CN115136928A

CN115136928A - Rapid modeling method for tree shrew type II diabetes

Info

Publication number: CN115136928A
Application number: CN202210785467.6A
Authority: CN
Inventors: 代解杰; 邱敏; 罕园园; 陆彩霞; 孙晓梅; 王文广; 李娜; 仝品芬; 郝佩琪
Original assignee: Institute of Medical Biology of CAMS and PUMC
Current assignee: Institute of Medical Biology of CAMS and PUMC
Priority date: 2022-07-05
Filing date: 2022-07-05
Publication date: 2022-10-04
Anticipated expiration: 2042-07-05
Also published as: CN115136928B

Abstract

The invention relates to a rapid modeling method for type II diabetes of tree shrews, which comprises the steps of selecting tree shrews, feeding the tree shrews for 12-15 days by adopting high-fat and high-sugar feed, and freely taking food and freely drinking water; on the 7 th to 9 th days of feeding, the tree shrews are dosed with streptozotocin at a dose of 90 to 110mg/kg; the high-fat high-sugar feed comprises the following components in parts by weight: 68-70 parts of basic feed, 9-11 parts of lard, 9-11 parts of white sugar, 9-11 parts of fructose and 0.9-1.2 parts of cholesterol. The method can make the tree shrew show persistent hyperglycemia, fasting blood glucose is more than or equal to 11.1mmol/L, urine glucose ++++, and can be used as a model for researching the pathogenesis of diabetes and complications thereof and the action mechanism of drugs. The method has the advantages of short period, simple molding method, low mortality and the like, can simulate the attack process of type II diabetes, has a phenotype close to that of human type II diabetes, and is easy to popularize and apply.

Description

Rapid modeling method for type II diabetes of tree shrews

Technical Field

The invention belongs to the technical field of animal model construction, and particularly relates to a rapid modeling method for type II diabetes of tree shrews.

Background

Type ii diabetes (T2 DM) is a multifactorial, metabolic disease characterized by hyperglycemia, poses a serious threat to human health, is associated with adequate compensation of insulin resistance and beta cell deficiency, resulting in a relative insulin deficiency, and chronic hyperglycemia causes a variety of complications, such as neuropathy, nephropathy, and retinopathy, and increases the risk of cardiovascular disease. The construction of the diabetes animal model has very important significance for researching the pathogenesis, preventing and diagnosing the diabetes animal model and screening new treatment medicines.

At present, the diabetes modeling method comprises diet induction, drug induction, genetic engineering construction and the like. The diet induction is mainly high-fat and high-sugar feed for feeding animals, is similar to the spontaneous diabetes of human beings, but has longer modeling time; the drug induction mainly adopts Alloxan (ALX) to induce islet beta cell death, but the ALX has great damage to liver and kidney and has high animal mortality; the animal model of type II diabetes constructed by genetic engineering is mainly single gene simulation, while type II diabetes is caused by a plurality of gene expression changes combined environment, genetic factors and the like, so that the generation and development process of type II diabetes in clinical practice is difficult to be completely simulated.

At present, animals for establishing diabetes comprise big mice, non-human primates, new Zealand rabbits, pigs, dogs and the like, and various directionally-related researches are carried out on the basis of diabetes of different species. Tree shrews, a small animal of the order Paniperus, have the characteristics of small size and rapid reproduction, are evolutionarily closer to nonhuman primates than rodents, are similar to humans in the nervous system, the visual system, the immune system and the metabolic system, and have been used for the study of models of ocular, optic nerve and metabolic related diseases and viral diseases. The basic physiological and biochemical indexes of the tree shrew blood are close to that of humans, the tree shrew has a spontaneous glycolipid metabolic abnormality, the metabolic system is similar to that of humans, the phenotypes of the tree shrew, such as ketosis, cataract and the like, are consistent with the human diabetes phenotype, and the pathological examination shows that beta cells of pancreatic islets of the tree shrew are deleted and are consistent with the pathogenesis of human diabetes, which indicates that the tree shrew is a potential experimental animal which can replace non-human primates in the research of metabolic related diseases.

The current tree shrew II type diabetes mellitus modeling method is shown in table 1, and as can be seen from table 1, a II type diabetes mellitus animal model which is simple in modeling method, rapid in modeling time, low in mortality and capable of stably simulating a human glycolipid metabolic pathway is not available at present, so that how to overcome the defects of the prior art is a problem which needs to be solved urgently in the technical field of animal model construction at present.

