Application of Caenorhabditis elegans in constructing animal model for detecting combined toxicity of antibiotics and metal ions

Abstract

The application relates to the field of biotechnology, in particular to the application of Caenorhabditis elegans in constructing an animal model for detecting combined toxicity of antibiotics and metal ions. The application provides the application of Caenorhabditis elegans in constructing an animal model for detecting combined toxicity of antibiotics and metal ions. The application takes Caenorhabditis elegans as a model organism, and provides a simple, fast, low-cost, short-cycle and reproducible method for constructing an animal model for detecting combined toxicity. The application provides technical support for combined toxicity of antibiotics and metal ions in a natural environment and specific action aspects, and has practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of Caenorhabditis elegans in constructing an animal model for detecting the combined toxicity of antibiotics and metal ions. BACKGROUND

[0002] Antibiotics are drugs that can inhibit the growth and activity of fungi, bacteria, pathogens and other microorganisms. Due to its excellent performance, it has been widely used in the treatment and prevention of various infections in humans and in the field of livestock diseases in the livestock industry. After nearly a hundred years of development, scientists have developed tens of thousands of methods for producing and using antibiotics, which have made great contributions to public health safety. Therefore, it is of great practical significance to study the impact of antibiotics on the environment.

[0003] Among them, synthetic antibiotics-fluoroquinolone (FQs) drugs have the advantages of broad-spectrum antibacterial properties, high production efficiency, etc. and have been listed by the World Health Organization as an important antibiotic. It has been proven to have a negative impact on the growth and development of nematodes, cardiovascular health of zebrafish, etc. Ciprofloxacin (CIP) as a third-generation FQs drug is often used to treat skin and respiratory infections, and is a white crystalline powder that is slightly soluble in water and easily soluble in dilute hydrochloric acid. Therefore, it is often prepared into ciprofloxacin hydrochloride. Due to its continuous use and incomplete management standards, CIP has been detected at the level of milligrams per liter in surface water, sediments, especially hospital wastewater. Literature has shown that CIP exhibits varying degrees of toxicity in rice, fruit flies, and duckweed, but there are few reports on its toxicity to soil nematodes.

[0004] At the same time, nickel, as a relatively abundant transition metal element in the earth's crust, is widely present in soil, water and air. Due to its excellent physical and chemical properties, nickel is widely used in modern industrial manufacturing fields, such as refining, welding; especially in recyclable nickel-cadmium battery manufacturing and electroplating industries. However, the mining and smelting of nickel ore, alloy manufacturing and processing, nickel-containing fuel combustion emissions, and the discharge of electroplating nickel-containing wastewater will continuously increase the concentration of environmental nickel, causing serious environmental problems. Although nickel is a trace element necessary for plant growth, excessive nickel in soil will cause metabolic disorders, poor development and inhibit growth in plants. At the same time, nickel can also enter the human body through skin contact and food intake and accumulate in human tissues to produce toxic effects.

[0005] At present, there is no article that uses Caenorhabditis elegans as a research model to evaluate the combined toxicity of nickel ions and ciprofloxacin in terms of growth and reproduction. SUMMARY

[0006] Therefore, the application provides an application of Caenorhabditis elegans in constructing an animal model for detecting combined toxicity of antibiotics and metal ions.

[0007] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] The application provides an application of Caenorhabditis elegans in constructing an animal model for detecting combined toxicity of antibiotics and metal ions.

[0009] In some embodiments of the application, the antibiotics include ciprofloxacin, and the metal ions include nickel ions.

[0010] In some embodiments of the application, the concentration of the antibiotics is 20 μM, and the concentration of the ciprofloxacin is 300-500 μM.

[0011] In some embodiments of the application, the concentration of the antibiotics is 20 μM, and the concentration of the ciprofloxacin is 300 μM or 500 μM.

[0012] The application further provides a construction method of the animal model, comprising the following steps.

[0013] S1: taking wild-type Caenorhabditis elegans for culture, lysing after oogenesis, and obtaining worm eggs;

[0014] S2: taking the worm eggs for culture, and obtaining L1 stage nematodes;

[0015] S3: taking the L1 stage nematodes for exposure to a to-be-tested sample, and obtaining the animal model;

[0016] The to-be-tested sample contains antibiotics and / or metal ions.

[0017] In some embodiments of the application, the antibiotics include ciprofloxacin, and the metal ions include nickel ions.

[0018] In some embodiments of the application, the concentration of the antibiotics is 20 μM, and the concentration of the ciprofloxacin is 300-500 μM.

[0019] In some embodiments of the application, the concentration of the antibiotics is 20 μM, and the concentration of the ciprofloxacin is 300 μM or 500 μM.

[0020] In some embodiments of the present application, in the above construction method, the cleavage employs a cleavage solution; the cleavage solution comprises: a sodium hydroxide solution and a sodium hypochlorite solution; the volume ratio of the sodium hydroxide solution to the sodium hypochlorite solution is 1: (0.1-10).

