A method for establishing a model of cancer-related fatigue in zebrafish

Abstract

The application relates to the field of drug evaluation, and discloses a method for establishing a zebrafish cancer-related fatigue model to solve the problems of the existing technology, such as difficulty in controlling the dosage of a chemotherapeutic drug, more cumulative toxicity of experimental animals, easy death, long modeling time, small flux and difficult operation, and the method comprises the following steps: selecting normal development zebrafish 4 days after fertilization, placing the zebrafish in a microwell plate, dividing the zebrafish into a normal control group and a model group, injecting the zebrafish in the model group with vinorelbine tartrate injection through intravenous injection, and not treating the normal control group; culturing the normal control group and the model group in a zebrafish culture solution at a constant temperature; comparing the total movement distance of the zebrafish in the control group and the model group; if the total movement distance of the zebrafish in the model group is significantly less than that of the normal control group, then the zebrafish cancer-related fatigue model is successfully established. The model is short in time required for establishment, simple in operation, and can realize efficient and high-flux evaluation of the anticancer-related fatigue effect of a drug.

Description

Technical Field

[0001] This invention relates to the field of drug evaluation, and more particularly to a method for establishing a zebrafish cancer-related fatigue model. Background Technology

[0002] Cancer-related fatigue (CRF) is one of the most common symptoms among cancer patients. Many reports indicate that 30%-60% of cancer patients experience moderate to severe fatigue during treatment, which can even lead to treatment interruption in some cases. CRF is persistent and significantly impacts work, mood, and daily life, resulting in impaired overall quality of life during and after treatment. Symptoms include fatigue, drowsiness, mood swings, weakness, and memory decline, and CRF cannot be relieved by rest alone. While extensive research has been conducted on CRF both domestically and internationally, its physiological and pathological mechanisms and effective treatments remain unclear. Therefore, establishing animal models of CRF is crucial for studying its mechanisms and screening related drugs.

[0003] There is still no standardized method for establishing animal models of cancer-related fatigue. Currently, the most common models are mouse and rat models. For example, Sorensen et al. used 6-week-old male BALB / c mice as subjects, establishing a cancer-related fatigue model by intraperitoneal injection of oxaliplatin. Loman et al. used multiple intraperitoneal injections of paclitaxel in 7-8-week-old female BALB / c mice to establish the model. These mouse models require multiple injections of chemotherapeutic drugs such as antibiotics, antimetabolites, alkaloids, alkylating agents, and cytotoxic agents to complete the modeling process. Because of the small size of mice, it is difficult to control the dosage of chemotherapeutic drugs, which can easily cause excessive cumulative toxicity and lead to death. Furthermore, mouse and rat models have certain limitations in research, such as low throughput, slow speed, and difficult operation. Summary of the Invention

[0004] To overcome the problems of difficulty in controlling chemotherapy drug dosage, high cumulative toxicity and mortality in experimental animals in existing animal models of cancer-related fatigue, as well as long modeling time, low throughput and difficult operation, this invention provides a method for establishing a zebrafish cancer-related fatigue model. This model requires less time to establish, is simple to operate, and can efficiently and with high throughput evaluate the efficacy of drugs in treating cancer-related fatigue.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for establishing a zebrafish cancer-related fatigue model includes the following steps:

[0007] A. Select zebrafish that are developing normally 4 days after fertilization and place them in microplates. Divide the zebrafish into a normal control group and a model group. The zebrafish in the model group were injected intravenously with vinorelbine tartrate injection, while the normal control group was not treated.

[0008] B. The normal control group and the model group were cultured at a constant temperature in zebrafish culture medium;

[0009] C. Comparing the total movement distance of zebrafish in the control group and the model group, the total movement distance of the model group was significantly less than that of the normal control group, thus the zebrafish cancer-induced fatigue syndrome was successfully established.