TABLE 1 Tree shrew II type diabetes mellitus modeling method summary table

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides a rapid modeling method for type II diabetes of tree shrews. The method can ensure that the tree shrew has persistent hyperglycemia; feeding the tree shrews with high-fat and high-sugar feed for 1 week, then feeding the tree shrews with STZ for 1 week to obtain II diabetic tree shrews; the method has high molding rate and short molding period.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

a rapid modeling method for type II diabetes of tree shrews is characterized in that the tree shrews are fed with high-fat and high-sugar feed for 12 to 15 days, and freely take food and drink water; on the 7 th to 9 th days of feeding, the tree shrews are dosed with streptozotocin at a dose of 90 to 110mg/kg;

the high-fat high-sugar feed comprises the following components in parts by weight: 68-70 parts of basic feed, 9-11 parts of lard, 9-11 parts of white sugar, 9-11 parts of fructose and 0.9-1.2 parts of cholesterol.

Further, it is preferable that the tree shrews are selected from healthy male tree shrews of 2 to 3 years old, and have a weight of 150g to 170g.

Further, it is preferable that the administration of streptozotocin to tree shrews is carried out after the tree shrews are fasted for 11-13 hours on days 6-8.

Further, preferably, the tree shrews are fed with high-fat and high-sugar feed for 14 days, and freely take the food and freely drink the water; fasting was started on the evening of day 7 after feeding, and after fasting for 12h, the tree shrews were dosed with streptozotocin at a dose of 100mg/kg.

Further, it is preferable that streptozotocin is formulated into a 2% solution and administered.

Further, it is preferable that STZ is dissolved in 0.1mmol/L citric acid buffer solution to prepare a 2% STZ solution, which is mixed, filtered through a 0.22 μm filtration membrane, and then administered.

Further, preferably, healthy male tree shrews of 2-3 years old are selected, the weight of the tree shrews is 150g-170g, and the tree shrews are fed with high-fat and high-sugar feed for 2 weeks; the high-fat high-sugar feed comprises the following components in parts by weight: 69 parts of basic feed, 10 parts of lard oil, 10 parts of white sugar, 10 parts of fructose and 1 part of cholesterol;

feeding the tree shrew with high-fat high-sugar feed for 1 week, and then feeding 100mg/kg of STZ medicine for no more than 10 minutes;

when the fasting blood sugar of the tree shrew is more than or equal to 11.1mmol/L and lasts for 4 weeks (the calculation is started when the blood sugar is more than or equal to 11.1), the urine sugar is more than or equal to + +, and the success establishment of the tree shrew type II diabetes model is determined.

Further, preferably, in the basic feed, crude protein is more than or equal to 160g/kg, crude fat is more than or equal to 40g/kg, crude fiber is less than or equal to 40g/kg, crude ash is less than or equal to 70g/kg, calcium is 8-12 g/kg, total phosphorus is 6-8 g/kg, and the weight ratio of calcium: the mass ratio of phosphorus is 1.2.

The result shows that after 2 weeks, the fasting blood sugar of the experimental group is more than or equal to 11.1mmol/L, the blood sugar of the experimental group is obviously different from the blood sugar of the control group, and the model of the type II diabetes is determined to be successfully established due to persistent hyperglycemia and urine sugar + + +. The weight is in a descending trend, and the comparison difference between the weight and the initial value of the weight per se in 10 weeks has statistical significance; the insulin level was increased and was statistically significant compared to the control group. After 4 weeks, the fur of the animal is dull, polydipsia, polyphagia and polyuria appear, and ulcer is formed at the tail and the feet in the later period; therefore, the rapid modeling of the type II diabetes of the tree shrew by adopting the high-fat high-sugar feed and the STZ medicine is feasible and can be used as an animal model for researching the pathogenesis, preventing and diagnosing the diabetes and screening new therapeutic medicines.

The high-fat high-sugar feed adopted by the invention is a 69% basic feed (wherein the basic feed comprises the following components of crude protein more than or equal to 160g/kg, crude fat more than or equal to 40g/kg, crude fiber less than or equal to 40g/kg, crude ash less than or equal to 70g/kg, calcium 8-12 g/kg, total phosphorus 6-8 g/kg, calcium is phosphorus 1.2. The high-fat high-sugar feed has good palatability, and increases the feed intake rate of animals. The high-fat high-sugar feed can not only induce the insulin resistance of animals, but also keep the animals in a hyperglycemia state. The high-fat high-sugar feed has the characteristics of low cost, simple feeding and processing and strong operability.

