CN114342875A - Method for establishing novel gestational diabetes animal model - Google Patents
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Abstract
The invention discloses a method for establishing a novel gestational diabetes animal model, which divides C57BL/6J female mice with 6 weeks of age into 4 groups, wherein the two groups of C57BL/6J female mice are fed with basic feed for 4 weeks; feeding two groups of C57BL/6J female mice with high-fat feed containing 45% fat for 4 weeks to establish a diet-induced obesity model; after the group feeding for 4 weeks, C57BL/6J female mice are mated in cages and the pregnancy condition is observed; from the 7 th pregnancy of the C57BL/6J female mouse, the insulin receptor antagonist S96120nmol/kg is injected subcutaneously every day to establish the gestational diabetes animal model. Different from the characteristics of long model building time, low success rate, high animal mortality rate, damage effect of model building mode on offspring and the like of the prior gestational diabetes mouse or rat model, the invention establishes a stable and reliable mouse model which is similar to human pathogenesis, has short model building time and high success rate, has no obvious toxic effect on both a mother mouse and an embryo, and accords with the characteristics of human gestational diabetes.
Description
Technical Field
The invention relates to the technical field of diabetes research, in particular to a method for establishing a novel gestational diabetes animal model.
Background
Gestational diabetes is a hyperglycemic phenomenon caused by relatively insufficient insulin secretion in the middle and late pregnancy or insulin resistance due to physiological changes in pregnancy, and mainly manifests as reduced insulin sensitivity in pregnancy and increased blood sugar caused by insufficient insulin utilization. Most of patients with gestational diabetes can recover normal blood sugar after the placenta is delivered, but gestational diabetes can induce poor pregnancy outcome and cause negative effects on long-term health of mothers and offspring, and the risk of getting type 2 diabetes after the mothers and the offspring become adults is increased. Gestational diabetes mellitus requires effective treatment to protect the health of both mothers and offspring.
Treatment of gestational diabetes is slow due to the lack of animal models that effectively mimic the pathogenesis of gestational diabetes in pregnant women. Therefore, the establishment of a reliable and easy-to-operate animal model of gestational diabetes would greatly facilitate the treatment of gestational diabetes, including the screening and evaluation of protective drugs. Although methods of inducing gestational diabetes models have been reported, including induction of streptozotocin, long-term high-sugar high-fat diet natural induction or streptozotocin induction with high-fat diet. At present, the establishment methods of the gestational diabetes mellitus have some defects. For example, streptozotocin injection mainly simulates a type I diabetes model but not a typical type gestational diabetes insulin relatively insufficient secretion model, and the streptozotocin has toxicity, can cause death of experimental animals, has embryotoxicity and is not suitable for the research on the influence of gestational diabetes on the offspring health. The model of the gestational diabetes induced by the high-sugar high-fat induced obesity needs long-time feeding of high-sugar high-fat feed, the molding rate is low, a plurality of reports show that about 50 percent of the model is obtained, and the method simulates the condition that the gestational diabetes induced by the diet induced obesity can not simulate normal weight but single insulin resistance.
Disclosure of Invention
The invention provides a method for establishing a novel gestational diabetes animal model, which has wide application value in the aspects of screening and evaluating gestational diabetes treatment medicines, and researching the influence of diabetes on offspring health and protective medicines.
The purpose of the invention is realized by the following technical scheme:
a method for establishing a novel gestational diabetes animal model comprises the step of carrying out subcutaneous injection of an insulin receptor antagonist S96120nmol/kg once a day from 7 days of gestation of animals to establish the gestational diabetes animal model.
Further, the animals are C57BL/6J female mice.
Further, the method specifically comprises:
1) grouping feeding of female mice: dividing 6-week-old C57BL/6J female mice into 4 groups, wherein two groups of C57BL/6J female mice are given basal feed for 4 weeks; feeding two groups of C57BL/6J female mice with high-fat feed containing 45% fat for 4 weeks to establish a diet-induced obesity model;
2) after the group feeding for 4 weeks, C57BL/6J female mice are mated in cages and the pregnancy condition is observed;
3) insulin receptor antagonist S961 induces gestational diabetes: from the 7 th pregnancy of the C57BL/6J female mouse, the insulin receptor antagonist S96120nmol/kg is injected subcutaneously every day to establish the gestational diabetes animal model.
