamic evaluation, neuroendocrine test indicators, and pathological histological analysis.

[0025] Furthermore, the behavioral tests include a sucrose preference test, a tail suspension test, and a forced swimming test.

[0026] Furthermore, the anti-metastatic pharmacodynamic evaluation includes one or more of the following: neuroendocrine index detection, in vivo imaging detection, and metastatic nodule counting.

[0027] Furthermore, the neuroendocrine marker detection includes the detection of norepinephrine and glucocorticoid-related markers.

[0028] Furthermore, the pathological histological analysis includes hematoxylin-eosin staining of lung or peritoneal tissue and statistical analysis of the area percentage of metastatic lesions based on their morphological characteristics.

[0029] The preferred implementation method is as follows: Male 615 inbred mice aged 3-4 weeks and weighing 18-22g are selected and acclimatized for 7 days; 50mL perforated centrifuge tubes are used as restraint devices, and chronic restraint stress is applied for 8 hours daily for 14 consecutive days; on the 14th day, 1.25×10^6 MFC cells are injected via tail vein, with an injection volume of 0.2mL / mouse; chronic restraint stress is continued for 21 days after inoculation; behavioral tests are performed on the 28th day of the experiment, in vivo imaging is performed on the 34th day, and final sampling, pathological analysis, and comprehensive evaluation are performed on the 35th day.

[0030] Furthermore, day 35 can be considered as a preferred endpoint time that balances the stability of load assessment with the survival status of animals.

[0031] In some implementations, the tail vein injection protocol is easier to standardize in terms of quantifying the pathological area of ​​lung metastases, and is therefore suitable as a preferred implementation.

[0032] Intraperitoneal injection can be used for peritoneal transfer studies, but its quantitative consistency is relatively weak, so it can be used as an extended implementation method.

[0033] A fourth aspect of the present invention also provides a method for screening candidate drugs for the prevention or treatment of chronic stress-related gastric cancer metastasis, the method comprising the following steps:

[0034] 1) The test candidate drug was administered to the mouse model constructed using the above method;

[0035] 2) Dynamically monitor changes in metastatic lesions in mouse models using in vivo imaging;

[0036] 3) Screening candidate drugs by changes in in vivo imaging signals and pathological histological indicators.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] This invention provides a method for constructing a chronic stress-induced gastric cancer metastasis model based on the immunocompetent 615 inbred mouse strain. Compared with existing models, this method can simulate the process of chronic stress promoting gastric cancer metastasis under a intact immune background. The model construction process is relatively clear and can stably obtain a chronic stress-induced gastric cancer metastasis model characterized by behavioral changes, neuroendocrine changes, and increased metastatic burden. Based on existing results, the preferred scheme has good model stability and endpoint evaluation feasibility, and can be used for related mechanism research and the screening and evaluation of drugs or interventions. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the model construction process described in this invention;

[0040] Figure 2The following is a statistical graph of the weight change curve, behavioral test results, and neuroendocrine-related ELISA test results in Example 1 of this invention (wherein, A, weight change curve; B, sucrose preference test results; C, tail suspension test results; D, forced swimming test results; E, plasma glucocorticoid-related index test results; F, plasma norepinephrine test results).

[0041] Figure 3 The images show in vivo imaging and statistical analysis of lung metastasis under the tail vein injection protocol (where A includes representative images of small animal in vivo imaging and quantitative statistical charts of imaging signals; B includes a representative gross view of lung tissue after final sampling, a representative image of lung tissue stained with hematoxylin and eosin, and a statistical chart of the percentage of metastatic area).

[0042] Figure 4 The images show in vivo imaging and metastatic nodule counting statistics of peritoneal metastasis under intraperitoneal injection protocol (where A includes representative in vivo imaging images of small animals and quantitative statistics of imaging signals; B includes representative gross peritoneum images after final tissue sampling, labeled peritoneal metastatic nodules, and statistics of the number of metastatic nodules).

