阅读说明：本技术 一种利用斑马鱼评价高浓度葡萄糖发育毒性的方法 (Method for evaluating developmental toxicity of high-concentration glucose by using zebra fish ) 是由 金立波 彭仁义 李瑶琦 嵇豪 林苏娥 孙达 于 2021-08-03 设计创作，主要内容包括：本发明公开了一种利用斑马鱼评价高浓度葡萄糖发育毒性的方法,其通过将斑马鱼受精卵置于不同浓度的葡萄糖培养液中进行培育,对不同浓度的葡萄糖对斑马鱼幼鱼脊柱畸形率和死亡率进行统计,并对斑马鱼生长形态进行观察,且结合幼鱼的活性氧产生量及细胞凋亡程度等多方面考察高浓度葡萄糖的发育毒性。本发明提供了一种操作简便、评价周期短,可高效、直观地利用斑马鱼评价高浓度葡萄糖发育毒性的方法,不仅能对高浓度葡萄糖对斑马鱼的发育毒性进行评价,且可为高糖对其他的生物体特别是人的发育毒性的病理机制研究提供参考。(The invention discloses a method for evaluating the developmental toxicity of high-concentration glucose by using zebra fish, which comprises the steps of culturing zebra fish fertilized eggs in glucose culture solutions with different concentrations, counting the spinal deformity rate and the death rate of zebra fish juvenile fish by using glucose with different concentrations, observing the growth form of the zebra fish, and inspecting the developmental toxicity of the high-concentration glucose in combination with the active oxygen generation amount, the apoptosis degree and other aspects of the juvenile fish. The method provided by the invention is simple and convenient to operate, short in evaluation period, and capable of efficiently and intuitively evaluating the developmental toxicity of the high-concentration glucose by using the zebra fish, not only can evaluate the developmental toxicity of the high-concentration glucose on the zebra fish, but also can provide a reference for the research on the pathological mechanism of the developmental toxicity of the high-glucose on other organisms, particularly human beings.)

1. A method for evaluating the developmental toxicity of high-concentration glucose by using zebra fish is characterized by comprising the following steps:

s1, preparing a glucose solution with the mass concentration of 0.5 to 3.5 percent by using the embryo culture solution;

s2: mating and laying eggs by using at least 3 pairs of male and female wild zebra fishes (AB type), collecting and selecting fertilized eggs and uniformly mixing the fertilized eggs together;

s3: respectively putting the glucose solution and the embryo culture solution into 6-hole plates, putting 30 fertilized eggs into each hole, and culturing under normal conditions; the fertilized eggs put into the embryo culture solution are used as a control group, and the fertilized eggs put into the glucose solution are used as an experimental group;

s4: replacing the culture solution every 12 hours in the culture process, continuously replacing the glucose solution in the experimental group, and continuously replacing the embryo culture solution in the control group; feeding the zebra fish juvenile fish feed when the culture solution is changed every day from the fifth day, observing the growth form of the zebra fish, and counting the spinal deformity rate and the death rate of the zebra fish juvenile fish;

s5: staining the zebra fish juvenile fish with the curved spine by using an active oxygen fluorescent probe, washing the stained zebra fish juvenile fish by using PBS buffer solution, and placing the zebra fish juvenile fish on a glass slide to observe the staining condition under a fluorescent microscope; staining zebrafish juvenile fish with curved spine with acridine orange staining solution, and then staining with ddH₂And O, washing the juvenile fish clean, and observing the juvenile fish staining condition under a fluorescence microscope.

2. The method for evaluating the developmental toxicity of high-concentration glucose by using zebrafish as claimed in claim 1, which comprises the following steps:

s1, preparing glucose solutions with mass concentrations of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3% and 3.5% respectively by using the embryo culture solution;

s2: mating and laying eggs by using at least 3 pairs of male and female wild zebra fishes (AB type), collecting and selecting fertilized eggs and uniformly mixing the fertilized eggs together;

s3: respectively putting the prepared glucose solution with 7 concentrations and the embryo culture solution into 6-hole plates, adding 10ml into each hole, putting 30 fertilized eggs into each hole, repeating the steps in parallel by taking each three holes as a group, and culturing under normal conditions; the fertilized eggs put into the embryo culture solution are used as a control group, and the fertilized eggs put into the glucose solution are used as an experimental group;

s4: replacing the culture solution every 12 hours in the culture process, continuously replacing the glucose solution in the experimental group, and continuously replacing the embryo culture solution in the control group; feeding the zebra fish juvenile fish feed when the culture solution is changed every day from the fifth day, observing the growth form of the zebra fish, and counting the spinal deformity rate and the death rate of the zebra fish juvenile fish;

s5: staining the zebra fish juvenile fish with the curved spine by using an active oxygen fluorescent probe, washing the stained zebra fish juvenile fish by using PBS buffer solution, and placing the zebra fish juvenile fish on a glass slide to observe the staining condition under a fluorescent microscope; staining zebrafish juvenile fish with curved spine with acridine orange staining solution, and then staining with ddH₂And O, washing the juvenile fish clean, and observing the juvenile fish staining condition under a fluorescence microscope.