[0010] This invention uses zebrafish as a model organism and selects vinorelbine tartrate to induce cancer-related fatigue in zebrafish. Vinorelbine tartrate is an anti-tumor drug that can cause neurotoxicity and hematologic toxicity during treatment, leading to fatigue in patients. This invention uses a single injection for modeling, resulting in relatively low and controllable cumulative toxicity in zebrafish. After culture, the total movement distance of the model group zebrafish was significantly reduced compared to the normal control group; a statistically significant reduction (p<0.05) indicates successful model establishment.

[0011] Preferably, in the model group of step A, the injection volume of vinorelbine tartrate injection is 1-20 ng / tail.

[0012] To reduce the difficulty of injection, anesthetic can be dripped into the microplate before injection.

[0013] Preferably, in step B, the zebrafish embryo culture medium comprises 5 mmol / L NaCl, 0.17 mmol / L KCl, 0.4 mmol / L CaCl2, and 0.16 mmol / L MgSO4.

[0014] Preferably, in step B, the normal control group and the model group are cultured at 26-28℃ for 6-44 hours.

[0015] Preferably, in step C, a behavior analyzer is used to determine the total movement distance of the zebrafish.

[0016] Preferably, in step C, the sensitivity Max parameter of the behavior analyzer is set to 28-32.

[0017] Preferably, step C further includes measuring the ATP content of zebrafish in the normal control group and the model group.

[0018] Cancer-related fatigue can also lead to a decrease in ATP levels, therefore, this invention also evaluates the degree of cancer-related fatigue in zebrafish by measuring their ATP levels.

[0019] Application of the zebrafish cancer-related fatigue model in evaluating the efficacy of drugs against cancer-related fatigue.

[0020] Preferably, the application includes the following steps:

[0021] (1) The model group was randomly divided into a model control group and a drug group. The zebrafish culture medium of the normal control group, the model control group and the drug group were cultured at 26-28℃ for 6-44h. The zebrafish culture medium of the drug group also contained the drug to be tested.

[0022] (2) Detect and compare the total movement distance and ATP content of zebrafish in each group.

[0023] When the total exercise distance and ATP content in the model control group were significantly reduced compared to the normal control group (p<0.05), and the total exercise distance and ATP content in the drug group were significantly increased compared to the model control group (p<0.05), it indicates that the drug has an effect on cancer-related fatigue. When the total exercise distance and ATP content in the drug group were not significantly increased compared to the model control group, it indicates that the drug does not have an effect on cancer-related fatigue.

[0024] Therefore, this invention proposes a new method for establishing a cancer-related fatigue model, which is of great significance for the study of the mechanism of cancer-related fatigue and the treatment of cancer-related fatigue. Compared with mammalian models, the model of this invention has the following advantages: 1) Relatively fewer drug-induced side effects – Because mouse and rat models require multiple and prolonged injections of chemotherapy drugs, the panic reaction and cumulative drug side effects caused by multiple injections cannot be ignored; zebrafish models are established with a single injection, and an anesthetic is added to the water before injection, so the injection operation has no significant impact on zebrafish, and the cumulative side effects caused by chemotherapy drugs are relatively small; 2) Short time and high efficiency – This model can be established within 6-24 hours, and the pharmacodynamic evaluation can be completed in only 26-44 hours, with a short experimental cycle; and because zebrafish are small in size, they can be analyzed in a standard 6, 12, 24, 48 or 96-well plate, making zebrafish an ideal model for high-throughput automated in vivo drug sensitization evaluation; 3) Simple operation – The CRF model of mice and rats takes at least 3 weeks to establish, and multiple injections of drugs are required; in this invention, zebrafish only requires one injection, and the model can be successfully established within 6-24 hours after injection. Detailed Implementation

[0025] The present invention will be further described below with reference to specific implementation methods.

[0026] Example 1

[0027] Establishment of a zebrafish model of cancer-related fatigue:

[0028] A. Determination of Injection Volume

[0029] (1) Five pairs of zebrafish parents were mated, and the embryos were hatched according to Westerfield's method. Four days after fertilization, the zebrafish were observed under a dissecting microscope. Normally developed zebrafish were selected and transferred into a 6-well microplate with 30 zebrafish in each well and a capacity of 3 mL per well.