Further, the step 1) specifically includes: after the C57BL/6J female mice aged 6 weeks are adaptively fed for 1 week, randomly dividing the mice into four groups, wherein the number of each group is the same, and measuring the basic fasting blood glucose; two groups of C57BL/6J female mice were given basal diet, and the other two groups of C57BL/6J female mice were given high fat diet containing 45% fat, fed for 4 weeks, and fasting blood glucose was measured after 4 weeks.
Further, in the step 1), the two groups of C57BL/6J female mice given basal feed were further divided into basal control group (CON) and basal injection S961 group (CS); the two groups of C57BL/6J female mice given high fat diet were further divided into a high fat control group (HF) and a high fat injected S961 group (HFs).
Further, the basal feed in step 1) is a 10% fat sterile basal feed.
Further, the step 2) is specifically as follows: after 4 weeks of group feeding, C57BL/6J female mice were mated with male mice in 2:1 cages and the presence of vaginal emboli was observed, which was scored as day 0 gestation.
Further, the step 3) is specifically as follows: measuring fasting blood glucose for 7 days of gestation when C57BL/6J female mice are pregnant for 7 days; starting from 7 days of pregnancy of a C57BL/6J female mouse, an insulin receptor antagonist S96120nmol/kg is subcutaneously injected every day according to the weight of an animal, the solution concentration is 5nmol/ml, and the total injection amount is less than 150ul, so that a gestational diabetes animal model is established.
Further, in the step 3), each C57BL/6J female mouse is continuously fed with the same feed before pregnancy during the pregnancy period, and the feeding mode adopts a free feeding mode.
Further, in step 3), the insulin receptor antagonist S961 is administered at 5 PM every day until the day before production.
The invention has the following beneficial effects:
different from the characteristics of long model building time, low success rate, high animal mortality rate, damage effect of model building mode on offspring and the like of the prior gestational diabetes mouse or rat model, the invention establishes a stable and reliable mouse model which is similar to human pathogenesis, has short model building time and high success rate, has no obvious toxic effect on both a mother mouse and an embryo, and accords with the characteristics of human gestational diabetes.
Drawings
FIG. 1 is an initial baseline fasting plasma glucose in C57BL/6J mice according to an embodiment of the present invention;
FIG. 2 is fasting blood glucose of C57BL/6J mice after being fed with high-fat diet for 4 weeks according to the embodiment of the present invention;
FIG. 3 is fasting blood glucose of C57BL/6J mice for 7 days of pregnancy in an example of the present invention;
FIG. 4 is an oral glucose tolerance test 7 days after administration of C57BL/6J mice in an example of the invention.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments and drawings. It should be understood that the examples are illustrative only and should not be construed as limiting the scope of the invention.
A method for establishing a novel gestational diabetes animal model comprises the step of carrying out subcutaneous injection of an insulin receptor antagonist S96120nmol/kg once a day from 7 days of gestation of animals to establish the gestational diabetes animal model.
Further, the animals are C57BL/6J female mice.
Further, the method specifically comprises:
1) grouping feeding of female mice: dividing 6-week-old C57BL/6J female mice into 4 groups, wherein two groups of C57BL/6J female mice are fed with basic feed for 4 weeks, and the other two groups of C57BL/6J female mice are fed with high-fat feed containing 45% fat for 4 weeks, and establishing a diet-induced obesity model;
2) after the group feeding for 4 weeks, C57BL/6J female mice are mated in cages and the pregnancy condition is observed;
3) insulin receptor antagonist S961 induces gestational diabetes: from the 7 th pregnancy of the C57BL/6J female mouse, the insulin receptor antagonist S96120nmol/kg is injected subcutaneously every day to establish the gestational diabetes animal model.
