Construction and application of recombinant inbred type 2 diabetes rat model

Abstract

This invention belongs to the field of experimental animal model construction technology, specifically relating to the construction method and application of a recombinant inbred type 2 diabetic rat model. The parent rats are SD (fa+ / +) or Wistar (fa+ / +) females and Zucker (fa+ / -) males. Heterozygous (fa+ / -) female offspring are backcrossed with the fathers until the F6 generation. Then, F6 heterozygous (fa+ / -) female rats are crossed with F6 heterozygous (fa+ / -) male rats. The resulting homozygous (fa- / -) SD / Zucker or Wistar / Zucker rats are the recombinant inbred type 2 diabetic rats. The rats obtained by this invention do not express functional leptin receptors, exhibit hyperphagia, and spontaneous polydipsia and polyphagia, which closely resembles the real-world scenario of type 2 diabetes caused by overnutrition under normal dietary conditions in humans.

Description

Technical Field

[0001] This invention belongs to the field of experimental animal model construction technology, specifically relating to the construction method and application of a recombinant inbred type 2 diabetic rat model. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, primarily including type 1 diabetes (T1D) and type 2 diabetes (T2D). In recent years, the number of people with diabetes worldwide has continued to rise, posing a serious threat to human health. To gain a deeper understanding of the pathogenesis of diabetes and develop effective intervention strategies, it is of great significance to construct stable, reproducible animal models that highly resemble the pathological process of human diabetes.

[0003] As mammals, rats are remarkably similar to humans in growth, development, and metabolism. With over 85% genetic similarity, rats are considered ideal model organisms among mammals due to their large litter size, moderate size, and ease of experimental handling. They are widely used in diabetes research. Common rodent models of type 2 diabetes include spontaneous diabetes models (such as Zucker diabetic fatty (ZDF) rats, Goto-Kakizaki (GK) rats, db / db mice, and ob / ob mice), diet-induced models (such as high-fat diets, high-carbohydrate diets, and high-fat combined with high-carbohydrate diets), and chemically induced models (such as streptozotocin and alloxan).

[0004] Existing models used to study type 2 diabetes are typically diet-induced models. These models work by inducing insulin resistance through extreme diets (such as high-fat diets), leading to type 2 diabetes. Chemically induced models, on the other hand, damage the insulin-secreting β-cells in the rat pancreas, causing insufficient insulin secretion and decreased function. Simultaneously, feeding rats a high-fat, high-carbohydrate diet induces type 2 diabetes. Furthermore, in studies of food, drugs, and nutrients, animal interventions such as gavage, intraperitoneal injection, and subcutaneous injection are used. However, the metabolic characteristics of rats induced with type 2 diabetes using special diets differ significantly from those in humans, do not conform to the pathological process of human type 2 diabetes, and fail to reflect the synergistic progression of insulin resistance and β-cell dysfunction. This is because the vast majority of human type 2 diabetes cases are not caused by short-term consumption of high-fat, high-carbohydrate diets, but rather by accumulated nutritional excess under normal dietary conditions.

[0005] Therefore, it is urgent to construct a rat model that conforms to the real-life scenario of type 2 diabetes caused by overnutrition under normal dietary conditions in humans. Only in this way can we simulate the natural evolution of human diabetes from metabolic syndrome to diabetes. This model is of great significance for exploring the mechanisms of action of food, drugs and precision nutritional intervention on type 2 diabetes and its complications.

[0006] Meanwhile, the rat model established by this invention will autonomously drink and eat more, which is a natural development of type 2 diabetes, thus avoiding the harm to the rats caused by treatment methods such as gavage, intraperitoneal injection and subcutaneous injection. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a method for constructing a type 2 diabetic rat model. The resulting rats do not express functional leptin receptors. Due to the absence of the leptin signaling system, these rats exhibit hyperphagia and spontaneous polydipsia, allowing them to show significant weight and fat gain around 5 weeks of age without damaging pancreatic islet cells, even under normal maintenance diet conditions. Hyperglycemia develops at 8-10 weeks of age. This closely mirrors the real-world scenario of type 2 diabetes in humans caused by overnutrition under normal dietary conditions.

