CN115136928B

CN115136928B - Rapid modeling method for tree shrew type II diabetes

Info

Publication number: CN115136928B
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: 2023-07-28
Anticipated expiration: 2042-07-05
Also published as: CN115136928A

Abstract

The invention relates to a rapid modeling method for tree shrew type II diabetes, which comprises the steps of selecting tree shrew, feeding the tree shrew for 12-15 days by adopting high-fat high-sugar feed, and freely taking food and drinking water; on days 7-9 of feeding, the tree shrew is dosed with streptozotocin at a dose of 90-110mg/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 cause the tree shrew to show persistent hyperglycemia, the fasting blood glucose is more than or equal to 11.1mmol/L, and the urine glucose is++, and can be used as a model for researching pathogenesis of diabetes and complications thereof and drug action mechanism. The method has the advantages of short period, simple modeling method, low mortality rate and the like, can imitate the disease course of type II diabetes, has a phenotype close to human type II diabetes, and is easy to popularize and apply.

Description

Rapid modeling method for tree shrew type II diabetes

Technical Field

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

Background

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

The current diabetes modeling method comprises diet induction, drug induction, genetic engineering construction and the like. The diet induction is mainly that the animals are fed with high-fat and high-sugar feed, which is similar to the spontaneous diabetes of human beings, but the modeling time is longer; the medicine induction mainly adopts tetraoxypyrimidine (ALX) to induce islet beta cell death, but ALX has larger damage to liver and kidney and higher animal death rate; the animal model of type II diabetes constructed by genetic engineering is mainly single gene simulation, and the type II diabetes is caused by the combination of a plurality of gene expression changes, the combined environment, genetic factors and the like, so that the occurrence and development processes of the type II diabetes in clinical practice are difficult to completely simulate.

Animals for which diabetes mellitus is established at present are big and small mice, non-human primates, new Zealand rabbits, pigs, dogs and the like, and various direction-related researches are carried out based on diabetes mellitus of different species. The tree shrew, which is a small animal belonging to the order of the climbing shrew, has the characteristics of small size and rapid propagation, is evolutionarily closer to non-human primates than rodents, is similar to human in nervous system, visual system, immune system and metabolic system, and has been used for research on eye, optic nerve and metabolic related diseases and viral disease models. The physiological and biochemical indexes of the blood base of the tree shrew are close to those of human beings, the tree shrew has spontaneous abnormal glycolipid metabolism, the metabolic system is similar to that of human beings, the phenotypes of the tree shrew, such as ketosis, cataract and the like, are consistent with that of human diabetes, pathological examination shows that beta cells of islets of the tree shrew are absent, and the pathological examination shows that the tree shrew is consistent with pathogenesis of human diabetes, and the tree shrew is an experimental animal which is potentially possible to replace non-human primates in research of metabolic related diseases.

The existing tree shrew type II diabetes modeling method is shown in table 1, and as can be seen from table 1, the existing tree shrew type II diabetes animal model which has the advantages of simple modeling method, rapid modeling time, low mortality and stability and simulates the human glycolipid metabolic pathway is not available, so how to overcome the defects of the prior art is a problem which needs to be solved in the technical field of the existing animal model construction.

TABLE 1 summary of tree shrew type II diabetes modeling methods

Disclosure of Invention

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

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

a rapid modeling method for tree shrew type II diabetes comprises feeding the tree shrew with high-fat and high-sugar feed for 12-15 days, and freely taking food and drinking water; on days 7-9 of feeding, the tree shrew is dosed with streptozotocin at a dose of 90-110mg/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 preferred that the tree shrew is selected from healthy male tree shrews of 2-3 years old, and has a weight of 150g-170g.

Further, it is preferred that the tree shrew is administered with streptozotocin after the tree shrew has been fasted for 11-13 hours on days 6-8 of feeding.

Further, preferably, the tree shrew is fed with the high-fat and high-sugar feed for 14 days, and is free to eat and drink water; the feeding is started to be fasted at night on the 7 th day, and after 12 hours of fasting, the tree shrew is dosed with 100mg/kg by streptozotocin.