[0030] (2) Zebrafish were divided into a normal control group and a model group. The zebrafish in the model group were injected intravenously with vinorelbine tartrate injection at a dose of 1-20 ng / fish. The normal control group was not treated.

[0031] (3) The normal control group and the model group were cultured in zebrafish culture medium at 28℃ for 24h. The zebrafish were then removed and the total movement distance of the zebrafish was measured using a behavior analyzer (Zebra Lab 3.22.3.31, Viewpoint, France). The results are shown in Table 1.

[0032] Table 1. Total movement distance of zebrafish under different injection volumes

[0033]

[0034] Compared with the normal control group, **p<0.01, ***p<0.001.

[0035] As shown in Table 1, when the injection dose is 1-2 ng / fish, the survival rate of zebrafish is high and the total movement distance is significantly reduced. Therefore, 1-2 ng / fish was selected to construct the model.

[0036] B. Determining the cultivation time

[0037] (1) Five pairs of zebrafish parents were mated, and the embryos were hatched according to Westerfield's method. Four days after fertilization, the zebrafish were observed under a dissecting microscope. Normally developed zebrafish were selected and transferred into a 6-well microplate with 30 zebrafish in each well and a capacity of 3 mL per well.

[0038] (2) Zebrafish were divided into a normal control group and a model group. The zebrafish in the model group were injected intravenously with vinorelbine tartrate injection at a dose of 2 ng / tail. The normal control group was not treated.

[0039] (3) The normal control group and the model group were cultured in zebrafish culture medium at 28℃ for 6-72h. The zebrafish were taken out and the total movement distance of the zebrafish was measured using a behavior analyzer (Zebra Lab 3.22.3.31, Viewpoint, France). The results are shown in Table 2.

[0040] Table 2. Total movement distance of zebrafish at different culture times

[0041] project Incubate for 6 hours 24h incubation 48h of incubation Cultured for 72 hours normal control group 2811±110 2872±126 2890±128 2585±180 Model group 1833±43*** 1813±31*** 1800±41*** 1185±96***

[0042] Compared with the normal control group, ***p<0.001.

[0043] As shown in Table 2, after culturing for 6-72 hours following injection of vinorelbine tartrate, the zebrafish in the model group all exhibited obvious fatigue symptoms, indicating that the model was successfully constructed. To reduce time costs, culturing for 6-24 hours is the optimal time to construct the model.

[0044] Example 2

[0045] An evaluation method for the anti-cancer-related fatigue effect of Bailin capsules includes the following steps:

[0046] (1) Five pairs of zebrafish parents were mated, and the embryos were hatched according to Westerfield's method. Four days after fertilization, the zebrafish were observed under a dissecting microscope. Normally developed zebrafish were selected and transferred into a 6-well microplate with 30 zebrafish in each well and a capacity of 3 mL per well.

[0047] (2) Zebrafish were divided into normal control group, model control group and Bailin capsule group. Zebrafish in the model control group and Bailin capsule group were injected intravenously with vinorelbine tartrate injection at a dose of 2 ng / tail. The normal control group was not treated.

[0048] (3) The normal control group, model control group, and Bailin capsule group were cultured in zebrafish culture medium at 28℃ for 6 hours. 50-150 μg / mL Bailin capsules were added to the zebrafish culture medium of the Bailin capsule group, while the other two groups remained unchanged. The zebrafish were then cultured at 28℃ for another 20 hours. (4) The total movement distance (S) of the zebrafish was measured using a behavior analyzer (Zebra Lab 3.22.3.31, Viewpoint, France). The formula for calculating the efficacy against cancer-related fatigue is as follows:

[0049]

[0050] (5) ATP content was measured using a multi-functional microplate reader according to the instructions of the ATP content assay kit (Promega, USA). The formula for calculating the efficacy against cancer-related fatigue is as follows:

[0051]

[0052] Table 3. Evaluation of the anti-cancer fatigue effect of Bailin capsules

[0053]

[0054] Compared with the normal control group, ***p<0.001; compared with the model control group, ### p<0.001.