[0008] The recombinant inbred type 2 diabetic rat model was constructed as follows: Female rats were selected from either SD (fa+ / +) or Wistar (fa+ / +) as parental rats, and male rats were selected from Zucker (fa+ / -) as parental rats. The female and male rats were crossed to obtain the F1 generation. The heterozygous (fa+ / -) female rats from the F1 generation were backcrossed with the male rats to obtain the F2 generation. The heterozygous (fa+ / -) female rats from the F2 generation were backcrossed with the male rats to obtain the F3 generation. The heterozygous (fa+ / -) female rats from the F3 generation were then backcrossed with the male rats to obtain the F3 generation. Female rats from the - group were backcrossed with male paternal rats to obtain the F4 generation. Female rats from the F4 generation heterozygotes (fa+ / -) were backcrossed with male paternal rats to obtain the F5 generation. Female rats from the F5 generation heterozygotes (fa+ / -) were backcrossed with male paternal rats to obtain the F6 generation. Female rats from the F6 generation heterozygotes (fa+ / -) were then crossed with male rats from the F6 generation heterozygotes (fa+ / -). The resulting SD / Zucker rats or Wistar / Zucker rats with homozygous (fa- / -) genotypes are the recombinant inbred type 2 diabetic rats.

[0009] Furthermore, the specific steps are as follows:

[0010] Step 1. Select parent rats, with female rats being either SD (fa+ / +) or Wistar (fa+ / +) and male rats being Zucker (fa+ / -).

[0011] Step 2: Using female SD (fa+ / +) rats and male Zucker (fa+ / -) rats as parents, crossbreed to obtain F1 generation SD / Zucker rats; F1 generation SD / Zucker rats have only two genotypes, namely wild type (fa+ / +) and heterozygous (fa+ / -), and the resulting F1 generation SD / Zucker rats carry half of the chromosomal genetic material of the maternal SD (fa+ / +) rats, with all the mitochondrial genetic material of the female rats coming from the maternal parent;

[0012] Alternatively, female Wistar (fa+ / +) rats and male Zucker (fa+ / -) rats can be used as parents to cross and obtain F1 generation Wistar / Zucker rats. The F1 generation Wistar / Zucker rats have only two genotypes: wild type (fa+ / +) and heterozygous (fa+ / -). The F1 generation Wistar / Zucker rats carry half of the chromosomal genetic material of the maternal Wistar (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0013] Step 3. Select female rats from the F1 generation heterozygous (fa+ / -) rats, i.e., female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F2 generation SD / Zucker rats. The F2 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F2 generation SD / Zucker rats carry one-quarter of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0014] Alternatively, female Wistar / Zucker (fa+ / -) rats from the F1 generation heterozygous (fa+ / -) can be selected and backcrossed with male Zucker (fa+ / -) rats to obtain F2 generation Wistar / Zucker rats. The F2 generation Wistar / Zucker rats have three genotypes: wild-type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F2 generation Wistar / Zucker rats carry one-quarter of the chromosomal genetic material from the maternal Wistar (fa+ / +) rats, with all mitochondrial genetic material of the female rats coming from the maternal parent.

[0015] Step 4. Select female rats from the F2 generation heterozygous (fa+ / -) rats, i.e., female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F3 generation SD / Zucker rats. The F3 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F3 generation SD / Zucker rats carry one-eighth of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0016] Alternatively, female Wistar / Zucker (fa+ / -) rats from the F2 generation heterozygous (fa+ / -) can be selected and backcrossed with male Zucker (fa+ / -) rats to obtain F3 generation Wistar / Zucker rats. The F3 generation Wistar / Zucker rats have three genotypes: wild-type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F3 generation Wistar / Zucker rats carry one-eighth of the chromosomal genetic material from the maternal Wistar (fa+ / +) rats, with all the mitochondrial genetic material of the female rats coming from the maternal parent.

