CN111066728A - Construction method of hyperglycemia animal model - Google Patents
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- 201000001421 hyperglycemia Diseases 0.000 title claims description 11
- 238000010276 construction Methods 0.000 title abstract description 12
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- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 claims abstract description 40
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 31
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a construction method of a hyperglycemic animal model, which comprises the steps of selecting an SPF-grade C57BL/6 mouse, carrying out intraperitoneal injection on 38-42mg/kg BW streptozotocin solution for 5 consecutive days, and fasting for 3-5h before injection; and fasting for 18-22h after 3 days, and injecting 95-105mg/kg BW streptozotocin solution into abdominal cavity once; on the 3 rd day after the injection, the fasting blood glucose value is measured, and the mice with the blood glucose value more than 12mmol/L are I type hyperglycemic animals. The injection mode of the streptozotocin solution is designed, the injection is divided into multiple times for administration, the injection dosage, the time interval and the fasting time with different sizes are adopted, high-fat high-sugar feed and STZ combined induction are not needed, the animal survival rate is high, the molding period is short, the molding success rate is high, the blood sugar value of an animal model is stable, the cost is relatively low, and the injection can be used as a research model of a hypoglycemic medicament and a diabetic pathogenesis.
Description
Technical Field
The invention relates to the field of biological model construction, in particular to a construction method of a hyperglycemic animal model.
Background
The incidence of diabetes is high, and the complications are many, which brings heavy burden to human health and social development. The current treatment method mainly reduces blood sugar by increasing insulin secretion through medicines or insulin injection, and has large side effects and high cost, so that the research on the treatment method of diabetes becomes the key research content of scientists. The animal model is an indispensable important means and method for researching diabetes, and is helpful for further disclosing pathogenesis and intervention measures of diabetes and testing and screening various hypoglycemic drugs.
The preparation of the diabetes animal model is related to the mode and dosage of STZ injection, large-dosage injection can directly cause the wide damage of islet β cells to cause the diabetes model, but the mortality rate is high, the small-dosage STZ can only damage the function of a part of islet β cells to cause that peripheral tissues are insensitive to insulin, high-calorie feed needs to be simultaneously fed, and the experimental period of the drug combination high-lipid induction method is long.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a construction method of a hyperglycemic animal model, which designs a proper injection mode of streptozotocin solution, and adopts multiple times of administration, different injection doses, time intervals and fasting time, does not need to adopt high-fat high-sugar feed and STZ for combined induction, and has the advantages of high animal survival rate, short molding period, high molding success rate, stable blood sugar value of the animal model and relatively low cost.
The purpose of the invention is realized by adopting the following technical scheme:
a construction method of a hyperglycemia animal model comprises the following steps:
s1: selecting SPF grade C57BL/6 mouse, and measuring fasting blood glucose as basic blood glucose value;
s2: injecting 38-42mg/kg BW streptozotocin solution into abdominal cavity of mice for 5 days continuously, fasting for 3-5h before each injection, and freely drinking water;
s3: on the 3 rd day after the step of S2 is finished, detecting fasting blood sugar of the mouse, fasting for 18-22h, freely drinking water, and then injecting 95-105mg/kg BW streptozotocin solution once in the abdominal cavity;
s4: and 3 days after the step S3 is finished, fasting the mice for 5-6h, carrying out venous blood collection, and detecting the fasting blood glucose value, wherein the mice with the blood glucose value more than 12mmol/L are I-type hyperglycemic animals.
Preferably, in step S2, the injection amount of streptozotocin solution is 40mg/kg BW, and the fasting time before injection is 4 h.
Preferably, in step S3, the fasting time is 20h, and the injection amount of streptozotocin solution is 100 mg/kg. BW.
Further, the body weight of the mouse is 22.5-25.5 g.
Preferably, the body weight of the mouse is 24.1 ± 1.2 g.
The C57BL/6 mouse is a male C57BL/6 mouse.
The vein blood collection is tail vein blood collection.
Further, the streptozotocin solution is prepared by dissolving streptozotocin in 0.1mol/l citric acid-sodium citrate buffer solution with pH of 4.2-4.5.
The method specifically comprises the following steps: the streptozotocin solution in step S2 is obtained by dissolving 3.8-4.2mg of streptozotocin in 1ml of citric acid-sodium citrate buffer solution, and the streptozotocin solution in step S3 is obtained by dissolving 9.5-10.5mg of streptozotocin in 1ml of citric acid-sodium citrate buffer solution.