[0055] Bailin capsules are a traditional Chinese medicine tonic with the effects of tonifying the lungs and kidneys and replenishing essence and qi. According to literature, it can be used to treat chemotherapy-related fatigue in lung cancer (Zhou Fang, Yang Liu. Observation on the efficacy of Bailin capsules in treating chemotherapy-related fatigue in lung cancer [J]. Chinese Community Doctor: Medical Professional, 2011(19):1.). As shown in Table 3, the total movement distance of zebrafish in the Bailin capsule group increased significantly, and the ATP content in their bodies also increased significantly.

[0056] Example 3

[0057] An evaluation method for the anticancer-related fatigue effect of Zhonghua Dieda Pills is provided. The steps are the same as in Example 2, except that the Bailin Capsule group is replaced with the Zhonghua Dieda Pills group. The test results are shown in Table 4.

[0058] Table 4. Evaluation of the anti-cancer-related fatigue effect of Zhonghua Dieda Pills

[0059]

[0060] Compared with the normal control group, ***p<0.001; compared with the model control group, ### p<0.001.

[0061] Table 4 shows that there was no significant difference in total movement distance and ATP content between the zebrafish in the Zhonghua Dieda Pill group and the model control group. Therefore, Zhonghua Dieda Pill has no effect on cancer-related fatigue. The comprehensive evaluation of the anti-cancer-related fatigue effect of Bailin Capsules indicates that the model of this invention can effectively evaluate the anti-cancer-related fatigue effect of drugs.

Claims

1. The application of a zebrafish cancer-related fatigue model in evaluating the efficacy of drugs against cancer-related fatigue, characterized in that... The method for establishing the zebrafish cancer-related fatigue model includes the following steps: A. Selected zebrafish that were in normal development 4 days after fertilization and placed them in microplates. The zebrafish were divided into a normal control group and a model group. The zebrafish in the model group were injected intravenously with vinorelbine tartrate injection at a dose of 1-2 ng / fish. The normal control group was not treated. B. The normal control group and the model group were cultured at a constant temperature in zebrafish culture medium; C. Comparing the total movement distance of zebrafish in the control group and the model group, the total movement distance of the model group was significantly less than that of the normal control group, thus the zebrafish cancer-induced fatigue syndrome was successfully established; D. After successfully establishing zebrafish cancer-related fatigue syndrome, the model group was randomly divided into a model control group and a drug group. The zebrafish culture medium of the normal control group, the model control group, and the drug group were cultured at 26-28℃ for 6-44 h. The zebrafish culture medium of the drug group also contained the drug to be tested. E. Detect and compare the total movement distance and ATP content of zebrafish in each group.

2. The application according to claim 1, characterized in that, In step A, an anesthetic agent is dripped into the microplate before injection to reduce the difficulty of injection.

3. The application according to claim 1, characterized in that, In step B, the zebrafish culture medium includes 5 mmol / L NaCl, 0.17 mmol / L KCl, 0.4 mmol / L CaCl2, and 0.16 mmol / L MgSO4.

4. The application according to any one of claims 1-3, characterized in that, In step B, the normal control group and the model group were cultured at 26-28℃.

5. The application according to claim 4, characterized in that, In step B, the culture time for the normal control group and the model group is 6-44 h.

6. The application according to claim 1, characterized in that, In step C, a behavior analyzer is used to determine the total distance the zebrafish traveled.

7. The application according to claim 6, characterized in that, In step C, the sensitivity Max parameter of the behavior analyzer is set to 28-32.

8. The application according to claim 1, characterized in that, Step C also includes measuring the ATP content in zebrafish from the normal control group and the model group.

Images