Of these, cholesterol is a conventional commercial product available from Macklin. Citric acid, sodium citrate and STZ are conventional commercial products, with citric acid and sodium citrate being available from aladdin and STZ from sigma.

The administration mode of the present invention is not particularly limited, and may be oral administration, injection, infusion, spray, etc.

The large dose of STZ (120 mg/kg) is administered before the team, the administration is carried out on 0 day, 7 days and 28 days, the blood sugar is increased after 3 weeks, the time consumption is long, and the experiment efficiency is greatly influenced. Prior to the present invention, comparative example 1 was fed with a high-fat and high-sugar diet and then STZ (100 mg/kg) was administered after 6 weeks, but the STZ was administered 2 to 3 times and the elevation of blood sugar occurred after 3 weeks, which took a long time. By improving the method of the comparative example 1, the invention adopts 1 week high-fat high-sugar feed to feed, then the STZ (100 mg/kg) is fed, then the 1 week high-fat high-sugar feed is fed, the animal glucose metabolism is disturbed in the 1 st week, the STZ medicine is immediately fed, and the 1 week high-fat high-sugar feed is fed again, so that the STZ medicine can not be self-regulated, the continuous hyperglycemia can be realized, 16 animals are selected in total, the molding is successful, the molding rate is 88%, the death rate is 0, the molding time is short, and the experimental efficiency can be improved. The STZ configuration method comprises the following steps: the solution of STZ in 0.1mmol/L citric acid buffer solution was prepared as a 2% STZ solution, mixed well and filtered through a 0.22 μm filtration membrane.

The STZ adopted by the invention has relatively low toxicity and higher molding rate than ALX, and can maintain a stable hyperglycemia state after molding, and the obtained model accords with the morbidity process of type II diabetes.

Compared with the prior art, the invention has the following beneficial effects:

the tree shrew II type diabetes mellitus animal model constructed by the invention has the advantages of simple modeling method, short modeling period, low mortality and the like, the blood sugar can be more than or equal to 11.1mmol/L in 3 weeks, the urine sugar is + + + + + + +, the modeling rate is 88 percent, the mortality is 0, the model can simulate the attack process of II type diabetes mellitus, the phenotype is close to that of human II type diabetes mellitus, and the model is easy to popularize and apply.

Drawings

FIG. 1 is a graph showing the results of fasting plasma glucose values of two groups of comparative example 1, wherein, compared with the experimental group and the control group, the value of Tp is less than 0.01;

FIG. 2 is a graph showing the results of the fasting body weight values of the two groups of comparative example 1, wherein, compared with the initial values of the experimental group, the value of P is less than 0.05 and the value of P is less than 0.01; tp is less than 0.0001;

FIG. 3 is a morphological observation (X100) of pancreatic pathological tissues in the experimental group of comparative example 1;

FIG. 4 is a graph showing the result of fasting blood glucose value of two groups of tree shrews in example 1, wherein, twai represents that Twai P is less than 0.0001 compared with the control group;

FIG. 5 is a graph showing the results of fasting body weight values for two groups in example 1, wherein P is less than 0.05 compared to the initial value for the test group;

FIG. 6 is a graph showing the results of two groups of 2-week urine glucose in example 1;

FIG. 7 is a graph showing the results of 2-month two fasting insulin levels of example 1, wherein, in comparison to the control group, P is less than 0.05;

FIG. 8 is the morphological observation (. Times.100) of pancreatic pathological tissues of the control group in example 1;

FIG. 9 is the morphological observation (. Times.100) of pancreatic pathological tissues in the experimental group of example 1;

FIG. 10 shows the morphological observation (. Times.100) of the kidney histopathology of the control group in example 1;

FIG. 11 shows the morphological observation (. Times.100) of the kidney histopathology in the experimental group of example 1.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.

In the examples, the methods were carried out in the conventional manner unless otherwise specified, and the materials or reagents used were commercially available reagents or reagents prepared in the conventional manner (edible lard and fructose were purchased from supermarkets) unless otherwise specified, and the percentages in the examples were by mass (75% alcohol cotton balls). In the embodiment, the tree shrew is provided by a germplasm resource center of the tree shrew in institute of medical biology of academy of medical sciences of China.

Comparative example 1

Combined STZ drug administration was performed using 6 weeks high fat high sugar diet feeding.