[0017] Step 5. Select female rats from the F3 generation heterozygous (fa+ / -) rats, i.e. female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F4 generation SD / Zucker rats. The F4 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F4 generation SD / Zucker rats carry one-sixteenth of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0018] Alternatively, female Wistar / Zucker (fa+ / -) rats from the F3 generation heterozygous (fa+ / -) can be selected and backcrossed with male Zucker (fa+ / -) rats to obtain F4 generation Wistar / Zucker rats. The F4 generation Wistar / Zucker rats have three genotypes: wild-type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F4 generation Wistar / Zucker rats carry one-sixteenth of the chromosomal genetic material from the maternal Wistar (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0019] Step 6. Select female rats from the F4 generation heterozygous (fa+ / -) rats, i.e., female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F5 generation SD / Zucker rats. The F5 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F5 generation SD / Zucker rats carry 1 / 32 of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0020] Alternatively, female Wistar / Zucker (fa+ / -) rats from the F4 generation heterozygous (fa+ / -) can be selected and backcrossed with male Zucker (fa+ / -) rats to obtain the F5 generation Wistar / Zucker rats. The F5 generation Wistar / Zucker rats have three genotypes: wild-type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F5 generation Wistar / Zucker rats carry 1 / 32 of the chromosomal genetic material from the maternal Wistar (fa+ / +) rats, with all the mitochondrial genetic material of the female rats coming from the maternal parent.

[0021] Step 7. Select female rats from the F5 generation heterozygous (fa+ / -) rats, i.e. female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F6 generation SD / Zucker rats. The F6 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F6 generation SD / Zucker rats carry 1 / 64 of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0022] Alternatively, female Wistar / Zucker (fa+ / -) rats from the F5 generation heterozygous (fa+ / -) can be selected and backcrossed with male Zucker (fa+ / -) rats to obtain F6 generation Wistar / Zucker rats. The F6 generation Wistar / Zucker rats have three genotypes: wild-type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F6 generation Wistar / Zucker rats carry 1 / 64 of the chromosomal genetic material of the maternal Wistar (fa+ / +) rats, with all the mitochondrial genetic material of the female rats coming from the maternal parent.

[0023] Step 8. Select female rats from the F6 generation heterozygotes (fa+ / -) and cross them with male rats from the F6 generation heterozygotes (fa+ / -) to obtain SD-Zucker recombinant inbred rats;

[0024] Alternatively, female rats from the F6 generation heterozygotes (fa+ / -) can be crossed with male rats from the F6 generation heterozygotes (fa+ / -) to obtain the Wistar-Zucker recombinant inbred rat line;

[0025] Step 9. Among the SD-Zucker recombinant inbred rats obtained in Step 8, there are three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). Among them, the SD / Zucker rats with the homozygous genotype (fa- / -) cannot express the functional leptin receptor, which are the SD / Zucker recombinant inbred type 2 diabetic rats.

[0026] Alternatively, the Wistar-Zucker recombinant inbred rats obtained in step 8 have three genotypes: wild-type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). Among them, the Wistar / Zucker rats with the homozygous genotype (fa- / -) cannot express a functional leptin receptor, which are the Wistar / Zucker recombinant inbred type 2 diabetic rats.

[0027] The method for constructing the recombinant inbred type 2 diabetic rat model described herein is applied in the construction of diabetic rat models.

[0028] Furthermore, when the recombinant inbred type 2 diabetic rats were fed a normal diet, hyperglycemia appeared at 8-10 weeks of age, with non-fasting blood glucose levels of 200-500 mg / dL at 8-10 weeks of age and 400-600 mg / dL after 10 weeks. The main nutritional components of the normal diet were: crude protein ≤20.7%, crude fiber ≤2.31%, crude fat ≤4.15%, carbohydrates ≤40%, calcium ≤1.24%, and phosphorus ≤0.83%.

[0029] Compared with existing technologies, the beneficial effects of this technical solution are as follows:

[0030] This study provides a method for constructing a type 2 diabetic rat model and successfully constructed SD / Zucker recombinant inbred type 2 diabetic rats and Wistar / Zucker recombinant inbred type 2 diabetic rats. The offspring of these two rat models reproduce stably and can spontaneously develop diabetes under normal diet. They can be used for research on the biology, pathogenesis, and intervention methods of type 2 diabetes. Attached Figure Description

[0031] Figure 1 A schematic diagram illustrating the construction of a type 2 diabetic rat model using the SD / Zucker recombinant inbred strain;

[0032] Figure 2 This is a schematic diagram showing the changes in blood glucose in SD / Zucker recombinant inbred type 2 diabetic rats under free-feeding conditions.

[0033] Figure 3 This is a schematic diagram showing the changes in body weight of SD / Zucker recombinant inbred type 2 diabetic rats during their growth process.

