CN114617104B

CN114617104B - Method for establishing II-type diabetes bee model and application thereof

Info

Publication number: CN114617104B
Application number: CN202210288424.7A
Authority: CN
Inventors: 郑浩; 仲召鹏; 齐悦; 王京奥; 王小斐
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-03-23
Filing date: 2022-03-23
Publication date: 2023-06-27
Anticipated expiration: 2042-03-23
Also published as: CN114617104A

Abstract

The application discloses a method for establishing a type II diabetes bee model, which comprises the following steps: animal selection: selecting mature bees as the object for establishing a type II diabetes bee model; preparing high-fat bee bread: preparing high-fat bee bread by using oil, protein-containing substances and saccharides; and (3) establishing a model: feeding the developed bees with the high fat bee bread for more than 5 days to establish a type II diabetes bee model. Also discloses the application of the type II diabetes bacterial bee model and/or the type II diabetes aseptic bee model established by the method. The method is simple and convenient to operate, low in cost, short in modeling period, remarkable in type II diabetes symptoms, stable in model, simple in experimental flow, and capable of effectively improving experimental efficiency of the animal model and reducing experimental cost.

Description

Method for establishing II-type diabetes bee model and application thereof

Technical Field

The present application relates to the field of biotechnology. In particular to a method for establishing a type II diabetes bee model and application thereof.

Background

Diabetes mellitus is a global epidemic metabolic disease, the prevalence of which tends to increase year by year and becomes an important cause of death and disability. In recent years, along with the improvement of living standard, the prevalence of diabetes in China is increased explosively in the past twenty years due to unhealthy diet and lack of exercise, and newly released global diabetes map of 2021IDF (10 th edition) shows that China is the most countries of diabetes patients in the world, and the number of diabetes patients in China is increased from 9000 to 1 hundred million to 4000 million in the past 10 years (2011-2021), and the increase of diabetes patients in the country is up to 56% in the first world.

Diabetes is a life-long metabolic disease which is caused by multiple causes and is characterized by chronic hyperglycemia, and causes great threat to human life health, however, the cause of diabetes is unknown until now, and more than 90% of diabetes patients are type II diabetes, also called non-insulin dependent diabetes, and the patients can not respond to the diabetes, so that insulin is relatively lack, the induction factors are numerous and complex, and therefore, the research of pathogenesis of type II diabetes becomes a focus of scientific research and medical staff at present. The traditional animal models of type II diabetes comprise rabbits and mice, and the animal models can carry out scientific research experiments and simulate human type II diabetes, but most of the animal models have the disadvantages of high construction cost, long time period, small quantity and complex operation.

Disclosure of Invention

In order to overcome the defects of the prior art and reduce the cost of model establishment, the application aims to provide a stable and efficient establishment method of a type II diabetes bee model, which can be used for screening or evaluating drugs for treating type II diabetes and can be applied to exploring specific mechanisms of diabetes related diseases.

The specific technical scheme of the application is as follows:

1. the method for establishing the type II diabetes bee model is characterized by comprising the following steps of:

animal selection: selecting mature bees as the object for establishing a type II diabetes bee model;

preparing high-fat bee bread: preparing high-fat bee bread by using oil, protein-containing substances and saccharides;

and (3) establishing a model: feeding the developed bees with the high fat bee bread for more than 5 days to establish a type II diabetes bee model.

2. The method according to item 1, wherein the type II diabetes bee model is a type II diabetes bacterial bee model, and in the animal selection step, intestinal tract bacteria liquid is prepared by using intestinal tract contents of normal bees, and then the eclosion bees are fed with a mixed solution containing the intestinal tract bacteria liquid to obtain mature bees, namely bacterial bees;

preferably, the mixed solution further comprises sterile pollen;

preferably, the intestinal bacterial solution is prepared by using the intestinal contents of more than 20 normal bees.

3. The method according to item 1, wherein the type II diabetes bee model is a type II diabetes aseptic bee model, and in the animal selection step, the eclosion bees are fed with an aseptic sucrose solution to obtain mature bees, namely aseptic bees;

preferably, the concentration of the sterile sucrose solution is 40w/v% to 60w/v%, preferably 50w/v%;

preferably, the aseptic sucrose solution is sterilized by filtration using a filter membrane having a pore size of 0.22. Mu.m.

4. The method according to any one of claims 1 to 3, wherein the high fat bee bread is a liquid bee bread, and the liquid bee bread contains 2 to 7w/v% of oils and fats, preferably 4 to 6 w/v%;

preferably, the first liquid bee bread contains 1-3 w/v% protein-containing substance, preferably 1.5-2.5 w/v%;

preferably, the liquid bee bread contains 40-60 w/v% sugar, preferably 45-55 w/v%.