[0043] Figure 5 Figures showing the survival observation results (where A is a schematic diagram of the experimental procedure for the independent survival observation cohort; B is the survival curve of the mouse lung metastasis model under tail vein injection; and C is the survival curve of the peritoneal metastasis model under intraperitoneal injection). Detailed Implementation

[0044] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0045] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, and all instruments used in the examples are commercially available.

[0046] Example 1:

[0047] A method for constructing a chronic stress gastric cancer metastasis model based on immune-healthy 615 inbred mice includes the following steps:

[0048] 1) Select immune-healthy 615 inbred mice and subject them to adaptive rearing;

[0049] 2) The mice were subjected to chronic restraint stress pretreatment;

[0050] 3) After the chronic restraint stress pretreatment was completed, the mice were inoculated with mouse forestomy cancer MFC cells;

[0051] 4) After tumor cell inoculation, the mice were subjected to chronic restraint stress to obtain a chronic stress gastric cancer metastasis animal model.

[0052] The chronic restraint stress treatment is carried out using a restraint device that has the function of restricting movement and has good ventilation, preferably a 50mL centrifuge tube with a total of 9 ventilation holes on the side wall and tip, and is treated for 8 hours a day, with a pretreatment time of 14 days.

[0053] The mice were SPF-grade male 615 inbred mice, aged 3-4 weeks, weighing 18-22g, and acclimatized for 5-7 days, preferably 7 days.

[0054] The total cycle of the construction method is 5 weeks, with the first 14 days being a chronic restraint stress pretreatment phase, followed by 21 days of chronic restraint stress treatment after MFC cell inoculation.

[0055] The MFC cells are revived and cultured before inoculation, preferably cells that have been passaged no more than 5 times after revival, and are inoculated 1 day after passage and when the cell confluence reaches 70% to 80%.

[0056] The MFC cells were inoculated using serum-free culture medium or PBS after resuspension, and the inoculation dose was 1.0×10^6 to 1.5×10^6 cells per mouse, preferably 1.25×10^6 cells; the inoculation volume was 0.1 to 0.2 mL per mouse, preferably 0.2 mL per mouse.

[0057] Step 1) Select SPF-grade male 615 inbred mice, aged 3 to 4 weeks (including 3 weeks, 4 weeks and the intermediate age), weighing 18 to 22g (including 18g, 22g and the intermediate weight), and acclimatize them for 5 to 7 days (including 5 days, 7 days and the intermediate number of days). In this example, the mice were acclimatized for 7 days.

[0058] Step 2) A 50mL centrifuge tube with a total of 9 vent holes on the side wall and tip was used as a chronic restraint stress device. The model group mice were subjected to chronic restraint stress treatment for 8 hours a day for 14 consecutive days as the chronic restraint stress pretreatment stage.

[0059] Step 3) On day 14 of the experiment, after the chronic restraint stress pretreatment, mice were inoculated with mouse forestomal cancer MFC cells. The MFC cells were resuscitated and cultured before inoculation, specifically cells passaged up to 5 times after resuscitation, and cultured for 1 day after passage until cell confluence reached 70%–80% before inoculation. The MFC cells were resuspended in serum-free medium. The inoculation dose was 1.0 × 10^6–1.5 × 10^6 cells per mouse, covering 1.0 × 10^6, 1.5 × 10^6 cells, and intermediate doses. In this example, the specific inoculation dose was 1.25 × 10^6 cells / mouse; the inoculation volume was 0.1–0.2 mL / mouse, covering 0.1 mL, 0.2 mL, and intermediate volumes. In this example, the specific inoculation volume was 0.2 mL / mouse; the inoculation method was tail vein injection.

[0060] Step 4) After tumor cell inoculation, the model group mice continued to be subjected to chronic restraint stress treatment for 8 hours a day for 14 to 21 consecutive days, covering 14 days, 21 days and the intermediate days. In this example, the treatment was carried out for 21 consecutive days to obtain a chronic stress gastric cancer lung metastasis animal model.

