A method for constructing a zebrafish model for testing glycemic index and application thereof

Abstract

The application provides a method for constructing a zebrafish model for testing a glycemic index, and the steps include breeding zebrafish, collecting zebrafish eggs, culturing the zebrafish and model construction experiments. The application selects zebrafish for model construction, and the zebrafish has high fecundity, short testing time, high throughput, and can test a plurality of samples at the same time; and the culture conditions are simple and the cost is low. The model construction cycle of the zebrafish model is short, 6-day-old zebrafish are selected for testing, and the model construction and the testing of the glycemic index can be completed in 1 day, the glycemic sensitivity is high, the measured blood glucose index is stable, the testing result and the known sample glycemic index have good positive correlation, and the obtained zebrafish glycemic test data can ideally reflect the sample glycemic level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological model construction, and in particular to a construction method of a zebrafish model for testing glycemic index and application thereof. BACKGROUND

[0002] For a long time, zebrafish has been one of the preferred species for developmental biology research. Recently, the use of zebrafish has grown exponentially in neurobehavioral research. Due to its small size (4 cm long in adult stage), high fecundity (200 eggs per female), prolificacy, low cost, easy maintenance, rapid development, transparent embryo, and other advantages, it has become an important model organism, and the completed zebrafish genome sequencing results show that the homology of zebrafish and human genes is as high as 87%.

[0003] The zebrafish model for human disease is a body research, which can serve as a huge biological fault between non-vertebrate animals (cells, etc.) and mammalian (mice, etc.) experiments, and perfect the existing research and development system. So far, by taking zebrafish as the object, biological models of diabetes, hyperlipidemia, hyperuricemia, insomnia, etc. can be successfully constructed, and targeted drug screening and drug safety and toxicology evaluation research can be effectively carried out.

[0004] In order to facilitate people to judge the speed of postprandial blood glucose rise caused by different foods, in 1981, Dr. Jenkins, a Canadian scholar, first proposed the concept of glycemic index (glycemic index, GI for short). Specifically, it refers to the relative speed of blood glucose rise within 2 hours after the intake of carbohydrate-rich food into the human body. Generally divided into low, medium and high three levels, the food with GI value lower than 55 can be called "low GI food". After eating, it can increase satiety and delay the occurrence time of hunger, avoid obesity, diabetes, etc. such as oil and fat food, high-protein food; while high GI food is on the contrary, such as sugar and starch food.

[0005] GI is the glycemic index of food, which is the ratio of the area under the blood glucose response curve of the test food containing 50g of carbohydrate to the area under the blood glucose response curve of the standard reference material (glucose or white bread) with the same amount of carbohydrate.

[0006] Therefore, establishing a good model for testing glycemic index is conducive to better testing and monitoring the glycemic index of food, facilitating people to judge the speed of postprandial blood glucose rise caused by different foods, and facilitating the selection of food by diabetic patients or other people who have requirements for sugar intake. SUMMARY

[0007] In view of the above problems, the present application provides a construction method of a zebrafish model for testing glycemic index, which specifically comprises the following steps:

[0008] Step S1: breeding of zebrafish: healthy zebrafish parents are selected, and the breeding is carried out at a temperature of 25-28.5℃ to obtain zebrafish for breeding and spawning;

[0009] Step S2: zebrafish egg collection: male and female fish are placed in a spawning tank in a certain proportion, and the zebrafish are allowed to mate and spawn, and the zebrafish eggs are collected and cleaned with fish embryo culture solution;

[0010] Step S3: zebrafish culture: healthy zebrafish embryos are selected from the collected zebrafish eggs, and the zebrafish are cultured at a temperature of 25-28.5℃ for not less than 5 days to obtain zebrafish for testing the glycemic index;

[0011] Step S4: model construction experiment: test samples are set, and the zebrafish exposure experiment is carried out using the test samples, the free sugar level in the zebrafish is tested, and the free sugar level in the zebrafish is positively correlated with the glycemic index of the test substance, so that the success of the zebrafish model for testing the glycemic index is determined.

[0012] The parent fish selected in the present application has no obvious infection and disease characteristics, and has not undergone drug treatment within 2 months.

[0013] Preferably, the breeding density of zebrafish in step S1 is controlled to be not less than 1 fish per liter of breeding solution, and the breeding is carried out with 12-16 hours of light per day, and a filtration system is configured.

[0014] More preferably, the breeding density is 1-3 fish per liter of breeding solution.

[0015] Further preferably, the zebrafish are fed at least twice a day during breeding, including feeding at least once with brine shrimp.