[0021] In some embodiments of the present application, in the above construction method, the volume ratio of the sodium hydroxide solution to the sodium hypochlorite solution is 1:4.

[0022] In some embodiments of the present application, in the above construction method, the concentration of the sodium hydroxide solution is 0.5-5 mol / L.

[0023] In some embodiments of the present application, in the above construction method, the concentration of the sodium hydroxide solution is 0.5 mol / L.

[0024] In some embodiments of the present application, in the above construction method, the cleavage time is 18-22 h.

[0025] In some embodiments of the present application, in the above construction method, the exposure time is 66-72 h.

[0026] In some embodiments of the present application, in the above construction method, the exposure employs a culture medium; the culture medium comprises: K liquid and Escherichia coli.

[0027] The K liquid comprises: 1-4 g / L of KCl and 1-4 g / L of NaCl.

[0028] In some embodiments of the present application, in the above construction method, the K liquid comprises: 2-3 g / L of KCl aqueous solution and / or 2-3 g / L of NaCl aqueous solution.

[0029] In some embodiments of the present application, in the above construction method, the Escherichia coli is: uracil-deficient Escherichia coli E. coli OP50.

[0030] In some embodiments of the present application, in the above construction method, when the sample to be tested contains antibiotics and metal ions, it is a combined exposure group; when the sample to be tested contains antibiotics or metal ions, it is a single exposure group; when the sample to be tested does not contain antibiotics and / or metal ions, it is an experimental control group.

[0031] In some embodiments of the present application, the above construction method further comprises the step of detecting the growth index, motor nerve index, reproductive capacity index and gonadal development index of the wild-type Caenorhabditis elegans.

[0032] In some embodiments of the present invention, in the above construction method, the growth indicators include: body length and / or food intake rate.

[0033] In some embodiments of the present invention, in the above-mentioned construction method, detecting the body length comprises the following steps: after exposing the L1 stage C. elegans for 72 hours, collecting and washing the nematodes, placing the nematodes in a water bath at 60°C for 10 minutes, removing the nematodes and transferring them to solid culture medium, capturing images of the nematodes using a CCD camera through a stereo microscope, and measuring the body length of the adult nematodes using Image J. Starting from the head of the nematode, a broken line is drawn along the midline of its body to the tail of the nematode, and the total length of the broken line is the body length of the nematode;

[0034] The solid culture medium can be NGM medium, which specifically consists of: 400 mL deionized water, 1.2 g NaCl, 6.8 g agar, 1 g peptone, 10 mL K3PO4 buffer solution (100 mL deionized water, 10.83 g KH2PO4, 4.66 g K2HPO4∙3H2O), 0.4 mL 1 M CaCl, 0.4 mL 1M MgSO4, and 0.4 mL 5 mg / mL cholesterol ethanol solution.

[0035] In some embodiments of the present invention, in the above-mentioned construction method, detecting the feeding rate comprises the following steps: recording the OD6001 of the unexposed control group, setting up an experimental control group, and recording the OD6002 of the experimental control group, the OD6003 of the blank control group, the OD6004 of the single exposure group, and the OD6005 of the combined exposure group after culturing at 20°C for 48 hours, and calculating the feeding rate according to the formula: food clearance rate = (OD6003 or OD6004 or OD6005 - OD6002) ÷ OD6001.

[0036] In some embodiments of the present invention, in the above construction method, the motor nerve index includes: head shaking.

[0037] In some embodiments of the present invention, in the above-mentioned construction method, detecting the head swing comprises the following steps: after the exposure is completed, each group of nematodes is transferred to a 24-well plate containing only 1 mL of K solution; after stable standing for 1 minute, a high-resolution video of each group of nematodes is captured for 30 seconds under a stereo microscope equipped with a CCD camera; the number of nematode head swings in a fixed time period is recorded; a single head swing is defined as the nematode head swinging from the original direction to the other side and then returning to the original direction.

[0038] In some embodiments of the present invention, in the above construction method, the reproductive capacity indicators include: one or more of: number of eggs, egg hatching rate, number of offspring and / or generation time.

[0039] In some embodiments of the present application, in the above method, detecting the number of eggs in the oocytes comprises the following steps: after the L1 stage C. elegans are exposed for 72 hours, part of the C. elegans are collected and washed, the C. elegans are transferred to solid culture medium, the oocyte-carrying C. elegans are lysed using a lysis solution, and the number of eggs in each C. elegans is counted.

[0040] In some embodiments of the present application, in the above method, detecting the egg hatching rate comprises the following steps: after the L1 stage C. elegans are exposed for 72 hours, the C. elegans are collected and washed, the eggs laid by the C. elegans in the first hour are transferred to solid culture medium without food, and then incubated at 20°C for 24 hours. Subsequently, the L1 larvae and any un-hatched eggs remaining on the culture medium are counted, and the hatching rate is determined by calculating the ratio of the number of L1 larvae to the total number of L1 larvae and the number of un-hatched eggs.