[0061] Step 5) Behavioral tests were conducted on day 28 of the experiment, including the sucrose preference test, tail suspension test, and forced swimming test. Results showed that the percentage of mice in the model group who preferred sucrose was lower than that in the control group, indicating anhedonia-like behavior. Figure 2 B; The model group mice had a longer tail suspension immobility time than the control group, corresponding to Figure 2 C; The model group mice had a longer forced swimming immobility time than the control group, corresponding to Figure 2 D. Figure 2 B to Figure 2 D cross-validated the same type of behavioral changes using different experimental methods, jointly demonstrating that the model group mice developed a stable chronic stress phenotype.

[0062] Step 6) On day 34 of the experiment, in vivo imaging was performed on the small animals to collect luminescent signals from the lung regions. The results showed that the total number of photons in the lung luminescent signals of the model group mice was higher than that of the control group, indicating an increased burden of lung metastases. Figure 3 A.

[0063] Step 7) Terminal sampling was performed on day 35 of the experiment to observe the gross appearance of lung tissue. Pathological sections of the lung tissue were prepared and stained with hematoxylin and eosin. The percentage of metastatic area to total lung tissue area was determined using image analysis software. Results showed that the model group mice had an increased number of metastatic lesions in their lung tissue, and the percentage of metastatic area was higher than that in the control group. Figure 3 B. Figure 3 A and Figure 3 B evaluates lung metastasis burden at both the in vivo and histological levels, forming an evidence loop of "in vivo imaging screening - pathological verification".

[0064] Step 8) Simultaneously, plasma was collected on day 35 of the experiment for enzyme-linked immunosorbent assay (ELISA) to detect plasma cortisol and norepinephrine levels. Results showed that the plasma cortisol concentration was elevated in the model group mice, corresponding to... Figure 2 E indicates that the hypothalamic-pituitary-adrenal axis is in a state of stress activation; the plasma norepinephrine concentration in the model group mice is elevated, corresponding to... Figure 2 F indicates that the sympathetic-adrenal medullary system is activated. Figure 2 E and Figure 2 F and Figure 2 B to Figure 2 The behavioral results of D corroborate each other, indicating that the model group mice not only received chronic restraint stress treatment, but also formed a relatively stable chronic stress state.

[0065] Step 9) Monitor mouse body weight changes during the experiment. The weight gain trend of the model group mice was slower than that of the control group. Figure 2 A reflects the lasting impact of chronic restraint stress on the body's overall condition. Figure 2 A to Figure 2 F together constitute a multi-dimensional verification system for chronic stress, progressing from external physiological state to behavioral manifestations and then to internal neuroendocrine mechanisms, demonstrating that this embodiment successfully constructed a chronic stress-related gastric cancer lung metastasis model under a complete immune background, and the pathological area of ​​the lung metastasis region showed good quantitative consistency, which is convenient for standardized evaluation.

[0066] Example 2:

[0067] Step 1) Select SPF-grade male 615 inbred mice, aged 3 to 4 weeks (including 3-week-old, 4-week-old and intermediate-week-old mice), weighing 18 to 22g (including 18g, 22g and intermediate-weight mice), and acclimatize them for 5 to 7 days. In this example, acclimatize them for 7 days.

[0068] Step 2) Use 50mL perforated centrifuge tubes as chronic restraint stress devices, treat for 8 hours a day for 14 consecutive days as the pretreatment stage of chronic restraint stress.

[0069] Step 3) On day 14 of the experiment, MFC cells that have been revived and passaged up to 5 times and cultured for 1 day until the cell confluence reaches 70% to 80% are resuspended in serum-free medium. The inoculation dose is 1.0×10^6 to 1.5×10^6 cells per mouse, covering 1.0×10^6, 1.5×10^6 cells and intermediate doses. In this example, it is 1.25×10^6 cells / mouse. The inoculation volume is 0.1 to 0.2 mL / mouse, covering 0.1 mL, 0.2 mL and intermediate volumes. In this example, it is 0.2 mL / mouse. The inoculation method is intraperitoneal injection.