Selecting male healthy tree shrews of about 2-3 years old, weighing 150-170 g, freely taking food and drinking water, placing two water cups, keeping the room at 18-22 ℃ and 50-70% of humidity, and keeping the room ventilated and clean.

(1) After 1 week of adaptive feeding, the tree shrews were randomly divided into two groups, a control group and an experimental group.

(2) The control group was fed with a general feed (dog growth and reproduction feed, purchased from Australian cooperative feed Co., ltd., beijing, department of Fabaceae).

(3) The experimental group was fed with high-fat high-sugar diet (formula of high-fat high-sugar diet: 69% basal diet +10% lard +20% white sugar +1% cholesterol) twice a day, heated to 60 deg.C and fed for 6 weeks. The basic feed is experiment monkey maintenance feed purchased from cooperative pharmaceutical bioengineering, LLC of Jiangsu province.

(4) After 6 weeks, animals were fasted for 12h (8 pm in the previous day with feed removed 30 to 8 am the next day with no water deprivation, fasting glucose and body weight were measured, followed by the dosing of (5) and (6).

(5) The experimental group was prepared with STZ at a dose of 100mg/kg, and the administration was started after fasting blood glucose and body weight were measured and completed within 10 minutes. STZ configuration method: the solution of STZ in 0.1mmol/L citric acid buffer solution was prepared as a 2% STZ solution, mixed well and filtered through a 0.22 μm filtration membrane.

(6) The control group is given with 0.1mmol/L citrate buffer solution, and the administration is started after the fasting blood glucose and the body weight are measured; the preparation method of 0.1mmol/L citric acid buffer solution comprises the following steps: solution A: adding double distilled water into 2.1g of citric acid to 100ml; and B, liquid B: 2.94g of sodium citrate is added with double distilled water to 100ml; solution A and solution B were mixed in a ratio of 1:1, the pH was measured using an FE20K pH meter, and the pH was adjusted to 4.2 to 4.3 by adding the solution A or the solution B, sterilized by filtration at 0.22 μm, and stored in a refrigerator at 4 ℃ until use.

(7) The animals of the experimental group were fed a glass of brown sugar water (300 mg brown sugar plus 10ml water, 3%) in the afternoon, and some hours after the STZ administration, hypoglycemia was likely to occur.

(8) After administration, the experimental groups were fed with normal feed.

(9) The control group was fed with normal feed throughout the whole course.

(10) At the later stage, when the blood glucose did not rise to 11.1mmol/L, the STZ drug was continuously administered, and 2% of the STZ solution was dispensed at a dose of 100mg/kg, and the drug was administered within 10 minutes. The experimental group was given 2 times, with fasting glucose measured at week 7, with part of animals having fasting glucose below 11.1mmol/L, and with STZ immediately, and with part of animals having fasting glucose below 11.1mmol/L, with STZ immediately.

Example 1

Feeding with high-fat and high-sugar feed for 2 weeks, and administering with STZ medicine.

(2) The control group was fed with normal feed (dog growth and reproduction feed, purchased from Australian cooperative feed Co., ltd., beijing, department of Peking).

(3) The experimental group was fed with high-fat high-sugar diet (formula of high-fat high-sugar diet: 69% basal diet +10% lard +10% white sugar +10% fructose +1% cholesterol) twice a day, heated to 60 ℃ and fed with the diet for 1 week. The basic feed is a maintenance feed for experimental monkeys, and is purchased from cooperative medical bioengineering, LLC of Jiangsu province.

(4) After 1 week, animals were fasted for 12h (8 pm in the previous day with feed removed 30 to 8 am the next day with no water deprivation, fasting glucose and body weight were measured, followed by the dosing of (5) and (6).

(5) The experimental group was prepared with STZ at a dose of 100mg/kg, and the administration was started within 10 minutes after the fasting blood glucose and body weight were measured. STZ configuration method: the solution of STZ in 0.1mmol/L citric acid buffer solution was prepared as a 2% STZ solution, mixed well and filtered through a 0.22 μm filtration membrane.

(6) The control group was administered with an equivalent amount of 0.1mmol/L citrate buffer solution, and administration was started after fasting glucose and body weight were measured; the preparation method of the 0.1mmol/L citric acid buffer solution comprises the following steps: solution A: adding double distilled water into 2.1g of citric acid to 100ml; and B, liquid B: 2.94g of sodium citrate is added with double distilled water to 100ml; the ratio of solution A to solution B is 1:1, the FE20K pH meter measures the pH, and the pH is adjusted to 4.2-4.3 by adding the solution A or the solution B, sterilized by filtration at 0.22 μm, and stored in a refrigerator at 4 ℃ for later use.