5. The method according to any one of claims 1 to 3, wherein the high fat bee bread comprises a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread contains 2-7w/v% of grease, preferably 4-6w/v%;

preferably, the second liquid bee bread contains 1-3 w/v% protein-containing substance, preferably 1.5-2.5 w/v%;

preferably, the second liquid bee bread contains 40-60 w/v% sugar, preferably 45-55 w/v%;

preferably, the solid bee bread contains 2-7 w/v% of grease, preferably 4-6 w/v%;

preferably, the solid bee bread contains protein-containing substances.

6. The method according to any one of claims 1 to 5, wherein the oils and fats are edible oils;

preferably, the mass percentage of saturated fatty acid in the edible oil is 30% -50%;

preferably, the edible oil is selected from one or two or three of palm oil, soybean oil and corn oil, preferably palm oil;

preferably, the protein-containing substance may be selected from one or two or three of casein hydrolysate, soybean protein powder and pollen.

7. The method according to any one of claims 1 to 6, wherein in the model building step, the developed bees are fed with the high fat bee bread in an incubator at a temperature of 30 to 40 ℃ and a humidity of 45 to 55%;

preferably, in the model building step, the developed bees are fed the high fat bee bread for more than 7 days to build a type II diabetes bee model.

8. The method according to any one of claims 1 to 7, wherein the mature bees are 2 to 15 day old bees, preferably 5 to 9 day old bees;

preferably, the mature bees are bees of the family Apidae, further preferably bees or bumblebees.

9. Use of a type II diabetes mellitus bacterial bee model established according to the method described in item 2 or 3, and/or a type II diabetes mellitus sterile bee model established according to the method described in item 4, in screening or evaluating a medicament for treating type II diabetes mellitus.

10. Use of a type II diabetes mellitus bacterial bee model established according to the method described in item 2 or 3, and/or a type II diabetes mellitus sterile bee model established according to the method described in item 4, for studying the mechanisms of diabetes-related diseases.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the method for establishing the II-type diabetes bee model, developed bees are used as objects for establishing the II-type diabetes bee model, the II-type diabetes bacterial bee model and the II-type diabetes aseptic bee model can be established respectively, the II-type diabetes of bees can be effectively initiated, the average glucose and trehalose content in blood stranguria of bees is increased, the weight is increased, and the original intestinal flora of bees is destroyed. Compared with other common animal models in the prior art, the method has the advantages of simple operation, low cost, short modeling period and obvious symptom of the II type diabetes, is suitable for a large-scale test of the II type diabetes model, can be used for screening a novel method or a novel medicament for preventing or treating the II type diabetes related diseases, and can be applied to exploring a specific mechanism of the II type diabetes related diseases.

Drawings

FIG. 1 is a graph of average glucose content data in blood strangles of each experimental group of sterile and bacterial bees.

FIG. 2 is a graph of average trehalose content data in blood strangles of each experimental group of sterile and bacterial bees.

FIG. 3 is a graph of average weight gain data for each experimental group of sterile and bacterial bees.

Fig. 4 is a graph of HE sections of fat bodies from experimental group 3 and experimental group 4 of bacterial bees.

Fig. 5 is a graph of average glucose content data in blood strangles of each experimental group of bacterial bumblebees.

FIG. 6 is a graph of average trehalose content data in blood strangles of various experimental groups of bacterial bumblebees.

Figure 7 is a graph of average weight gain data for each experimental group of bacterial bumblebees.

Detailed Description

Exemplary embodiments of the present application are described below, including various details of embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Accordingly, one of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the present application.

In one aspect, the present application provides a method for establishing a type II diabetes bee model, comprising the steps of:

The method can successfully construct the type II diabetes bee model in a short time at low cost, is simple and convenient, has the advantages that raw materials used for inducing the model are simple and easy to obtain, and the constructed model is stable, so that researchers can search and research type II diabetes.

The method further comprises the step of judging whether modeling is successful after the modeling step is established, wherein after feeding the high-fat bee bread, after freezing and dizziness each bee at 3-5 ℃, whether modeling is successful is judged by measuring the weight, the glucose content in blood, the trehalose content, the fat body fat drop accumulation amount and the intestinal flora change.

In a specific embodiment, after the high-fat bee bread is fed to the bacterial bees, after each bacterial bee is frozen and stunned at the temperature of 3-5 ℃, the weight gain rate of the bacterial bees is measured to be more than 160%, the blood glucose content is more than 5.3 mug/mu l, and the trehalose content is more than 11 mug/mu l, so that the modeling is judged to be very successful.

In a specific embodiment, after the high-fat bee bread is fed to the bacterial bees, after each bacterial bee is frozen and stunned at the temperature of 3-5 ℃, the weight gain rate of the bacterial bees is measured to be more than 100%, the blood glucose content is more than 2 mug/mu l, and the trehalose content is more than 5 mug/mu l, and the judgment and modeling are successful.