[0070] Step 4) After tumor cell inoculation, the model group mice continued to be subjected to chronic restraint stress treatment for 8 hours a day for 14 to 21 consecutive days, covering 14 days, 21 days and the intermediate days. In this embodiment, the treatment was carried out for 21 consecutive days to obtain a chronic stress gastric cancer peritoneal metastasis animal model.

[0071] Step 5) Behavioral testing was conducted on day 28 of the experiment. The model group showed behavioral changes consistent with those in Example 1.

[0072] Step 6) On day 34 of the experiment, in vivo imaging was performed on the small animals to collect luminescence signals from the peritoneal region. The results showed that the total number of photons in the peritoneal region of the model group mice was higher than that of the control group, indicating an increased tumor burden within the peritoneal cavity. Figure 4 A.

[0073] Step 7) Terminal tissue sampling was performed on day 35 of the experiment to observe the gross appearance of the peritoneum, and peritoneal metastatic nodules were marked and counted. Results showed that the number of peritoneal metastatic nodules in the model group was higher than that in the control group. Figure 4 B. Figure 4 A and Figure 4 B evaluated peritoneal metastasis from both in vivo imaging and endpoint gross observation perspectives, both indicating an increased peritoneal metastasis burden in the model group. It should be noted that the number of peritoneal nodules is affected by factors such as nodule fusion, size variation, and observation angle, resulting in relatively weaker quantitative consistency compared to lung tissue pathological area proportion analysis. Therefore, the intraperitoneal injection protocol is more suitable as an extended implementation method under current evidence.

[0074] Example 3:

[0075] Step 1) Construct a chronic stress-induced gastric cancer lung metastasis model according to the method described in Example 1, and use this model to study the mechanism by which chronic stress promotes gastric cancer metastasis.

[0076] Step 2) On day 28 of the experiment, behavioral tests were performed on mice in the model group and the control group. The results showed that mice in the model group exhibited decreased sugar water preference, prolonged immobility time during tail suspension, and prolonged immobility time during forced swimming, displaying depressive-like behavioral changes, corresponding to... Figure 2 B. Figure 2 C and Figure 2 D. The above results were cross-validated using different experimental methods on the same type of behavioral changes, which enhanced the credibility of the results.

[0077] Step 3) Plasma was collected at the end of day 35 of the experiment for ELISA testing. Results showed that plasma cortisol levels in the model group mice were significantly elevated, corresponding to... Figure 2 E; Plasma norepinephrine levels were significantly elevated, corresponding to Figure 2F. The above neuroendocrine indicators suggest that chronic stress causes persistent neuroendocrine disorders, which corroborates the behavioral findings.

[0078] Step 4) Simultaneously, on day 35 of the experiment, hematoxylin-eosin staining was performed on the lung tissue for pathological analysis. The results showed that the number of metastatic lesions and the proportion of metastatic area in the lung tissue of the model group mice increased. Figure 3 B provides direct histological evidence that chronic restraint stress promotes the formation and spread of lung metastases.

[0079] Step 5) Synthesis Figure 2 B to Figure 2 F and Figure 3 The results of B show that the aforementioned behavioral, neuroendocrine, and pathological histological indicators logically progress from external manifestations to internal mechanisms, indicating that the model can stably simulate the process of chronic stress promoting gastric cancer metastasis in a complete immune background, and simultaneously presents characteristics such as behavioral changes, neuroendocrine changes, and increased metastatic burden, making it suitable for studying the mechanism of chronic stress promoting gastric cancer metastasis.

[0080] Example 4: MFC cells were seeded via tail vein injection to construct a chronic stress-related gastric cancer lung metastasis model.

[0081] Step 1) Construct a chronic stress-related gastric cancer lung metastasis model according to the method described in Example 1, and use the model to screen or evaluate drugs or interventions that inhibit chronic stress-related gastric cancer metastasis.

[0082] Step 2) During model construction, on day 34 of the experiment, the changes in imaging signals of lung metastases were dynamically monitored using in vivo small animal imaging to obtain total photon count data for the lung region. Figure 3 A. This indicator reflects the overall and dynamic changes in transferred load at the living organism level.