[0041] In some embodiments of the present application, in the above method, detecting the number of offspring comprises the following steps: after the L1 stage C. elegans are exposed for 72 hours, the C. elegans are washed with K solution and transferred to solid culture medium, the culture medium is placed with the C. elegans to allow them to naturally produce offspring, the parent C. elegans are transferred to a fresh solid culture plate, adult C. elegans are transferred to a new culture plate every day, the number of larvae hatched on the solid culture medium is counted, and the above operation is repeated until the C. elegans adult no longer lays eggs, and the number of successfully hatched larvae produced by each C. elegans is the number of offspring.

[0042] In some embodiments of the present application, in the above method, detecting the generation time comprises the following steps: after the L1 stage C. elegans are exposed for 72 hours, the time of laying the first egg is recorded and the C. elegans are cultured to adulthood, the time of laying the first egg by the adult C. elegans is recorded, and the difference between the two times is the generation time.

[0043] In some embodiments of the present application, in the above method, the gonad development indicator comprises the number of apoptotic germ cells and / or the number of gonadal cells.

[0044] In some embodiments of the present application, in the above construction method, the detecting the number of germ cell apoptosis comprises the following steps: after exposure, the C. elegans are collected in a centrifuge tube, washed with K liquid 3 times, and then placed in a 24-well plate, 500 μL of K liquid and 5 μL of 50 times E. coli solution are added, and after about 10 h of recovery, 100 μL of fluorescent dye is added to each well for 1 h of dark staining. After staining, the C. elegans are collected and washed twice, and then transferred to an agar plate for 40 min of recovery; a glass slide is taken, 10 μL of fixing solution is dropped in the center, about 30 C. elegans are picked into the levamisole solution drop, and after the C. elegans stop moving, the drop is covered with a cover glass, and the number of cell apoptosis in the unilateral gonadal arm of the C. elegans is observed under a fluorescence microscope; the dye is 150 g / mL acridine orange; and the fixing solution is 60 μg / mL levamisole solution.

[0045] In some embodiments of the present application, in the above construction method, the detecting the number of gonadal cells comprises the following steps: after exposure, the C. elegans are collected in a centrifuge tube, washed with anhydrous ethanol 3 times to kill all cells of the C. elegans; a glass slide is taken, and each group of worm liquid is dropped on the glass slide, and after the ethanol is naturally air-dried, 10 μL of dye is dropped in the center, and after 20 min of dark staining, the number of all cells in the gonadal arm of the C. elegans is observed under a fluorescence microscope; and the dye is preferably DAPI dye produced by Biyun Tian Company.

[0046] In some embodiments of the present application, in the above construction method, the detection standard is:

[0047] When the endpoint indicators of the combined exposure group and the single exposure group have no difference (P>0.05), it is determined that ciprofloxacin has no effect on the toxicity effect of nickel ions;

[0048] When the endpoint indicators of the combined exposure group and the single exposure group have a difference (P<0.05), it is determined that ciprofloxacin has an effect on the toxicity effect of nickel ions.

[0049] The present application also provides an animal model obtained by the above construction method.

[0050] The present application also provides an application of the animal model in detecting the combined toxicity of antibiotics and metal ions.

[0051] In some embodiments of the present application, in the above application, the antibiotics comprise ciprofloxacin; and the metal ions comprise nickel ions.

[0052] The present application provides an application of C. elegans in constructing an animal model for detecting the combined toxicity of antibiotics and metal ions.

[0053] The application provides an application of Caenorhabditis elegans in detection of combined toxicity of an antibiotic ciprofloxacin and a metal nickel ion. BRIEF DESCRIPTION OF DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced.

[0055] Figure 1 A representation of effects of nickel and ciprofloxacin on body length of the nematode in the application is shown in the figure;

[0056] Figure 2 A representation of effects of nickel and ciprofloxacin on feeding rate of the nematode in the application is shown in the figure;

[0057] Figure 3 A representation of effects of nickel and ciprofloxacin on head swing of the nematode in the application is shown in the figure;

[0058] Figure 4 A representation of effects of nickel and ciprofloxacin on the number of eggs carried by the nematode in the application is shown in the figure;

[0059] Figure 5 A representation of effects of nickel and ciprofloxacin on the egg hatching rate of the nematode in the application is shown in the figure;

[0060] Figure 6 A representation of effects of nickel and ciprofloxacin on the number of offspring of the nematode in the application is shown in the figure;

[0061] Figure 7 A representation of effects of nickel and ciprofloxacin on the generation time of the nematode in the application is shown in the figure;

[0062] Figure 8 A representation of effects of nickel and ciprofloxacin on the number of reproductive cell apoptosis of the nematode in the application is shown in the figure;

[0063] Figure 9 A representation of effects of nickel and ciprofloxacin on the number of gonadal cells of the nematode in the application is shown in the figure. DETAILED DESCRIPTION

[0064] The application discloses application of Caenorhabditis elegans in construction of an animal model for detecting combined toxicity of antibiotics and metal ions.

