Method for constructing zebra fish 137Cs nuclide biological contamination model

Abstract

The invention provides a construction method of a zebra fish 137Cs nuclide biological contamination model. The construction method comprises the following steps: preparation of mother liquor: adding a certain volume of distilled water into liquid 137Cs radioactive sources with different volumes, and uniformly mixing; preparing a 137Cs nuclide solution, namely adding aerated and disinfected tap water into the prepared mother solution, and uniformly mixing; determining the concentration of a 137Cs radioactive source in the 137Cs nuclide solution by adopting a gamma spectrometer; the method comprises the following steps: selecting zebrafish in a breeding period for domestication, dividing the domesticated zebrafish or zebrafish in an embryonic period into 5 groups, putting the 5 groups of zebrafish into a 137Cs nuclide solution for contamination and observation, and counting the death rate. The method can be applied to aquatic organism radiation biological effect research of nuclide 137Cs, and can be used for evaluating the potential influence of 137Cs contained in ocean or other water bodies on human health.

Description

Technical Field

[0001] This invention relates to the field of radioactive biological model technology, and particularly to a zebrafish. 137 Methods for constructing Cs radionuclide biological exposure models. Background Technology

[0002] Radionuclide contamination has become a global environmental problem, especially around nuclear power plants and in areas affected by nuclear accidents. Nuclear power plant leaks and nuclear wastewater discharges can have serious radiation consequences. The highest concentration of radionuclides released into the sea during a nuclear accident is found in… 137 Cs, 137 Cs, as a product of nuclear fission, has a half-life of approximately 30 years. 137 Cs enters oceans and lakes through various pathways and migrates and spreads in oceans or rivers. It accumulates in fish at high trophic levels through the food chain, and once it is caught in fisheries and enters the market, it will ultimately endanger human health.

[0003] Zebrafish, one of the 12 reference organisms proposed by the ICRP (Integrated Chemical Reproductive Technology and Research Program), is an ideal model organism for studying the biological effects of ionizing radiation. Its advantages include ease of experimental culture, short lifespan, high reproductive capacity, sensitivity to environmental stresses, near-transparent embryos that can be observed using a stereomicroscope, meeting the visualization needs of developmental processes, small size, limited range of movement, convenient in vitro embryonic development, and relatively clear characteristics of different phases of embryonic development. Existing techniques have largely focused on the radiobiological effects of acute exposure and high-dose irradiation on living organisms, while models for low- and moderate-dose exposures are less common. Regarding... 137 Radiation biological exposure models of cesium (Cs) affecting zebrafish at various life stages remain limited, especially regarding exposure methods. External irradiation is the primary method, and studies on the radiation biological effects of live zebrafish cultured in cesium-containing solutions are even scarcer. Current model construction lacks radiation research models specifically for breeding zebrafish, and even more so, basic data on zebrafish at various age stages. Establishing a zebrafish radiation biological exposure model is crucial. 137 The Cs radionuclide biological exposure model and experimental research on the radiation biological effects of aquatic organisms are of great significance for formulating my country's standards for evaluating the radiation impact on non-human species, as well as for the construction, operation, environmental safety supervision of nuclear facilities, and public opinion response.

[0004] The above problems urgently need to be addressed. Summary of the Invention

[0005] This invention discloses a zebrafish 137 The method for constructing a Cs radionuclide biological exposure model aims to address the challenges of radionuclide biotoxicity in existing technologies. 137Technical issues of zebrafish biological models at different life stages (Cs).

[0006] The present invention adopts the following technical solution: This invention provides a zebrafish 137 A method for constructing a Cs radionuclide biological exposure model, comprising the following steps: Preparation of the stock solution: mixing different volumes of liquid... 137 A certain volume of distilled water was added to the Cs radioactive source and mixed well. 137 Preparation of Cs radionuclide solution: Add aerated and disinfected tap water to the prepared stock solution and mix well; Gamma spectrometer was used to measure 137 Cs nuclide solution 137 Concentration of Cs radioactive source; Select zebrafish in their breeding season for domestication. Divide the zebrafish that have completed domestication or are in their embryonic stage into 5 groups and release them into the [unclear - likely referring to a specific location or process]. 137 The individuals were exposed to Cs radionuclide solution, observed, and the mortality rate was recorded.