[0016] Preferably, the breeding solution is replaced daily during the breeding of zebrafish.

[0017] Further preferably, the indoor temperature is controlled to be 20-25℃ during the breeding of zebrafish.

[0018] Preferably, the salinity of the breeding solution is 0.25-0.50‰, the saturation of dissolved oxygen is ≥80%, and the pH value is 6.5-8.5.

[0019] Further preferably, the salinity of the breeding solution is 0.25-0.50‰, the saturation of dissolved oxygen is ≥80%, the pH value is 6.5-8.5, the conductivity is 400-800µS / cm, and the hardness is 30-300mg / L CaCO3.

[0020] The specific raw material components of the breeding liquid in the present application are not limited. In one embodiment, the raw material components of the breeding liquid for breeding the parent fish in step S1 include deionized water and artificial sea salt. More preferably, the concentration of the artificial sea salt in the breeding liquid is 350-450 mg / L.

[0021] In the present application, when the zebrafish is bred and spawned, the zebrafish is passed as much as possible by side crossing to maintain genetic diversity, and after 5 generations of breeding of the pure strain parent fish, a new batch of parent fish needs to be introduced for population rejuvenation.

[0022] The zebrafish in step S2 can be spawned using a small spawning tank or a large spawning tank. If a small spawning tank is used for spawning, the zebrafish is put into the spawning tank 1-2 hours before the light is turned off the day before spawning, and to avoid genetic bias, the fish eggs collected in at least 3 spawning tanks are mixed and then selected for standby. If a large tank is used to collect fish eggs, the egg collection box is put into the fish tank where the fish eggs are to be collected the day before spawning or before the light is turned on the day of spawning; to avoid the fish eggs being eaten by adult fish, the egg collection box is covered with an inert mesh. Artificial green plants made of plastic or glass can be fixed on the mesh screen as needed to stimulate spawning. Mating, spawning and fertilization are completed within about 30 minutes after the light is turned on, at which time the egg collection box can be removed from the fish tank, and after the fish eggs are taken out of the egg collection box, the fish embryos are cleaned with fish embryo culture solution.

[0023] Preferably, the zebrafish embryos in step S3 are cultured at a density of not more than 1 embryo per 200 μL of fish embryo culture solution at 28°C, and the culture solution is replaced and unhealthy zebrafish embryos are removed every day.

[0024] Under the condition of 28°C, the fertilized fish embryos complete the first division in about half an hour, and then divide into 4, 8, 16, 32… cells, at which time the fertilized fish embryos are clearly distinguishable.

[0025] Preferably, in step S3, the zebrafish embryos are cultured at 25°C-28.5°C for 6 days to obtain zebrafish for testing the glycemic index.

[0026] The 6-day-old zebrafish in the present application is more sensitive than other age groups. It can test samples that do not need to be digested, such as sugar, and is also suitable for testing food that needs to be digested, such as rice and flour. If a younger zebrafish, such as a 4-day-old zebrafish, is used, the test results are also stable, but the glycemic sensitivity is reduced, and since the zebrafish does not open its mouth for 5 days, it cannot be ingested for digestion and conversion of glycemic index. If an older zebrafish, such as a 7-day-old or 8-day-old zebrafish, is used, the individual differences will be larger as the zebrafish needs to start feeding to supplement energy due to less yolk retention, and the stability of the test results will decrease.

[0027] Preferably, the pH value of the fish embryo culture solution is 6.5-8.5.

[0028] The specific raw material components of the fish embryo culture solution are not limited in the present application. In one embodiment, the raw material components of the fish embryo culture solution are: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, 55 mg of potassium chloride, and 10 L of water.

[0029] The culture conditions, such as density and temperature, of zebrafish embryos can affect the stability of the free sugar level test results if not properly controlled. This may be due to the fact that the culture conditions can affect the growth and development of zebrafish, resulting in differences in the tested zebrafish and thus affecting the test results.

[0030] Preferably, the test samples in step S4 are divided into a blank control group, a model control group, and a test substance group. The blank control group uses a zebrafish embryo culture solution, the model control group uses a 4% glucose solution, and the test substance group uses 4% glucose and a test substance.

[0031] Preferably, the test method of the exposure experiment is as follows: randomly select zebrafish for the glucose-raising index test, place the zebrafish in a 27-29°C incubator at a density of no more than 1 fish per 200 μL of fish embryo culture solution for 15-17 hours, and observe and record the number of dead fish embryos at the end of the exposure.