In a specific embodiment, after the aseptic bees are fed with the high-fat bee bread, after each aseptic bee is frozen and stunned at the temperature of 3-5 ℃, the weight gain rate of the aseptic bees is measured to be more than 130%, the blood glucose content is more than 3 mug/mu l, and the trehalose content is more than 5 mug/mu l, so that the modeling is judged to be very successful.

In a specific embodiment, after the aseptic bees are fed with the high-fat bee bread, after each aseptic bee is frozen and stunned at the temperature of 3-5 ℃, the weight gain rate of the aseptic bees is measured to be more than 90%, the blood glucose content is more than 2 mug/mu l, and the trehalose content is more than 3 mug/mu l, and the judgment modeling is successful.

In one specific embodiment, the circulating glucose content and the trehalose content in blood are measured by taking blood from the bee after the frozen corona, wherein the blood taking step comprises the following steps:

a small gap is cut on the neck of each bee, a 10 mu L pipette is used for sucking the hemolymph, 2 to 5 bees of hemolymph is collected, and 2 to 5 mu L of hemolymph is taken for detection;

and detecting and quantifying the collected haemolymph by using a glucose kit and a trehalose kit.

In the model creation step of the present application, for example, the developed and matured bees may be fed with the high fat bee bread for 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, etc., preferably 7 days or more, to create a type II diabetes bee model.

In one embodiment, the mature bees are 2 to 15 days old bees, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., preferably 5 to 9 days old bees.

The term "bee" in the present application refers to a collective term for other insects of the order hymenoptera except for termite insects, and the species of bees in the present application are not limited, and may belong to, for example, the families of bees, the families of phyllosphidae, the families of agalmatoidae, the families of trichogramma, the families of wasps, the families of Sha Fengke, and the like, and further may belong to the genera bees, bumblebees, bees, thornless bees, wallbees, cut bees, and the like under the family of bees.

In a specific embodiment, the mature bee is a bee of the family Apidae, further preferably a bee or bumblebee.

The 9 insects of the genus Apis of the family Apidae are generally called Apis, and are typically characterized by collecting nectar and pollen and making nectar into honey, wherein the most common are Apis cerana in Apis cerana and Apis italica, carnix E Lafeng, apis cerana, and the like in Apis cerana, apis sappan, apis Su Weila Apis cerana, apis lupin, and the like. The bee species in the present application is not limited, but is preferably a bee species that is easy to purchase and obtain.

More than 500 social insects of bumblebee genus of Apidae are generally called bumblebee, and are typically characterized by thick and strong body and dense whole body and black, yellow or long hairs with various colors alternately distributed in various regions of the world except Antarctic and Dayang, and have completely the same nest life and bee colony structure, and take pollen and nectar of the festive plants as food, and are mainly of the species of heavy bumblebee, short-headed bumblebee, etc. The bumblebee species in the present application is not limited, and is preferably a bumblebee species that is easy to purchase and obtain.

In a specific embodiment, the type II diabetes bee model is a type II diabetes bacterial bee model, and in the animal selecting step, intestinal tract bacterial liquid is prepared by using intestinal tract contents of normal bees, and then the eclosion bees are fed with a mixed solution containing the intestinal tract bacterial liquid to obtain mature bees, namely the bacterial bees.

In a specific embodiment, the normal bees are bees with normal intestinal flora and perfect development, and the normal bees can be the same type as bees used for establishing the type II diabetes bee model or different types, and are preferably bees of the same type as bees used for establishing the type II diabetes bee model.

In a specific embodiment, more than 20 normal bees are used to prepare intestinal bacterial solutions, for example, 20, 22, 24, 26, 28, 30, 40, 50, etc.

In a specific embodiment, the method for collecting the intestinal bacterial liquid comprises the following steps: adding glycerol PBS buffer solution into a sterile centrifuge tube I, taking 20-30 normal bees, dissecting the normal bees to obtain a complete intestinal tract, putting the complete intestinal tract into the centrifuge tube I, fully grinding and homogenizing, sucking out a mixture of intestinal contents in the sterile centrifuge tube and the glycerol PBS buffer solution (discarding intestinal tissues) by using a pipette, and collecting the mixture in the centrifuge tube II to obtain the intestinal bacterial solution. The intestinal flora of bees also has differences among bees, and taking 20-30 normal bees can minimize the influence of the individual differences.

After the intestinal bacterial liquid is collected, the intestinal bacterial liquid is subpackaged into a plurality of sterile centrifuge tubes, and the sterile centrifuge tubes are frozen at the temperature of minus 80 ℃ for standby. In this way, the packaging is carried out, and the small tube is taken when in use, so that repeated freezing and thawing are avoided.