[0007] In one possible implementation, the liquid 137 The activity of the Cs radioactive source is 1.213 × 10⁻⁶. 6 Bq / g.

[0008] In one possible implementation, the aforementioned 137 Cs nuclide solution 137 The concentration of the Cs radioactive source is 1.5 × 10⁻⁶. 2 1.6×10 3 7.5×10 3 1.6×10 4 Bq / L.

[0009] In one possible implementation, the stock solution is prepared by using a pipette to aspirate 0.16, 1.54, 7.69, and 15.38 mL of liquid, respectively. 137 Pour the Cs radioactive source into the volumetric flask, and draw distilled water to bring the volumetric flask to a final volume of 50 ml.

[0010] In one possible implementation, the domestication process of the zebrafish is as follows: in fresh water at 28±1℃, under a 14h light-10h dark cycle, fresh fish food is fed twice a day for at least 7 days.

[0011] In one possible implementation, the observation period is 0 days, 7 days, 14 days, 21 days, and 28 days.

[0012] In one possible implementation, the mortality rate calculation formula is: Mortality rate = (Number of dead fish at this time point ÷ Total number of fish in the group) × 100%.

[0013] The technical solution adopted in this invention can achieve the following beneficial effects: This invention mainly provides a zebrafish 137 A method for constructing a Cs radionuclide biological exposure model, comprising the following steps: Preparation of the stock solution: mixing different volumes of liquid... 137 A certain volume of distilled water was added to the Cs radioactive source and mixed well. 137 Preparation of Cs radionuclide solution: Add aerated and disinfected tap water to the prepared stock solution and mix well; determine the concentration using a gamma spectrometer. 137 Cs nuclide solution 137 The concentration of the Cs radioactive source; select zebrafish in their breeding period for domestication, and divide the domesticated zebrafish or zebrafish in their embryonic stage into 5 groups, and put them into 137 This invention involves exposure to Cs radionuclide solutions, observation, and mortality rate analysis. It utilizes radionuclide biocontamination of zebrafish during their breeding and embryonic stages, filling a gap in current radionuclide-based fish exposure models that only use embryos and toxicological fish. This method can be used to assess the presence of Cs radionuclides in marine or other water bodies. 137 The potential impact of Cs on human health. Attached Figure Description

[0014] Figure 1 This is an image of a zebrafish during its breeding period, provided in Embodiment 1 of the present invention. Figure 2 This is a diagram illustrating the breeding process of zebrafish provided in Embodiment 2 of the present invention; Figure 3 This is a diagram illustrating the zebrafish embryonic development process provided in Embodiment 2 of the present invention; Figure 4 This is a diagram of a zebrafish deformity provided in Embodiment 2 of the present invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0016] Example 1 1. Preparation of radionuclide solutions 1) Material preparation 137 Cs source solution: Activity 1.213×10 6Bq / g, 0.805 g. Two beakers (500ml each, lined with cotton to prevent spillage of the source solution), one volumetric flask (50ml), two beakers (100ml each), two pipettes (5ml each), several pipette tips, and a certain amount of tap water after aeration and sterilization.

[0017] 2) The concentration gradient is 1×10 2 1×10 3 5×10 3 1×10 4 Bq / L formulation (1) Prepare four 50 ml beakers and fill them with 20 mL of distilled water. Place the dry 50 ml volumetric flasks inside the 500 ml volumetric beakers with cotton padding at the bottom. (2) According to the calculation, the source solutions to be added in sequence for the above four concentrations are 0.16, 1.54, 7.69 and 15.38 mL. Use a pipette to pick up the source solutions in sequence and transfer them to a 50 mL volumetric flask. (3) Draw 50 ml of distilled water into the beaker to rinse the syringe 3 times. Pour the rinsing solution into the 50 ml volumetric flask. Finally, make up the volumetric flask to 50 ml and shake well before use. (4) According to the concentration to be prepared, use a pipette to draw the source solution according to step (2) and add it to the prepared tap water, and mix well; (5) The concentration of the solution was determined using a gamma spectrometer. The concentration of the sample after measurement was 1.5 × 10⁻⁶. 2 1.6×10 3 7.5×10 3 1.6×10 4 Bq / L.