Further, the step 1) specifically includes: after the C57BL/6J female mice aged 6 weeks are adaptively fed for 1 week, randomly dividing the mice into four groups, wherein the number of each group is the same, and measuring the basic fasting blood glucose; two groups of C57BL/6J female mice were given basal diet, and two other groups of C57BL/6J female mice were given high fat diet containing 45% fat for 4 weeks, and fasting blood glucose was measured after 4 weeks.
Further, in the step 1), the two groups of C57BL/6J female mice given basal feed were further divided into basal control group (CON) and basal injection S961 group (CS); the two groups of C57BL/6J female mice given high fat diet were further divided into a high fat control group (HF) and a high fat injected S961 group (HFs).
Further, the basal feed in step 1) is a 10% fat sterile basal feed.
Further, the step 2) is specifically as follows: after 4 weeks of group feeding, C57BL/6J female mice were mated with male mice in 2:1 cages and the presence of vaginal emboli was observed, which was scored as day 0 gestation.
Further, the step 3) is specifically as follows: measuring fasting blood glucose for 7 days of gestation when C57BL/6J female mice are pregnant for 7 days; starting from 7 days of pregnancy of a C57BL/6J female mouse, an insulin receptor antagonist S96120nmol/kg is subcutaneously injected every day according to the weight of an animal, the solution concentration is 5nmol/ml, and the total injection amount is less than 150ul, so that a gestational diabetes animal model is established.
Further, in the step 3), each C57BL/6J female mouse is continuously fed with the same feed before pregnancy during the pregnancy period, and the feeding mode adopts a free feeding mode.
Further, in step 3), the insulin receptor antagonist S961 is administered at 5 PM every day until the day before production.
Examples
The embodiment relates to a method for establishing a novel gestational diabetes animal model, which comprises the following steps:
1 materials of the experiment
80 SPF grade 6 week old C57BL/6J female mice, purchased from Henan Spxbeck Biotech, Inc. The experimental animals are raised in an SPF-level laboratory, the environmental temperature is controlled at 24 +/-2 ℃, the humidity is maintained at 40-70%, the light and shade period is 1:1, the experimental animals are randomly divided into 4 groups, and each group comprises 20 animals. 45% high fat diet and 10% fat containing sterile basal diet were purchased from Beijing Huafukang laboratory animals Co.
2 Primary reagent
Insulin receptor antagonist (S961): purchased from Nanjing Jettish peptide Biotech Ltd.
3 preparation of the main solution
PBS. 5nmol/ml S961 solution: weighing 1mg of S961 powder, dissolving in 1ml of PBS, diluting to 5nmol/ml after all the S961 powder is dissolved, subpackaging, and storing in a refrigerator at 4 ℃; the required dose was calculated as 20nmol/kg of animal body weight and administered by subcutaneous injection at 5 pm daily.
4 Main instrument
A blood glucose meter: yapei blood sugar detector, Yapei blood sugar test paper.
5 concrete Experimental method
5.1C57BL/6J female mice basal blood glucose
(1) After 6-week-old female mice are adaptively fed for 1 week, the female mice are randomly divided into four groups, 20 mice in each group are selected, 10 mice in each group are randomly selected, the basal fasting blood glucose is measured, and the animals are fasted for 10-12 hours without water prohibition.
(2) The tail vein is adopted for blood sampling, and an Yapeh glucometer and matched blood glucose test paper are used for measuring the basic fasting blood glucose.
5.2C57BL/6J female mice were high-fat fed for 4 weeks
(1) Of the four groups of animals, two groups were basal diet groups and two groups were high fat diet groups. Two groups of female mice in the basal diet group were given basal diet (fat-energized 10%), two groups of female mice in the high fat diet group were given high fat diet (fat-energized 45%), and fed for 4 weeks.
(2) Of the two basal feed groups, one was basal Control (CON) and the other was basal injection S961 (CS). In two groups of high fat diet, one group was high fat control (HF) and the other group was high fat injection S961 (HFs).
