A method for constructing a gestational diabetes mouse model with humanized intestinal flora by fecal microbiota transplantation

Abstract

The present invention relates to the technical field of diabetes research, and provides a method for constructing a humanized gestational diabetes mouse model by fecal microbiota transplantation. The present invention uses fecal microbiota suspension of overweight and obese pregnant women with gestational diabetes to perform fecal microbiota transplantation on sterile mice to simulate the intestinal flora of GDM patients. The present invention solves the phenomenon of mouse death after fecal microbiota transplantation, and the blood sugar of mice during pregnancy increases significantly; and successfully solves the phenomenon of dystocia and death of mother mice and death of pups due to inability to carry pups after delivery, and the mother mice give birth normally. The present invention provides an ideal animal model for gestational diabetes research.

Description

Technical Field

[0001] The present invention relates to the technical field of diabetes research, and in particular to a method for constructing a gestational diabetes mouse model with humanized intestinal flora through fecal microbiota transplantation. Background Art

[0002] Gestational diabetes (GDM) traditionally refers to impaired glucose tolerance that develops or is first discovered during pregnancy. Gestational diabetes generally occurs in the second or third trimester of pregnancy and is not clearly diagnosed as overt diabetes before pregnancy. Studies have shown that overweight is one of the compounding factors for pregnant women to develop gestational diabetes, and increased weight increases the risk of GDM. Research on pregnant women with gestational diabetes and non-gestational diabetes will promote our understanding of the disease.

[0003] In recent years, the methods for establishing GDM animal models mainly include experimental animal models, spontaneous animal models and transgenic animal models. Spontaneous animal diseases occur and develop under natural conditions, which are very similar to human diseases, but the source of such models is limited and it is impossible to apply them in large quantities; transgenic animal models are expensive and take a long time to reproduce. Because they are caused by genes, they cannot reflect the characteristics of gestational diabetes caused by endocrine disorders caused by abnormal metabolism. They are difficult to use for the efficacy evaluation of food, medicine, etc., and are not suitable for general experimental research; experimental animal models are often used in experimental research. At present, the experimental animal models chosen by many researchers are to use physical, chemical, biological and other pathogenic factors to cause certain damage to animal tissues, organs and even the whole body, thereby inducing certain functions, metabolism or toxic side effects similar to human diseases. Animal models provide an attractive alternative for studying the mechanisms behind GDM and possible treatments. At present, the commonly used animal models are mice or rats. The commonly used modeling methods are high-dose STZ, alloxan and other chemical drugs, which cause serious insulin damage, and high-sugar and high-fat diets with long cycles and low success rates. In addition, these models cannot simulate the characteristics of the intestinal flora of GDM patients, and the intestinal flora plays an important role in the occurrence and development of gestational diabetes. There is currently a lack of mature technology for fecal microbiota transplantation to construct a humanized gestational diabetes sterile mouse model. In addition, humanized fecal microbiota transplantation is difficult, and there is no unified standard for the optimal time period for intervention, which requires more in-depth research. The current methods for constructing gestational diabetes mouse models mainly have the following problems: 1) There is no significant change in blood sugar during pregnancy, and the modeling is unsuccessful; 2) The experimental mice suffer from severe weight loss and a high mortality rate, making it difficult to carry out related research such as intervention; 3) Due to dystocia, failure to carry the offspring, and failure to breastfeed, the offspring die, making it difficult to study the effects of pregnancy intervention on the offspring; 4) The intestinal flora of GDM patients cannot be simulated, reducing the reliability of the research results. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a gestational diabetes mouse model with humanized intestinal flora by fecal microbiota transplantation.

[0005] The present invention is conceived as follows: Germ-free (GF) mice refer to mice without bacteria, viruses, fungi, protozoa and other saprophytic or parasitic life forms. Fecal microbiota transplantation (FMT) refers to transplanting the functional flora in the feces of healthy people into the gastrointestinal tract of patients to rebuild new intestinal flora and realize the humanization of intestinal flora. The present invention innovatively selects fecal bacterial suspensions of overweight and obese women with gestational diabetes who meet the standards to gavage sterile mice, exerts its destructive metabolism to promote the occurrence of gestational diabetes, and simulates the intestinal flora during pregnancy to realize the humanization of intestinal flora. At the same time, combined with the effect of high-fat diet on endocrine disorders, and low-dose, short-term STZ injection slightly destroys the function of pancreatic islet B cells, constructs a humanized intestinal flora gestational diabetes sterile mouse model, and ensures that its genes, intestinal flora and other organs simulate the state of endocrine disorders on the premise of improving the success of modeling (determined by the results of oral glucose tolerance test during pregnancy), improves the similarity of model simulation, and the reliability of evaluating the intervention efficacy of food, drugs, etc.