[0083] Step 3) After the final sampling on day 35 of the experiment, the anti-metastatic pharmacodynamics was evaluated through gross observation of lung tissue, identification of metastatic nodules in lung tissue, and determination of the proportion of metastatic area in lung tissue pathological sections. Figure 3 B. This indicator provides a more intuitive pathological basis at the histological level.

[0084] Step 4) By comparing the changes in in vivo imaging signal intensity and the proportion of lung tissue metastasis area between the drug administration group and the model control group, the inhibitory effect of the test drug or intervention on chronic stress-related gastric cancer metastasis can be quantitatively evaluated. Figure 3 A and Figure 3 The two work together to form an evidence loop of "in vivo imaging screening - pathological verification", which makes the evaluation results both dynamic and histologically empirical, providing a reliable experimental tool for the screening and evaluation of anti-metastatic drugs.

[0085] Example 5: MFC cells were seeded via intraperitoneal injection to construct a chronic stress-related gastric cancer peritoneal metastasis model for drug screening.

[0086] Step 1) Provides a method for screening candidate drugs for the prevention or treatment of chronic stress-related gastric cancer metastasis. A chronic stress-related gastric cancer peritoneal metastasis model is constructed according to the method described in Example 2, specifically including: selecting 3-4 week old, 18-22g male 615 inbred mice and acclimatizing them for 7 days; using 50mL perforated centrifuge tubes as restraint devices, subjecting them to 8 hours of chronic restraint stress daily for 14 consecutive days; on the 14th day, injecting 1.25×10^6 MFC cells intraperitoneally at a volume of 0.2mL / mouse; continuing chronic restraint stress treatment for 21 days after inoculation.

[0087] Step 2) Administer the candidate drug to the mouse model constructed above. The administration method can be selected according to the characteristics of the drug, such as intraperitoneal injection, gavage, or intravenous injection. The dosage and frequency are determined based on the drug's half-life and preliminary experimental results. Administration begins after tumor cell inoculation, once daily for 21 consecutive days.

[0088] Step 3) On day 34 of the experiment, the changes in peritoneal metastases in the mouse model were dynamically monitored using in vivo small animal imaging, and luminescence signals in the peritoneal region were collected. The inhibitory effect of the drug on the overall burden of peritoneal metastases was evaluated by comparing the total number of photons in the peritoneal region of the drug-treated group and the model control group (administered the same volume of solvent). Figure 4 A.

[0089] Step 4) Terminal sampling was performed on day 35 of the experiment. The peritoneum was observed grossly, peritoneal metastatic nodules were counted, and peritoneal histopathological analysis (hematoxylin-eosin staining) was performed when necessary to comprehensively evaluate the anti-peritoneal metastasis effect of the drug. Figure 4 B. By comparing the differences in the number of peritoneal metastatic nodules between the treatment group and the model control group, candidate drugs with the potential to inhibit peritoneal metastasis of chronic stress-related gastric cancer were screened.

[0090] Step 5) If the drug administration group shows a significant decrease in the above indicators (abdominal in vivo imaging signal, number of peritoneal metastatic nodules) compared with the model control group, and the behavioral indicators (sucrose preference, tail suspension, forced swimming) and neuroendocrine indicators (cortisol, norepinephrine) do not show significant deterioration, it suggests that the candidate drug has the potential to prevent or treat chronic stress-related gastric cancer metastasis, and further validation studies can be conducted.

[0091] Comparative Example 1:

[0092] Based on Example 1, the age of the experimental animals was changed from 3-4 weeks to 4-5 weeks, while other modeling conditions remained unchanged. Observations showed that under the same chronic restraint stress conditions, the difference in transfer load between the model group and the control group was not significant in 4-5 week old mice, therefore it was not considered the preferred option for further development. Combined with observations during the modeling process, the struggling and resistance exhibited by 4-5 week old mice during chronic restraint stress treatment were relatively weaker than those in 3-4 week old mice. Based on the above results, 3-4 weeks of age can be considered the preferred age range under the current evidence conditions of this invention.