Further, it is preferable that, at the time of administration, streptozotocin is formulated as a 2% solution and then administered.

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

Further, preferably, healthy male tree shrew of 2-3 years old is selected, the weight of the tree shrew is 150-170 g, and the tree shrew is 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, 10 parts of white sugar, 10 parts of fructose and 1 part of cholesterol;

after feeding the tree shrew high-fat high-sugar feed for 1 week, 100mg/kg of STZ drug is given for no more than 10 minutes;

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

Further, it is preferable that in the basic feed, crude protein is not less than 160g/kg, crude fat is not less than 40g/kg, crude fiber is not less than 40g/kg, crude ash is not more than 70g/kg, calcium is 8-12 g/kg, total phosphorus is 6-8 g/kg, calcium: the mass ratio of the phosphorus is 1.2:1-1.5:1.

The results show that after 2 weeks, the fasting blood glucose of the experimental group is more than or equal to 11.1mmol/L, the fasting blood glucose is obviously different from that of the control group, and the continuous hyperglycemia and the urine glucose++ are used for determining that the type II diabetes model is successfully established. The weight is in a descending trend, and the comparison difference between the weight of 10 weeks and the initial value of the weight has statistical significance; the increase in insulin levels was statistically significant compared to the control group. After 4 weeks, the animal fur is matted, polydipsia, polyphagia and diuresis occur, and ulcers are formed at the tail and foot of the later period; it can be seen that the rapid modeling of tree shrew type II diabetes by combining high-fat high-sugar feed with STZ drugs is feasible, and can be used as an animal model for researching pathogenesis of diabetes, preventing, diagnosing and screening new therapeutic drugs.

The formula of the high-fat high-sugar feed adopted by the invention is 69% basic feed (wherein the basic feed comprises the components of crude protein not less than 160g/kg, crude fat not less than 40g/kg, crude fiber not less than 40g/kg, crude ash not more than 70g/kg, calcium 8-12 g/kg, total phosphorus 6-8 g/kg, calcium: phosphorus 1.2:1-1.5:1), 10% lard, 10% white sugar, 10% fructose and 1% cholesterol. The high-fat high-sugar feed has good palatability and increases the feeding rate of animals. The high-fat and high-sugar feed can induce insulin resistance of animals and also can keep the animals in a hyperglycemic state. The high-fat high-sugar feed has the characteristics of low cost, simple feeding processing and strong operability.

Among them, 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 purchased from aladin and STZ being purchased from sigma.

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

The administration of large dose STZ (120 mg/kg) before team, administration on days 0, 7 and 28, and blood sugar rise after 3 weeks, which takes a little longer time, and the experimental efficiency is greatly affected. Prior to the present invention, comparative example 1 was fed with an excessively high-fat high-sugar feed for 6 weeks, STZ (100 mg/kg) was administered, but STZ was administered 2 to 3 times, and blood sugar elevation occurred after 3 weeks, for a long period of time. According to the method for improving the comparative example 1, STZ (100 mg/kg) is fed after feeding with 1-week high-fat high-sugar feed, then the 1-week high-fat high-sugar feed is fed, animal sugar metabolism is disturbed in week 1, STZ medicine is immediately fed, and the 1-week high-fat high-sugar feed is fed again, so that the animal sugar metabolism can not be regulated by oneself, continuous hyperglycemia can be caused, 16 animals can be selected in total, 14 animals are successfully molded, 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: STZ was dissolved in 0.1mmol/L citric acid buffer to prepare a 2% STZ solution, which was then mixed and filtered through a 0.22 μm filter membrane.

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

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

the tree shrew type II diabetes animal model constructed by the invention has the advantages of simple modeling method, short modeling period, low death rate and the like, can lead the blood sugar to be more than or equal to 11.1mmol/L, the urinary sugar to be++, the modeling rate to be 88 percent and the death rate to be 0 in the 3 rd week, can imitate the incidence of type II diabetes, has a phenotype close to human type II diabetes, and is easy to popularize and apply.

