WO2014202022A1

WO2014202022A1 - Method for preparing rhesus macaque diabetes mellitus type 2 model

Info

Publication number: WO2014202022A1
Application number: PCT/CN2014/080412
Authority: WO
Inventors: 陈又南; 程惊秋; 陆燕蓉; 刘敬平; 李新丽; 杨光; 张�杰; 廖光能; 李红霞; 王莉; 任艳; 田浩明; 钟治晖
Original assignee: 四川大学华西医院; 四川康城生物科技有限公司
Priority date: 2013-06-21
Filing date: 2014-06-20
Publication date: 2014-12-24
Also published as: CN103314925A

Abstract

A method for preparing a Rhesus macaque diabetes mellitus type 2 model, comprising the following steps: (1) a Rhesus macaque is fed a high-fat and high-cholesterol feed twice per day at a feeding amount of 0.3-0.4 kg/instance/animal, where the feed comprises raw materials of the following proportions by weight: 78 parts of a standard monkey feed, 15 parts of an animal fat, 5 parts of a sugar, and 2 parts of cholesterol; and, (2) after 24 months, streptozotocin of a dosage of 25 mg/kg is administered to the Rhesus macaque once per week for 2 successive instances. By employing a scheme of long-term feeding of the high-fat and high-cholesterol feed and intravenous injection of a small dose of streptozotocin for joint modeling, a Rhesus macaque diabetes mellitus type 2 model is successfully induced.

Description

Method for preparing rhesus monkey type 2 diabetes model

The invention relates to a method for preparing a rhesus monkey type 2 diabetes model.

Background technique

In recent years, the global incidence of diabetes has grown rapidly. According to the latest data from WHO, the number of people with diabetes worldwide has exceeded 177 million, and it is expected to reach 370 million by 2025. The incidence of diabetes in China is also increasing at an alarming rate. The recent epidemiological survey of the Diabetes Branch of the Chinese Medical Association found that the national incidence of diabetes has rapidly increased from 4% in 2002 to 11% now, and 15% in addition. People have abnormal blood sugar. The number of people with diabetes in China has reached 91 million, ranking first in the world. More than 90% of patients with diabetes mellitus are type 2 Diabetes Mellitus (T2DM). It is of great clinical and social value to continuously explore new modes of T2MD treatment and develop new drugs.

The pathogenesis of T2DM has many influencing factors, the process is long, and the individual responses are significantly different. In rodents, T2DM models with insulin resistance have been successfully established through genetic screening, genetic modification and diet induction, but more and more studies have shown that small animals have huge fat and inflammatory responses. Differences, can not well simulate the pathophysiological characteristics of human metabolic diseases. The establishment of large animals, especially the highly anthropomorphic non-human primate T2DM model, is of great significance for the clinical transformation of T2DM research.

However, it is quite difficult to establish a large animal T2DM model and to develop standard induction programs and evaluation criteria. Screening for spontaneously diabetic animals in primates is rare and rare, and transgenic technology has no mature technology in primates and is costly.

Summary of the invention

In order to solve the above problems, the present invention provides a method for preparing a rhesus monkey type 2 diabetes model.

The preparation method of the rhesus monkey type 2 diabetes model of the present invention comprises the following steps:

(1) Feeding the high-heavy and high-cholesterol feed to the rhesus monkeys, feeding twice a day, feeding 0.3-0.4kg/time. The feed contains the following weight ratio raw materials: 78 standard monkey feeds. 15 parts of animal fats, 5 parts of sugar, 2 parts of cholesterol;

(2) After 24 months, streptozotocin at a dose of 25 mg/kg was administered to the monkey once a week for 2 consecutive times.

Preferably, the animal fat is lard; the sugar is sucrose.

The standard monkey feed refers to the monkey compound feed specified in the National Standard of the Chinese Republic of China GB14924.8-2001. Animal fat refers to fat derived from animals, such as butter, sheep oil, and lard.

In the step (2), the administration method is intravenous injection.

The present invention establishes a feed for the Ganger type 2 diabetes model, which comprises the following raw materials by weight ratio: 78 parts of standard monkey feed, 15 parts of animal fat, 5 parts of sugar, and 2 parts of cholesterol.

Preferably, the animal fat is lard; the sugar is sucrose.

The present invention also provides a rhesus monkey type 2 diabetes model prepared by the aforementioned method and its use in screening for a drug for treating a type 2 diabetes model.