In a specific embodiment, the mixed solution further comprises sterile pollen.

In a specific embodiment, the type II diabetes bee model is a type II diabetes aseptic bee model, and in the animal selecting step, the emerging bees are fed with an aseptic sucrose solution to obtain mature bees, namely aseptic bees.

In one embodiment, the concentration of the sterile sucrose solution is 40w/v% to 60w/v%, for example, 40w/v%, 42w/v%, 44w/v%, 46w/v%, 48w/v%, 50w/v%, 52w/v%, 54w/v%, 56w/v%, 58w/v%, 60w/v%, etc., preferably 50w/v%; the aseptic sucrose solution was sterilized by filtration using a filter membrane having a pore size of 0.22. Mu.m.

In the present application, the term "1w/v%" of a substance contained in a liquid or a solid means that 10g of the substance is contained in 1L of the liquid or the solid. In the present application, the liquid concentration is "1w/v%", which means that the mass concentration of the liquid is 10g/L.

In a specific embodiment, in the process of preparing the type II diabetes aseptic bee model, after mature bees are obtained, aseptic bee verification is also carried out, and the specific process is as follows: taking out 2-5 bees as verification bees, taking out the intestinal contents of each verification bee and measuring the total number of bacteria, and verifying that the developed bees are aseptic bees.

In one embodiment, the specific process of sterile bee verification is: taking out 2-5 bees as verification bees, dissecting the verification bees, placing the intestinal tracts of the verification bees into 100 mu L of 50% glycerol (PBS configuration), grinding with a grinding pestle, diluting 10 mu L of the verification bees with 10 mu L of glycerol ² 10 ³ Multiple (denoted as 10) ^-3 、10 ^-4 ) 100. Mu.L of the mixture was spread and cultured (35 ℃ C., 5% CO) ₂ ) Counting, if the total bacterial count of the final bee intestinal tract content is less than 10 ⁵ Can be considered as a sterile bee. Field planting water of intestinal bacteria in different bee bodiesThere may be differences in the levels, but the method of coating counting itself may also be somewhat occasional, so here diluted 10 separately ² 10 ³ The doubling is to ensure that reliable data can be obtained from the final coating results.

In one embodiment, the process of breeding the emerging bees is: selecting a honeycomb, taking out the bee pupa which is darker and has no growing hair, placing the bee pupa in an incubator with the temperature of 30-40 ℃ and the humidity of 60-80%, culturing the bee pupa by using a sterile sucrose solution for overnight, observing the emergence condition of the bee pupa the next day (culturing for one night if the bee pupa does not emerge), and culturing to obtain the emergence bee.

In a specific embodiment, the high-fat bee bread is a liquid bee bread, and the liquid bee bread contains 2w/v% -7 w/v% of grease, for example, 2w/v%, 2.5w/v%, 3w/v%, 3.5w/v%, 4w/v%, 4.5w/v%, 5w/v%, 5.5w/v%, 6w/v%, 6.5w/v%, 7w/v%, etc., preferably 4w/v% -6 w/v%.

The amount of the protein-containing substance contained in the high fat bee bread in the present application is not particularly limited as long as the protein required for bees can be provided.

In a specific embodiment, the high fat bee bread is a liquid bee bread, and the liquid bee bread contains 1 to 3w/v% of protein-containing substances, for example, 1 to 2.4 w/v%, 1.6w/v%, 1.8w/v%, 2w/v%, 2.5w/v%, 3w/v%, and the like, preferably 1.5 to 2.5w/v%.

In a specific embodiment, the high fat bee bread is a liquid bee bread, and the liquid bee bread contains 40 to 60w/v% of saccharides, for example, 40 to 55w/v% of saccharides, such as 40 to 43w/v%, 45 to 48w/v%, 50 to 52w/v%, 55 to 58w/v%, 60w/v%, and the like, preferably 45 to 55 w/v%.

In a specific embodiment, the high-fat bee bread comprises a second liquid bee bread and a solid bee bread, wherein the solid bee bread contains protein-containing substances, and the second liquid bee bread contains saccharides.

In a specific embodiment, the high-fat bee bread comprises a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread and/or the solid bee bread contain oil, the solid bee bread contains protein-containing substances, and the second liquid bee bread contains saccharides.

In a specific embodiment, the high-fat bee bread consists of a liquid bee bread II and a solid bee bread, wherein the liquid bee bread does not contain protein-containing substances, and the solid bee bread contains protein-containing substances.

In a specific embodiment, the high-fat bee bread consists of a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread and the solid bee bread both contain protein-containing substances.