Drawings

FIG. 1 is a graphical representation of results of two sets of fasting blood glucose values for comparative example 1, showing P < 0.01 compared to the experimental and control sets;

FIG. 2 is a graph showing the results of two sets of fasting weight values of comparative example 1, where P < 0.05 and P < 0.01, respectively, compared to the initial values of the experimental set; p is less than 0.0001;

FIG. 3 is a morphological observation of pancreatic pathology of the experimental group of comparative example 1 (. Times.100);

FIG. 4 is a graphical representation of the results of fasting blood glucose values for two sets of tree shrew according to example 1, showing P < 0.0001 compared to the control;

FIG. 5 is a graph showing the results of two sets of fasting body weight values of example 1, with P < 0.05 compared to the initial values of the experimental set;

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

FIG. 7 is a graphical representation of results of two sets of fasting insulin levels at 2 months of example 1, showing P < 0.05 compared to the control;

FIG. 8 is a morphological observation of pancreatic pathology of the control group of example 1 (. Times.100);

FIG. 9 is a morphological observation of pancreatic pathology of the experimental group of example 1 (. Times.100);

FIG. 10 is a morphological observation of kidney pathology of the control group of example 1 (. Times.100);

FIG. 11 is a morphological observation of kidney pathology in the experimental group of example 1 (. Times.100).

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 present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.

The methods in the examples are all conventional methods unless otherwise specified, and the materials or reagents used are all conventional commercial reagents or reagents prepared by conventional methods (edible lard and fructose are purchased from supermarkets) unless otherwise specified, and the percentages in the examples are all mass percent (75% alcohol cotton balls). In the examples, the tree shrew is provided by the tree shrew germplasm resource center of the medical biology institute of academy of medical science.

Comparative example 1

The drug administration is carried out by adopting 6 weeks of high-fat high-sugar feed feeding and combining STZ drugs.

Selecting male healthy tree shrew of about 2-3 years old, taking 150-170 g of body weight, freely taking food and drinking water, placing two water cups, keeping room temperature at 18-22 ℃ and humidity at 50-70%, and keeping indoor ventilation and clean.

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

(2) The control group was fed with normal diet (canine growth and reproduction diet, available from beijing australian synergetic diet limited).

(3) The experimental group is fed with high-fat high-sugar feed for 6 weeks (the formula of the high-fat high-sugar feed is 69% basic feed, 10% lard, 20% white sugar and 1% cholesterol), and the feed is fed twice a day and heated to 60 ℃. The basic feed is an experimental monkey maintenance feed and is purchased from Jiangsu province collaborative medical bioengineering Limited liability company.

(4) After 6 weeks, animals were fasted for 12 hours (8:30 withdrawal of feed from the evening of the previous day to 8:30 the morning of the next day), were not deprived of water, and were tested for fasting blood glucose and body weight, followed by administration of (5) and (6).

(5) The experimental group was now dosed with STZ at 100mg/kg, and dosing was started after fasting blood glucose and body weight were measured, and was completed within 10 minutes. The STZ configuration method comprises the following steps: STZ was dissolved in 0.1mmol/L citric acid buffer to prepare a 2% STZ solution, which was then mixed and filtered through a 0.22 μm filter membrane.

(6) The control group is given with equal amount of 0.1mmol/L citric acid buffer solution, and administration is started after the fasting blood glucose and the body weight are measured; preparation method of 0.1mmol/L citric acid buffer solution: and (3) solution A: 2.1g of citric acid and double distilled water are added to 100ml; and (2) liquid B: sodium citrate 2.94g is added with double distilled water to 100ml; the solution A and the solution B are prepared according to the following proportion of 1:1, and the pH is measured by using an FE20K pH meter, and the pH is adjusted to be 4.2-4.3,0.22 mu m by supplementing the solution A or the solution B, filtered and sterilized, and stored in a refrigerator at 4 ℃ for later use.

(7) Surglycemic levels may occur several hours after STZ administration by placing a cup of brown sugar water (300 mg brown sugar plus 10ml water, 3%) in the afternoon of the experimental animals.