The present invention screens a method for treating a drug of a type 2 diabetes model, which comprises the following steps: a. Establishing a rhesus monkey type 2 diabetes model according to the aforementioned method;

b. administering the candidate drug to the animal model;

c. Use animal models to evaluate potential drugs for the treatment of type 2 diabetes.

The modeling method of the present invention can induce hyperinsulinemia, impaired glucose tolerance and insulin resistance in rhesus monkeys, and the clinical manifestations of type 2 diabetes are simple, and the method of administration is simple and reproducible.

It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention without departing from the spirit and scope of the invention.

The above description of the present invention will be further described in detail by way of specific embodiments of the embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

DRAWINGS

Figure 1 Rhesus monkey weight abdominal circumference test;

Figure 2 Rhesus liver and muscle tissue oil red 0 staining;

Figure 3 HE staining of liver tissue of rhesus monkeys, A. HE staining of liver in control group; B. HE staining of liver in experimental group (arrow indicates inflammatory cell infiltration); C. Liver HE staining in experimental group (arrow indicates hepatic cell edema) ;

Figure 4 Detection of lipid metabolism indicators in rhesus monkeys;

Figure 5 Rhesus monkey sugar metabolism related indicators;

Figure 6 IVGTT and high insulin blood-normal blood glucose clamp test results after 18m of high-fat feeding; Figure 7 Related indicators of islet function damage.

detailed description

Example 1 Preparation of Rhesus Monkey Type 2 Diabetes Model of the Invention

1 experimental materials and instruments

1.1 Experimental materials

Eight 6-month-old rhesus monkeys (male and female), no B virus, monkey retrovirus, monkey leukemia virus and immunodeficiency virus infection, purchased from Chengdu Pingan Animal Breeding Base. Experimental group: STZ group (HF+STZ group, 4 rats) After giving a rouge high cholesterol diet 24 months after intravenous injection of small doses of streptozotocin induced type 2 diabetes, high fat group (HF group, 4) only given high Cholesterol diet feeding.

Control group: monkey standard monkey feed

Feeding method: Feed 2 times a day, feeding 0.3-0.413⁄4/time*.

Feed formula:

Control group: Monkey standard monkey feed - Commercialized monkey pellet feed (purchased from the Animal Research Institute of Sichuan Medical Science Institute, the formula is the same as the monkey compound feed specified in the National Standard of the Chinese Republic of China GB14924.8-2001).

Experimental group: High fat and high cholesterol feed: 2% (w/w) Edible cholesterol (Anhui Tianqi Chemical Technology Co., Ltd.), 15% (w/w) refined lard (commercially available), 5% (w/w) sucrose (commercially available), 78% (w/w) monkey standard monkey feed.

1.2 main reagents

Streptozotocin (STZ): purchased from Chengdu Yuyang High-Tech Development Co., Ltd.

1.3 Preparation of the main solution

Sodium citrate buffer preparation:

Solution A (0.1 M citric acid solution): Weigh 2.1 g of citric acid (C6H807-H20 FW: 210.14) dissolved in 100 ml of double distilled water. Solution B (0.1 M sodium citrate solution): 2.94 g of sodium citrate (C6H5Na307-2H20 FW: 294.10) was weighed and dissolved in 100 ml of double distilled water.

Working solution: Take 28 ml of A solution and 22 ml of B solution (according to the ratio of 1.32:1), dilute to 100 ml with double distilled water, adjust pH=4.5.

Streptozotocin solution: STZ is weighed according to animal weight ratio, and STZ is dissolved at 1% concentration with the above AB mixed working solution. The solution is required to be stored and stored at 4 ° C. The injection is completed within 30 minutes. .

1.4 Main instruments

Blood glucose detector: Luo Kang full vitality blood glucose detector, Luo Kang full vitality blood glucose test strips Other tests are completed in the 4th Division of the University of West China Hospital Laboratory.

2 Experimental methods

2. 1 induced diabetes model

The experimental group (including STZ group and HF group) was given a rouge high cholesterol diet, and the control group was fed with regular monkey feed, fed twice a day, and the feeding amount was 0.3-0.413⁄4/time* only;

After giving a rouge high-cholesterol diet for 24 months, the STZ group received a small dose of STZ intravenously: 25 mg/kg each time, once a week for 2 consecutive times. The HF group and the control group were left untreated.