In a specific embodiment, the high-fat bee bread includes a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread contains 2w/v% -7 w/v% of grease, for example, 2w/v%, 2.5w/v%, 3w/v%, 3.5w/v%, 4w/v%, 4.5w/v%, 5w/v%, 5.5w/v%, 6w/v%, 6.5w/v%, 7w/v%, and the like, and preferably 4w/v% -6 w/v%.

In a specific embodiment, the high-fat bee bread includes a liquid bee bread and a solid bee bread, wherein the liquid bee bread contains 1w/v% to 3w/v% of protein-containing substances, for example, 1w/v%, 1.2w/v%, 1.4w/v%, 1.6w/v%, 1.8w/v%, 2w/v%, 2.5w/v%, 3w/v%, and the like, and preferably 1.5w/v% to 2.5w/v%.

In a specific embodiment, the high-fat bee bread includes a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread contains 40w/v% to 60w/v% of saccharides, for example, 40w/v%, 43w/v%, 45w/v%, 48w/v%, 50w/v%, 52w/v%, 55w/v%, 58w/v%, 60w/v%, and the like, preferably 45w/v% to 55w/v% of saccharides.

In one embodiment, the oils and fats are edible oils, and the mass percentage of saturated fatty acids in the edible oils is 30% -50%, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, etc.

In a specific embodiment, the edible oil is selected from one or two or three of palm oil, soybean oil and corn oil, preferably palm oil.

The protein-containing substance of the present application is not particularly limited as long as it is a protein required by bees. In a specific embodiment, the protein-containing substance may be selected from one or two or three of casein hydrolysate, soybean protein powder and pollen.

The term "casein hydrolysate (Lactum)" as used herein refers to a casein hydrolysate obtained by using skim milk as a raw material and performing procedures such as casein separation, hydrolysis, and spray drying. The typical chemical components are as follows: 75% of protein, 1% of fat, 5% of moisture, 15% of ash and 1% of lactose. Can be dissolved in water, has no bitter taste, is stable when the pH is 2-9, is thermally stable, and can resist the high temperature of 180 ℃ for 50min. The αs1-Cn (f 91-100) obtained after casein hydrolysis is a three-dimensional structural polypeptide containing 10 amino acids, and the typical content in casein hydrolysate is 1.8%.

In a specific embodiment, in the step of preparing the high-fat bee bread, edible oil, protein-containing substances and saccharides are dissolved in water, and after mixing, filtering and sterilizing are carried out by using a filter membrane with the caliber of 0.22 mu m, so that the high-fat bee bread is prepared.

In a specific embodiment, in the modeling step, the mature bees are fed with the high fat bee bread in an incubator at a temperature of 30-40 ℃ and a humidity of 45% -55%, for example, the temperature may be 30 ℃, 32 ℃, 34 ℃, 36 ℃, 38 ℃, 40 ℃, etc., and the humidity may be 45%, 47%, 49%, 50%, 52%, 54%, 55%, etc.

On the other hand, the application also provides the II-type diabetes mellitus bacterial bee model established by the method and/or the application of the II-type diabetes mellitus sterile bee model established by the method in screening or evaluating the drug for treating the II-type diabetes mellitus. Chemical and/or biological agents may be included in the medicaments of the present application.

In still another aspect, the application further provides a type II diabetes bacterial bee model established by the method and/or application of the type II diabetes sterile bee model established by the method in researching diabetes related disease mechanism. It should be appreciated that the study was not therapeutic.

The application provides a method for respectively establishing a type II diabetes bacterial bee model and a type II diabetes aseptic bee model, which can efficiently trigger type II diabetes of bees, increase the average glucose and trehalose content in blood stranguria of bees, increase the weight and destroy the original intestinal flora of bees.

Aiming at a special bee model, three specific components of edible oil, casein hydrolysate and sucrose are selected to be successfully induced and modeled, and the concentrations of the edible oil, the casein hydrolysate and the sucrose in the high-fat bee bread used for the induction and modeling are controlled simultaneously to build a more stable model.

There are significant differences in the signs of the bacterial or aseptic bee-induced type II diabetes model, mainly including weight gain rate, blood glucose content and trehalose content. Aiming at experimental requirements, if the function of intestinal bacteria needs to be explored, a type II diabetes aseptic bee model can be selected for experiments; the bacterial bee model is suitable for all researches of diabetic model animals.

Examples

The method for establishing the type II diabetes bee model of the present application will be specifically described with reference to the drawings in the embodiments of the present application. The described embodiment is one preferred embodiment, but not all embodiments, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments of the present application without making any inventive effort, are intended to be within the scope of the present application.