(7) The animals of the experimental group were placed in a glass of brown sugar water (300 mg brown sugar plus 10ml water, 3%) in the afternoon, and a hypoglycemic condition was likely to occur several hours after STZ administration.

(8) After administration, the experimental groups were continued to be fed with high-fat high-sugar feed for 1 week, and then with normal feed after 1 week.

(9) The control group was fed with normal feed throughout the course.

The high-fat high-sugar feed provided by the invention is added with 10% of lard, 10% of white sugar, 10% of fructose and 1% of cholesterol except for 69% of basic feed of a germplasm resource center of a tree shrew. Different from the formula of the high-fat high-sugar feed used in the comparative example 1, the proportion of white sugar is reduced, and 10% of fructose is added, so that the palatability of the tree shrews is improved; compared with conventional high-fat high-sugar feed, the 10% lard added can cause blood fat level disorder, fatty acid is accumulated in liver, cholesterol synthesis is promoted, excessive free radicals are induced, and mitochondrial function is damaged and insulin resistance is caused; the feed is added with 10% of fructose, the fructose participates in lipid metabolism pathway of liver, the fatty acid synthesis efficiency is high, the liver inflammatory reaction is increased, the liver cell aging is accelerated, and the liver insulin sensitivity is reduced; the addition of 1% cholesterol can induce inflammation, oxidative stress, and promote disease formation.

And (3) evaluating a model:

(1) Measuring fasting blood glucose and body weight every week, using a fish leap 580 type blood glucose meter and matched test paper for measuring fasting blood glucose, after fixing an animal, cutting off hairs at a blood sampling part, wiping by using an alcohol cotton ball, wiping off alcohol by using a dry cotton ball, using a disposable blood taking needle, before using, firstly screwing off a protective cap (after using, placing the blood taking needle in a sharp instrument box), and after wiping off first drop of blood by using the dry cotton ball, measuring blood glucose, wherein the specific measurement reference method refers to the use instruction of the blood glucose meter.

(2) After 2 weeks, two groups of urine glucose were detected, ordinary urine glucose test paper was used for urine glucose determination, animal morning urine was dropped on the test paper, and the results were interpreted after 60 seconds. (3) After 2 months, 0.4mL of tail venous blood is taken after the experimental group and the control group are fasted for 12 hours, the experimental group and the control group are placed in an incubator at 37 ℃ for half an hour and then placed in a refrigerator at 4 ℃ for 2 hours, centrifugation (3000rpm, 10min) is carried out, serum and red blood cells are rapidly and carefully separated, an enzyme-linked immunosorbent assay (ELISA) is used for insulin determination, a tree shrew Insulin (INS) kit is purchased from Shanghai research Biotech limited company, the kit adopts a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA), INS is added into coated micropores which are coated with antibodies in advance, a specimen (the experimental group and the control group serum), an INS standard product and an INS detection antibody marked by HRP are sequentially added, the specimen is incubated and thoroughly washed, a substrate TMB is used for color development, the TMB is catalyzed to be blue by peroxidase and is converted into yellow under the action of acid, the color shade is positively correlated with the Insulin (INS) in the sample, and the sample concentration is calculated by measuring the absorbance (OD value under the wavelength of 450nm by an enzyme-labeling instrument. The method comprises the following specific steps: the desired panel was removed from the aluminum foil bag after equilibration for 20min at room temperature, and the remaining panels were sealed with a zip-lock bag and placed back at 4 ℃. All liquid components were thoroughly shaken before use. Setting standard substance holes and sample holes, wherein 50 mu L of standard substances with different concentrations are added into the standard substance holes, 10 mu L of samples are added into the sample holes to be detected, and 40 mu L of sample diluent is added into the sample holes to be detected (namely, the samples are diluted by 5 times); blank wells were not added. In addition to the blank wells, 100. Mu.L of detection antibody labeled with horseradish peroxidase (HRP) was added to each of the standard wells and the sample wells, the reaction wells were sealed with a sealing plate film, and incubated in an incubator at 37 ℃ for 60min. The liquid was discarded, blotted on absorbent paper, each well was filled with wash solution (dilution of 20 × wash buffer: distilled water diluted 1. Add stop solution 50. Mu.L to each well, measure the OD value of each well at a wavelength of 450nm within 15min. Drawing a standard curve: and in an Excel worksheet, drawing a linear regression curve of the standard substance by taking the concentration of the standard substance as an abscissa and taking the corresponding OD value as an ordinate, and calculating the concentration value of each sample according to a curve equation. Note: 20 XWash buffer, substrates A, B, stop buffer, etc. were all reagents in the Tree shrew Insulin (INS) kit.