3. The method for evaluating the developmental toxicity of high-concentration glucose by using zebrafish according to claim 1 or 2, wherein the composition of the embryo culture solution is as follows: ddH per liter₂O contains 5g of KCl and 10g of Ca²⁺2.5g of NaHCO₃And 26ml of methyl blue

4. The method for evaluating the developmental toxicity of high-concentration glucose by using zebrafish according to claim 1 or 2, wherein the normal culture conditions of step S3 are as follows: controlling the temperature at 28.5 ℃; the photoperiod is: light/dark 14/10 hours.

5. The method for evaluating the developmental toxicity of high-concentration glucose by using zebrafish according to claim 1 or 2, wherein the step of staining with an active oxygen fluorescent probe in the step S5 comprises the following specific steps: the zebra fish with the curved spine is firstly stained for 30min at 37 ℃ by using a 10 mu M/L active oxygen fluorescent probe, and the stained zebra fish juvenile fish is washed clean by using PBS buffer solution.

6. The method for evaluating the developmental toxicity of high-concentration glucose by using zebrafish according to claim 1 or 2, wherein the step of staining with acridine orange staining solution in the step S5 comprises the following specific steps: zebrafish larvae with a curved spine were stained with acridine orange staining solution at a concentration of 400. mu.g/L at 25 ℃ for 30min and rinsed clean with ddH 2O.

Technical Field

The invention relates to the technical field of toxicological detection, in particular to a method for evaluating development toxicity of high-concentration glucose by using zebra fish.

Background

With the continuous abundance of material life, the incidence of pregnancy complicated with diabetes is obviously increased, which harms the health of pregnant women and fetuses, and the incidence of pregnancy-induced hypertension, premature birth, newborn asphyxia and giant infants is obviously increased due to gestational diabetes. High concentrations of glucose have neurotoxic effects, causing excessive oxygen free radicals and the production of glycosylated end products, which can cause significant damage to maternal health and early fetal development, and even direct development of the larvae or death. At present, the pathological mechanism research of the high-sugar toxic and side effects is mostly researched by adopting young mice, rats, rabbits or in-vitro cultured cells, and although the method is widely used, the method has high cost, long time, complex operation and difficult observation of phenomena.

Zebrafish, a small tropical freshwater fish native to southern asia, has become a widely used vertebrate animal worldwide due to its characteristics of easy feeding, strong fertility, rapid growth, transparent fertilized eggs, easy observation, etc. Zebrafish, as a representative of lower vertebrates, has 87% homology with human genes, is similar to human in morphological structure, physiological function and pathological response in a plurality of important tissues and organs including cardiovascular system and nervous system, and is rapidly and widely applied in the fields of developmental biology, compound toxicity evaluation, human disease model research and the like.

Disclosure of Invention

Therefore, based on the background, the invention provides a method for evaluating the developmental toxicity of high-concentration glucose, which is simple and convenient to operate, short in evaluation period and capable of efficiently and intuitively evaluating the developmental toxicity of the high-concentration glucose by using zebra fish.

The technical scheme provided by the invention is as follows:

a method for evaluating the developmental toxicity of high-concentration glucose by using zebra fish comprises the following steps:

s1, preparing a glucose solution with the mass concentration of 0.5 to 3.5 percent by using the embryo culture solution;

s2: mating and laying eggs by using at least 3 pairs of male and female wild zebra fishes (AB type), collecting and selecting fertilized eggs and uniformly mixing the fertilized eggs together;

s3: respectively putting the glucose solution and the embryo culture solution into 6-hole plates, putting 30 fertilized eggs into each hole, and culturing under normal conditions; the fertilized eggs put into the embryo culture solution are used as a control group, and the fertilized eggs put into the glucose solution are used as an experimental group;

s4: replacing the culture solution every 12 hours in the culture process, continuously replacing the glucose solution in the experimental group, and continuously replacing the embryo culture solution in the control group; feeding the zebra fish juvenile fish feed when the culture solution is changed every day from the fifth day, observing the growth form of the zebra fish, and counting the spinal deformity rate and the death rate of the zebra fish juvenile fish;

s5: staining the zebra fish juvenile fish with the curved spine by using an active oxygen fluorescent probe, washing the stained zebra fish juvenile fish by using PBS buffer solution, and placing the zebra fish juvenile fish on a glass slide to observe the staining condition under a fluorescent microscope; staining zebrafish juvenile fish with curved spine with acridine orange staining solution, and then staining with ddH₂And O, washing the juvenile fish clean, and observing the juvenile fish staining condition under a fluorescence microscope.