[0065] It should be understood that the expression "one or more of" individually includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0066] The use of the term "include", "have" or "contain", including grammatical equivalents thereof, should generally be understood to be open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0067] It should be understood that the order of steps or order of performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0068] The use of any and all examples, or exemplary language herein, for example, "such as" or "including", is intended merely to better illustrate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0069] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and the numerical values set forth in the detailed description are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Therefore, unless otherwise specified, all ranges disclosed herein are to be understood to be approximations, and the numerical values underlying any such ranges are intended to be precise. Herein, "about" is understood to encompass a range of plus or minus 10% of the indicated value, or plus or minus 5%, or plus or minus 1%, or plus or minus 0.5%.

[0070] The application provides an evaluation method for combined toxicity effect of nickel ions and ciprofloxacin, which exposes the same period of synchronized Caenorhabditis elegans to liquid culture medium containing ciprofloxacin and nickel ions, and evaluates whether the sample has combined toxicity effect according to at least one index of growth indexes of Caenorhabditis elegans, such as body length and feeding rate; motor nerve indexes, such as head swing; reproductive capacity indexes, such as egg number, egg hatching rate, offspring number and generation time; and gonad development indexes, such as number of germ cells and number of gonad cells.

[0071] Preferably, the eggs of the C. elegans in the egg stage are lysed by the lysis solution to obtain sufficient eggs, and the hatched C. elegans in the L1 stage is transferred into the control medium and the experimental medium after being cultured for 18-22 h to perform exposure experiments, and the exposure time is 66-72 h.

[0072] In the present application, the lysis solution comprises sodium hydroxide solution and sodium hypochlorite stock solution in a volume ratio of 1:0.1-10, and the concentration of the sodium hydroxide solution is 0.5-5 mol / L. Preferably, in some specific embodiments, the lysis solution comprises 0.5 mol / L NaOH and NaClO stock solution in a volume ratio of 4:1.

[0073] The control medium in the present application comprises K liquid and E. coli;

[0074] The experimental medium in the present application comprises a single exposure experimental medium and a combined exposure experimental medium.

[0075] The single exposure experimental medium in the present application comprises one of nickel ions and ciprofloxacin, K liquid and E. coli.

[0076] The combined exposure medium in the present application comprises ciprofloxacin and nickel ions, K liquid and E. coli.

[0077] Preferably, the concentration of nickel ions in the single exposure medium is 20 μM, and the concentration of ciprofloxacin is 300 μM.

[0078] The concentration of nickel ions in the combined exposure medium is 20 μM, and the concentration of ciprofloxacin is 10-500 μM.

[0079] The K liquid is 1 g / L-4 g / L KCl and 1 g / L-4 g / L NaCl, and preferably comprises 2-3 g / L KCl aqueous solution and / or 2-3 g / L NaCl aqueous solution.

[0080] The E. coli is uracil-deficient E. coli E. coli OP50.

[0081] Further, in some specific embodiments, the measurement of the body length of the nematode is specifically as follows: after the C. elegans in the L1 stage is exposed for 72 h, the nematodes are collected and washed, the nematodes are placed in a water bath at 60℃ for 10 min, the nematodes are taken out and transferred to a solid culture medium, a CCD camera is used to capture a picture of the nematodes through a stereomicroscope, and Image J is used to measure the body length of the adult nematodes, a broken line is drawn along the body midline of the nematode from the head to the tail, and the total length of the broken line is the body length of the nematode.

[0082] The solid culture medium can be NGM medium, and the specific composition is: 400 mL deionized water, 1.2 g NaCl, 6.8 g agar, 1 g peptone, 10 mL K3PO4 buffer solution (100 mL deionized water, 10.83 g KH2PO4, 4.66 g K2HPO4·3H2O), 0.4 mL 1 M CaCl, 0.4 mL 1 M MgSO4, 0.4 mL 5 mg / mL cholesteryl ethanol solution.

[0083] The acquisition method of the nematode feeding rate is: recording OD6001 of the unexposed control group, setting the experimental control group, and recording OD6002 of the experimental control group, OD6003 of the blank control group, OD6004 of the single exposure group and OD6005 of the combined exposure after 48 h of culture at 20 ℃, and the feeding rate is obtained according to the formula food removal rate = (OD6003 or OD6004 or OD6005- OD6002) ÷ OD6001.