(4) Pathological HE staining of pancreas and kidney: at week 10, animals were euthanized, animal pancreases were bluntly isolated and fixed with 4% paraformaldehyde 2After 4h, the liquid is changed, and after further fixing for 24h, the pancreas and kidney are respectively cut into 0.2X 0.3cm ³ Tissue blocks, routinely dehydrated, paraffin embedded. The method comprises the following specific steps: preparing a paraffin section: soaking the fixed specimen in 80% alcohol for 4h,95% alcohol I and II for 4h respectively, 100% alcohol for 2h respectively, and dimethylbenzene I and II for 15min respectively; paraffin embedding: placing the specimen in liquid paraffin I for 15min, paraffin II for 20min and paraffin III for 30-40min, and finally embedding the tissue block with paraffin; slicing: the slices were cut successively and the thickness of the slices was 4 μm. Drying in 60 deg.C oven for use. HE staining: dewaxing and hydrating: 100% dimethylbenzene I and II solution for 5min respectively; washing with distilled water, absolute ethanol for 1min,95% ethanol for 1min,85% ethanol for 1min,75% ethanol for 1min, washing with distilled water for 2min, hematoxylin for 5min, and washing with running water; separating color with 75% hydrochloric acid ethanol for 30s, and washing with running water; 1% eosin, 4min; performing gradient dehydration on 95% alcohol I and II and 100% alcohol I and II; the dimethylbenzenes I, II and III are transparent; taking out the slices from xylene, slightly drying, and sealing the slices with neutral gum; and placing the dyed and sealed section under a microscope for microscopic observation.

And (3) model verification: the fasting blood sugar is more than or equal to 11.1mmol/L and lasts for 4 weeks, the urine sugar is more than or equal to + + and insulin resistance is taken as the diagnostic standard of type II diabetes.

Results and analysis

(1) Comparative example 1 fasting blood glucose measurement results are shown in fig. 1, and it can be seen from fig. 1 that, compared with the control group, blood glucose of the test group tends to increase during the period of high-fat high-sugar feed feeding, but blood glucose returns to a normal value at

week

2 and 3, and blood glucose does not increase after the STZ drug is administered 3 days after the STZ drug is administered, so that blood glucose is significantly increased at week 9 (. About.p < 0.01) after the STZ drug is administered 1 time at a dose of 100mg/kg and no blood glucose is increased at week 8, and the STZ drug is continuously administered 1 time at a dose of 100mg/kg. Although the method can increase the blood sugar of the tree shrew, the early period consumes long time and the molding time is long, and the method is not beneficial to researching the diabetes-related complications.

(2) Comparative example 1 fasting body weight measurement results are shown in fig. 2, and as can be seen from fig. 2, the body weight of the control group did not change significantly; compared with the initial value of the weight of the tree shrew in the empty stomach, the weight of the tree shrew is reduced at the 2 nd week, the difference has statistical significance (P is less than 0.05), the palatability of the first high-fat high-sugar feed is not strong, the animal remains more, and the weight is rapidly reduced, so the tree shrew is fed by a common feed (a dog growth and reproduction feed purchased from Australian cooperative feeds Co., ltd., beijing Ke.) in the afternoon to ensure the normal survival of the animal, and the weight is reduced after the STZ medicament is fed in the 7 th week, and the difference has statistical significance.

(3) Comparative example 1 HE staining observation of pancreatic pathology is shown in fig. 3, and fig. 3 shows that islets are rare, and transudates are seen in the lumen of the pancreatic duct, showing that the STZ drug destroys the islet cells.

(4) The results of fasting blood glucose measurement of the two groups of experiments in example 1 of the invention are shown in fig. 4, and as can be seen from fig. 4, compared with the control group, the blood glucose of the experimental group is significantly increased, and the value is more than or equal to 11.1mmol/L, which has significant difference, that is, the blood glucose of the tree shrew can be increased by combining high-fat high-sugar feed and STZ administration.