[0018] 2. Zebrafish farming Purchase 4-6 month old zebrafish from the China Zebrafish Resource Center. At this age, zebrafish are in their breeding period and can lay eggs. Then, acclimate them for 7 days in fresh water at 28±1℃ with a 14-hour light-10-hour dark cycle. You can gradually increase or decrease the light intensity to simulate sunrise and sunset. Feed them fresh fish food twice a day.

[0019] 3. Validation of biological exposure models Figure 1 This is an image of a zebrafish during its breeding period, provided in Embodiment 1 of the present invention. Figure 1 The left side shows male zebrafish, and the right side shows female zebrafish. During the breeding season, zebrafish were divided into five groups: a control group of 40 zebrafish (20 males and 20 females), a dosage group of 42 zebrafish (21 males and 21 females), and a daily culture medium containing [missing information - likely a specific ingredient or substance]. 137 Cs concentrations were 0 Bq / L (control group) and 1.5 × 10⁻⁶ Bq / L, respectively. 2 Bq / L, 1.6×103 Bq / L, 7.5×10 3 Bq / L, 1.6×10 4 Bq / L. As shown in Tables 1 and 2, after the start of the poisoning, except for the control group, zebrafish began to die. The observation period was 28 days, and the number of deaths was counted weekly. The mortality rate of each group was calculated according to the formula: (number of dead fish at this time point ÷ total number of fish in the group) × 100%.

[0020] Table 1. Number of zebrafish infected with the virus and dying at different times during the breeding season.

[0021] Table 2. Mortality rate of zebrafish exposed to the virus at different times during the breeding season.

[0022] As shown in Tables 1 and 2, at the same time point, the mortality rate of zebrafish gradually increased with the increase of radionuclide concentration. Within the same concentration group, the mortality rate increased significantly starting from the third week, indicating that the radionuclide concentration was significantly higher than that of the zebrafish. 137 Exposure time and concentration of Cs are important indicators affecting the biological effects of radiation.

[0023] Example 2 1. Preparation of radionuclide solutions 1) Material preparation 137 Cs source solution: Activity 1.213×10 6 Bq / g, 0.805 g. Two beakers (500ml each, lined with cotton to prevent spillage of the source solution), one volumetric flask (50ml), two beakers (100ml each), two pipettes (5ml each), several pipette tips, and a certain amount of tap water after aeration and sterilization.

[0024] 2) The concentration gradient is 1×10 2 1×10 3 5×10 3 1×10 4 Bq / L formulation (1) Prepare four 50 ml beakers and fill them with 20 mL of distilled water. Place the dry 50 ml volumetric flasks inside the 500 ml volumetric beakers with cotton padding at the bottom. (2) According to the calculation, the source solutions to be added in sequence for the above four concentrations are 0.16, 1.54, 7.69 and 15.38 mL. Use a pipette to pick up the source solutions in sequence and transfer them to a 50 mL volumetric flask. (3) Draw 50 ml of distilled water into the beaker to rinse the syringe 3 times. Pour the rinsing solution into the 50 ml volumetric flask. Finally, make up the volumetric flask to 50 ml and shake well before use. (4) According to the concentration to be prepared, use a pipette to draw the source solution according to step (2) and add it to the prepared tap water, and mix well; (5) The concentration of the solution was determined using a gamma spectrometer. The concentration of the sample after measurement was 1.5 × 10⁻⁶. 2 1.6×10 3 7.5×10 3 1.6×10 4 Bq / L.

[0025] 2. Zebrafish farming Purchase 4-6 month old zebrafish from the China Zebrafish Resource Center. At this age, zebrafish are in their breeding period and can lay eggs. Then, acclimate them for 7 days in fresh water at 28±1℃ with a 14-hour light-10-hour dark cycle. You can gradually increase and decrease the light intensity to simulate sunrise and sunset. Feed them fresh fish food twice a day to help them adapt to the current environment.