[0032] Preferably, the culture time is 16 hours.

[0033] Further preferably, in the exposure experiment, no less than 3 groups of parallel experiments are set for each sample test, and each group has no less than 20 fish.

[0034] In one embodiment, the test method of the exposure experiment is as follows: randomly select 60 zebrafish into a 6-well plate, with 20 zebrafish and 5 mL of test sample per well, place the exposed zebrafish in a 28°C incubator for 16 hours, and observe and record the number of dead fish embryos at the end of the exposure.

[0035] The exposure time of the present application is selected to be 16 hours, which is convenient for experimental time arrangement, and the sensitivity and stability will decrease if the exposure time is too long or too short.

[0036] Preferably, the specific test method of the free sugar level test is as follows: collect the surviving zebrafish in each well in a test tube, freeze and anesthetize to death in ice water, wash the zebrafish with fish embryo culture solution for 3 times, and homogenize the zebrafish with a manual homogenizer; filter, then centrifuge at 15,000 rpm for 10 min; take 1.5 μL of the supernatant and drop it on the blood glucose meter test paper to read the free sugar level, test twice per tube, and take the average value, which is the free sugar level in the zebrafish.

[0037] Another aspect of the present application provides the use of the zebrafish model constructed by the above-mentioned construction method, which can be used for testing and evaluating the glycemic index of food.

[0038] The food includes non-digestive food and digestive food.

[0039] The non-digestive food includes glucose, white sugar (containing 99% sugar), brown sugar (containing 88% sugar), monoono-oligosaccharide, honey sugar, sugar substitute, fructose and various drinks.

[0040] The digestive food is, for example, rice, flour, bread, cake, biscuit and the like.

[0041] Beneficial effects:

[0042] 1. The present application constructs a zebrafish model for testing the glycemic index, which is constructed by using zebrafish, has high fecundity, short testing time, high flux and can test a large number of samples at the same time, and has simple culture conditions and low cost.

[0043] 2. The zebrafish model of the present application has a short model construction period, and 6-day-old zebrafish are used for testing, and the model construction and the testing of the glycemic index can be completed in 1 day, the glycemic sensitivity is high, the measured blood glucose index is stable, the testing result has good positive correlation with the known sample glycemic index, and the obtained zebrafish glycemic testing data can ideally reflect the glycemic level of the sample. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 A representative diagram of normal zebrafish embryo early development;

[0045] Figure 2 A flow chart of the zebrafish model construction method of Example 1 of the present application;

[0046] Figure 3 An experimental diagram when the zebrafish exposure experiment of Example 1 of the present application is carried out;

[0047] Figure 4 An experimental diagram when the zebrafish is cultured in the present application. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0049] EMBODIMENT

[0050] The wild type AB strain zebrafish used in the present application is purchased from the National Zebrafish Resource Center of China.

[0051] The specific test method of free sugar level test is as follows: the zebrafish in each well is collected in a test tube, frozen and anesthetized to death in ice water, washed with fish embryo culture solution for 3 times, homogenized with a manual homogenizer, filtered, and centrifuged at 15,000 rpm for 10 min; 1.5 μL supernatant is dropped on the blood glucose meter test paper to read the free sugar level, and 2 samples are tested for each tube, and the average value is obtained, which is the free sugar level in the zebrafish.

[0052] Example 1

[0053] The present embodiment provides a method for constructing a zebrafish model for testing the glycemic index, which specifically comprises the following steps:

[0054] Step S1: breeding of zebrafish: zebrafish parents with fish age of 6-12 months are selected, the selected parents have no obvious visible infection and disease characteristics, and have not experienced drug treatment within 2 months; the breeding liquid temperature is controlled at 26-28.5°C, and the indoor temperature is controlled at 20-25°C for more than 14 days; the breeding density of zebrafish is controlled at 1-2 fish per liter of water, and the light condition is maintained at 12-16 hours per day during breeding, and a filtration system is configured; the zebrafish is fed twice a day, and at least once with abundant shrimps; and zebrafish for breeding and spawning is obtained.

[0055] The components of the breeding liquid include deionized water, artificial sea salt and NaHCO3; 400 mg of artificial sea salt is added per liter of water. The artificial sea salt is purchased from Yier Company in 5 kg.

[0056] Step S2: collection of zebrafish eggs: 3 spawning tanks are set, and the zebrafish is put into the spawning tank 2 hours before the light is turned off the day before spawning, 2-3 pairs of zebrafish are put in each spawning tank, and the ratio of male and female fish is 1:1 to make the zebrafish mate and spawn, and the zebrafish eggs in the 3 spawning tanks are collected and cleaned with fish embryo culture solution.