(5) The results of the two fasting body weight measurements of example 1 of the present invention are shown in fig. 5, and it can be seen from fig. 5 that the body weight of the control group does not fluctuate much, the body weight of the experimental group is in a decreasing trend, and the difference between the body weight of the experimental group and the initial value in 10 weeks has statistical significance (. About.P < 0.05), which is consistent with the body weight decrease in the late stage of diabetes.

(6) The results of the two sets of urine glucose measurements of example 1 are shown in FIG. 6. As can be seen in FIG. 6, the control set is negative (-) and the experimental set is positive (+++).

(7) The results of the fasting insulin levels in the two groups of example 1 are shown in fig. 7, and it can be seen from fig. 7 that the fasting insulin levels in the experimental group were significantly increased compared to the control group, and the difference was statistically significant (. P < 0.05), indicating that the tree shrews were in an insulin resistant state.

(8) The results of the histological morphology of the pancreas of the control group in example 1 are shown in fig. 8, and fig. 8 shows that the pancreas of the control group has normal islet morphology, abundant acinar cells, close arrangement, clear boundary and normal cell morphology.

(9) The morphological observation results of the pancreas tissues of the experimental group of example 1 are shown in FIG. 9, and FIG. 9 shows the pancreas tissues of the experimental group, the islet occasional necrosis of islet cells in the visual field, the deep staining of the karyocyte, and the irregular shape (black arrows).

(10) The histological observation result of the kidney of the control group in example 1 is shown in fig. 10, and fig. 10 shows that the kidney of the control group has the tissue morphology, uniform glomerulus distribution, uniform cell number and stroma in the glomerulus, rounded and plump renal tubular epithelial cells, and regular brush-like edges.

(11) The histological observation of kidney in the experimental group of example 1 is shown in fig. 11, and fig. 11 shows the morphology of kidney tissue in the experimental group, focal renal tubular atrophy, increased cytoplasmic alkalophilicity, luminal narrowing (black arrows), more tubular dilation, and flattened epithelium (white arrows) were observed in the kidney tissue.

In conclusion, the method used by the invention can successfully and rapidly establish the type II diabetes of the tree shrew.

Example 2

A method for quickly modeling type II diabetes of tree shrews is characterized in that the tree shrews are fed with high-fat and high-sugar feed for 13 days, and freely take food and freely drink water; on the 8 th day of feeding, the tree shrews were dosed with 100mg/kg of streptozotocin;

the high-fat high-sugar feed comprises the following components in parts by weight: 68-70 parts of basic feed, 10 parts of lard oil, 10 parts of white sugar, 10 parts of fructose and 1 part of cholesterol.

Example 3

A method for quickly modeling type II diabetes of tree shrews is characterized in that the tree shrews are fed with high-fat and high-sugar feed for 12 days, and freely take food and freely drink water; on day 7 of feeding, the tree shrews were dosed with streptozotocin at a dose of 90mg/kg;

the high-fat high-sugar feed comprises the following components in parts by weight: 68 parts of basic feed, 9 parts of lard, 9 parts of white sugar, 9 parts of fructose and 0.9 part of cholesterol.

Selecting healthy male tree shrews of 2-3 years old with the weight of 150-170 g.

Before administration, the tree shrews are administered with streptozotocin after fasting for 11 h.

Example 4

A rapid modeling method for type II diabetes of tree shrews is characterized in that the tree shrews are fed with high-fat and high-sugar feed for 15 days, and freely take food and drink water; on the 9 th day of feeding, the tree shrews are dosed with streptozotocin at a dose of 110mg/kg;

the high-fat high-sugar feed comprises the following components in parts by weight: 70 parts of basic feed, 11 parts of lard oil, 11 parts of white sugar, 11 parts of fructose and 1.2 parts of cholesterol.

Before administration, the tree shrews are administered with streptozotocin after fasting for 13 hours.

When the drug is administered, streptozotocin is formulated into a 2% solution and then administered.

The solution of STZ was dissolved in 0.1mmol/L citric acid buffer solution to prepare a 2% STZ solution, mixed well, filtered through a 0.22 μm filtration membrane, and then administered.

In the basic feed, the crude protein is more than or equal to 160g/kg, the crude fat is more than or equal to 40g/kg, the crude fiber is less than or equal to 40g/kg, the crude ash content is less than or equal to 70g/kg, the calcium content is 8g/kg, and the total phosphorus content is 6g/kg.