Further, it comprises the following steps:

s1, preparing glucose solutions with mass concentrations of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3% and 3.5% respectively by using the embryo culture solution;

s2: mating and laying eggs by using at least 3 pairs of male and female wild zebra fishes (AB type), collecting and selecting fertilized eggs and uniformly mixing the fertilized eggs together;

s3: respectively putting the prepared glucose solution with 7 concentrations and the embryo culture solution into 6-hole plates, adding 10ml into each hole, putting 30 fertilized eggs into each hole, repeating the steps in parallel by taking each three holes as a group, and culturing under normal conditions; the fertilized eggs put into the embryo culture solution are used as a control group, and the fertilized eggs put into the glucose solution are used as an experimental group;

s4: replacing the culture solution every 12 hours in the culture process, continuously replacing the glucose solution in the experimental group, and continuously replacing the embryo culture solution in the control group; feeding the zebra fish juvenile fish feed when the culture solution is changed every day from the fifth day, observing the growth form of the zebra fish, and counting the spinal deformity rate and the death rate of the zebra fish juvenile fish;

s5: staining the zebra fish juvenile fish with the curved spine by using an active oxygen fluorescent probe, washing the stained zebra fish juvenile fish by using PBS buffer solution, and placing the zebra fish juvenile fish on a glass slide to observe the staining condition under a fluorescent microscope; staining zebrafish juvenile fish with curved spine with acridine orange staining solution, and then staining with ddH₂And O, washing the juvenile fish clean, and observing the juvenile fish staining condition under a fluorescence microscope.

Further, the composition of the embryo culture solution is as follows: ddH per liter₂O contains 5g of KCl and 10g of Ca²⁺2.5g of NaHCO₃And 26ml of methyl blue

Further, the normal culture conditions of step S3 are: controlling the temperature at 28.5 ℃; the photoperiod is: light/dark 14/10 hours.

Further, the step S5 of staining with the reactive oxygen species fluorescent probe specifically includes: the zebra fish with the curved spine is firstly stained for 30min at 37 ℃ by using a 10 mu M/L active oxygen fluorescent probe, and the stained zebra fish juvenile fish is washed clean by using PBS buffer solution.

Further, the step S5 of staining with acridine orange staining solution specifically comprises the following steps: zebrafish larvae with a curved spine were stained with acridine orange staining solution at a concentration of 400. mu.g/L at 25 ℃ for 30min and rinsed clean with ddH 2O.

By adopting the technical scheme, the method has the following beneficial effects:

the method utilizes the zebra fish with short growth period to evaluate the developmental toxicity of the high-concentration glucose, can finish evaluation within 10 days, has short period, is simple and convenient to operate, is quick and efficient, and can greatly reduce the cost; and the developmental toxicity of the high-concentration glucose is inspected from multiple aspects such as the teratogenesis rate, the survival rate, the active oxygen generation amount of the juvenile fish, the apoptosis degree and the like, the accuracy is high, and the evaluation result is ensured; the method not only can evaluate the developmental toxicity of high-concentration glucose to the zebra fish, but also can provide reference for the pathological mechanism research of the developmental toxicity of high-concentration glucose to other organisms, particularly human beings.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

Fig. 1 shows a spinal curvature of a zebra fish juvenile fish in an embodiment of the present invention.

FIG. 2 is a graph showing the statistics of spinal deformity and mortality of 8 th day of zebra fish cultured with glucose at different concentrations according to an embodiment of the present invention;

FIG. 3 is a fluorescent picture of zebrafish stained with a ROS fluorescent probe according to an embodiment of the present invention;

FIG. 4 is a fluorescent picture of zebrafish stained with acridine orange according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention is further described below with reference to the accompanying drawings.