[0034] Figure 4 A comparison of heterozygous (fa+ / -) and homozygous (fa- / -) SD / Zucker rats at week 11 in the SD / Zucker recombinant inbred rat line;

[0035] Figure 5 A schematic diagram showing the total cholesterol content in the serum of SD / Zucker recombinant inbred type 2 diabetic mice at 3 weeks, 5 weeks, 8 weeks, and 11 weeks.

[0036] Figure 6 A schematic diagram showing the total triglyceride content in the serum of SD / Zucker recombinant inbred type 2 diabetic rats at 3 weeks, 5 weeks, 8 weeks, and 11 weeks.

[0037] Figure 7 A schematic diagram showing the changes in blood glucose in Wistar / Zucker recombinant inbred type 2 diabetic rats under free-feeding conditions;

[0038] Figure 8 This is a schematic diagram showing the changes in body weight of Wistar / Zucker recombinant inbred type 2 diabetic rats during their growth process.

[0039] Figure 9 A comparison of heterozygous (fa+ / -) Wistar / Zucker rats and homozygous (fa- / -) Wistar / Zucker rats at week 11 in the Wistar Zucker recombinant inbred rat line;

[0040] Figure 10 A schematic diagram showing the total cholesterol content in the serum of Wistar / Zucker recombinant inbred type 2 diabetic rats at 3 weeks, 5 weeks, 8 weeks, and 11 weeks.

[0041] Figure 11 This is a schematic diagram showing the total triglyceride content in the serum of Wistar / Zucker recombinant inbred type 2 diabetic rats at 3 weeks, 5 weeks, 8 weeks, and 11 weeks. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. However, the scope of the present invention is not limited to the following embodiments.

[0043] The principle behind this invention for constructing a recombinant inbred type 2 diabetic rat model is as follows: SD rats (or Wistar rats) have a stable genetic background, good health, and strong reproductive capacity. Zucker rats are an ideal model for studying obesity and type 2 diabetes. Simultaneously, their mitochondrial genetic material is maternally inherited, and changes in mitochondrial function are associated with many metabolic diseases. Crossing female SD (or Wistar) rats with male Zucker rats produces offspring carrying both SD (or Wistar) mitochondrial material and Zucker and SD chromosomal genetic material. When the two strains are crossed, the F1 generation receives half of the chromosomal genetic material from each parent. Through repeated backcrossing of female heterozygotes with the father, the maternal chromosomal genetic material is diluted (by the sixth generation, the maternal chromosomal genetic material is reduced to one-sixtieth of its original value, minimizing the influence of the maternal genetic set on the offspring), while preserving the maternal mitochondrial genetic material. This allows the leptin receptor gene mutation to be carried in strains other than Zucker rats, making it possible to study the contribution of mitochondrial genetic material in type 2 diabetes. Through similar studies on this strain of animals, we found that: 1. The leptin receptor gene in SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) retains mutations in the new mitochondrial background. 2. Feeding SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) with a standard rat maintenance diet can induce diabetes. 3. SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) primarily exhibit severe hyperglycemia in adulthood, with no further weight gain. 4. Leptin receptor mutations in SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) lead to polydipsia, polyphagia, and diabetes, making them an ideal animal model for studying diabetes caused by overnutrition.

[0044] Taking the commonly used type 2 diabetic ZDF rat model as an example, research data shows that ZDF rats typically require a Purina Rodent LabDiet 5008 diet to develop hyperglycemia, and the non-fasting blood glucose level of ZDF rats is 200-400 mg / dL at 8 weeks of age and 400-600 mg / dL at 12 weeks of age. In this invention, after constructing a recombinant inbred type 2 diabetic rat model, feeding them with a normal rat maintenance diet can induce diabetes, with blood glucose levels of 200-500 mg / dL at 8-10 weeks of age and reaching 400-600 mg / dL after 10 weeks. Specific diet comparisons are shown in Table 1.

[0045] Table 1: Comparison of main nutrient components between Purina Rodent LabDiet 5008 feed used to feed ZDF rats and ordinary rat maintenance feed used in this invention.