5.3C57BL/6J female mice fasting plasma glucose after high fat feeding for 4 weeks
(1) Animals were fasted for 10-12 hours without water deprivation. Tail vein blood sampling is adopted, and an yapei glucometer and matched blood glucose test paper are used for measuring the fasting blood glucose of the mice after the mice are fed with high fat for 4 weeks.
5.4C57BL/6J female mice coop
(1) C57BL/6J female mice and male mice are mated in 2:1 cages, the vaginal embolus condition is observed at 6 o 'clock and 20 o' clock in the morning, and the observation of the vaginal embolus is marked as 0 day of pregnancy.
(2) The mice were weighed daily and observed for weight gain during pregnancy.
5.5 pregnant 7 sky belly blood sugar
(1) After the mice had an empty stomach for 5 hours at 7 days of gestation, blood glucose was measured by tail vein blood sampling.
5.6S961 Induction of gestational diabetes
(1) For C57BL/6J female mice in CS group and HFS group, 20nmol/kg of S961 solution is injected subcutaneously according to the weight of the animals at 7 days of gestation, the concentration of the solution is 5nmol/ml, the total injection amount is less than 150ul, and the tolerance of the animals is facilitated.
(2) The injection time was fixed daily, approximately 17:00 a.m. per day.
5.7 oral glucose tolerance test
(1) 7 days after administration, the animals were subjected to an oral glucose tolerance test at 14 days of gestation. The glucose solution was gavaged at 1.2 g/kg.
(2) After the pregnant mice fasting for 5 hours, tail vein blood sampling is carried out to measure fasting blood glucose, then glucose solution is given for intragastric administration, and blood glucose is measured 30 minutes, 60 minutes, 120 minutes and 180 minutes after intragastric administration, which are all tail vein blood sampling methods.
5.8 weight gain of mother mouse in pregnancy period and weight of fetal mouse
(1) When the mother mouse is pregnant for 18 days, part of the mother mouse is euthanized, blood and liver tissues are left for taking, and the weight of the fetal mouse is weighed.
5.9 birth weight and lactation weight of the child mouse
(1) The remaining female mice were delivered naturally, the birth weight of the child mice was measured, and the female mice were fed water ad libitum.
(2) The mice were continued to be breastfed for 4 weeks and the body weight of the mice was measured weekly.
The experimental results of this example are as follows:
the mouse insulin receptor antagonist S96120nmol/kg is administered by subcutaneous injection at regular intervals for 7 days of pregnancy until the production method can successfully induce the gestational diabetes animal model characterized by insulin resistance. Abnormal glucose tolerance occurs in the middle and late pregnancy of the mice.
Successful modeling criteria included (1) abnormal glucose tolerance in the late gestation phase of the female mouse; (2) the offspring is prone to acquired obesity after birth. The experimental modeling model has the advantages that the basic feed administration group and the high-fat feed administration group both meet the modeling standard and have no obvious side effect or complication.
The body weight gain of the female mice before and after 4 weeks of high fat diet feeding is shown in table 1:
TABLE 1 weight gain of the female mice before and after 4 weeks of high fat diet feeding (adaptive feeding period of 0-1 week)
Note: basal control group (CON), basal injection S961 group (CS), high fat control group (HF), and high fat injection S9Group 61 (HFS);arepresents P < 0.05 compared to CON group,bindicates P < 0.05 compared to the CS group.
Table 1 shows that there was no difference in initial body weight and body weight after 1 week of adaptive feeding after animals were randomly divided into 4 groups, the body weight was significantly higher in the high fat control group HF and the high fat dosed group HFs after being fed with 45% high fat diet than in the basal diet group and the basal dosing group, and after 4 weeks, the average body weight was 10.02% higher in the high fat group than in the basal group, which resulted in overweight level.
As shown in fig. 1, the baseline fasting plasma glucose was found in 4 mice before and after high fat feeding, and there was no difference in baseline plasma glucose between the basal control group (CON), basal injection S961 group (CS), high fat control group (HF), and high fat injection S961 group (HFs)4 groups before and after high fat feeding.