Example 5

A method for quickly modeling type II diabetes of tree shrews is characterized in that the tree shrews are fed with high-fat and high-sugar feed for 14 days, and freely take food and freely drink water; on the 7 th to 9 th days of feeding, the tree shrews are dosed with 100mg/kg of streptozotocin;

the high-fat high-sugar feed comprises the following components in parts by weight: 69 parts of basic feed, 10 parts of lard oil, 10 parts of white sugar, 10 parts of fructose and 1 part of cholesterol.

Feeding the tree shrews with high-fat and high-sugar feed for 14 days, and freely taking the tree shrews and freely drinking water; fasting was started on the evening of day 7 after feeding, and after fasting for 12h, the tree shrews were dosed with streptozotocin at a dose of 100mg/kg.

Dissolving STZ in 0.1mmol/L citric acid buffer solution to 2%.

In the basic feed, crude protein is more than or equal to 160g/kg, crude fat is more than or equal to 40g/kg, crude fiber is less than or equal to 40g/kg, crude ash is less than or equal to 70g/kg, calcium is 12g/kg, and total phosphorus is 8g/kg.

Example 6

A quick modeling method for type II diabetes of tree shrews comprises the following specific steps:

selecting healthy male tree shrews of 2-3 years old, weighing 150-170 g, and feeding for 2 weeks by adopting high-fat high-sugar feed; the high-fat high-sugar feed comprises the following components in parts by weight: 69 parts of basic feed, 10 parts of lard oil, 10 parts of white sugar, 10 parts of fructose and 1 part of cholesterol;

when the fasting blood glucose of the tree shrew is more than or equal to 11.1mmol/L and lasts for 4 weeks, the urine glucose is more than or equal to + +, and the success establishment of the tree shrew type II diabetes model is determined.

In the basic feed, the crude protein is more than or equal to 160g/kg, the crude fat is more than or equal to 40g/kg, the crude fiber is less than or equal to 40g/kg, the crude ash content is less than or equal to 70g/kg, the calcium content is 8.4g/kg, and the total phosphorus content is 7g/kg.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A rapid modeling method for tree shrew type II diabetes is characterized in that: feeding the tree shrews with high-fat and high-sugar feed for 12-15 days, and freely taking the tree shrews and freely drinking water; on the 7 th to 9 th days of feeding, the tree shrews are dosed with streptozotocin at a dose of 90 to 110mg/kg;

2. The rapid modeling method for tree shrew type II diabetes mellitus according to claim 1, characterized in that: selecting healthy male tree shrews of 2-3 years old with the weight of 150-170 g.

3. The rapid modeling method for type II diabetes of tree shrew according to claim 1, which is characterized in that: after the tree shrews are fed on the 6 th to 8 th days and the fasting time is 11 to 13 hours, the tree shrews are dosed with streptozotocin.

4. The rapid modeling method for tree shrew type II diabetes mellitus according to claim 1, characterized in that: feeding the tree shrews with high-fat and high-sugar feed for 14 days, and freely taking the tree shrews and freely drinking water; after feeding for 12 hours on an empty stomach, the tree shrews were administered with streptozotocin at a dose of 100mg/kg.

5. The rapid modeling method for tree shrew type II diabetes mellitus according to claim 1, characterized in that: when the drug is administered, streptozotocin is formulated into a 2% solution and then administered.

6. The rapid modeling method for tree shrew type II diabetes mellitus according to claim 5, characterized in that: dissolving STZ in 0.1mmol/L citric acid buffer solution to 2%.

7. The rapid modeling method for type II diabetes of tree shrew according to claim 1, which is characterized in that:

feeding the tree shrew with the high-fat and high-sugar feed for 1 week, and then feeding 100mg/kg of STZ medicine for no more than 10 minutes;

when the fasting blood sugar of the tree shrew is more than or equal to 11.1mmol/L and lasts for 4 weeks, the urine sugar is more than or equal to + +, and the success establishment of the tree shrew II type diabetes model is determined.

8. The rapid modeling method for tree shrew type II diabetes mellitus according to claim 1, characterized in that: in the basic feed, crude protein is more than or equal to 160g/kg, crude fat is more than or equal to 40g/kg, crude fiber is less than or equal to 40g/kg, crude ash is less than or equal to 70g/kg, calcium is 8-12g/kg, total phosphorus is 6-8g/kg, and calcium: the mass ratio of phosphorus is 1.2 to 1.5.