(8) After administration, the experimental group was fed with normal feed.

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

(10) And if the later blood sugar is not increased to 11.1mmol/L, continuing to administer the STZ medicament, preparing a 2% STZ solution, and completing administration within 10 minutes at a dosage of 100mg/kg. The experimental group was supplemented with 2 times, and the fasting blood glucose was measured at week 7, and the STZ drug was immediately supplemented with a fraction of animals having a fasting blood glucose below 11.1 mmol/L.

Example 1

The feed is fed for 2 weeks by adopting high-fat and high-sugar feed, and is combined with STZ medicine for administration.

(2) The control group was given normal feed feeding (using canine growth and reproduction feed, purchased from beijing australian synergetic feed limited).

(3) The experimental group is fed with high-fat high-sugar feed for 1 week (the formula of the high-fat high-sugar feed is 69% basic feed, 10% lard, 10% white sugar, 10% fructose and 1% cholesterol), and the feed is fed twice a day and heated to 60 ℃. The basic feed is an experimental monkey maintenance feed and is purchased from Jiangsu province collaborative medical bioengineering Limited liability company.

(4) After 1 week, animals were fasted for 12 hours (8:30 withdrawal of feed from the evening of the previous day to 8:30 the morning of the next day), were not deprived of water, and were tested for fasting blood glucose and body weight, followed by administration of (5) and (6).

(6) The control group is given with equal amount of 0.1mmol/L citric acid buffer solution, and administration is started after the fasting blood glucose and the body weight are measured; preparation method of 0.1mmol/L citric acid buffer solution: and (3) solution A: 2.1g of citric acid and double distilled water are added to 100ml; and (2) liquid B: sodium citrate 2.94g is added with double distilled water to 100ml; the solution A and the solution B are prepared according to the following proportion of 1:1, and the pH value is measured by an FE20K pH meter, and the pH value is adjusted to be 4.2-4.3,0.22 mu m by supplementing the solution A or the solution B, filtered and sterilized, and stored in a refrigerator at 4 ℃ for later use.

(7) Animals of the experimental group were put in a glass of brown sugar water (300 mg brown sugar in 10ml water, 3%) in the afternoon, and hypoglycemia was likely to occur after STZ administration for several hours.

(8) After administration, the experimental group was fed with high-fat and high-sugar feed for 1 week, and after 1 week, with normal feed.

(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% lard, 10% white sugar, 10% fructose and 1% cholesterol except 69% basic feed of the tree shrew germplasm resource center. Compared with the formula of the high-fat high-sugar feed used in the comparative example 1, the formula is different in that the proportion of white sugar is reduced, 10% of fructose is added, and the palatability of the tree shrew is improved; compared with the conventional high-fat and high-sugar feed, the addition of 10% lard can cause disturbance of blood lipid level, accumulation of fatty acid in liver, promotion of cholesterol synthesis, induction of excessive free radicals, and damage of mitochondrial function and insulin resistance; the feed is added with 10% of fructose, the fructose participates in lipid metabolism pathways of livers, the fatty acid synthesis efficiency is high, and the liver inflammatory reaction can be increased, the liver cell aging can be accelerated, and the liver insulin sensitivity can be reduced; 1% cholesterol is added to induce inflammation and oxidative stress, and promote disease formation.

Model evaluation:

(1) The method comprises the steps of measuring fasting blood glucose and body weight every week, measuring the fasting blood glucose by using a fish-jump 580 type blood glucose meter and matched test paper, cutting wool at a blood sampling part after an animal is fixed, wiping an alcohol cotton ball, wiping the alcohol cotton ball, using a dry cotton ball to wipe the alcohol, using a disposable blood taking needle, screwing a protective cap (putting the blood taking needle into a sharp instrument box after using), measuring blood glucose after the dry cotton ball wipes first blood drop, and referring to a blood glucose meter using instruction by a specific measurement reference method.