[0084] The determination method of the head swing is: after the exposure is completed, the nematodes in each group are transferred to a 24-well plate containing only 1 mL K solution. After stable standing for 1 minute, high-resolution video of each group of nematodes is captured under a body microscope equipped with a CCD camera for 30 seconds. The number of head swings of the nematodes in a fixed time period is recorded. A single head swing is defined as the nematode head swinging from the original direction to the other side and then returning to the original direction.

[0085] The determination of the number of eggs carried by the nematode is specifically: after L1 stage C. elegans is exposed for 72 h, part of the nematodes are collected and washed, the nematodes are transferred to a solid culture medium, the egg-carrying nematodes are lysed using a lysis solution, and the number of eggs in each nematode is counted;

[0086] The determination of the egg hatching rate is specifically: after L1 stage C. elegans is exposed for 72 h, the nematodes are collected and washed, the eggs laid by the nematodes in the first hour are transferred to a solid culture medium without food, and then incubated at 20 ℃ for 24 hours. Subsequently, the L1 larvae and any un-hatched eggs remaining on the culture medium are counted. The hatching rate is determined by calculating the ratio of the number of L1 larvae to the total number of L1 larvae and the number of un-hatched eggs;

[0087] The number of offspring of C. elegans is specifically measured according to the following method: after L1 stage C. elegans is exposed for 72 h, the C. elegans are washed with K solution and transferred to a solid culture medium, the culture medium is placed with the nematodes to naturally produce offspring, the parent nematodes are transferred to a fresh solid culture plate, the adult nematodes are transferred to a new culture plate every day, the hatched larvae on the solid culture medium are counted, and the above operation is repeated until the adult nematodes no longer lay eggs. The number of successfully hatched larvae produced by each nematode is the number of offspring.

[0088] The nematode generation time is measured according to the following method: after L1 stage C. elegans is exposed for 72 h, the time of laying the first egg is recorded, and the time of laying the first egg of the adult is recorded, and the difference between the two times is the generation time;

[0089] The number of germ cell apoptosis is measured according to the following method: after L1 stage C. elegans is exposed for 66 h, it is transferred to blank medium for recovery, and the number of cell apoptosis in the unilateral gonadal arm of the nematode is observed after recovery. The specific steps are as follows: after the exposed C. elegans is collected in a centrifuge tube, it is washed with K solution for 3 times, then placed in a 24-well plate, 500 μL of K solution and 5 μL of 50 times of E. coli solution are added, and after about 10 h of recovery, 100 μL of fluorescent staining agent is added to each well for 1 h of dark staining. After staining, the nematodes are collected and washed twice, and then transferred to an agar plate for 40 min of recovery. A glass slide is taken, 10 μL of fixing solution is dropped in the center, and about 30 nematodes are picked into the levamisole solution drop. After the nematodes stop moving, the drop is covered with a cover glass, and the number of cell apoptosis in the unilateral gonadal arm of the nematode is observed under a fluorescence microscope; the staining agent is 150 g / mL concentration of acridine orange; and the fixing solution is 60 μg / mL concentration of levamisole solution.

[0090] The number of gonadal cells is obtained according to the following steps: the adult C. elegans is placed under a fluorescence microscope, and the cells in the gonadal arm are counted. The specific steps are as follows: after exposure, the nematodes are collected in a centrifuge tube, washed with K solution, and the supernatant is discarded. Anhydrous ethanol is added for washing three times to kill all cells of the nematodes. A glass slide is taken, and the worm solution of each group is dropped on the glass slide. After the ethanol is naturally dried, 10 μL of staining agent is dropped in the center, and after 20 min of staining in the dark, the number of all cells in the gonadal arm of the nematode is observed under a fluorescence microscope; the staining agent is preferably DAPI staining agent produced by Biyun Tian Company.

[0091] The evaluation indexes of the control group nematodes and the experimental group nematodes are measured respectively, and the combined toxicity effect of the antibiotic and the metal ion is obtained according to the evaluation indexes. The evaluation method of the present application is as follows:

[0092] When there is no difference (P>0.05) in the end point index between the combined exposure group and the single exposure group, it is determined that ciprofloxacin has no effect on the toxicity effect of nickel ions;

[0093] When there is a difference (P<0.05) in the end point index between the combined exposure group and the single exposure group, it is determined that ciprofloxacin has an effect on the toxicity effect of nickel ions.

[0094] In Examples 1 to 10 of the present invention, the wild-type strain of Caenorhabditis elegans was derived from wild-type (Bristol N2) Caenorhabditis elegans donated by the Caenorhabditis Genetics Center (CGC) at the University of Minnesota.