[0093] Comparative Example 2:

[0094] Based on Example 1, the MFC cell inoculation time point was adjusted to day 7 and day 21 of chronic restraint stress treatment, while other modeling conditions remained unchanged. Observations showed that when MFC cells were inoculated on day 7, some mice died earlier, and the overall metastatic burden in the terminal stage was high, which was not conducive to stably reflecting the difference in metastasis promoted by chronic stress. When MFC cells were inoculated on day 21, although the metastatic burden between the model group and the control group showed a certain trend of difference, the difference was not significant, suggesting insufficient tumor growth window, which was not conducive to forming a stable model difference. In contrast, inoculating MFC cells on day 14 of chronic restraint stress treatment was more conducive to forming a relatively stable stress background while preserving a sufficient tumor growth window, thus making it more suitable as the preferred inoculation time point of this invention.

[0095] Comparative Example 3:

[0096] Based on Example 1, with a total modeling period of 5 weeks and MFC cell inoculation time determined to be day 14 after chronic restraint stress treatment, the inoculation effects of 1.0 × 10^6, 1.25 × 10^6, and 1.5 × 10^6 MFC cells / animal were further compared, while other modeling conditions remained unchanged. The results showed that when the inoculation dose was 1.0 × 10^6 cells / animal, some animals showed insufficient tumor formation or metastasis, and the difference between the model group and the control group was not stable enough. When the inoculation dose was 1.5 × 10^6 cells / animal, the tumor burden was high, and the risk of early death in animals increased, which was not conducive to the stable presentation of model differences. In contrast, the inoculation dose of 1.25 × 10^6 cells / animal was more suitable in terms of tumor formation stability, metastasis burden control, and the presentation of differences between groups, and therefore can be considered as the preferred inoculation dose under the current evidence conditions of this invention.

[0097] Comparative Example 4:

[0098] Based on Example 1, behavioral tests were conducted in weeks 2, 3, and 4 of the experiment, with all other modeling conditions remaining unchanged. The results showed that the behavioral differences between the model group and the control group were not ideal in week 2; although some differences were observed in week 3, the overall effect was still weaker than in week 4; and the behavioral differences were most significant and stable in week 4. Therefore, week 4, i.e., day 28, is more suitable as the preferred time point for behavioral testing in this invention.

[0099] Comparative Example 5:

[0100] Based on Example 1, an independent survival observation cohort was established using a "5-week restraint, tumor inoculation on day 14" protocol. From week 6 onwards, no further manipulation of the mice was performed; only survival observation was conducted. The experimental procedure for this survival observation cohort is as follows: Figure 5 As shown in Figure A. The results showed that within the total modeling period of 5 weeks, mice typically did not die due to tumor burden and were able to complete behavioral testing, in vivo imaging, and endpoint sampling relatively completely; however, mortality began to occur in the model group after the 6th week of the experiment. Figure 5 As shown in Figure B, under tail vein injection conditions, the survival curves of the model group mice decreased earlier than those of the control group, and were generally lower than those of the control group; Figure 5 As shown in Figure C, under intraperitoneal injection conditions, the survival curves of the model group mice also decreased faster and the terminal survival rate was lower than that of the control group. These results suggest that if the experimental period is further extended, the model will gradually enter a significant mortality phase, thus affecting the sample integrity and quality of tissue collection during endpoint evaluation. Therefore, day 35 better balances endpoint evaluation quality and animal survival status, making it a suitable preferred experimental endpoint for this invention. Figure 5 B. Figure 5 C and the aforementioned Figure 3 , Figure 4 The metastatic load detection maps complement each other, and the aforementioned results mainly demonstrate that chronic restraint stress promotes the formation and expansion of metastatic lesions, while Figure 5 This further demonstrates that the effect has a clear outcome significance, thereby enhancing the application value of the model of this invention in mechanism research, drug screening, and intervention evaluation.