The citric acid-sodium citrate buffer solution is prepared by the following method: preparing 0.1mol/l citric acid solution and 0.1mol/l sodium citrate solution, and mixing the citric acid solution and the sodium citrate solution according to a volume ratio of 1: 1, adjusting the pH value to 4.2-4.5, and filtering and removing impurities by using a filter membrane with the diameter of 0.22 mu m or 0.45 mu m to obtain the citric acid-sodium citrate buffer solution.
The breeding method of the type I hyperglycemic animal comprises the following steps: feeding in a weak light and dry environment, changing water for 4-5 times per day, and adding feed for 3-4 times per day.
Compared with the prior art, the invention has the beneficial effects that:
1. the hyperglycemia animal model construction method adopts a drug delivery method of injecting the streptozotocin in the abdominal cavity at a small dose for 5 days continuously and injecting the streptozotocin in the abdominal cavity at a medium dose after 3 days, and the streptozotocin is injected in the abdominal cavity at two times and different doses; meanwhile, only by adopting streptozotocin injection, the invention shortens the molding time and reduces the molding cost, but the conventional multiple administration mode can be realized by combining high-fat high-sugar feed and STZ injection for induction, the experimental period is long, and the blood sugar value of the animal model is unstable.
2. In order to avoid death and slow damage of the islets, the invention firstly adopts small-dose injection and short fasting time to destroy the islets, so that the blood sugar is slowly increased, and the blood sugar does not rise to an ideal value, so that the once STZ injection with long fasting time and large dose is matched, so that the islet β cell is seriously damaged, the blood sugar rises obviously, and the high blood sugar is maintained for a long time.
3. The C57BL/6 mouse hyperglycemic model constructed by the invention has the advantages of simple and convenient modeling, high molding rate, short period, zero animal mortality, long hyperglycemic maintenance time and the like.
Drawings
FIG. 1 is a graph showing fasting plasma glucose values of mice on day 3 after the model construction of example 1 and comparative example 1 is completed;
FIG. 2 is a graph showing fasting plasma glucose values of mice on day 28 after completion of model construction in example 1 and comparative example 1.
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. It should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
A construction method of a hyperglycemia animal model comprises the following steps:
s1: selecting SPF male C57BL/6 mouse with weight of 22.5-25.5g, and measuring fasting blood glucose as basic blood glucose value;
s2: injecting 38-42mg/kg BW streptozotocin solution into abdominal cavity of mice for 5 days continuously, fasting for 3-5h before each injection, and freely drinking water;
s3: on the 3 rd day after the step of S2 is finished, detecting fasting blood sugar of the mouse, fasting for 18-22h, freely drinking water, and then injecting 95-105mg/kg BW streptozotocin solution once in the abdominal cavity;
s4: and 3 days after the step S3 is finished, fasting the mice for 5-6h, carrying out venous blood collection, and detecting the fasting blood glucose value, wherein the mice with the blood glucose value more than 12mmol/L are I-type hyperglycemic animals.
The breeding method of the type I hyperglycemic animal comprises the following steps: feeding in a weak light and dry environment, changing water for 4-5 times per day, and adding feed for 3-4 times per day.
The weight of the mouse can influence the drug property of streptozotocin, if the weight of the mouse is over 35g, the drug resistance to STZ is strengthened, the molding rate is reduced, and the selection of the mouse with proper weight is more favorable for the success of modeling; studies have shown that when modeling a type I hyperglycemic animal model, females have poor modeling and may have a higher mortality rate than males, and therefore male mice are selected for this example.
The commonly used injection modes of the streptozotocin solution include tail vein injection and intraperitoneal injection, although the utilization rate of the tail vein injection medicine is high, the operation is troublesome, and the quick absorption of the medicine is easy to generate side effects, so that the death of animals is caused. The preparation method adopts intraperitoneal injection, is convenient to administer and simple to operate, the medicine has an absorption process, the toxicity of the streptozotocin is reduced, animals are relatively safe, and the death rate is reduced.
As a further preferable scheme, the vein blood sampling mode is tail vein blood sampling, the wound on animals is small, the stress is small, the blood volume required by a glucometer is small, and the blood volume of the tail vein blood sampling is met.