[0006] In order to achieve the purpose of the present invention, the present invention provides a method for constructing a humanized gestational diabetes mouse model with intestinal flora by fecal microbiota transplantation, comprising the following steps:

[0007] (1) 7-8 week old female C57BL / 6J germ-free mice that had been pregnant once were selected as experimental subjects; they were gradually transitioned from normal feed to high-fat feed for 3-14 days;

[0008] (2) Feces of women diagnosed with gestational diabetes were collected and bacterial suspension was prepared. 5 ~10 7 CFU / mL was administered orally to mice fed a high-fat diet, and then 10 CFU / mL was administered orally to mice fed a high-fat diet 3-10 days later. 7 ~10 9 CFU / mL was administered to mice by gavage at a high dose of 0.1-0.4 m / time;

[0009] (3) After intragastric administration, observe the condition of the mice. When the mice recover to normal, with no weight loss and a normal diet, mate them together and observe the pregnancy. OGTT is measured on days 13-16 (preferably day 15) of pregnancy to establish a humanized intestinal flora gestational diabetes mouse model.

[0010] In steps (2) and (3), the rats are fed with high-fat feed throughout the entire process.

[0011] Furthermore, step (3) further includes:

[0012] After intragastric administration, the mice were observed for their condition. When the mice recovered to normal, with no weight loss and normal diet, they were mated together and their pregnancy was observed. On days 2, 3, 4, and 5 of female gestation, STZ (streptozotocin) solution with a concentration of 15-25 mg / kg was intraperitoneally injected daily. The injection dose was 0.1-0.2 mL / mouse, and the injection time was controlled within 3-120 seconds. Before the first injection, the mice were fasted for 8-15 hours at night, and high-fat feed was added 1-3 hours after the injection. 5-10% glucose drinking water was added 3-5 hours after the injection. Normal drinking water was used 8-12 hours after the injection. Fasting was not required for the next few injections.

[0013] OGTT was measured on 13-16 days of pregnancy (preferably 15 days) to establish a humanized intestinal flora gestational diabetes mouse model.

[0014] Furthermore, the feces in step (2) comes from women with gestational diabetes who have not taken probiotic products, antibiotics or any drugs within three months, have no infectious diseases, and have no constipation, diarrhea or other physical discomfort.

[0015] Furthermore, the fat content of the high-fat feed is 40-80% (preferably 60%).

[0016] In the present invention, the high-fat feed is fed in an ad libitum manner.

[0017] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0018] This proposal addresses several technical issues in the modeling of gestational diabetes, as follows:

[0019] (I) Current studies have found that when germ-free mice were only transplanted with bacterial suspensions from pregnant women with GDM, their blood sugar levels did not rise significantly during pregnancy. The present invention solves the problem of no significant changes in blood sugar levels during the glucose tolerance test during pregnancy by intragastric administration of bacterial suspensions from overweight and obese women with gestational diabetes, combined with a high-fat diet and a small amount of STZ injection. The results of the glucose tolerance test during pregnancy showed that blood sugar levels rose significantly.

[0020] (ii) Current studies have found that after sterile mice were transplanted with bacterial suspension, they developed intolerance to fecal microbiota transplantation and even died, as well as weight loss after transplantation. The present invention adopts a method of mildly transplanting bacterial suspension, which shortens the recovery period of mice after fecal microbiota transplantation, improves the success rate of modeling and shortens the experimental period;

[0021] (iii) Using fecal intervention from volunteers who are overweight / obese, have gestational diabetes, and have not taken any medications that affect the intestinal flora to increase the interference of the intestinal flora on glucose metabolism and improve the success rate of the model;

[0022] (IV) Currently, the problem of germ-free mice not taking care of their offspring and eating their offspring makes it difficult to study the effects of pregnancy intervention on offspring. The present invention allows the mother mouse to become pregnant once before starting the intervention, which solves the problem of germ-free pregnant mice not taking care of their offspring after giving birth, making it impossible to study the intergenerational effects of pregnancy intervention;

[0023] (V) The present invention successfully achieved the construction of a germ-free mouse model of gestational diabetes with humanized intestinal flora, thereby providing an ideal animal model for gestational diabetes research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph of blood glucose concentration (OGTT) of mice at 15 days of pregnancy in the first embodiment of the preferred embodiment of the present invention;