[0095] Nematode growth medium (NGM) consists of: 400 mL deionized water, 1.2 g NaCl, 6.8 g agar, and 1 g peptone. Mix the above ingredients and sterilize them at 121°C for 20 min. Once the temperature drops to approximately 60°C, add 10 mL of K₃PO₄ buffer solution (10.83 g KH₂PO₄, 4.66 g K₂HPO₄∙3H₂O dissolved in 100 mL deionized water and sterilized at 121°C for 20 min) in a clean hood, 0.4 mL of 1 M CaCl₂, 0.4 mL of 1 M MgSO₄, and 0.4 mL of 5 mg / mL cholesterol in ethanol. Mix thoroughly and pour into a culture dish. Allow to air dry in the clean hood before use.

[0096] Nematode lysis buffer includes: 800 mL 0.5 M NaOH and 200 mL NaClO.

[0097] The buffer solution was K solution, which included 1.521 g NaCl and 1.192 g KCl.

[0098] The nickel (Ni) was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0099] Ciprofloxacin (CIP) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0100] All the raw materials and reagents used can be purchased from the market.

[0101] The present invention will be further described below in conjunction with the embodiments:

[0102] Example 1 Cultivation and Exposure of Caenorhabditis elegans

[0103] Wild-type Caenorhabditis elegans (N2) was cultured in Nematode Growth Medium (NGM) containing a lawn of Escherichia coli (E. coli OP50) at 20°C until the worms became fertile. Eggs were then lysed with a nematode lysis buffer to obtain the eggs. The eggs were then incubated in a buffered solution at 20°C for 18–24 hours to obtain L1 worms.

[0104] L1 nematodes were exposed to the control group, nickel group, ciprofloxacin group, combination group 1, combination group 2, and combination group 3, respectively. Escherichia coli was added as food and cultured at 20°C for 72 h. The culture medium for all groups was K solution, the nickel group contained 20 μM nickel ions, the ciprofloxacin group contained 300 μM ciprofloxacin, combination group 1 contained 20 μM nickel ions and 10 μM ciprofloxacin, combination group 2 contained 20 μM nickel ions and 300 μM ciprofloxacin, and combination group 3 contained 20 μM nickel ions and 500 μM ciprofloxacin.

[0105] Example 2 Determination of Caenorhabditis elegans body length

[0106] In this example, nematode body length was determined using the following steps: L1 C. elegans were exposed for 72 hours, then collected and washed. The nematodes were placed in a 60°C waterbath for 10 minutes, removed, and transferred to solid culture medium. Images of the nematodes were captured using a CCD camera under a stereomicroscope. Adult nematode body length was measured using Image J. A broken line was drawn along the midline of the nematode's body, starting from the head and ending at the tail. The total length of the broken lines was the nematode's body length. Thirty nematodes were included in each group.

[0107] The results are as follows Figure 1 As shown, from Figure 1 It can be seen that the body lengths of the combination 2 and combination 3 groups were significantly different from those of the group exposed to nickel ions (20 μM) alone (different letters in the two columns indicate a significant difference between the two test groups, P < 0.05; identical letters indicate no significant difference, P > 0.05). This indicates that combined exposure exhibited a weaker growth-inhibiting effect than nickel exposure alone, and that 300-500 μM ciprofloxacin weakened the toxic effect of 20 μM nickel ions on the shortened body length of nematodes.

[0108] Table 1

[0109]

[0110] Figure 1 The specific data corresponding to each group are shown in Table 1.

[0111] Example 3 Caenorhabditis elegans feeding rate

[0112] In this embodiment, the nematode feeding rate was obtained according to the following steps: the OD6001 of the unexposed control group was recorded, an experimental control group was set up, and after culturing at 20°C for 48 h, the OD6002 of the experimental control group, the OD6003 of the blank control group, the OD6004 of the single exposure group, and the OD6005 of the combined exposure group were recorded. The feeding rate was obtained according to the formula food clearance rate = (OD6003 or OD6004 or OD6005 - OD6002) ÷ OD6001.

[0113] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the body lengths of the combination 1 and combination 3 groups were significantly different from those of the group exposed to nickel ions (20 μM) alone (different letters in the two columns indicate a significant difference between the two test groups, P < 0.05; identical letters indicate no significant difference, P > 0.05). This indicates that combined exposure exhibited a weaker inhibitory effect on feeding rate than nickel exposure alone, and that 10-500 μM ciprofloxacin weakened the toxic effect of 20 μM nickel ions on the reduction of nematode feeding.

[0114] Table 2

[0115]

[0116] Figure 2 The specific data corresponding to each group are shown in Table 2.

[0117] Example 4 Head shaking in Caenorhabditis elegans

[0118] In this example, nematode head oscillations were measured using the following steps: After exposure, each group of nematodes was transferred to a 24-well plate containing only 1 mL of K solution. After stabilization for 1 minute, a 30-second high-resolution video of each group of nematodes was captured under a stereo microscope equipped with a CCD camera. The number of head oscillations during this fixed time period was recorded. A single head oscillation was defined as a nematode head oscillating from one direction to the other and then back to the original direction.