[0046] Main nutrients Animal feed formulation Animal feed formulation Feed Name Purina RodentLabDiet 5008 (for feeding Zucker diabeticfatty Rats) Standard rat maintenance diet (Wuhan Wanqian Jiaxing) (used to feed the SD / Zucker recombinant inbred type 2 diabetic rats and Wistar / Zucker recombinant inbred type 2 diabetic rats constructed in this invention) Crude protein (%) 23.0 20.7 Crude fiber (%) 4.0 2.31 Crude fat (%) 6.5 4.15 carbohydrate(%) 50 40 calcium(%) 1.2 1.24 phosphorus(%) 0.8 0.83

[0047] Table 1 shows that the Purina Rodent LabDiet 5008 diet formulation has higher proportions of major nutrients such as crude protein, crude fiber, crude fat, and carbohydrates than the standard rat maintenance diet used to feed the SD / Zucker recombinant inbred type 2 diabetic rats and Wistar / Zucker recombinant inbred type 2 diabetic rats constructed in this invention. This indicates that the SD / Zucker recombinant inbred type 2 diabetic rats and Wistar / Zucker recombinant inbred type 2 diabetic rats constructed in this invention are more likely to develop type 2 diabetes than traditional ZDF rats under lower dietary nutrient intake, making them ideal animal models for studying type 2 diabetes and its complications caused by overnutrition. This is because the homozygous mutation of the leptin receptor causes the SD / Zucker recombinant inbred type 2 diabetic rats and Wistar / Zucker recombinant inbred type 2 diabetic rats to spontaneously increase their food and water intake, which is more consistent with the real-world scenario of overnutrition and disease development in humans due to diet. This natural characteristic also avoids the harm to rats caused by other treatments such as gavage, intraperitoneal injection, and subcutaneous injection during studies of the effects of food, drugs, and nutrients on animals.

[0048] On the other hand, mitochondria are an important target for metabolic diseases. This diabetic rat model retains the mitochondrial genetic material carried by the maternal line (SD or Wistar rats) through backcrossing between offspring and parents, allowing the leptin receptor mutant gene to be carried in strains other than Zucker rats. This makes it possible to study the contribution of mitochondrial genetic material in type 2 diabetes, broadens the research field of animal models of metabolic diseases, and enriches the innovative aspects of Zucker rats as a model animal.

[0049] Example 1

[0050] The parental female rats were designated SD (fa+ / +) and the male rats were designated Zucker (fa+ / -) as examples. Figure 1 A schematic diagram illustrating the construction of the SD / Zucker type 2 diabetes recombinant inbred rat model. The construction method is as follows:

[0051] Step 1. Select parent rats, including female rats as SD (fa+ / +) and male rats as Zucker (fa+ / -);

[0052] Step 2: Using female SD (fa+ / +) rats and male Zucker (fa+ / -) rats as parents, crossbreed to obtain F1 generation SD / Zucker rats; F1 generation SD / Zucker rats have only two genotypes, namely wild type (fa+ / +) and heterozygous (fa+ / -), and the resulting F1 generation SD / Zucker rats carry half of the chromosomal genetic material of the maternal SD (fa+ / +) rats, with all the mitochondrial genetic material of the female rats coming from the maternal parent;

[0053] Step 3. Select female rats from the F1 generation heterozygous (fa+ / -) rats, i.e., female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F2 generation SD / Zucker rats. The F2 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F2 generation SD / Zucker rats carry one-quarter of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0054] Step 4. Select female rats from the F2 generation heterozygous (fa+ / -) rats, i.e., female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F3 generation SD / Zucker rats. The F3 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F3 generation SD / Zucker rats carry one-eighth of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0055] Step 5. Select female rats from the F3 generation heterozygous (fa+ / -) rats, i.e. female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F4 generation SD / Zucker rats. The F4 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F4 generation SD / Zucker rats carry one-sixteenth of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0056] Step 6. Select female rats from the F4 generation heterozygous (fa+ / -) rats, i.e., female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F5 generation SD / Zucker rats. The F5 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F5 generation SD / Zucker rats carry 1 / 32 of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0057] Step 7. Select female rats from the F5 generation heterozygous (fa+ / -) rats, i.e. female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) paternal rats to obtain F6 generation SD / Zucker rats. The F6 generation SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The F6 generation SD / Zucker rats carry 1 / 64 of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent.