[0025] Figure 2 This is a graph of blood glucose concentration (OGTT) of mice 1 day before cage-housing (left) and 15 days of pregnancy (right) in Scheme 2 of the preferred embodiment of the present invention;

[0026] Figure 3 This is a graph of blood glucose concentration (OGTT) of mice in comparative example 1 of the present invention at day 16 of pregnancy (left), 2 days before cage placement, and day 16 of pregnancy (right);

[0027] Figure 4 This is a graph of blood glucose concentration (OGTT) of mice in comparative example 2 of the present invention at day 16 of pregnancy;

[0028] Figure 5 These are the mouse AUC graphs for Scheme 1 (left) and Scheme 2 (right) in the preferred embodiments of the present invention. DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] The selection method of experimental mice in the following examples was: 7-8 week old C57BL / 6J germ-free mice, pregnant once.

[0031] The fecal microbiota transplantation technology used to construct a gestational diabetes mouse model uses fecal intervention from volunteers who are overweight / obese, have gestational diabetes, and have not taken any medications that affect the intestinal flora, to increase the interference of the intestinal flora on sugar metabolism and improve the success rate of the model;

[0032] The inclusion criteria are as follows:

[0033] Type 1:

[0034] 1) Single pregnancy, gestational age 24 weeks and above (late pregnancy);

[0035] 2) Pre-pregnancy BMI ≥ 24 kg / m 2 ;

[0036] 3) The patient had undergone gestational diabetes screening at 24-28 weeks of pregnancy and was diagnosed with gestational diabetes;

[0037] Type 2:

[0038] 1) BMI ≥ 24 kg / m before and during pregnancy 2 ;

[0039] 2) The patient had undergone gestational diabetes screening at 24 to 28 weeks of pregnancy and was diagnosed with gestational diabetes;

[0040] 3) Age ≤ 45 years old;

[0041] Type 3:

[0042] 1) Single pregnancy, gestational age 24 weeks and above (late pregnancy);

[0043] 2) The patient had undergone gestational diabetes screening at 24 to 28 weeks of pregnancy and was diagnosed with gestational diabetes;

[0044] All three of the above types must meet the following requirements:

[0045] 1) No probiotic products, antibiotics or any medications have been taken within three months;

[0046] 2) No infectious diseases (such as AIDS, hepatitis B, hepatitis C, etc.) and other diseases / illnesses;

[0047] 3) No constipation, diarrhea or other physical discomfort.

[0048] The moderated high-fat diet intervention method involved in the following embodiments is as follows: The high-fat diet intervention adopts a gradual addition method to gradually increase the proportion of high-fat diet in normal diet, and the transition time range from normal diet to complete high-fat diet is (3-14 days).

[0049] The mild fecal microbiota transplantation method involved in the following embodiments is as follows: low dose first, then high dose, with a certain interval (3-10 days) to avoid the toxicity of high-dose microbiota to germ-free mice. The dose range (10 5 ~10 9 CFU / mL).

[0050] Observe the status of mice through weight, food intake / hair, etc. For mice with abnormal behaviors, such as grooming, dull fur, etc., improve their health status and increase the success rate of modeling by feeding them in single cages.

[0051] The fluid replacement STZ injection technique involved in the following examples is as follows: 2 / 3 / 4 / 5 days of pregnancy, intraperitoneal injection of STZ (15-25 mg / kg) every day, the injection drug dosage should be 0.1-0.2mL; the STZ solution is injected intraperitoneally, and the injection speed should be slow. Before the first injection, fast for 8-15h at night, add feed 1-3h after injection, add 5-10% glucose drinking water 3-5h after injection, and change to normal drinking water 8-12h after injection. Fasting is no longer required for the next few injections.

[0052] Care techniques for female mice after conception: Sterile sunflower seeds and other nutrients are added separately, with the added amount being 3-5 seeds per mouse.

[0053] The high-fat feed used in the following examples was purchased from Changzhou Shuyi Shuer Biotechnology Co., Ltd. and had a fat content of 60%.

[0054] Example 1

[0055] 1. Preparation of fecal bacterial suspension

[0056] In an anaerobic incubator, all stool specimens were melted, mixed together, vortexed and shaken at room temperature, then allowed to stand for 3-20 minutes, filtered with gauze, and frozen in a -80°C refrigerator for later use.