[0026] Figure 2 This is a diagram illustrating the breeding process of zebrafish provided in Embodiment 2 of the present invention; Figure 3 This is a diagram illustrating the zebrafish embryonic development process provided in Embodiment 2 of the present invention; as shown. Figure 2-3 As shown, zebrafish reach sexual maturity at 4 months of age and are ready for spawning experiments. Fill the spawning tank with aerated water, and separate the tank into two compartments using a partition. Place female and male zebrafish in one compartment, with a female-to-male ratio of 2:1. Select 3 males and 6 females for breeding, and then cover the spawning tank with the lid. The next morning at 8:00 AM, turn on the lights, feed the fish, and remove the partition. You will see the male and female fish chasing and bumping into each other, with the females laying eggs and the males releasing sperm, forming fertilized eggs. After breeding in a quiet environment for half an hour, a large number of fertilized eggs will be visible at the bottom of the spawning tank. Carefully remove the fertilized eggs. After breeding for 2 hours, separate the males and females and place them in the zebrafish breeding system. After the breeding tank has settled for a few minutes, slowly pour out the water and collect the fertilized eggs through a filter. Place the collected fertilized eggs in a glass dish. At this point, the fertilized eggs will be transparent; the opaque white areas are unfertilized eggs or dead eggs. Use a pipette to remove unfertilized egg cells, necrotic fertilized eggs (egg agglutination), feces, and other impurities. Then wash the mixture three times with culture medium before using it for subsequent experiments.

[0027] 3. Validation of biological exposure models The collected fertilized eggs were placed in 6-well plates, and five groups were set up with different dosage groups, each containing 100 fertilized eggs. The daily culture medium for exposure to the toxin contained... 137 Cs concentrations were 0 Bq / L (control group) and 1.5 × 10⁻⁶ Bq / L (control group).2 Bq / L, 1.6×10 3 Bq / L, 7.5×10 3 Bq / L, 1.6×10 4 Bq / L. Since embryonic development is completed within 72 hours after fertilization, the hatching rate is calculated for 72 hours. Zebrafish hatching was observed, and the hatching rate was calculated as the percentage of hatched embryos among the live embryos in each group. 100 embryos were used in each group. Hatching rate = (Total number of zebrafish embryos successfully hatched / Total number of zebrafish embryos) × 100%. Table 3 shows the hatching rate of zebrafish during the embryonic stage. ** in Table 3 represents the hatching rate compared to the control group. p value( p -value) < 0.01.

[0028] The deformity rate is calculated as the percentage of deformed embryos out of the viable embryos in each group. Deformities are observed under a stereomicroscope. Figure 4 The image shows a deformed zebrafish as provided in Embodiment 2 of the present invention. Figure 4 As shown, the main deformities in zebrafish include yolk sac cysts, scoliosis, microphthalmia, microcephaly, pericardial edema, tail developmental abnormalities, and developmental delays. This example observed the deformity rate over 72 hours, using 100 embryos per group. The deformity rate is calculated as: (Number of zebrafish embryos with deformities / Total number of zebrafish embryos) × 100%. Table 4 shows the deformity rate of zebrafish during the embryonic period. ** in Table 4 represents the deformity rate compared to the control group. p value( p -value) < 0.01.

[0029] Since the development of juvenile zebrafish is completed within 120 hours after fertilization, the mortality rate is calculated at this 120-hour mark. Embryonic death is defined as the disappearance of heartbeat and egg condensation (the absence of heartbeat and blood flow is used as the criterion for death, and detachment from the chorion is used as the hatching criterion). The mortality rate for each group is calculated as the percentage of dead embryos out of the total number of embryos, with 100 embryos used in each group. Mortality rate = (Number of dead zebrafish embryos / Total number of zebrafish embryos) × 100%. Table 5 shows the mortality rate of zebrafish during the embryonic period; ** in Table 5 represent the mortality rate compared to the control group. p value( p -value) < 0.01.