[0057] Step S3: culture of zebrafish: healthy zebrafish embryos are selected from the collected zebrafish eggs, and cultured at a density of not more than 1 fish embryo per 200 μL of fish embryo culture solution at 28°C for 6 days, and the culture solution is replaced and unhealthy zebrafish embryos are removed every day, to obtain zebrafish for testing the glycemic index; under the condition of 28°C, the fertilized fish embryos complete the first division in about half an hour, and then divide into 4, 8, 16, 32……cells, at which time the fertilized fish embryos are clearly distinguishable. As shown in FIG. 1, it is a representative diagram of the early development of normal zebrafish embryos. Figure 1 ​

[0058] The pH value of the fish embryo culture solution is 6.5-8.5, and the fish embryo culture solution is prepared by dissolving 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride in 10 L of water.

[0059] Step S4: model construction experiment: set test samples, the test samples are divided into a blank control group, a model control group and a test substance group, the blank control group uses a zebrafish embryo culture solution, the model control group uses a 4% glucose solution, and the test substance group uses 4% glucose and a test substance; the test substance is 4% white sugar (containing 99% sugar).

[0060] The test sample is used for zebrafish exposure experiment, 60 zebrafish are randomly selected into a 6-well plate, 20 zebrafish and 5 mL of test sample per well, the exposed zebrafish are placed in a 28°C incubator for 16 hours, and the number of dead fish embryos is observed and recorded at the end of the exposure; the free sugar level in the zebrafish is tested, and the free sugar level in the zebrafish is positively correlated with the glycemic index of the test substance, so that the construction of the zebrafish model for testing the glycemic index is determined to be successful.

[0061] Example 2

[0062] The embodiment provides a method for constructing a zebrafish model for testing the glycemic index, which is different from the embodiment 1 in that the test substance of the embodiment is 4% brown sugar (containing 88% sugar).

[0063] Example 3

[0064] The embodiment provides a method for constructing a zebrafish model for testing the glycemic index, which is different from the embodiment 1 in that the test substance of the embodiment is 4% fructose.

[0065] Example 4

[0066] The embodiment provides a method for constructing a zebrafish model for testing the glycemic index, which is different from the embodiment 1 in that the test substance of the embodiment is 4% Luo Han Guo sugar (erythritol, Luo Han Guo disaccharide).

[0067] Example 5

[0068] The embodiment provides a method for constructing a zebrafish model for testing the glycemic index, which is different from the embodiment 1 in that the test substance of the embodiment is ordinary supermarket rice.

[0069] Example 6

[0070] The embodiment provides a method for constructing a zebrafish model for testing the glycemic index, which is different from the embodiment 1 in that the test substance of the embodiment is a resistant starch rice marked with low GI (GI is 48.9).

[0071] Example 7

[0072] This example provides a method for constructing a zebrafish model for testing the glycemic index, which is different from Example 1 in that the test substance in this example is rice marked as low GI (GI≤55).

[0073] Example 8

[0074] This example provides a method for constructing a zebrafish model for testing the glycemic index, which is different from Example 1 in that the test substance in this example is buckwheat noodles marked as low GI (GI is 42.7).

[0075] Example 9

[0076] This example provides a method for constructing a zebrafish model for testing the glycemic index, which is different from Example 1 in that the zebrafish in this example is 4-day-old zebrafish used for glycemic index testing, and the test sample is a 4% glucose solution.

[0077] Example 10

[0078] This example provides a method for constructing a zebrafish model for testing the glycemic index, which is different from Example 1 in that the exposure time of the zebrafish in this example is 6 hours, and the test sample is a 4% glucose solution.

[0079] Example 11

[0080] This example provides a method for constructing a zebrafish model for testing the glycemic index, which is different from Example 1 in that the exposure time of the zebrafish in this example is 24 hours, and the test sample is a 4% glucose solution.

[0081] The zebrafish models of Examples 1-4 were subjected to test substance experiments, and the measured free sugar levels of zebrafish embryos are summarized in Table 1 below.

[0082] Table 1 Summary of changes in free sugar levels of zebrafish embryos (Examples 1-4)

[0083]

[0084] The zebrafish models constructed according to the present application were used to test the glycemic index of glucose, white sugar (containing 99% sugar), brown sugar (containing 88% sugar), monoono sugar, fructose, etc., and the changes in free sugar of zebrafish embryos were measured. As can be seen from Table 1 above, the zebrafish glycemic test data can ideally reflect the glycemic level of the sample.