Example 1: a method for evaluating the developmental toxicity of high-concentration glucose by using zebra fish comprises the following specific operation steps:

s1, preparing glucose solutions with mass concentrations of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3% and 3.5% respectively by using the embryo culture solution;

s2: mating and laying eggs by using at least 3 pairs of male and female wild zebra fishes (AB type), collecting and selecting fertilized eggs and uniformly mixing the fertilized eggs together;

s3: respectively putting the prepared glucose solution with 7 concentrations and the embryo culture solution into 6-hole plates, adding 10ml into each hole, putting 30 fertilized eggs into each hole, repeating the steps in parallel by taking each three holes as a group, and culturing under normal conditions; the fertilized eggs put into the embryo culture solution are used as a control group, and the fertilized eggs put into the glucose solution are used as an experimental group; the normal culture conditions were: controlling the temperature at 28.5 ℃; the photoperiod is: light/dark 14/10 hours.

S4: replacing the culture solution every 12 hours in the culture process, continuously replacing the glucose solution in the experimental group, and continuously replacing the embryo culture solution in the control group; feeding the zebra fish juvenile fish feed when the culture solution is changed every day from the fifth day, observing the growth form of the zebra fish, and counting the spinal deformity rate and the death rate of the zebra fish juvenile fish; statistics of spine deformity and mortality of zebra fish juvenile fish on day eight are shown in fig. 2. Referring to fig. 1, the spine distortion rate and death rate of zebra fish juvenile fish are remarkably increased with the increase of the sugar concentration of the glucose solution.

S5: staining the zebra fish juvenile fish with the curved spine by using an active oxygen fluorescent probe, washing the stained zebra fish juvenile fish with a PBS buffer solution, and observing the staining condition on a glass slide under a fluorescent microscope (see the attached figure 3); staining zebrafish juvenile fish with curved spine with acridine orange staining solution, and then staining with ddH₂And O, washing the juvenile fish clean, and observing the juvenile fish staining condition under a fluorescence microscope (see attached figure 4).

The specific steps of staining with the active oxygen fluorescent probe in step S5 are as follows: the zebra fish with the curved spine is firstly stained for 30min at 37 ℃ by using a 10 mu M/L active oxygen fluorescent probe, and the stained zebra fish juvenile fish is washed clean by using PBS buffer solution.

The step S5 of dyeing with the acridine orange staining solution comprises the following specific steps: zebrafish larvae with a curved spine were stained with acridine orange staining solution at a concentration of 400. mu.g/L at 25 ℃ for 30min and rinsed clean with ddH 2O.

The treatment group (i.e., experimental group) of fig. 3 is a fluorescent photograph of zebrafish juvenile fish after being stained with the ROS fluorescent probe, which was continuously exposed to a 3% glucose solution for 6 days; the normal group is a fluorescence picture of the zebra fish juvenile fish dyed by the ROS fluorescent probe under the normal growth condition. As can be seen from the fluorescence photographs of the control group and the treated group in FIG. 3, the fluorescence intensity in the photograph of the treated group is significantly stronger than that of the control group, and the stronger the fluorescence intensity, the stronger the brightness, the more ROS are accumulated.

The treatment group (i.e., experimental group) of fig. 4 is a fluorescent photograph of zebrafish larvae after acridine orange staining, which were continuously exposed to a 3% glucose solution for 6 days; the normal group is a fluorescent picture of zebra fish juvenile fish stained with acridine orange under normal growth conditions. The acridine orange can stain early apoptotic cells, the stronger the brightness is, the more serious the apoptosis is, and the stronger the developmental toxicity of high sugar is, and it can be obviously seen from fig. 4 that the area with brightness and the intensity of the zebra fish juvenile fish cultured by the high-concentration glucose solution are both obviously higher than those of the control group, which indicates that the apoptosis degree of the zebra fish juvenile fish cultured by the high-concentration glucose is obviously far higher than that of the control group.

The invention utilizes zebra fish to evaluate the developmental toxicity of high-concentration glucose, in the evaluation process, the spinal deformity of the zebra fish juvenile fish cultured by the high-concentration culture solution is obvious in characteristic, and the spinal deformity is higher along with the higher concentration of the glucose, which shows that the correlation of the glucose concentration on the developmental influence of the zebra fish spinal is very large, the genome sequence of the zebra fish is highly similar to the human genome, and a plurality of important tissue organs including a cardiovascular system and a nervous system are similar to human in morphological structure, physiological function and pathological reaction, so the evaluation result of the invention and the glucose concentration have great reference significance on the developmental toxicity of the human.

The present invention and its embodiments have been described above, and the description is not intended to be limiting, and the drawings are only one embodiment of the present invention, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.