[0058] Step 8. Select female rats from the F6 generation heterozygotes (fa+ / -) and cross them with male rats from the F6 generation heterozygotes (fa+ / -) to obtain SD-Zucker recombinant inbred rats;

[0059] Step 9. Among the SD-Zucker recombinant inbred rats obtained in Step 8, there are three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). Among them, the SD / Zucker rats with the homozygous genotype (fa- / -) cannot express the functional leptin receptor, which are the SD / Zucker recombinant inbred type 2 diabetic rats.

[0060] like Figure 2As shown, under normal maintenance diet, the blood glucose levels of lean rats remained within the normal range (blood glucose ≤200 mg / dL) during the free-feeding period after weaning, with minimal fluctuations. In contrast, the blood glucose levels of obese rats exceeded normal levels from week 8-9 onwards under free-feeding conditions, indicating a diabetic state. Figure 3 As shown, under normal rat maintenance diet, obese rats gained weight rapidly from 3 to 15 weeks, and almost stopped gaining weight after 15 weeks; while lean rats gained weight steadily from 2 to 22 weeks, and the weights of obese and lean rats were comparable at week 22. Figure 4 This is a comparison photo of lean rats (left) and obese rats (right) at week 11. Figure 5 As shown, under normal maintenance diet, the changes in serum total cholesterol levels in lean and obese rats at weeks 3, 5, 8, and 11 were observed. At the same time points, the serum total cholesterol levels in obese rats were significantly higher than those in lean rats. Furthermore, the serum total cholesterol levels in obese rats gradually increased with the development of diabetes, while those in lean rats remained almost stable. Figure 6 As shown, under normal maintenance diet, the changes in serum total triglyceride content in lean and obese rats at weeks 3, 5, 8 and 11 were observed. At the same time, the serum total cholesterol content in obese rats was significantly higher than that in lean rats. Furthermore, the serum triglyceride content in obese rats gradually increased with the development of diabetes, while that in lean rats remained almost stable.

[0061] Note: Figure 2 , 3 In categories 4, 5, and 6, ZL (Zucker Lean) is a lean rat, which is an SD / Zucker rat with wild-type (fa+ / +) and heterozygous (fa+ / -) genotypes from the SD-Zucker recombinant inbred rat line obtained in step 8; ZDF (Zucker Diabetes Fatty) is an obese rat, which is an SD / Zucker rat with homozygous (fa- / -) genotype from the SD-Zucker recombinant inbred rat line obtained in step 8.

[0062] Example 2

[0063] When the parental female rats were Wistar (fa+ / +) and the male rats were Zucker (fa+ / -), the construction and experimental methods were the same as above, and the experimental results were as follows. Figure 7As shown, under normal maintenance diet, the blood glucose levels of lean rats remained within the normal range (blood glucose ≤200 mg / dL) during the free-feeding period after weaning, with minimal fluctuations. In contrast, the blood glucose levels of obese rats exceeded normal levels from week 7-9 onwards under free-feeding conditions, indicating a diabetic state. Figure 8 As shown, under normal rat maintenance diet, obese rats gained weight rapidly from 3 to 15 weeks, and almost stopped gaining weight after 15 weeks; while lean rats gained weight steadily from 2 to 21 weeks. Figure 9 This is a comparison photo of lean rats (left) and obese rats (right) at week 11. Figure 10 As shown, under normal maintenance diet, the changes in serum total cholesterol levels in lean and obese rats at weeks 3, 5, 8, and 11 were observed. At the same time points, the serum total cholesterol levels in obese rats were significantly higher than those in lean rats. Furthermore, the serum total cholesterol levels in obese rats gradually increased with the development of diabetes, while those in lean rats remained almost stable. Figure 11 As shown, under normal maintenance diet, the changes in serum total triglyceride content in lean and obese rats at weeks 3, 5, 8 and 11 were observed. At the same time, the serum total cholesterol content in obese rats was significantly higher than that in lean rats. Furthermore, the serum triglyceride content in obese rats gradually increased with the development of diabetes, while that in lean rats remained almost stable.