[0085] The zebrafish models of Examples 5-8 were subjected to test substance experiments, and the measured free sugar levels of zebrafish embryos are summarized in Table 2 below.

[0086] Table 2 Summary of free sugar level changes in zebrafish embryos (Examples 5-8)

[0087]

[0088] * Compared with the blank control group.

[0089] According to the data in Table 2, the zebrafish model constructed according to the present application was used to test the glycemic index of common rice (Jinfeng brand), low GI rice (Zhongnenghaier rice, anti-childhood diabetes resistant starch rice and rice institute scholar rice), and low GI buckwheat noodles (Zhonglan 70 buckwheat noodles). After the samples were cooked, they were tested at a uniform original sample concentration (4% by mass fraction). Glucose at the same concentration was used as a positive control. The results showed that the higher the glycemic index of the sample, the higher the free sugar level induced in the zebrafish. The free sugar level in the zebrafish was positively correlated with the glycemic index of the sample. Among the samples with a glycemic index GI > 70 (high GI), glucose and Jinfeng brand rice had a glycemic rate in the zebrafish of > 200%, and the samples with a GI of 50-70 (low GI) had a glycemic rate in the zebrafish of < 150%. It can be seen that the zebrafish glycemic test data can ideally reflect the glycemic level of the sample.

[0090] The free sugar levels of the zebrafish in Examples 9-11 were tested, and the mortality rate of the fish embryos after exposure was recorded. The test data are shown in Table 3.

[0091] Table 3

[0092]

Claims

1. A method for constructing a zebrafish model for testing the glycemic index, characterized by, Specifically comprising the following steps: Step S1: breeding of zebrafish: healthy zebrafish parents are selected, and the breeding liquid temperature is controlled at 25-28.5°C to obtain zebrafish for breeding and spawning; Step S2: zebrafish egg collection: male and female fish are placed in the spawning tank according to the proportion, and the zebrafish are allowed to mate and spawn, and the zebrafish eggs are collected and cleaned with fish embryo culture solution; Step S3: zebrafish culture: healthy zebrafish embryos are selected from the collected zebrafish eggs, and cultured at 25-28.5°C for 6 days to obtain zebrafish for testing the glycemic index; Step S4: model construction experiment: test samples are set, and the zebrafish exposure experiment is performed using the test samples, the free sugar level in the zebrafish is tested, and the free sugar level in the zebrafish is positively correlated with the glycemic index of the test substance, that is, the zebrafish model for testing the glycemic index is successfully constructed, In step S3, the zebrafish embryos are cultured at a density of not more than 1 embryo per 200 μL of fish embryo culture solution at 25-28.5°C.

2. The method of constructing a zebrafish model for testing the glycemic index according to claim 1, wherein, In step S1, the breeding density of zebrafish is controlled to be not less than 1 fish per liter of breeding liquid, and the light is kept on for 12-16 hours per day during breeding, and a filtration system is configured.

3. The method for constructing a zebrafish model for testing glycemic index according to claim 2, characterized in that, The breeding density is 1-3 fish per liter of breeding liquid.

4. The method of claim 2, wherein the zebrafish model for testing the glycemic index is constructed by, The salinity of the breeding liquid is 0.25‰-0.50‰, the saturation of dissolved oxygen is ≥80%, and the pH value is 6.5-8.

5.

5. A method for constructing a zebrafish model for testing glycemic index according to claim 4, characterized in that, The salinity of the breeding liquid is 0.25‰-0.50‰, the saturation of dissolved oxygen is ≥80%, the pH value is 6.5-8.5, the conductivity is 400-800 μS / cm, and the hardness is 30-300 mg / L CaCO3.

6. The method of constructing a zebrafish model for testing the glycemic index according to claim 1, wherein, The pH value of the fish embryo culture solution is 6.5-8.

5.

7. The method of constructing a zebrafish model for testing the glycemic index according to claim 1, wherein, The test method of the exposure experiment is: randomly selecting zebrafish for testing the glycemic index, placing the zebrafish in a 27-29°C constant temperature incubator at a density of not more than 1 fish per 200 μL of fish embryo culture solution for 15-17 hours, and observing and recording the number of dead fish embryos at the end of the exposure.

8. Use of a zebrafish model constructed according to the method of any one of claims 1 to 7 for testing a glycemic index. The zebrafish model for testing the glycemic index can be used to test and evaluate the glycemic index of food.

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