The streptozotocin solution is prepared by adopting 0.1mol/l citric acid-sodium citrate buffer solution with the pH value of 4.2-4.5. Streptozotocin needs to be stored away from light and placed on ice as it is prepared. The method specifically comprises the following steps: the streptozotocin solution in step S2 is prepared by dissolving 3.8-4.2mg of streptozotocin in 1ml of citric acid-sodium citrate buffer solution, and the streptozotocin solution in step S3 is prepared by dissolving 9.5-10.5mg of streptozotocin in 1ml of citric acid-sodium citrate buffer solution.
Wherein, the citric acid-sodium citrate buffer solution is prepared by the following method:
preparing 0.1mol/l citric acid solution and 0.1mol/l sodium citrate solution, and mixing the citric acid solution and the sodium citrate solution according to a volume ratio of 1: 1, adjusting the pH value to 4.2-4.5, and filtering and removing impurities by using a filter membrane with the diameter of 0.22 mu m or 0.45 mu m to obtain the citric acid-sodium citrate buffer solution.
Example 1
Selecting 40 SPF male C57BL/6 mice of 8 weeks, and distinguishing the mice by using ear tags; at the moment, the mice are mature in sex, the organisms are mature, and the tissues and organs are mature, so that the stability of experimental data is facilitated. Detecting fasting blood glucose of the mouse by using a blood glucose meter (Roche) as a basic blood glucose value;
the mice of this example were injected intraperitoneally with 40 mg/kg. BW streptozotocin solution (4mg streptozotocin/1 ml citric acid-sodium citrate buffer) for 5 consecutive days, fasted for 4h before each injection, and water was freely drunk; after the injection is completed, the mice are fasted for 6 hours on the 3 rd day, the tail is cut to collect venous blood, the fasting blood sugar value is detected, the blood sugar value does not rise to the ideal value, the mice are fasted for 20 hours, water is freely drunk in the period, and then 100mg/kg BW streptozotocin solution (10mg streptozotocin/1 ml citric acid-sodium citrate buffer solution) is injected into the abdominal cavity once. On day 3 after the completion of the above 6 injections, the mice were fasted for 6 hours, and venous blood was collected by tail snipping, and fasting blood glucose values and mouse conditions were measured as shown in table 1. The mice with the blood sugar value of more than 12mmol/L are type I hyperglycemic model animals. Feeding in a weak light and dry environment, changing water for 4-5 times/day, and adding feed for 3-4 times/day to ensure sufficient water and feed.
TABLE 1 EXAMPLE 1 blood glucose level and death status in mice after molding
As can be seen from Table 1, in the 40 molding mice of the present example, only one mouse had a blood sugar lower than 10mmol/L, and 37 mice had a blood sugar of more than 12mmol/L, which belongs to the model I hyperglycemic animal, i.e., the molding success rate is as high as 92.5%, and the animal did not die during the whole molding process, and the survival rate is 100%. Therefore, the C57BL/6 mouse hyperglycemic model constructed by utilizing streptozotocin is feasible and can be used as a hypoglycemic medicament and a research model of a diabetic pathogenesis.
Comparative example 1
10 SPF-grade male C57BL/6 mice of 8 weeks were selected and distinguished from the example mice by ear tags. Measuring fasting blood glucose of the comparative mouse by adopting a blood glucose meter (Roche) as a basic blood glucose value;
the mice of the comparative example were injected with 40mg/kg & BW normal saline intraperitoneally for 5 consecutive days, fasted for 4 hours before each injection, and had free water; after the injection is finished, the mice are fasted for 6h on the 3 rd day, tail is cut to collect venous blood, fasting blood glucose value is detected, the mice are continuously fasted for 20h, water is freely drunk in the period, and then 100mg/kg & BW physiological saline is injected into the abdominal cavity once. On the 3 rd day after the above 6 injections were completed, the mice were fasted for 6 hours, and venous blood was collected by cutting the tail, measuring the fasting blood glucose value, and observing the growth of the mice.
Test example 1
Blood glucose monitoring was performed on the hyperglycemic model mouse of example 1 and the mouse of comparative example 1, and the blood glucose values and the mouse growth conditions on the 3 rd, 14 th, 28 th, 35 th, and 50 th days after completion of the test modeling were measured, and the results are shown in table 2.