[0064] Note: Figure 7 , 8 In categories 9, 10, and 11, ZL (Zucker Lean) is a lean rat, which is a Wistar / Zucker rat with wild-type (fa+ / +) and heterozygous (fa+ / -) genotypes from the Wistar-Zucker recombinant inbred rat line obtained in step 8; ZDF (Zucker Diabetes Fatty) is an obese rat, which is a Wistar / Zucker rat with homozygous (fa- / -) genotype from the Wistar-Zucker recombinant inbred rat line obtained in step 8.

Claims

1. A method for constructing a recombinant inbred type 2 diabetic rat model, characterized in that: Female rats were selected from either SD fa+ / + or Wistar fa+ / + parental rats, and male rats were selected from Zucker fa+ / - parental rats. The female and male rats were crossed to obtain the F1 generation. Heterozygous female fa+ / - rats from the F1 generation were backcrossed with male rats to obtain the F2 generation. Heterozygous female fa+ / - rats from the F2 generation were backcrossed with male rats to obtain the F3 generation. Heterozygous female fa+ / - rats from the F3 generation were backcrossed with male rats to obtain the F4 generation. Finally, heterozygous female fa+ / - rats from the F4 generation were backcrossed with... Male rats were backcrossed to obtain the F5 generation. Female rats from the F5 generation heterozygotes fa+ / - were backcrossed with male rats from the paternal line to obtain the F6 generation. Female rats from the F6 generation heterozygotes fa+ / - were then crossed with male rats from the F6 generation heterozygotes fa+ / -. The resulting SD / Zucker rats or Wistar / Zucker rats with homozygous fa- / - genotypes were fed with normal feed until they were 8-10 weeks old and became recombinant inbred type 2 diabetic rats.