[0057] 2. Construction of a humanized germ-free mouse model of gestational diabetes mellitus using fecal microbiota transplantation

[0058] (1) Option 1: Fecal microbiota transplantation + high-fat diet + STZ injection

[0059] Eight-week-old female C57BL / 6J germ-free mice were selected for model construction. They were given free access to water and food. They were conceived once and fed adaptively after delivery. After the mice recovered their weight, they were randomly divided into a control group and a model group to ease the adaptation to high-fat diet. The model was gavaged with fecal bacterial suspension from overweight / obese women with gestational diabetes. A low dose (about 10 7 CFU / mL) after high dose (10 8 CFU / mL) was gently gavaged at a dose of 0.2 mL / time / mouse. The control group was gavaged with sterile phosphate solution (PBS) twice, with a dose of 0.2 mL / time / mouse. After the mice recovered (no weight loss, normal condition, normal diet), breeding began, and STZ was injected during pregnancy for four consecutive injections (25 mg / kg STZ solution was injected intraperitoneally every day on days 2, 3, 4, and 5 of female gestation; fasting for 12 hours at night before the first injection, high-fat feed was added 2 hours after the injection, 5% glucose drinking water was added 4 hours after the injection, and normal drinking water was replaced 10 hours after the injection. Fasting was not required for the next few injections). The control group had a normal diet throughout the whole process, and the model group had a high-fat diet throughout the whole process. OGTT was measured on the 15th day of pregnancy.

[0060] (2) Option 2: Fecal microbiota transplantation + high-fat diet

[0061] Eight-week-old female C57BL / 6J germ-free mice were selected for model construction. They were given free access to water and food. They were conceived once and fed adaptively after delivery. After the mice recovered their weight, they were randomly divided into a control group and a model group to ease the adaptation to high-fat diet. The model group was gavaged with fecal bacterial suspension from overweight / obese women with gestational diabetes. A low dose (about 10 7 CFU / mL) after high dose (10 8 CFU / mL) was gently gavaged at a dose of 0.2 mL / time / mouse. The control group was gavaged with sterile phosphate solution (PBS) twice, 0.2 mL / time / mouse. After the mice recovered (no weight loss, normal condition, normal diet), breeding began. The control group had a normal diet throughout the whole process, and the model group had a high-fat diet throughout the whole process. OGTT was measured on the 15th day of pregnancy.

[0062] Comparative Example 1

[0063] Eight-week-old female C57BL / 6J germ-free mice were selected for model construction. Water and food were freely available and adaptive feeding was carried out for one week. The mice were randomly divided into a control group and a model group. The control group was gavaged with sterile PBS solution at a dose of 0.2 mL / time / mouse. The model group G1 was gavaged with fecal flora fluid from patients with gestational diabetes. The flora was derived from feces of overweight and obese pregnant women with gestational diabetes. The gavage was performed on D1, D3, and D5, respectively, with a dose of 0.2 mL / time / mouse and a fecal flora concentration of 10 8 CFU / mL. The mice were fed a high-fat diet during the fecal microbiota transplantation. Male mice did not need to be modeled. The G1 group continued to be fed a high-fat diet for 2 weeks after fecal microbiota transplantation. OGTT was measured two days before cage placement and on the 16th day of pregnancy.

[0064] The results showed that some mice died after fecal microbiota transplantation in the G1 group, and their blood sugar levels during pregnancy decreased compared to before pregnancy. In addition, their blood sugar levels during pregnancy did not increase significantly.

[0065] Comparative Example 2

[0066] Eight-week-old female C57BL / 6J germ-free mice were selected for model construction. They were given free access to water and food and were adaptively fed for one week. The mice were randomly divided into a control group and a model group. The control group was gavaged with sterile PBS solution at a dose of 0.2 mL / time / mouse. The model group G2 was gavaged with fecal flora from patients with gestational diabetes. The flora was derived from feces of overweight and obese pregnant women with gestational diabetes. The gavage was performed on D1, D3, and D5, respectively, with a dose of 0.2 mL / time / mouse and a fecal flora concentration of 10 8 CFU / mL. The mice were fed a high-fat diet during the fecal microbiota transplantation. Male mice did not need to be modeled. The G2 group continued to be fed a high-fat diet for 4 weeks after fecal microbiota transplantation. OGTT was measured two days before cage placement and on the 16th day of pregnancy.

[0067] The results showed that some mice in the G2 group died after fecal microbiota transplantation, there was no significant increase in blood sugar during pregnancy, and the pregnancy rate of mice in the G2 group was low (pregnancy rate 22.22%).

