Construction method of mouse model for generating endogenous metal flavor through induction of cis-platinum

Abstract

The invention relates to a construction method of a mouse model for generating endogenous metal flavor through cis-platinum induction, and belongs to the technical field of animal models. 6-week-old BALB / c female mice with the body weight of 20-22 g are selected, and mice in an aversion state to a 3.2 mg / L ferrous sulfate solution are screened through a double-bottle preference experiment; preparing a 3.5 mg / kg cis-platinum solution; the cis-platinum solution is injected into the body of the mouse through the abdominal cavity of the mouse for three weeks continuously, the injection is performed twice per week, and the injection dosage is 0.1 ml / 10 g; and after the injection process is finished, screening the mice in a preference state on the ferrous sulfate solution by using a double-bottle preference experiment. The mouse model for generating the endogenous metal flavor through cisplatin induction is successfully constructed by adopting a double-bottle preference experiment screening method and a cisplatin intraperitoneal injection method, and the problem that the research basis of a treatment method for generating the endogenous metal flavor for a patient after cisplatin chemotherapy is lacked is solved.

Description

Technical Field

[0001] The invention relates to a method for constructing a mouse model for generating endogenous metallic taste induced by cisplatin, and belongs to the technical field of animal models. Background Art

[0002] Cisplatin (CP) is a drug widely used in chemotherapy for cancer patients. During chemotherapy, cancer patients often suffer from impaired taste perception, which is mainly manifested as a metallic bitter taste in the mouth, affecting appetite and hindering physical recovery.

[0003] Currently, there is no effective way to treat and improve the long-term recurring endogenous metallic taste problem in chemotherapy patients. This is mainly because the potential mechanism by which cisplatin causes the production of endogenous metallic taste in the body is still unclear. Therefore, it is necessary to establish an effective animal model for research. Summary of the invention

[0004] The purpose of the present invention is to solve the problem of lack of research basis for the treatment of endogenous metallic taste produced by patients after cisplatin chemotherapy, and provide a method for constructing a mouse model of cisplatin-induced endogenous metallic taste. The present invention adopts a double-bottle preference experiment as a screening criterion for the first time. After intraperitoneal injection of cisplatin, the double-bottle preference experiment is used for screening again, and a mouse model of cisplatin-induced endogenous metallic taste is successfully constructed, which paves the way for finding a method to improve the endogenous metallic taste produced by patients after cisplatin chemotherapy.

[0005] Technical Solution

[0006] A method for constructing a mouse model of endogenous metallic taste induced by cisplatin comprises the following steps:

[0007] (1) BALB / c female mice (SPF grade) weighing 20-22 g and aged 6 weeks were selected as experimental subjects, and a two-bottle preference test was used to screen mice that showed an aversion to 3.2 mg / L ferrous sulfate solution, a metallic solution;

[0008] (2) dissolving cisplatin in physiological saline to prepare a 3.5 mg / kg cisplatin solution; injecting the cisplatin solution into mice intraperitoneally for three consecutive weeks, twice a week, with an injection dose of 0.1 ml / 10 g;

[0009] (3) After the injection process is completed, a double-bottle preference experiment is used to screen mice that prefer ferrous sulfate solution, a metallic-tasting solution, thereby obtaining a mouse model of cisplatin-induced endogenous metallic taste.

[0010] Furthermore, in step (1) and step (3), the method for screening mice by the double bottle preference experiment is as follows: the mice are separated and raised in cages, water bottles are placed on both sides, and they are fed normally and ultrapure water. After 48 hours of adaptation, water bottles containing ultrapure water and 3.2 mg / L ferrous sulfate solution are placed on both sides of the cage cover, and the ferrous sulfate solution is reconfigured and the consumption is recorded after 24 hours, and the position of the double bottles is changed, and the consumption of the double bottles is recorded again after 24 hours. The calculation formula of the ferrous sulfate solution preference index (PI): preference index = ferrous sulfate solution consumption / total consumption. A preference index greater than 0.5 indicates that the experimental mice are in a state of preference for ferrous sulfate solution; a preference index less than 0.5 indicates that they are in a state of disgust. The larger the absolute value of the difference between the preference index and 0.5, the higher the degree of preference or disgust.