Example 1

Establishment of type II diabetes mellitus bacterial bee model and type II diabetes mellitus aseptic bee model

(1) Early preparation of experiment

Several sterile 1.5mL centrifuge tubes were prepared and 200 μl glycerol PBS buffer (50% glycerol: 1 x PBS buffer=1:1) was added. Taking normal worker bees in 20-30 beehives, dissecting bees to obtain complete intestinal tracts, respectively placing the complete intestinal tracts into centrifuge tubes, homogenizing the complete intestinal tracts for 30s on ice at the rotating speed of 6000rpm by using an electric grinding pestle, and ensuring the sufficient grinding of intestinal tissues. Taking a sterile 50mL centrifuge tube, sucking out intestinal contents in all 1.5mL centrifuge tubes (discarding intestinal tissues) by using a pipettor, collecting the intestinal contents in the 50mL centrifuge tubes, sub-packaging the mixed intestinal bacterial liquid into a plurality of new sterile 1.5mL centrifuge tubes again, and freezing the mixed intestinal bacterial liquid at the temperature of-80 ℃ for later use. When in use, the small tube is taken, and repeated freezing and thawing are avoided.

(2) Experimental bee selection and culture

Selecting a proper honeycomb, randomly selecting a plurality of points on the honeycomb by using sterilized awls or tweezers, gently poking a bee pupa cover, observing the mature condition of the bee pupa, continuously poking the cover around the bee pupa which is darker in color and has no long hair, and paying attention to cleaning the periphery as much as possible when poking the pupa cover, so that the carrying of honeycomb impurities during taking the bee pupa is avoided. The sterilized tweezers are tightly combined, the tweezers are penetrated from the upper part of the bee pupae, then the tweezers are opened to the two sides of the bee pupae, the pupae is taken out by lightly pinching the chest of the bee pupae, and the physical injury to the bee pupae is avoided as much as possible. Note also that bee pupae that have developed wings are not available. The picked bee pupas are put into plastic square boxes sterilized by 84 sterilizing liquid and alcohol in advance, 2mL centrifuge tubes (tube wall punching) filled with sterile sucrose solution are inserted at the side or upper part of the box body, and 150-200 bee pupas can be placed in each square box. Placing the square box containing the bee pupa in an incubator with the temperature of 35 ℃ and the humidity of 60-80% for culturing overnight. The following day, the emergence status of the bee pupae is observed (if the bee pupae are not emerged, the bee pupae are cultured for one night), and 200 bee pupae are selected.

Mixing 40 μl of intestinal bacteria solution, 4000 μl of insect physiological saline and 0.3g of sterile rape pollen to obtain mixed solution, and planting 100 emerging bees (worker bees) to obtain 3 day old bacterial bees.

Taking the rest 100 bees (worker bees) which are eclosion to feed 50w/v% sterile sucrose solution, and obtaining the sterile bees of 3 days old.

(3) Animal grouping and experimental design

200 bacterial bees and aseptic bees of 3 days old obtained above were divided into 4 major groups: experimental group 1, experimental group 2, experimental group 3 and experimental group 4; each group contains 25 sterile bees and 25 bacterial bees, 25 sterile bees in each group are fed into one cup, and 25 bacterial bees in each group are fed into one cup.

Feeding the experimental group 1 with liquid high-fat bee bread containing 1w/v% soybean oil, 2w/v% casein hydrolysate, 50w/v% sucrose and water, and freely feeding; experimental group 2 was fed liquid high fat bee bread containing 5w/v% soybean oil, 2w/v% casein hydrolysate, 50w/v% sucrose and water; experiment group 3 was fed with liquid high fat bee bread containing 1w/v% palm oil, 2w/v% casein hydrolysate, 50w/v% sucrose and water, and was fed with free feed; experimental group 4 was fed liquid high fat bee food containing 5w/v% palm oil, 2w/v% casein hydrolysate, 50w/v% sucrose and water, and was fed ad libitum.

The rape pollen used in this experiment was rape pollen from Qinghai province (30 kGy electron beam sterilization), palm oil was Polylobus majoris super oil (product standard number: Q/02A 0796S), and soybean oil was produced by Zhongliang Fujinyan (product standard number: Q/02A 3211S).

(4) Experimental observation, sampling and experimental results

After feeding bees for 7 days, each bee measures the weight after freezing and dizziness at 4 ℃, and blood lymph and fat bodies are collected, and whether the insulin resistance type II diabetes model of the experimental construction bee is successfully modeled is judged by measuring the content of glucose and trehalose, the accumulation of fat body fat drops, the weight growth rate and the intestinal flora change condition. Wherein body weight is determined by a ten-thousandth balance and glucose and trehalose content is determined by a kit.

For the bacterial bees, if the weight gain rate of the bacterial bees is measured to be more than 160%, and the blood glucose content is more than 5.3 mug/mu l, and the trehalose content is more than 11 mug/mu l, the modeling is judged to be very successful. If the weight gain rate of the bacterial bees is measured to be more than 100%, and the blood glucose content is more than 2 mug/mu l and the trehalose content is more than 5 mug/mu l, judging that the modeling comparison is successful.