[0119] The results are as follows Figure 3 As shown, from Figure 3 It can be seen that the body lengths of the combination 1 and combination 3 groups were significantly different from those of the group exposed to nickel ions (20 μM) alone (different letters in the two columns indicate a significant difference between the two test groups, P < 0.05; identical letters indicate no significant difference, P > 0.05). The combined exposure demonstrated a stronger inhibitory effect on head shaking than nickel exposure alone, and 10 μM and 500 μM ciprofloxacin enhanced the neurotoxic effects of 20 μM nickel ions on nematode locomotion.

[0120] Table 3

[0121]

[0122] Figure 3 The specific data corresponding to each group are shown in Table 3.

[0123] Example 5 Determination of the number of eggs in Caenorhabditis elegans

[0124] In this example, the number of nematode eggs was determined using the following steps: L1-stage Caenorhabditis elegans were collected, washed, and transferred to a nematode growth medium (NGM) containing a lawn of E. coli (OP50) bacteria to determine the number of eggs. Five to six egg-laden adult worms were selected and placed under a stereomicroscope. Lysing buffer was used, and after several minutes, the mother worms were broken apart, allowing the number of eggs within each worm to be clearly observed. The number of eggs within each worm was observed and recorded. Each group consisted of 20 nematodes.

[0125] The results are as follows Figure 4 As shown, from Figure 4 It can be seen that the number of eggs in the combined two groups was significantly different from that in the group exposed to nickel ions (20 μM) alone (different letters in the two columns indicate a significant difference between the two test groups, P < 0.05; identical letters indicate no significant difference, P > 0.05). This indicates that combined exposure exhibited a weaker growth and development inhibitory effect than nickel exposure alone, and that 300 μM ciprofloxacin weakened the toxic effect of 20 μM nickel ions on the reduction of nematode egg number.

[0126] Table 4

[0127]

[0128] Figure 4 The specific data corresponding to each group are shown in Table 4.

[0129] Example 6 Determination of the hatching rate of Caenorhabditis elegans eggs

[0130] In this example, the hatchability of nematode eggs was determined using the following procedures: After 72 hours of exposure, L1-stage C. elegans were collected and washed. Eggs laid during the first hour were transferred to solid culture medium without food and incubated at 20°C for 24 hours. The number of L1 larvae and any unhatched eggs remaining on the culture medium were then counted. The hatchability was determined by dividing the number of L1 larvae by the total number of L1 larvae and the number of unhatched eggs. Each group consisted of 20 nematodes.

[0131] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that the egg hatching rates in combination groups 1 and 2 did not show significant differences compared to the egg hatching rate in the group exposed to nickel (20 μM) alone (different letters in two columns indicate significant differences between the two test groups, P < 0.05; identical letters indicate no significant differences, P > 0.05). The egg hatching rate in combination group 3 showed a significant difference compared to the egg hatching rate in the group exposed to nickel (20 μM) alone. This indicates that 500 μM ciprofloxacin enhances the toxic effect of 20 μM nickel ions on the reduced nematode egg hatching rate.

[0132] Table 5

[0133]

[0134] Figure 5 The specific data corresponding to each group are shown in Table 5.

[0135] Example 7 Determination of the number of progeny in Caenorhabditis elegans

[0136] In this example, the number of C. elegans progeny was measured using the following method: After 72 hours of exposure, L1 C. elegans were rinsed with K solution and transferred to solid culture medium. The worms were left in the culture medium to produce offspring naturally. The parent worms were then transferred to a fresh solid culture plate. Adult worms were then transferred to a new culture plate daily, and the larvae that hatched from the solid culture medium were counted. This procedure was repeated until the adult worms stopped laying eggs. The number of larvae that successfully hatched from each worm was considered the number of progeny. Each group consisted of 20 worms.

[0137] The results are as follows Figure 6 As shown, from Figure 6 The offspring numbers in Combination 1, Combination 2, and Combination 3 groups were not significantly different from those in the group exposed to nickel (20 μM) alone (different letters in two columns indicate a significant difference between the two test groups, P < 0.05; identical letters indicate no significant difference, P > 0.05). However, all groups showed significant differences from the control group. Ciprofloxacin (10-500 μM) did not significantly alter the toxic effect of 20 μM nickel ions on the reduction in offspring number in nematodes.

[0138] Table 6

[0139]

[0140] Figure 6 The specific data corresponding to each group are shown in Table 6.

[0141] Example 8 Determination of Generation Time of Caenorhabditis elegans

[0142] In this example, generation time was measured using the following method: After 72 hours of exposure, the time of the first egg laid by L1 C. elegans was recorded. The worms were cultured until they became adults, and the time of their first egg laying was recorded. The difference between the two times was calculated as the generation time. Each group consisted of 20 worms.