As shown in FIG. 2, the fasting plasma glucose of the mice after 4 weeks of high fat feeding is shown, wherein a represents P < 0.05 compared with CON group and b represents P < 0.05 compared with CS group. After the animals are fed with the high-fat feed for 4 weeks, the fasting blood sugar of the animals in the high-fat feed group is obviously higher than that of the animals in the basal feed group, but the fasting blood sugar of the animals in the high-fat feed group does not reach the diabetes diagnosis standard, which shows that the blood sugar of the female mice can be increased by feeding the animals with the high-fat feed for 4 weeks, but the blood sugar of the female mice does not reach the diabetes standard.
As shown in fig. 3, the mice were pregnant with 7 days of fasting plasma glucose, and the plasma glucose tended to be higher in the high-fat group than in the basal group.
As shown in FIG. 4, oral glucose tolerance of 14 days of pregnancy mice is shown, wherein a indicates that CON compared with CS group P < 0.05, # indicates that CON compared with HFS group P < 0.05,. and £ indicates that CS compared with HF group P < 0.05. After 7 days of administration, glucose tolerance of mice is abnormal due to the S961+ basal diet and the S961+ high-fat diet, and the glucose tolerance of animals is not abnormal due to the induction of the simple 45% high-fat diet.
The effects of S961 and high lipid induction on body weight during pregnancy are shown in table 2:
TABLE 2S 961 Effect of high fat induction on body weight of mice during pregnancy
Note: basal control group (CON), basal injection S961 group (CS), high fat control group (HF), high fat injection S961 group (HFs);
arepresents P < 0.05 compared to CON group,bindicates that P < 0.05 compared to the CS group,cindicates P < 0.05 compared to the HF group.
Table 2 shows that simple 45% high fat feeding can cause the most weight gain in animals, but S961-induced gestational diabetes can cause a decrease in weight gain.
The effects of S961 and high lipid induction on the intrauterine growth development of the rats are shown in table 3:
TABLE 3 influence of S961 and high fat induction on growth and development of fetal mice
Note: basal control group (CON), basal injection S961 group (CS), high fat control group (HF), high fat injection S961 group (HFs);arepresents P < 0.05 compared to CON group,bindicates that P < 0.05 compared to the CS group,cindicates P < 0.05 compared to the HF group.
Table 3 shows that simple S961 or S961+ high fat feeding can cause a decrease in the intrauterine weight of mice.
The effects of S961 and high lipid induction on the growth and development of the mice during birth and lactation are shown in table 4:
TABLE 4 influence of S961 and high lipid induction on growth and development of rats
Note: basal control group (CON), basal injection S961 group (CS), high fat control group (HF), high fat injection S961 group (HFs);
arepresents P < 0.05 compared to CON group,bindicates that P < 0.05 compared to the CS group,cindicates P < 0.05 compared to the HF group.
Table 4 shows that simple S961 or S961+ high fat feeding can cause a reduction in birth weight in mice. However, after the animal is born, the S961-induced gestational diabetes group has higher obesity susceptibility, the weight gain is obviously higher than that of the control group, and in addition, the weight gain of the S961-combined high-fat feed-fed group is also obviously higher than that of the single high-fat feed-induced group.
The gestational diabetes model induced by subcutaneous injection of 20nmol/kg S961 daily starting 7 days of gestation in combination with high fat diet did not result in animal death nor cause serious complications or side effects.
The weight gain of the gestational diabetic animals induced by subcutaneous injection of 20nmol/kg S961 or combined with high fat feed every day starting from 7 days of gestation was reduced compared with the corresponding basal control group and high fat control group. The glucose tolerance abnormality of the animals affects the growth and development of the mice in utero and causes the mice to be easy to feel fat in the milk taking period.
Experimental results show that the method of the gestational diabetes mellitus model induced by subcutaneously injecting 20nmol/kg S961 every day from 7 days of pregnancy and combining a high-fat feed feeding method causes insulin resistance, insulin insufficiency of pregnant mice, impaired glucose tolerance and clinical manifestations of gestational diabetes mellitus, and the gestational diabetes mellitus mouse animal model is successfully established.