[0101] Comparative Example 6:

[0102] Based on Example 1, the MFC cell seeding method was changed from tail vein injection to intraperitoneal injection, while other modeling conditions remained unchanged. Observations showed that the intraperitoneal injection protocol could induce peritoneal metastasis-related phenotypes, but with a relatively large number of metastatic lesions, peritoneal nodule counting was relatively difficult and subjective. In contrast, the lung metastasis model constructed by the tail vein injection protocol was easier to identify metastatic areas and quantify their area proportions through HE staining of lung tissue, which was more beneficial for model evaluation, parameter screening, and subsequent mechanism research. Therefore, under the current evidence, tail vein injection is more suitable as the preferred embodiment of this invention, while intraperitoneal injection can be considered as an extended embodiment.

[0103] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a chronic stress gastric cancer metastasis model based on immune-intelligent 615 inbred mice, characterized in that, Includes the following steps: 1) Select immune-healthy 615 inbred mice and subject them to adaptive rearing; 2) The mice were subjected to chronic restraint stress pretreatment; 3) After the chronic restraint stress pretreatment was completed, the mice were inoculated with mouse forestomy cancer MFC cells; 4) After tumor cell inoculation, the mice were subjected to chronic restraint stress to obtain a chronic stress gastric cancer metastasis animal model. The chronic restraint stress treatment is carried out using a restraint device that has the function of restricting movement and has good ventilation, with 8 hours of treatment per day, and a pretreatment period of 14 days.

2. The method for constructing a chronic stress gastric cancer metastasis model based on immune-intelligent 615 inbred mice according to claim 1, characterized in that, The mice were SPF-grade male 615 inbred mice, 3-4 weeks old, weighing 18-22g, and acclimatized for 5-7 days.

3. The method for constructing a chronic stress gastric cancer metastasis model based on immune-intelligent 615 inbred mice according to claim 1, characterized in that, The chronic restraint stress treatment adopts a sequence of pretreatment, inoculation, and then continuous treatment; after tumor cell inoculation, chronic restraint stress treatment is continued for 14 to 21 days.

4. The method for constructing a chronic stress gastric cancer metastasis model based on immune-intelligent 615 inbred mice according to claim 1, characterized in that, The MFC cells are revived and cultured before inoculation, preferably cells that have been passaged no more than 5 times after revival, and are inoculated 1 day after passage and when the cell confluence reaches 70% to 80%.

5. The method for constructing a chronic stress gastric cancer metastasis model based on immune-intact 615 inbred mice according to claim 1, characterized in that, The MFC cells were inoculated using serum-free culture medium or PBS after resuspension, and the inoculation dose was 1.0×10^6 to 1.5×10^6 cells per mouse, with an inoculation volume of 0.1 to 0.2 mL per mouse.

6. The method for constructing a chronic stress gastric cancer metastasis model based on immune-intelligent 615 inbred mice according to claim 1, characterized in that, The MFC cells are inoculated via either tail vein injection or intraperitoneal injection.

7. The method for constructing a chronic stress gastric cancer metastasis model based on immune-intelligent 615 inbred mice according to claim 6, characterized in that, When tail vein injection is used, lung metastasis is the primary endpoint of observation; the evaluation indicators include lung in vivo imaging signal, number of metastatic nodules in lung tissue and / or the proportion of metastatic area in lung tissue pathological sections.

8. The method for constructing a chronic stress gastric cancer metastasis model based on immune-infected 615 inbred mice according to claim 6, characterized in that, When intraperitoneal injection is used, peritoneal metastasis is the primary endpoint of observation; the evaluation indicators include peritoneal in vivo imaging signal, number of peritoneal metastatic nodules and / or peritoneal pathological changes.

9. The application of the animal model obtained by the construction method according to any one of claims 1-8 in the study of the mechanism by which chronic stress promotes gastric cancer metastasis.

10. The use of animal models obtained by the construction method according to any one of claims 1-8 in screening or evaluating drugs or interventions that inhibit chronic stress-related gastric cancer metastasis.

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