[0143] The results are as follows Figure 7 As shown, from Figure 7It can be seen that the generation time of the combination 2 group has a significant difference compared with the generation time of the nickel (20 μM) single exposure group (different letters on the two columns represent a significant difference between the two groups, P <0.05; containing the same letter has no significant difference, P >0.05), which determines that 300 μM ciprofloxacin has a weakening effect on the toxicity effect of 20 μM nickel ions on the increase of generation time.

[0144] Table 7

[0145]

[0146] Figure 7 The specific data corresponding to each group is shown in Table 7.

[0147] Example 9 Measurement of the number of reproductive cell apoptosis of C. elegans

[0148] In this embodiment, the number of reproductive cell apoptosis is measured according to the following method: after the L1 stage C. elegans is exposed for 66 h, it is transferred to a blank culture medium for recovery, and the number of cell apoptosis in the unilateral arm of the ovary of the worm is observed after recovery. The specific steps are as follows: after the exposed C. elegans is collected in a centrifuge tube, it is washed with K liquid 3 times, then placed in a 24-well plate, 500 μL of K liquid is added, and 5 μL of 50 times of E. coli solution is added, after about 10 h of recovery, 100 μL of fluorescent dye is added to each well for 1 h of dark staining. After staining, the worms are collected and washed twice, and then transferred to an agar plate for 40 min of recovery. A glass slide is taken, 10 μL of fixing solution is dropped in the center, about 30 worms are picked into the levamisole drop, and after the worms stop moving, the drop is covered with a cover glass, and the number of cell apoptosis in the unilateral arm of the ovary of the worm is observed under a fluorescence microscope; the dye is 150 g / mL concentration of acridine orange; the fixing solution is 60 μg / mL concentration of levamisole solution.

[0149] The results are shown in Table 8. Figure 8 Figure 8 It can be seen that the number of offspring of the combination 1 group and the combination 3 group has a significant difference compared with the number of offspring of the nickel (20 μM) single exposure group (different letters on the two columns represent a significant difference between the two groups, P <0.05; containing the same letter has no significant difference, P >0.05), which determines that 10 μM and 500 μM ciprofloxacin have an enhancing effect on the toxicity effect of 20 μM nickel ions on reproductive cell apoptosis.

[0150] Table 8

[0151]

[0152] Figure 8 The specific data corresponding to each group is shown in Table 8.​

[0153] Example 10 Determination of the total number of gonadal cells in Caenorhabditis elegans

[0154] In this example, the total number of gonadal cells was measured using the following method: adult C. elegans were placed under a fluorescence microscope, and the cells within the gonadal arms were counted. The specific steps were as follows: After exposure, the nematodes were collected in a centrifuge tube, washed with K solution, the supernatant discarded, and then washed three times with anhydrous ethanol to kill all nematode cells. A glass slide was placed, and the worm fluid from each group was dropped onto the slide. After the ethanol dried naturally, 10 μL of stain was dripped into the center of the slide. After staining in the dark for 20 minutes, the total number of cells within the gonadal arms of the nematodes was observed under a fluorescence microscope. The stain was preferably DAPI stain produced by Biyuntian Pharmaceuticals.

[0155] The results are as follows Figure 9 As shown, from Figure 9 It can be seen that there are significant differences in the number of cells between the combination group 2 and the combination group 3 compared with the nickel (20 μM) single exposure group (different letters in the two columns represent significant differences between the two test groups, P<0.05; the same letters represent no significant differences, P>0.05), which indicates that 300 μM ciprofloxacin has a weakening effect on the toxic effect of 20 μM nickel ions on the number of gonadal cells.

[0156] Table 9

[0157]

[0158] Figure 9 The specific data corresponding to each group are shown in Table 9.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Caenorhabditis elegans in constructing an animal model for detecting the combined toxicity of antibiotics and metal ions; The antibiotics include: ciprofloxacin; the metal ions include: nickel ions; The concentration of the nickel ion is 20 μM; the concentration of the ciprofloxacin is 300-500 μM.

2. A method for constructing an animal model, characterized in that: The steps include: S1: Take wild-type Caenorhabditis elegans and culture them. After they are fertilized, they are lysed to obtain eggs. S2: After culturing the eggs, L1 stage nematodes are obtained; S3: exposing the L1 stage nematodes to a test sample to obtain the animal model; The sample to be tested contains: antibiotics and metal ions; The antibiotics include: ciprofloxacin; the metal ions include: nickel ions; The concentration of the nickel ion is 20 μM; the concentration of the ciprofloxacin is 300-500 μM.

3. The construction method according to claim 2, wherein: The lysis is performed using a lysis solution; the lysis solution comprises: sodium hydroxide solution and sodium hypochlorite solution; the volume ratio of the sodium hydroxide solution to the sodium hypochlorite solution is 1:(0.1-10).

4. The construction method according to claim 2 or 3, wherein: Also includes: The step of detecting the growth index, motor nerve index, reproductive ability index and gonad development index of the wild-type Caenorhabditis elegans.

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