(2) After 2 weeks, two groups of urine glucose were detected, and the results were interpreted 60 seconds later by dropping animal morning urine on a common urine glucose test paper. (3) After 2 months, taking 0.4mL of tail vein blood after the experimental group and the control group are fasted for 12 hours, placing the tail vein blood in a 37 ℃ incubator for half an hour, placing the incubator for 2 hours, centrifuging (3000 rpm,10 minutes), rapidly and carefully separating serum and red blood cells, performing insulin determination by using an enzyme-linked immunosorbent (ELISA), obtaining tree shrew Insulin (INS) kit from Shanghai research and development biotechnology company, obtaining the kit by using a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA), adding a specimen (serum of the experimental group and the control group), an INS standard substance and an INS detection antibody marked by HRP into coated micropores in advance, performing incubation and thorough washing, developing by using a substrate TMB, converting TMB into blue under the catalysis of peroxidase, converting the color to be finally yellow under the action of acid, and measuring absorbance (OD value) of the insulin in a sample at a wavelength of 450nm by using an ELISA, and calculating the concentration of the sample. The method comprises the following specific steps: the required strips were removed from the aluminium foil bags after 20min equilibration at room temperature and the remaining strips were sealed with a self-sealing bag and returned to 4 ℃. All liquid components were thoroughly shaken before use. Setting a standard substance hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are respectively added into the standard substance hole, 10 mu L of sample is added into a sample Kong Xian to be tested, and 40 mu L of sample diluent (namely 5 times of sample dilution) is added into the sample to be tested; blank holes are not added. In addition to the blank wells, 100. Mu.L of horseradish peroxidase (HRP) -labeled detection antibody was added to each of the standard wells and sample wells, and the reaction wells were sealed with a sealing plate membrane and incubated at 37℃for 60min. Removing liquid, beating on absorbent paper, filling washing liquid (dilution of 20 Xwashing buffer: distilled water is diluted according to 1:20, namely 1 part of 20 Xwashing buffer is added with 19 parts of distilled water), standing for 1min, throwing off the washing liquid, beating on absorbent paper, repeating the plate washing for 5 times (plate washing can be performed), adding 50 mu L of substrate A, B to each hole, and incubating for 15min at 37 ℃ in dark place. The OD of each well was measured at a wavelength of 450nm by adding 50. Mu.L of stop solution to each well for 15min. Drawing a standard curve: in an Excel worksheet, standard substance concentration is taken as an abscissa, a corresponding OD value is taken as an ordinate, a standard substance linear regression curve is drawn, and each sample concentration value is calculated according to a curve equation. And (3) injection: the 20 Xwashing buffer, the substrate A, B, the stop solution and the like are all reagents in the tree shrew Insulin (INS) kit.

(4) Pancreatic and renal pathology HE staining: at week 10, animals were euthanized, pancreas of animals was blunt-isolated, fixed with 4% paraformaldehyde for 24 hours, and after further fixation for 24 hours, pancreas and kidney were excised by 0.2X0.2X0.3 cm, respectively ³ Tissue mass, conventional dehydration, paraffin embedding. The method comprises the following specific steps: paraffin sections were prepared: soaking the fixed specimen in 80% alcohol for 4h,95% alcohol I and II for 4h,100% alcohol for 2h, and xylene I and II for 15min; and (3) paraffin embedding: placing the specimen in liquid paraffin I for 15min, paraffin II for 20min, and paraffin III for 30-40min, and embedding the tissue block with paraffin; slicing: the sections were serially cut, and the thickness of the sheet was 4. Mu.m. Drying in oven at 60deg.C. HE staining: dewaxing and hydrating: 100% xylene I and II solutions for 5min each; distilled water washing, absolute ethyl alcohol 1min,95% alcohol 1min,85% alcohol 1min,75% alcohol 1min, distilled water washing for 2min, hematoxylin washing for 5min, and running water washing; color separation for 30s with 75% ethanol hydrochloride, and flushing with running water; 1% eosin for 4min; gradient dehydration is carried out on 95% alcohol I and II and 100% alcohol I and II; the dimethylbenzene I, the dimethylbenzene II and the dimethylbenzene III are transparent; taking out the slice from the dimethylbenzene, airing the slice, and sealing the slice with neutral resin; and placing the stained and sealed slice under a microscope for microscopic observation.