[0068] 3. OGTT assay

[0069] OGTT assay method: The mother mice were fasted for 6 hours (during the daytime when the mice were sleeping, so as to avoid long fasting at night and premature birth), and were allowed to drink water freely. After measuring blood sugar at 0 min, the mice were weighed. After the mice adapted for 30 min, they were given oral glucose solution (2 g / kg body weight). After gavage, tail blood was collected at 15, 30, 60, 90, and 120 min to measure blood sugar. Blood sugar was measured twice.

[0070] The blood glucose levels of mice in the model groups of Scheme 1 and Scheme 2 on the 15th day of pregnancy were significantly higher than those in the control group, and the difference was statistically significant, which was consistent with the blood glucose characteristics of GDM, and the modeling was successful.

[0071] 4. Cycle and mortality rate

[0072] The experimental results are shown in Figure 1~Figure 4 As shown in Table 1, the experimental results show that neither Scheme 1 nor Scheme 2 resulted in mouse death, dystocia, or inability to carry offspring.

[0073] Table 1

[0074]

[0075] 5. Moulding rate detection

[0076] OGTT was measured on the 15th day of pregnancy. The blood glucose levels of mice in the model groups of Scheme 1 and Scheme 2 were significantly increased compared with those in the control group at 0min, 15min, 30min, 60min, 90min, and 120min, and the model success rate was 100%.

[0077] 6. Mouse AUC graph

[0078] On the 15th day of pregnancy, an oral glucose tolerance test (OGTT) was performed. The blood glucose levels at (0, 15, 30, 60, 90, 120) min after intragastric glucose administration were measured, and the area under the curve (AUC) was calculated. The results are shown in Figure 5 .

[0079] The present invention uses fecal bacterial suspension of overweight, obese pregnant women with gestational diabetes to perform fecal bacterial transplantation on germ-free mice, simulating the intestinal flora of GDM patients, thereby providing an ideal animal model for gestational diabetes research.

[0080] The present invention solves the problem of mouse death after fecal microbiota transplantation, and no mice died after fecal microbiota transplantation; the present invention solves the problem of no obvious increase in blood sugar during pregnancy, and the blood sugar of mice increased significantly during pregnancy; the present invention solves the problem of mother mice dying of dystocia and the inability to carry their pups after delivery, leading to the death of their pups, and the mother mice gave birth normally.

[0081] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a humanized gestational diabetes mouse model with intestinal flora by fecal microbiota transplantation, characterized in that: The following steps are involved: (1) 7-8 week old female C57BL / 6J germ-free mice that had been pregnant once were selected as experimental subjects; they were gradually transitioned from normal feed to high-fat feed for 3-14 days; (2) Feces of women diagnosed with gestational diabetes were collected and bacterial suspension was prepared. 5 ~10 7 CFU / mL was administered orally to mice fed a high-fat diet, and then 10 CFU / mL was administered orally to mice fed a high-fat diet 3-10 days later. 7 ~10 9 The mice were gavaged with a high dose of CFU / mL, and the gavage dose was 0.1-0.4 mL / time; (3) After intragastric administration, the mice were observed. When they recovered to normal, with no weight loss and a normal diet, they were mated together and their pregnancy was observed. OGTT was measured on days 13-16 of pregnancy to establish a humanized intestinal flora gestational diabetes mouse model. Wherein, steps (2) and (3) are all high-fat feeding; Step (3) also includes: After intragastric administration, the mice were observed for their condition. When the mice recovered to normal, their weight did not decrease, and their diet was normal, they were mated together and their pregnancy was observed. On days 2, 3, 4, and 5 of female gestation, STZ solution at a concentration of 15-25 mg / kg was injected intraperitoneally every day. The injection dose was 0.1-0.2 mL / mouse, and the injection time was controlled within 3-120 seconds. Before the first injection, they were fasted for 8-15 hours at night, and high-fat feed was added 1-3 hours after the injection. 5-10% glucose drinking water was added 3-5 hours after the injection. Normal drinking water was used 8-12 hours after the injection. Fasting was not required for the next few injections. OGTT was measured on gestational days 13-16, and a humanized intestinal flora gestational diabetes mouse model was established.

2. The method according to claim 1, characterized in that Step (3) Measure OGTT on the 15th day of pregnancy.

3. The method according to claim 1, characterized in that The feces in step (2) comes from women with gestational diabetes who have not taken probiotic products, antibiotics or any drugs within three months, have no infectious diseases, and have no constipation, diarrhea or other physical discomfort.

4. The method according to any one of claims 1 to 3, characterized in that: The fat content of the high-fat feed is 40-80%.

5. The method according to claim 4, characterized in that The high-fat diet was fed freely.

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