The modeling method can form a stable and normative gestational diabetes model, can be used for screening the gestational diabetes drugs and researching the influence of the gestational diabetes on the offspring growth and development, and has greater application value for screening the drugs for treating the gestational diabetes and researching the influence of the gestational diabetes on the offspring growth and development compared with other drug-induced gestational diabetes models.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention relates, several simple deductions or substitutions may be made without departing from the spirit of the invention, and all shall be considered as belonging to the scope of the invention.
Claims (10)
1. A method for establishing a novel gestational diabetes animal model is characterized in that the gestational diabetes animal model is established by subcutaneously injecting insulin receptor antagonist S96120nmol/kg once a day from 7 days of gestation of animals.
2. The method for creating a novel animal model of gestational diabetes according to claim 1, wherein the animal is a C57BL/6J female mouse.
3. The method for establishing the novel gestational diabetes animal model according to claim 2, wherein the method for establishing the gestational diabetes animal model comprises the following steps:
1) grouping feeding of female mice: dividing 6-week-old C57BL/6J female mice into 4 groups, wherein two groups of C57BL/6J female mice are given basal feed for 4 weeks; feeding two groups of C57BL/6J female mice with high-fat feed containing 45% fat for 4 weeks to establish a diet-induced obesity model;
2) after the group feeding for 4 weeks, C57BL/6J female mice are mated in cages and the pregnancy condition is observed;
3) insulin receptor antagonist S961 induces gestational diabetes: from the 7 th pregnancy of the C57BL/6J female mouse, the insulin receptor antagonist S96120nmol/kg is injected subcutaneously every day to establish the gestational diabetes animal model.
4. The method for establishing the novel gestational diabetes animal model according to claim 3, wherein the step 1) specifically comprises: after the C57BL/6J female mice aged 6 weeks are adaptively fed for 1 week, randomly dividing the mice into four groups, wherein the number of each group is the same, and measuring the basic fasting blood glucose; two groups of C57BL/6J female mice were given basal diet, and the other two groups of C57BL/6J female mice were given high fat diet containing 45% fat, fed for 4 weeks, and fasting blood glucose was measured after 4 weeks.
5. The method of claim 4, wherein in the step 1), the two groups of C57BL/6J female mice given basal diet are further divided into a basal control group (CON) and a basal injection group S961 (CS); the two groups of C57BL/6J female mice given high fat diet were further divided into a high fat control group (HF) and a high fat injected S961 group (HFs).
6. The method for establishing a novel animal model of gestational diabetes according to claim 3, wherein the basal feed in step 1) is a 10% fat sterile basal feed.
7. The method for establishing the novel gestational diabetes animal model according to claim 3, wherein the step 2) is specifically as follows: after 4 weeks of group feeding, C57BL/6J female mice were mated with male mice in 2:1 cages and the presence of vaginal emboli was observed, which was scored as day 0 gestation.
8. The method for establishing the novel gestational diabetes animal model according to claim 3, wherein the step 3) is specifically as follows: measuring fasting blood glucose for 7 days of gestation when C57BL/6J female mice are pregnant for 7 days; starting from 7 days of pregnancy of a C57BL/6J female mouse, an insulin receptor antagonist S96120nmol/kg is subcutaneously injected every day according to the weight of an animal, the solution concentration is 5nmol/ml, and the total injection amount is less than 150ul, so that a gestational diabetes animal model is established.
9. The method for creating a novel animal model of gestational diabetes mellitus according to claim 3, wherein in step 3), each C57BL/6J female mouse is continuously fed with the same feed before pregnancy in a free feeding manner.
10. The method for establishing a novel animal model of gestational diabetes according to claim 3, wherein in step 3), the insulin receptor antagonist S961 is administered at 5 pm daily until the day before birth.
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| US20170342097A1 (en) * | 2016-05-26 | 2017-11-30 | Cornell University | Methods and compositions to treat type-1 and type-2 diabetes |
| CN109922831A (en) * | 2016-08-30 | 2019-06-21 | 瑞泽恩制药公司 | Signal transduction by interfering glucagon receptor treats the method that severe insulin is resisted |
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