[0011] Furthermore, in step (2), the preparation method of cisplatin solution is as follows: the preparation amount and injection amount of cisplatin solution is 0.1 ml / 10 g based on the mouse body weight, the dose of cisplatin required each time is calculated according to the cisplatin concentration of 3.5 mg / kg, cisplatin powder approximately close to the required dose is weighed into a centrifuge tube, and the required volume of physiological saline calculated based on the weighed cisplatin dose is added to dissolve, and ultrasonicated at 25°C for 30 minutes to fully dissolve, and prepared before use.

[0012] Furthermore, in step (1), the mice were kept in an environment with a temperature of 25±0.5°C and a 12-h light-dark cycle, with free access to water and feed.

[0013] Furthermore, in step (2), the mice are kept in an environment with a temperature of 24±2°C and a humidity of 40%-60%, and are allowed to drink water and eat food freely.

[0014] The beneficial effects of the present invention are as follows: the present invention successfully constructs a mouse model of cisplatin-induced endogenous metallic taste by adopting a double-bottle preference experiment screening plus intraperitoneal injection of cisplatin, thereby making up for the lack of research basis for the treatment of endogenous metallic taste in patients after cisplatin chemotherapy and providing an animal model scheme. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The preference index of FeSO4 solution in the first screening and at the end of the experiment for mice in the control group;

[0016] Figure 2 It is the FeSO4 solution preference index of the model group mice at the first screening and at the end of the experiment;

[0017] Figure 3 The bar graph shows the significance analysis of the five differentially expressed genes selected for validation by real-time fluorescence quantitative PCR analysis. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0019] It is worth noting that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the materials or instruments used is not specified, they are all conventional products that can be purchased.

[0020] In the following embodiments:

[0021] Experimental Animals:

[0022] 6-week-old, BALB / c female mice (SPF grade), weighing 20-22 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., license number SCXK (Beijing) 2024-0001. They were kept in an air-conditioned environment, the temperature was maintained at (25 ± 0.5) ° C, in a 12-h light-dark cycle, with free access to water and feed (normal maintenance feed, Sibeifu (Beijing) Biotechnology Co., Ltd.). All mice were fed adaptively for 7 days before the experiment.

[0023] Experimental drugs:

[0024] Cisplatin was purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., CAS No.: 15663-27-1, purity> 98.0%. FeSO4·7H2O was purchased from Yisheng Food Additive Co., Ltd., production batch number: 2023052401, purity> 98.0%.

[0025] Example 1

[0026] A method for constructing a mouse model of endogenous metallic taste induced by cisplatin comprises the following steps:

[0027] (1) Screening of double-bottle preference experiment: After 7 days of adaptive feeding in the animal breeding room, the mice were separated and raised in cages with water bottles on both sides, and fed with normal food and ultrapure water. After 48 hours of adaptation, water bottles containing ultrapure water and 3.2 mg / L ferrous sulfate solution were placed on both sides of the cage cover. After 24 hours, the ferrous sulfate solution was re-prepared and the consumption was recorded. The position of the double bottles was changed, and the consumption of the double bottles was recorded again after 24 hours. The mice that were disgusted with ferrous sulfate solution were screened out in the first screening.

[0028] The calculation formula of the preference index (PI) of ferrous sulfate solution is: preference index = ferrous sulfate solution consumption / total consumption. A preference index greater than 0.5 indicates that the experimental mice like ferrous sulfate solution; a preference index less than 0.5 indicates that they dislike it. The greater the absolute value of the difference between the preference index and 0.5, the higher the degree of preference or dislike. Under normal circumstances, mice should be disgusted with ferrous sulfate solution, a metallic solution.

[0029] (2) Grouping and administration: The mice that passed the first screening were randomly divided into a control group and a model group. The model group was intraperitoneally injected with cisplatin twice a week for three weeks. Cisplatin was prepared with normal saline to a concentration of 3.5 mg / kg and the injection dose was 0.1 ml / 10 g. The control group was injected with 0.1 ml / 10 g normal saline in the same way. During the experiment, the mice were free to eat and drink.

[0030] (3) After the administration, the mice were housed in a single cage with two bottles. After 48 hours of adaptation, a two-bottle preference experiment was conducted to record the solution consumption and verify the preference of the model group and the control group mice for ferrous sulfate. Under normal circumstances, the preference index of the model group mice for ferrous sulfate solution should be greater than 0.5, and the preference index of the control group mice for ferrous sulfate solution should be less than 0.5.

[0031] All the mice in the groups were killed by cervical dislocation. The intact thymus and spleen of the mice were taken out and washed with saline. The weight was measured after drying with absorbent paper, and the organ index was calculated according to the following formula.