2. 2 Rhesus monkey intravenous glucose tolerance test

Through the intravenous glucose tolerance test (IVGTT) Test animal pancreatic function, understand the range of blood glucose fluctuations, and rule out the possibility of animals having spontaneous diabetes. Animals fasted for 10-12h, can not help but water;

According to the body weight at the time of purchase, ketamine (50 mg/ml) was intramuscularly injected at 15 mg/Kg _;

After the animals are anesthetized, the weight is accurately weighed and recorded;

The upper and lower limbs of the rhesus monkey are fixed on the operating table, and the saphenous vein of the lower leg of the lower leg is exposed to the saphenous vein, and the iodine fluoride is disinfected;

20G residual needle through the great saphenous vein, 5ml syringe blood 6ml (respectively biochemical, blood routine, blood glucose and fasting insulin, and marked as 0 minutes);

Immediately use a 5ml syringe to inject a 50% glucose hypertonic solution 0.5g / Kg (within 30 seconds), and start recording, then inject 5ml of normal saline, push the heparin (250 U / ml) into the stagnation needle lml 1.5 ml of blood was collected at the 1st, 3rd, 5th, 10th, and 30th minutes after the time of recording, and placed in the red blood collection tube to measure the insulin level; and at 0, 1, 3, 5, 10, 30, 60, respectively. Blood glucose was measured at 120 min;

At the end of the test, the animals were placed back in the cage and the animals were observed to awaken.

2.3 high insulin - normal blood glucose clamp test

Rhesus monkeys were fasted for more than 16 hours, routine anesthesia, weighing, and collecting 4 ml of lower extremity venous blood into a separate gel-promoting tube.

Take the supine position, put the arm vein in the opposite direction of the heart, put in the indwelling catheter, and maintain the channel with normal saline to prepare blood for blood sugar measurement. Slowly instill saline without blood collection, and temporarily close the infusion set before blood collection. Heat the pad by heating to maintain the temperature of the hand at 50 to 60 °C.

The venous cannula was placed from the median vein of the elbow and the two veins were used to construct two venous channels for infusion of insulin and glucose. Infusion of human insulin solution (Uberin, 40 U/mL; > at a rate of 4 mU / (kg · min) within 10 min of the clamp starts to increase the blood insulin concentration rapidly, followed by 2 mU / (kg * within 11 min) Mm) Continuous infusion of the rate and simultaneous input of 20% glucose solution with an infusion pump.

During this period, the venous blood glucose level was measured every 5 minutes, and the infusion rate of 200 mL/L glucose solution was adjusted according to the blood glucose level, so that the blood glucose level of the subject was maintained at about 4.5 mmol/L. Venous blood was measured every 30 min to determine serum insulin concentration.

When the blood glucose level changes by less than 10%, it is considered to reach a steady state, and the steady state lasts for about 60 minutes and the test is terminated. The blood samples of insulin and C-peptide were centrifuged and stored in a refrigerator at 20 ° C. The glucose disposal rate (M) of the peripheral tissues during the clamp test was calculated according to Steel's formula. The formula is: M = clamp The glucose infusion per kilogram of body weight per minute for the last 40 min of the test + (G80 - G120) X 0.0625, where G80 and G120 were the blood glucose concentrations (mg I dl) at 80 min and 120 min, respectively, in the clamp test. Calculate the insulin sensitivity index SIClamp = M / Δ I / fat-free mass (Kg) according to the M value, M is the glucose infusion rate at steady state, Δ Ι is stable The difference between the insulin level at the time and the basal insulin level before the experiment was 0 iIU/ml).

3 experimental results

3.1 High-fat diet can induce glucose metabolism and lipid metabolism disorder in rhesus monkeys

The experimental results are shown in Figures 1 to 6 and Table 1:

Table 1 IVGTT and clamp experiment (18m;> mean value of glucose metabolism variables in experimental group and control group

Variables Control (n = 8) HFHCD (n = 8)

K _Glu (%) 1.73±0.37 0.70±0.33***

AUC-insulin((JU/ L* min ^_1 ) 387.24±122.39 105.50±66.04***

M value(mg« kg— ¹ · min ^_1 ) 16.35±2.06 13.78±1.21

SIciamp (mg · kg - ¹ · min - ¹ /μΐυ / L) 0.12±0.05 0.07±0.03

HOMA-IR 0.85±0.55 1.55±1.12

Note: HFHCD vs. Control: *** P <0.001.HFD: high fat and high cholesterol word group, control: control group

According to Figures 1 to 6 and Table 1, it can be seen that the glucose metabolism and lipid metabolism of the control group were normal, and the glucose metabolism and lipid metabolism of the experimental group were abnormal:

1. The high-fat and high-cholesterol diet caused fat ectopic viscera, and obvious lipid droplet deposition appeared in the liver and skeletal muscle of rhesus monkeys in the experimental group.