[0030] Table 3 Hatching rate of zebrafish during the embryonic stage

[0031] Table 4. Deformity rate of zebrafish during the embryonic period

[0032] Table 5. Mortality rate of zebrafish during the embryonic period

[0033] Figure 2 This describes the entire process of a fertilized egg developing into a juvenile fish. Unfertilized eggs and embryos exhibiting egg agglutination were removed, and healthy embryos were selected for subsequent experiments. This was done to test... 137 The effects of Cs solution on early embryonic development in zebrafish were observed at different active concentrations. 137 Hatching rate, malformation rate, and mortality rate of zebrafish embryos after Cs treatment. Results are as follows: Figure 3 Figure 4 As shown in Table 3, the hatching rate of zebrafish increases with... 137 The concentration of Cs decreased in a dose-dependent manner with increasing concentration, which indicates that... 137 Cs can inhibit the development of zebrafish embryos. Table 4 shows that observations of zebrafish malformations revealed that… 137 Following Cs treatment, the rate of developmental abnormalities in zebrafish increased. The incidence of abnormalities increased with increasing concentration, primarily manifesting as scoliosis, tail curvature, and yolk sac enlargement. Table 5 shows that concentrations greater than or equal to 1.6 × 10⁻⁶... 3 of 137 Cs solution can induce the death of zebrafish juveniles, and the mortality rate increases with increasing solution concentration.

[0034] This invention uses 137 Cs source solution to be configured with concentration gradient 137 Cs nuclide solution; according to the intended concentration, in 137 Tap water was added to the Cs source solution and mixed well. The concentration of the solution was then measured using a gamma spectrometer to obtain the current concentration. 137 Cs radionuclide solution; prepare zebrafish culture medium, select 4-6 month old zebrafish and acclimate them for 7 days; divide the acclimatized zebrafish into 5 groups and place them in the current concentration of Cs radionuclide solution. 137 This invention involves exposure to Cs radionuclide solutions, observation, and mortality rate statistics. In this study, zebrafish in their breeding period were subjected to radionuclide biocontamination. As the concentration of radionuclide increased, the mortality rate of the zebrafish gradually rose. With increasing exposure time, the mortality rate increased dramatically, indicating that the exposure time of radionuclides is one of the important indicators affecting the biological effects of radiation. This invention fills a gap in current radionuclide exposure models for fish and for zebrafish. Most exposure methods only treat embryos or fish used for toxicology, without directly adding radionuclides to the culture medium. The zebrafish used are mostly toxicology fish (non-spawning, non-breeding zebrafish). This invention can assess the presence of radionuclides in marine or other water bodies. 137 The potential impact of Cs on human health.

[0035] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A type of zebrafish 137 The method for constructing a Cs radionuclide biological exposure model is characterized by, Includes the following steps: Preparation of mother liquor: Mixing different volumes of liquid... 137 A certain volume of distilled water was added to the Cs radioactive source and mixed well. 137 Preparation of Cs radionuclide solution: Add aerated and disinfected tap water to the prepared stock solution and mix well; Gamma spectrometer was used to measure 137 Cs nuclide solution 137 Concentration of Cs radioactive source; Select zebrafish in their breeding season for domestication. Divide the zebrafish that have completed domestication or are in their embryonic stage into 5 groups and release them into the [unclear - likely referring to a specific location or process]. 137 The individuals were exposed to Cs radionuclide solution, observed, and the mortality rate was recorded.

2. The construction method according to claim 1, characterized in that, The liquid 137 The activity of the Cs radioactive source is 1.213 × 10⁻⁶. 6 Bq / g.

3. The construction method according to claim 1, characterized in that, The 137 Cs nuclide solution 137 The concentration of the Cs radioactive source is 1.5 × 10⁻⁶. 2 1.6×10 3 7.5×10 3 1.6×10 4 Bq / L.

4. The construction method according to claim 3, characterized in that, The preparation method of the stock solution is as follows: use a pipette to pipette 0.16, 1.54, 7.69, and 15.38 mL of the liquid, respectively. 137 Pour the Cs radioactive source into the volumetric flask, and draw distilled water to bring the volumetric flask to a final volume of 50 ml.

5. The construction method according to claim 1, characterized in that, The ultraviolet disinfection time is 3 hours.

6. The construction method according to claim 5, characterized in that, The domestication process of the zebrafish is as follows: in fresh water at 28±1℃, under a 14h light-10h dark cycle, fresh fish food is fed twice a day for at least 7 days.

7. The construction method according to claim 1, characterized in that, The observation periods were 0 days, 7 days, 14 days, 21 days, and 28 days.

8. The construction method according to claim 7, characterized in that, The mortality rate calculation formula is: Mortality rate = (Number of dead fish at this time point ÷ Total number of fish in this group) × 100%.

Images