[0032]

[0033] The tongue was cut and placed in a spiral tube. The sample was quickly frozen with liquid nitrogen and stored at -80°C. We screened 5 genes (Tas2r130, Gnat3, TRPM5, CD74, H2-Ab1) for real-time fluorescence quantitative PCR analysis. The primers were synthesized by the Chengdu Branch of Beijing Qingke Biotechnology Co., Ltd., and their sequences are listed in Table 4. The analysis was performed on an analytikjena-qTOWER2.2 fluorescence quantitative PCR instrument (Germany), and each sample was measured 3 times. GAPDH was used as an internal reference, and 2 -ΔΔCt Relative quantification method.

[0034] Table 1 Effect of cisplatin on the preference index of FeSO4 solution in mice ( n=6)

[0035]

[0036] Table 2 Comparison of body weight of mice in the model group and control group at the end of the experiment ( n=6)

[0037]

[0038] Table 3 Effects of cisplatin on organ indexes of mice ( n=6)

[0039]

[0040] Table 4 Primer sequences used for qRT-PCR analysis

[0041]

[0042] Depend on Figure 1 , Figure 2 , Table 1, Table 2, Table 3, Figure 3 It can be seen that after intraperitoneal injection, the behavioral preference index of the model group of the present invention mice for the metallic taste solution was significantly higher than that of the first screening and the preference index was greater than 0.5, while the control group did not change much and the preference index was still less than 0.5, and the body weight and organ index were lower than those of the control group injected with normal saline. The tongue bitter receptors and downstream taste signals as well as immune-related genes in the model group were significantly downregulated.

Claims

1. A method for constructing a mouse model of endogenous metallic taste induced by cisplatin, characterized in that: The steps include: (1) BALB / c female mice weighing 20-22 g and aged 6 weeks were selected as experimental subjects, and mice that showed aversion to 3.2 mg / L ferrous sulfate solution were screened using a two-bottle preference test; (2) dissolving cisplatin in physiological saline to prepare a 3.5 mg / kg cisplatin solution; injecting the cisplatin solution into mice intraperitoneally for three consecutive weeks, twice a week, with an injection dose of 0.1 ml / 10 g; (3) After the injection process is completed, the double-bottle preference experiment is used to screen mice that have a preference for ferrous sulfate solution, thereby obtaining a mouse model of cisplatin-induced endogenous metallic taste.

2. The method for constructing a mouse model of endogenous metallic taste induced by cisplatin according to claim 1, characterized in that: In step (1) and step (3), the method for screening mice by the double bottle preference experiment is as follows: the mice are separated and raised in cages, water bottles are placed on both sides, and they are fed normally and ultrapure water; after 48 hours of adaptation, water bottles containing ultrapure water and 3.2 mg / L ferrous sulfate solution are placed on both sides of the cage cover, and the ferrous sulfate solution is reconfigured and the consumption is recorded after 24 hours, and the positions of the double bottles are changed, and the consumption of the double bottles is recorded again after 24 hours; the ferrous sulfate solution preference index PI is calculated as follows: preference index = ferrous sulfate solution consumption / total consumption; The preference index was greater than 0.5, indicating that the experimental mice preferred the ferrous sulfate solution; If the preference index is less than 0.5, it indicates a state of disgust; the greater the absolute value of the difference between the preference index and 0.5, the higher the degree of preference or disgust.

3. The method for constructing a mouse model of endogenous metallic taste induced by cisplatin according to claim 1, characterized in that: In step (2), the preparation method of cisplatin solution is as follows: the preparation amount and injection amount of cisplatin solution is 0.1 ml / 10 g based on the mouse body weight, and the dose of cisplatin required each time is calculated according to the cisplatin concentration of 3.5 mg / kg. Cisplatin powder is weighed into a centrifuge tube, and the required volume of physiological saline calculated based on the weighed cisplatin dose is added to dissolve it, and ultrasonicated at 25°C for 30 minutes to fully dissolve it, and prepared before use.

4. The method for constructing a mouse model of cisplatin-induced endogenous metallic taste according to claim 1 is characterized in that: In step (1), the mice were kept in an environment with a temperature of 25±0.5°C and a 12-h light-dark cycle, with free access to water and feed.

5. The method for constructing a mouse model of endogenous metallic taste induced by cisplatin according to claim 1 is characterized in that: In step (2), the mice were kept in an environment with a temperature of 24±2°C and a humidity of 40%-60%, and were allowed to drink water and eat freely.

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