2. The method for constructing a recombinant inbred type 2 diabetic rat model according to claim 1, characterized in that... The specific steps are as follows: Step 1. Select parent rats, where female rats are either SD fa+ / + or Wistar fa+ / +, and male rats are Zucker fa+ / -; Step 2: Using female SD fa+ / + rats and male Zucker fa+ / - rats as parents, crossbreed to obtain F1 generation SD / Zucker rats; F1 generation SD / Zucker rats have only two genotypes, namely wild-type fa+ / + and heterozygous fa+ / -. The F1 generation SD / Zucker rats carry half of the chromosomal genetic material of the maternal SD fa+ / + rats, and the mitochondrial genetic material of the female rats comes entirely from the maternal parent. Alternatively, female Wistar fa+ / + rats and male Zucker fa+ / - rats can be used as parents to cross and obtain F1 generation Wistar / Zucker rats. The F1 generation Wistar / Zucker rats have only two genotypes: wild-type fa+ / + and heterozygous fa+ / -. The F1 generation Wistar / Zucker rats carry half of the chromosomal genetic material of the maternal Wistar fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Step 3. Select female rats from the F1 generation heterozygous fa+ / -, i.e., female SD / Zucker fa+ / -, and backcross them with male Zucker fa+ / - paternal rats to obtain F2 generation SD / Zucker rats. The F2 generation SD / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F2 generation SD / Zucker rats carry one-quarter of the chromosomal genetic material of the maternal SD fa+ / + rats, and all mitochondrial genetic material of the female rats comes from the maternal parent. Alternatively, female Wistar / Zucker rats can be selected from the F1 generation heterozygous fa+ / -, i.e., female Wistar / Zucker fa+ / -, and backcrossed with male Zucker fa+ / - paternal rats to obtain F2 generation Wistar / Zucker rats. The F2 generation Wistar / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The resulting F2 generation Wistar / Zucker rats carry one-quarter of the chromosomal genetic material from the maternal Wistar fa+ / + rats, and all mitochondrial genetic material of the female rats comes from the maternal parent. Step 4. Select female rats from the F2 generation heterozygous fa+ / -, i.e., female SD / Zucker fa+ / -, and backcross them with the male paternal Zucker fa+ / - rats to obtain F3 generation SD / Zucker rats. The F3 generation SD / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F3 generation SD / Zucker rats carry one-eighth of the chromosomal genetic material of the maternal SD fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Alternatively, female rats from the F2 generation heterozygotes fa+ / -, i.e. female Wistar / Zucker fa+ / -, can be backcrossed with male Zucker fa+ / - rats to obtain F3 generation Wistar / Zucker rats; The F3 generation Wistar / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F3 generation Wistar / Zucker rats carry one-eighth of the chromosomal genetic material from the maternal Wistar fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Step 5. Select female rats from the F3 generation heterozygous fa+ / -, i.e., female SD / Zucker fa+ / -, and backcross them with the paternal male Zucker fa+ / - rats to obtain F4 generation SD / Zucker rats. The F4 generation SD / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F4 generation SD / Zucker rats carry one-sixteenth of the chromosomal genetic material of the maternal SD fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Alternatively, female Wistar / Zucker rats can be selected from the F3 generation heterozygous fa+ / -, i.e., female Wistar / Zucker fa+ / -, and backcrossed with male Zucker fa+ / - paternal rats to obtain F4 generation Wistar / Zucker rats. The F4 generation Wistar / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F4 generation Wistar / Zucker rats carry one-sixteenth of the chromosomal genetic material from the maternal Wistar fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Step 6. Select female rats from the F4 generation heterozygous fa+ / -, i.e., female SD / Zucker fa+ / -, and backcross them with the male paternal Zucker fa+ / - rats to obtain the F5 generation SD / Zucker rats. The F5 generation SD / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F5 generation SD / Zucker rats carry 1 / 32 of the chromosomal genetic material of the maternal SD fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Alternatively, female Wistar / Zucker rats can be selected from the F4 generation heterozygous fa+ / -, i.e., female Wistar / Zucker fa+ / -, and backcrossed with male Zucker fa+ / - paternal rats to obtain F5 generation Wistar / Zucker rats. The F5 generation Wistar / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F5 generation Wistar / Zucker rats carry 1 / 32 of the chromosomal genetic material from the maternal Wistar fa+ / + rats, and all the mitochondrial genetic material of the female rats comes from the maternal parent. Step 7. Select female rats from the F5 generation heterozygous fa+ / -, i.e., female SD / Zucker fa+ / -, and backcross them with male Zucker fa+ / - paternal rats to obtain F6 generation SD / Zucker rats. The F6 generation SD / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F6 generation SD / Zucker rats carry chromosomal genetic material from the maternal SD fa+ / + rats in one-sixty-fourth proportion, and all mitochondrial genetic material of the female rats comes from the maternal parent. Alternatively, female Wistar / Zucker rats can be selected from the F5 generation heterozygous fa+ / -, i.e., female Wistar / Zucker fa+ / -, and backcrossed with male Zucker fa+ / - paternal rats to obtain F6 generation Wistar / Zucker rats. The F6 generation Wistar / Zucker rats have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. The F6 generation Wistar / Zucker rats carry chromosomal genetic material from the maternal Wistar fa+ / + rats in one-sixty-fourth proportion, with all mitochondrial genetic material of the female rats coming from the maternal parent. Step 8. Select female rats from the F6 generation heterozygotes fa+ / - and cross them with male rats from the F6 generation heterozygotes fa+ / - to obtain SD-Zucker recombinant inbred rats; Alternatively, female rats from the F6 generation heterozygotes fa+ / - can be crossed with male rats from the F6 generation heterozygotes fa+ / - to obtain the Wistar-Zucker recombinant inbred rat line; Step 9. Among the SD-Zucker recombinant inbred rats obtained in Step 8, there are three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. Among them, the SD / Zucker rats with the homozygous fa- / - genotype cannot express functional leptin receptors, which are the SD / Zucker recombinant inbred type 2 diabetic rats. Alternatively, the Wistar-Zucker recombinant inbred rats obtained in step 8 have three genotypes: wild-type fa+ / +, heterozygous fa+ / -, and homozygous fa- / -. Among them, the Wistar / Zucker rats with the homozygous fa- / - genotype cannot express functional leptin receptors, which are Wistar / Zucker recombinant inbred type 2 diabetic rats.

3. The method for constructing a recombinant inbred type 2 diabetic rat model according to claim 2, characterized in that: The recombinant inbred type 2 diabetic rats were fed a normal diet and developed hyperglycemia at 8-10 weeks of age. Their non-fasting blood glucose levels were 200-500 mg / dL at 8-10 weeks of age and 400-600 mg / dL after 10 weeks. The main nutritional components of the normal diet were: crude protein ≤20.7%, crude fiber ≤2.31%, crude fat ≤4.15%, carbohydrates ≤40%, calcium ≤1.24%, and phosphorus ≤0.83%.

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