For the aseptic bees, if the weight gain rate of the aseptic bees is measured to be more than 130 percent, the blood glucose content is more than 3 mug/mu l, and the trehalose content is more than 5 mug/mu l, the modeling is judged to be very successful. If the weight gain rate of the sterile bees is measured to be more than 90%, and the blood glucose content is more than 2 mug/mu l and the trehalose content is more than 3 mug/mu l, the modeling comparison is judged to be successful.

Wherein, the average glucose content data graph of each experimental group of bee blood strangles is shown in figure 1; the average trehalose content data in bee blood strangles of each experimental group are shown in figure 2; the average weight gain rate of bees in each experimental group is shown in figure 3; the fat body HE slice diagrams of the bacterial bees in the experimental group 3 and the experimental group 4 are shown in the figure 4, cells in tissues are stained and then scanned into the diagram by an electron microscope, the blank part which is not colored is lipid drop, and the experimental group 4 is more abundant than the lipid drop content in the experimental group 3, so that the fat body is provided with lipid accumulation; the intestinal flora changes of the bacterial bees in the experimental group 3 and the experimental group 4 are shown in the following table 1, wherein the experimental groups 3-1 to 3-6 are respectively 6 samples of the experimental group 3, and the experimental groups 4-1 to 4-6 are respectively 6 samples of the experimental group 4.

TABLE 1 relative abundance of intestinal microbiota composition Table (%)

In experimental groups 1-4, the blood sugar and weight indexes of the aseptic bees and the bacterial bees are higher in 7 days, which indicates that the high-fat diet containing oil, protein substances and sugar can construct a type II diabetes bee model.

The 7-day later blood glucose of the bacterial bees of experimental group 3 and experimental group 4 was higher than that of experimental group 1 and experimental group 2, demonstrating that a diet containing palm oil-based high fat can successfully build a type II diabetes bee model.

The blood sugar and body weight of the aseptic and bacterial bees of the experimental group 4 are highest after 7 days, and the accumulation of lipid droplets in the fat body of the experimental group 4 is more than that of the experimental group 3, which shows that the high-fat diet containing the palm oil with higher proportion can be very successfully used for constructing the type II diabetes bee model.

Example 2

Establishment of type II diabetes fungus bumblebee model

(1) Experimental bumblebee selection and culture, animal grouping and experimental design

Referring to example 1, 36 eclosion bumblebees were selected, 14.4. Mu.L of intestinal bacteria liquid, 1440. Mu.L of insect physiological saline and 0.108g of sterile rape pollen were mixed to prepare a mixed solution, and the 36 eclosion bumblebees were planted in the mixed solution to obtain 3-day-old bacterial bumblebees.

The 36 3-day-old bacterial bumblebees were divided into 3 groups: control group, experimental group 5 and experimental group 6, 12 bumblebees in each group are evenly fed in 3 cups, and 4 bumblebees in each cup are fed in each group.

Feeding liquid bee bread containing 50w/v% sucrose and water and rape flower pollen solid bee bread to the control group, and freely feeding; experiment group 5 was fed with liquid bee bread containing 5w/v% corn oil and 50w/v% sucrose and water, and rape flower pollen solid bee bread containing 5w/v% corn oil, and was fed freely; experimental group 6 was fed liquid bee bread with 5w/v% palm oil and 50w/v% sucrose and water, and rape pollen solid bee bread with 5w/v% palm oil, and was free to feed. Wherein, the rape flower pollen solid bee bread containing 5w/v% of palm oil is solid bee bread composed of 0.05g of palm oil and 1ml of rape flower pollen; the rape flower pollen solid bee bread containing 5w/v% corn oil is solid bee bread composed of 0.05g corn oil and 1ml rape flower pollen. The rape pollen is rape pollen (30 kGy electron beam sterilization) of Qinghai province, the palm oil is Polydragon brand palm super liquid oil, and the corn oil is produced by Goldlong corporation (product standard number: Q/BBAH 0036S).

(2) Experimental observation, sampling and experimental results

After feeding bees for 7 days, measuring the weight of each bumblebee after freezing and dizziness at 4 ℃, collecting haemolymph, and judging whether the bumblebee insulin resistance type II diabetes model of each experimental group is successfully modeled by comparing the glucose and trehalose content difference of each experimental group and a control group.

The blood sugar data and the body weight of 7 days later of bumblebee worker bees in the experimental group 5 are different from those of the control group to a certain extent, which shows that the high-fat diet based on corn oil can be used for constructing a diabetes model; blood sugar data and body weight of bumblebee worker bees in the experimental group 6 are obviously increased compared with those in the control group, and the expected blood sugar data and body weight are satisfied, so that the palm oil-based high-fat diet can be used for successfully constructing the type II diabetes worker bee model.