Model verification: the fasting blood glucose is more than or equal to 11.1mmol/L, and the duration is 4 weeks, the urine glucose is more than or equal to++ and the insulin resistance is used as the diagnosis standard of the type II diabetes.

Results and analysis

(1) As shown in fig. 1, compared with the control group, the blood sugar of the experimental group had a tendency to rise during the feeding period of the high-fat and high-sugar feed, but the blood sugar was recovered to the normal value at week 2 and week 3, and we had no significant change in blood sugar of the control group due to the administration of STZ drug at week 7, and the blood sugar was not raised after 3 days of STZ drug administration, so that the dosage of STZ drug was 100mg/kg for 1 time, the blood sugar was not raised at week 8, the STZ drug was continued to be administered for 1 time, the dosage was 100mg/kg, and the blood sugar was significantly raised at week 9 (.times.p < 0.01). Although the method increases the blood sugar of the tree shrew, the early period is long in time consumption and long in modeling time, and is not beneficial to researching the related complications of diabetes.

(2) Comparative example 1 the results of the fasting body weight measurement are shown in fig. 2, and as can be seen from fig. 2, there was no significant change in the body weight of the control group; the fasting weight of the tree shrew is compared with the initial value of the tree shrew, the weight is reduced at the 2 nd week, the difference has statistical significance (P is less than 0.05), the palatability of the high-fat and high-sugar feed for the first time is not strong, the animal residue is more, and the weight is reduced rapidly, so that the tree shrew is fed by common feed (the dog growth and propagation feed is purchased from Australian feed Co., ltd. In Beijing) in the afternoon, the normal survival of the animal is ensured, the weight is reduced after the STZ drug is fed at the 7 th week, and the difference has statistical significance.

(3) Comparative example 1 pancreatic pathology HE staining the results are shown in fig. 3, and fig. 3 shows that islets are rare, the pancreatic duct lumen sees exudates, and STZ drug damages islet cells.

(4) The results of the two groups of fasting blood glucose measurement in the experiment of the embodiment 1 of the invention are shown in figure 4, and compared with the control group, the blood glucose of the experiment group is obviously increased, the numerical value is not less than 11.1mmol/L, and the blood glucose of the tree shrew can be increased by combining the administration of the high-fat high-sugar feed and the STZ.

(5) As can be seen from fig. 5, the results of the two fasting body weight measurements in example 1 of the present invention are shown in fig. 5, the body weight of the control group is not greatly fluctuated, 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 (P < 0.05) and accords with the body weight decrease in the later period of diabetes.

(6) The results of the two urine glucose determinations of example 1 are shown in fig. 6, and as can be seen from fig. 6, control urine glucose is negative (-), urine sugar of experimental group positive (+++).

(7) The results of the fasting insulin levels of the two groups of example 1 are shown in fig. 7, and it can be seen from fig. 7 that the fasting insulin levels of the experimental group are significantly elevated compared with the control group, and the difference has a statistical significance (P < 0.05), indicating that the tree shrew is in an insulin resistant state.

(8) The pancreas histomorphology observation result of the control group of the example 1 is shown in fig. 8, and fig. 8 shows that the pancreas of the control group has normal islet morphology, abundant acinar cells are arranged in a compact manner, clear boundaries and normal cell morphology.

(9) The results of the pancreatic histomorphology observation of the experimental group of example 1 are shown in fig. 9, and fig. 9 shows the pancreatic histomorphology of the experimental group, the islets in the visual field occasionally appear to have necrotic islet cells, and the nuclei are stained with deep nuclei, and the shapes are irregular (black arrows).

(10) The results of the kidney histology of the control group of example 1 are shown in FIG. 10, and FIG. 10 shows that the kidney tissue morphology of the control group is uniform, the glomeruli are uniformly distributed, the cell number and matrix in the glomeruli are uniform, the tubular epithelial cells are round and full, and the brush border is orderly arranged.