2. The experimental group showed obvious diet-dependent hypercholesterolemia. Sustained hypercholesterolemia triggered compensatory changes in the corresponding serum lipid metabolism parameters, which involved accelerated cholesterol transport (lipoprotein HDL, elevated LDL). , increased conversion and excretion, increased fat breakdown and elevated triglycerides.

3. The control group was normal. The fasting blood glucose of the experimental group increased gradually after 6 months of feeding, while the serum insulin level increased progressively, and HbAlC was significantly higher than the control group. Under the high glucose stress, the area under the glucose curve increased, the glucose clearance rate (Kglu) and the area under the insulin curve decreased. The M value and SIciamp of the clamp experiment were lower than those of the control group, and the insulin resistance index increased.

The experimental results showed that the islet β-cell function of the rhesus monkey in the experimental group was impaired, the glucose tolerance was impaired, and the exogenous and endogenous insulin sensitivity of the peripheral tissues decreased.

3.2 Low-dose STZ can induce islet function damage, accelerate the rhesus monkey type 2 diabetes after intravenous injection of STZ 1 month, the rhesus monkey islet function indicators as shown in Figure 7: compared with the control group, STZ group constant Rhesus monkeys showed significant elevation of blood glucose and reached the diagnostic criteria for type 2 diabetes;

The fasting insulin level in the STZ group, the area under the blood glucose curve of the IVGTT, and the insulin resistance index (HOMA-I) were significantly higher than those in the other two groups, demonstrating hyperinsulinemia, impaired glucose tolerance, and insulin resistance.

The experimental results show that the method of the present invention causes partial β cell cell damage and induces rhesus monkeys to be high. Insulinemia, impaired glucose tolerance and insulin resistance, the clinical manifestations of type 2 diabetes, successfully established a rhesus monkey type 2 diabetes model. Example 2 Screening for the treatment of type 2 diabetes using the model of the present invention

a. Rhesus type 2 diabetes model established according to the method of Example 1;

b. administering the candidate drug to the animal model;

c. Observe the effects of drug candidates on various indicators of type 2 diabetes and evaluate potential drugs for the treatment of type 2 diabetes. In summary, the modeling method of the present invention, based on high-fat and high-cholesterol feed feeding, is supplemented by a small dose of STZ, which causes partial β cell cell damage, satisfies the necessary conditions for type 2 diabetes, and induces insulin resistance through lipid metabolism disorder. The formation of a stable and standardized rhesus monkey T2DM model in a relatively short period has important practical application value. Industrial applicability

The modeling method of the invention forms a relatively stable and standardized rhesus monkey T2DM model in a relatively short period, and can be used for screening for type 2 diabetes drugs, which is suitable for industrial applications.

Claims

claims

1. A method for preparing a rhesus monkey type 2 diabetes model, which is characterized by: including the following steps:

(1) Feed high-fat and high-cholesterol feed to rhesus monkeys twice a day, and the feeding amount is 0.3-0.4kg/time·animal. The feed contains raw materials with the following weight ratio: 78 parts of standard monkey feed , 15 parts of animal fat, 5 parts of sugar, 2 parts of cholesterol;

(2) After 24 months, streptozotocin at a dose of 25 mg/kg was administered to the monkeys once a week for 2 consecutive times.

2. The preparation method according to claim 1, characterized in that: in step (1), the animal fat is lard; and the sugar is sucrose.

3. The preparation method according to claim 1, characterized in that: in step (2), the administration method is intravenous injection.

4. A feed for establishing a rhesus monkey type 2 diabetes model, characterized in that: it contains raw materials in the following weight ratio: 78 parts of standard monkey feed, 15 parts of animal fat, 5 parts of sugar, and 2 parts of cholesterol.

5. The feed according to claim 4, characterized in that: the animal fat is lard; and the sugar is sucrose.

6. The rhesus monkey type 2 diabetes model prepared by the method of any one of claims 1 to 3.

7. Use of the animal model of claim 6 in screening drugs for treating type 2 diabetes.

8. A method for screening drugs for treating type 2 diabetes model, characterized by: It includes the following steps:

a. Establish a rhesus monkey type 2 diabetes model according to the method described in any one of claims 1 to 3; b. Apply the candidate drug to the animal model;