The results of the above embodiments show that the method for establishing the type II diabetes model is particularly suitable for bees, especially bees of the family Apidae, such as bees and bumblebees, and the specific experimental conditions of different bees need to be finely adjusted to achieve a better modeling effect.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.

Claims

and (3) establishing a model: feeding the developed bees with the high-fat bee bread for more than 5 days to establish a type II diabetes bee model;

the high-fat bee bread is a liquid bee bread, and the liquid bee bread contains 2-7 w/v% of grease, 1-3 w/v% of protein-containing substances and 40-60 w/v% of saccharides; or the high-fat bee bread comprises a liquid bee bread II and a solid bee bread, wherein the liquid bee bread II contains 2-7w/v% of grease, 1-3w/v% of protein-containing substances and 40-60w/v% of saccharides, and the solid bee bread contains 2-7w/v% of grease and one or two or three protein-containing substances selected from casein hydrolysate, soybean protein powder and pollen;

the grease is edible oil, and the edible oil is one or two or three of palm oil, soybean oil and corn oil;

the type II diabetes bee model is a type II diabetes bacterial bee model, in the animal selection step, intestinal tract bacterial liquid is prepared by using intestinal tract contents of normal bees, and then the eclosion bees are fed with a mixed solution containing the intestinal tract bacterial liquid to obtain mature bees, namely the bacterial bees.

2. The method of claim 1, wherein the mixed solution further comprises sterile pollen.

3. The method of claim 1, wherein the intestinal bacterial fluid is prepared from the intestinal contents of more than 20 normal bees.

4. The method for establishing the type II diabetes bee model is characterized by comprising the following steps of:

the type II diabetes bee model is a type II diabetes aseptic bee model, and in the animal selection step, the eclosion bees are fed with an aseptic sucrose solution to obtain mature bees, namely aseptic bees.

5. The method of claim 4, wherein the concentration of the sterile sucrose solution is 40w/v% to 60w/v%.

6. The method of claim 4, wherein the concentration of the sterile sucrose solution is 50w/v%.

7. The method according to claim 4, wherein the aseptic sucrose solution is sterilized by filtration using a filter membrane having a pore size of 0.22. Mu.m.

8. The method of any one of claims 1-7, wherein the high fat bee bread is a liquid bee bread, and the liquid bee bread contains 4-6 w/v% of grease.

9. The method of any one of claims 1-7, wherein the high fat bee bread is a liquid bee bread, and the liquid bee bread contains 1.5-2.5 w/v% protein-containing substances.

10. The method of any one of claims 1-7, wherein the high fat bee bread is a liquid bee bread, and the liquid bee bread contains 45-55 w/v% of saccharides.

11. The method of any one of claims 1-7, wherein the high fat bee bread comprises a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread contains 4-6 w/v% of oil.

12. The method of any one of claims 1-7, wherein the high fat bee bread comprises a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread contains 1.5-2.5 w/v% protein-containing substances.

13. The method of any one of claims 1-7, wherein the high fat bee bread comprises a second liquid bee bread and a solid bee bread, wherein the second liquid bee bread contains 45-55 w/v% of saccharides.

14. The method of any one of claims 1-7, wherein the high fat bee bread comprises a liquid bee bread and a solid bee bread, wherein the solid bee bread contains 4-6 w/v% of oil.

15. The method according to any one of claims 1 to 7, wherein the edible oil is palm oil.

16. The method according to any one of claims 1 to 7, wherein in the modeling step, the mature bees are fed with the high fat bee bread in an incubator at a temperature of 30 to 40 ℃ and a humidity of 45 to 55%.

17. The method of claim 16, wherein in the model building step, the mature bees are fed the high fat bee bread for more than 7 days to build a type II diabetes bee model.

18. The method of any one of claims 1-7, wherein the mature bee is a 2-15 day old bee.

19. The method of any one of claims 1-7, wherein the mature bees are 5-9 day old bees.

20. The method of any one of claims 1-7, wherein the mature bee is a bee of the family honeybee.

21. The method of claim 20, wherein the mature bee is a bee or bumblebee.

22. Use of a bacterial type II diabetes bee model established according to the method of any one of claims 1-3 and/or a bacterial type II diabetes bee model established according to the method of any one of claims 4-7 in screening a medicament for treating type II diabetes.

23. Use of a bacterial bee model of type II diabetes established according to the method of any one of claims 1 to 3 and/or a bacterial bee model of type II diabetes established according to the method of any one of claims 4 to 7 for studying the mechanisms of diabetes related diseases.