(11) The results of the experimental group kidney histology of example 1 are shown in fig. 11, and fig. 11 shows the morphology of the kidney tissue of the experimental group, in which focal tubular atrophy, enhanced cytoplasmic alkalophilicity, lumen stenosis (black arrows), more tubular dilation, and flattened epithelium (white arrows) were seen in the kidney tissue.

In summary, the method used in the invention can successfully and rapidly establish the tree shrew type II diabetes.

Example 2

A rapid modeling method for tree shrew type II diabetes mellitus, tree shrew is fed by high-fat high-sugar feed for 13 days, and free to eat and drink water; on day 8 of feeding, the tree shrew is dosed with streptozotocin at a dose of 100mg/kg;

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

Example 3

A rapid modeling method for tree shrew type II diabetes mellitus, the tree shrew is fed by high-fat high-sugar feed for 12 days, and is free to eat and drink water; on day 7 of feeding, the tree shrew is 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.

The tree shrew is selected from healthy male tree shrews of 2-3 years old, and the weight of the tree shrew is 150-170 g.

Before the administration, the tree shrew is administrated by streptozotocin after the tree shrew is fasted for 11 hours.

Example 4

A rapid modeling method for tree shrew type II diabetes mellitus, tree shrew is fed by high-fat high-sugar feed for 15 days, and free to eat and drink water; on day 9 of feeding, the tree shrew is 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, 11 parts of white sugar, 11 parts of fructose and 1.2 parts of cholesterol.

Before the administration, the tree shrew is administrated by streptozotocin after 13h of empty stomach of the tree shrew.

For administration, streptozotocin is formulated as a 2% strength solution for administration.

STZ was dissolved in 0.1mmol/L citric acid buffer to prepare a 2% STZ solution, and the solution was uniformly mixed, and the mixture was filtered through a 0.22 μm filter membrane to prepare a drug.

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 8g/kg, and total phosphorus is 6g/kg.

Example 5

A rapid modeling method for tree shrew type II diabetes mellitus, tree shrew is fed by high-fat high-sugar feed for 14 days, and free to eat and drink water; on days 7-9 of feeding, the tree shrew is dosed with 100mg/kg with streptozotocin;

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

Feeding the tree shrew for 14 days by adopting a high-fat high-sugar feed, and freely taking food and drinking water; the feeding is started to be fasted at night on the 7 th day, and after 12 hours of fasting, the tree shrew is dosed with 100mg/kg by streptozotocin.

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 rapid modeling method for tree shrew type II diabetes comprises the following steps:

selecting healthy male tree shrew of 2-3 years old, and feeding the healthy male tree shrew with the weight of 150-170 g by adopting a 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, 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 for 4 weeks and the urine glucose is more than or equal to++, determining that the tree shrew type II diabetes model is successfully established.

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 8.4g/kg, and total phosphorus is 7g/kg.

The foregoing has shown and described the basic principles, principal 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, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. 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: selecting healthy male tree shrew of 2-3 years old, and taking 150-170 g of body weight, and freely feeding and drinking water; feeding the tree shrew with high-fat and high-sugar feed for 2 weeks, and feeding the tree shrew with streptozotocin at a dosage of 90-110mg/kg after the tree shrew is fasted for 11-13 hours on days 6-8;

the high-fat high-sugar feed comprises the following components in percentage by weight: 69% of basic feed, 10% of lard, 10% of white sugar, 10% of fructose and 1% of cholesterol;

2. The rapid prototyping method for tree shrew type ii diabetes of claim 1, wherein: streptozotocin is formulated as a 2% strength solution for administration for no more than 10 minutes.

3. The rapid prototyping method for tree shrew type ii diabetes of claim 2, wherein: streptozotocin is dissolved in 0.1mmol/L citric acid buffer solution to prepare 2% solution, and the solution is uniformly mixed, and is filtered by a 0.22 mu m filter membrane for administration.

4. The rapid prototyping method for tree shrew type ii diabetes of claim 1, wherein: 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 calcium: the mass ratio of the phosphorus is 1.2:1-1.5:1.