Table 2 example 1 blood glucose monitoring results in hyperglycemic animal models
As can be seen from Table 2, the blood glucose level of example 1 showed a significant upward trend from day 3 to day 14 after molding and gradually reached a steady level on day 35 after molding, with a significant difference (P <0.005) from that of comparative example 1. After continuous observation for 50 days, the blood sugar value is still maintained at a stable level, and the duration of the high blood sugar value is long, thereby being more beneficial to the research of diabetes. The mice of example 1 showed an upward trend in body weight during the monitoring period, but were not statistically different from the initial body weight. FIGS. 1 and 2 are graphs of fasting plasma glucose values of mice at day 3 and day 28 after completion of model construction in example 1 and comparative example 1, respectively, and the mice after STZ injection are significantly different from those in the control group.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of the present invention claimed in the present invention.
Claims (10)
1. A method for constructing a hyperglycemic animal model is characterized by comprising the following steps:
s1: selecting SPF grade C57BL/6 mouse, and measuring fasting blood glucose as basic blood glucose value;
s2: injecting 38-42mg/kg BW streptozotocin solution into abdominal cavity of mice for 5 days continuously, fasting for 3-5h before each injection, and freely drinking water;
s3: on the 3 rd day after the step of S2 is finished, detecting fasting blood sugar of the mouse, fasting for 18-22h, freely drinking water, and then injecting 95-105mg/kg BW streptozotocin solution once in the abdominal cavity;
s4: and 3 days after the step S3 is finished, fasting the mice for 5-6h, carrying out venous blood collection, and detecting the fasting blood glucose value, wherein the mice with the blood glucose value more than 12mmol/L are I-type hyperglycemic animals.
2. The method for constructing an animal model with hyperglycemia according to claim 1, wherein the injection amount of streptozotocin solution is 40 mg/kg-BW and the fasting time before injection is 4h in step S2.
3. The method for constructing an animal model with hyperglycemia according to claim 1, wherein the fasting time is 20 hours and the injection amount of streptozotocin solution is 100 mg/kg-BW in step S3.
4. The method of claim 1, wherein the body weight of the mouse is 22.5-25.5 g.
5. The method of claim 4, wherein the body weight of the mouse is 24.1 ± 1.2 g.
6. The method for constructing an animal model of hyperglycemia according to claim 1, wherein the C57BL/6 mouse is a male C57BL/6 mouse.
7. The method for constructing an animal model with hyperglycemia according to claim 1, wherein the venous blood collection is tail vein blood collection.
8. The method for constructing an animal model with hyperglycemia according to claim 1, wherein the streptozotocin solution is prepared by dissolving streptozotocin in 0.1mol/l citric acid-sodium citrate buffer solution having pH of 4.2-4.5.
9. The method for constructing an animal model with hyperglycemia according to claim 8, wherein the citric acid-sodium citrate buffer is prepared by the following method: firstly, preparing 0.1mol/l citric acid solution and 0.1mol/l sodium citrate solution, and then mixing the citric acid solution and the sodium citrate solution according to the volume ratio of 1: 1, adjusting the pH value to 4.2-4.5, and filtering and removing impurities by using a filter membrane with the diameter of 0.22 mu m or 0.45 mu m to obtain the citric acid-sodium citrate buffer solution.
10. The method for constructing the hyperglycemic animal model according to claim 1, wherein the method for raising the type I hyperglycemic animal comprises: feeding in a weak light and dry environment, changing water for 4-5 times per day, and adding feed for 3-4 times per day.
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| CN113575516A (en) * | 2021-08-27 | 2021-11-02 | 河北生命原点生物科技有限公司 | Method for establishing type II diabetes rat model |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011116501A1 (en) * | 2010-03-26 | 2011-09-29 | 四川大学华西医院 | Establishment of rhesus monkey model of autoimmunity type 1 diabetes |
| KR20110116518A (en) * | 2010-04-19 | 2011-10-26 | 강원대학교산학협력단 | A animal model having type 1 and type 2 diabetes mellitus and a manufacturing method thereof |
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2020
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| WO2011116501A1 (en) * | 2010-03-26 | 2011-09-29 | 四川大学华西医院 | Establishment of rhesus monkey model of autoimmunity type 1 diabetes |
| KR20110116518A (en) * | 2010-04-19 | 2011-10-26 | 강원대학교산학협력단 | A animal model having type 1 and type 2 diabetes mellitus and a manufacturing method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113575516A (en) * | 2021-08-27 | 2021-11-02 | 河北生命原点生物科技有限公司 | Method for establishing type II diabetes rat model |
| CN113575516B (en) * | 2021-08-27 | 2023-03-07 | 河北生命原点生物科技有限公司 | Method for establishing type II diabetes rat model |
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