Construction method and application of recombinant inbred line type 2 diabetes mellitus rat model

Abstract

The invention belongs to the technical field of construction of experimental animal models, and particularly relates to a construction method and application of a recombinant inbred line type 2 diabetes mellitus rat model, in parent rats, female rats are SD (fa + / +) or Wistart (fa + / +), male rats are Zucker (fa + / -), heterozygote (fa + / -) female rats in filial generations obtained through hybridization are backcrossed with male parents until an F6 generation, and the female rats and the male rats are subjected to cross breeding to obtain the recombinant inbred line type 2 diabetes mellitus rat model. And hybridizing the F6 generation heterozygote (fa < + / ->) female rat with the F6 generation heterozygote (fa < + / ->) male rat to obtain a homozygote (fa <- / ->) SD / Zucker or Wistart / Zucker rat which is the recombinant inbred line type 2 diabetes mellitus rat. The obtained rats cannot express functional leptin receptors, have hyperappetite and autonomous polydipsia and polyphagia, and are very in line with real occurrence scenes of type 2 diabetes mellitus caused by overnutrition of diet under normal diet conditions of human beings.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing experimental animal models, and in particular relates to a construction method and application of a recombinant inbred type 2 diabetic rat model. Background Art

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

[0003] As a mammal, rats closely resemble humans in growth, development, and metabolism. The genetic similarity between rats and humans exceeds 85%. Rats are considered an ideal model organism among mammals due to their large litter size, moderate size, and ease of experimental manipulation. They are widely used in diabetes research. Common rodent models of type 2 diabetes include spontaneous diabetic models (such as Zucker diabetic fatty (ZDF) rats; Goto-Kakizaki (GK) rats; db / db mice; ob / ob mice), diet-induced models (such as high-fat diet, high-carbohydrate diet, and high-fat and high-carbohydrate diets), and chemical-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, destroy insulin-secreting beta cells in the rat pancreas, resulting in insufficient insulin secretion and decreased function. Simultaneously, the rats are fed a high-fat, high-carbohydrate diet, leading to type 2 diabetes. Furthermore, when studying food, drugs, and nutrients, interventions with animals are often performed using gavage, intraperitoneal injection, and subcutaneous injection. However, the metabolic profiles of rats that develop type 2 diabetes on these specialized diets differ significantly from those of humans, misaligning with the human pathological process of type 2 diabetes and failing to reflect the coordinated progression of insulin resistance and beta-cell dysfunction. This is because the pathological process of type 2 diabetes in humans is not caused by a short-term high-fat, high-carbohydrate diet, but rather by the cumulative effects of overnutrition on a normal diet.

[0005] Therefore, there is an urgent need to construct a rat model that conforms to the actual occurrence scenario of type 2 diabetes caused by dietary excess nutrition under normal dietary conditions. Only in this way can the natural evolution process of human diabetes from metabolic syndrome to diabetes be simulated. This model can be used to explore the mechanism of action of food, medicine and precise nutritional intervention on type 2 diabetes and its complications, which is of great significance.

[0006] At the same time, the rat model established by the present invention will drink and eat more on its own, and utilizes its natural characteristics to develop into type 2 diabetes, which can avoid the harm to the rats themselves caused by treatment methods such as gavage, intraperitoneal injection and subcutaneous injection. Summary of the Invention

[0007] To address the problems of the prior art, the present invention provides a method for constructing a rat model of type 2 diabetes. The resulting rats are unable to express functional leptin receptors. Due to the lack of leptin signaling, these rats develop hyperphagia and spontaneous polyphagia. This allows these rats to show significant weight and fat gain around 5 weeks of age, and hyperglycemia by 8-10 weeks of age, without the need for islet cell destruction and simply by being fed a standard rat diet. This model closely matches the actual development of type 2 diabetes in humans due to dietary overnutrition on a normal diet.

[0008] The method for constructing the recombinant inbred type 2 diabetes rat model was as follows: SD (fa+ / +) or Wistar (fa+ / +) were selected as female parent rats, and Zucker (fa+ / -) were selected as male parent rats; the female rats from the maternal line were hybridized with the male rats from the paternal line to obtain the F1 generation, the female rats from the heterozygous (fa+ / -) of the F1 generation were backcrossed with the male rats from the paternal line to obtain the F2 generation, the female rats from the heterozygous (fa+ / -) of the F2 generation were backcrossed with the male rats from the paternal line to obtain the F3 generation, and the heterozygous (fa+ / -) are backcrossed with male rats from the father's side to obtain the F4 generation; female rats from the F4 generation heterozygotes (fa+ / -) are backcrossed with male rats from the father's side to obtain the F5 generation; female rats from the F5 generation heterozygotes (fa+ / -) are backcrossed with male rats from the father's side to obtain the F6 generation; female rats from the F6 generation heterozygotes (fa+ / -) are then hybridized with male rats from the F6 generation heterozygotes (fa+ / -); the resulting SD / Zucker rats or Wistar / Zucker rats with a homozygous (fa- / -) genotype are recombinant inbred type 2 diabetic rats.

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

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

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

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

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

[0014] Alternatively, female rats from the F1 generation heterozygous (fa+ / -), namely female Wistar / Zucker (fa+ / -), are selected and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F2 generation Wistar / Zucker rats. 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+ / +) rat, and all of the mitochondrial genetic material in the female rats comes from the maternal lineage.

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

[0016] Alternatively, female rats from the F2 generation heterozygous (fa+ / -), namely female Wistar / Zucker (fa+ / -), are selected and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F3 generation Wistar / Zucker rats. F3 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F3 generation Wistar / Zucker rats carry one-eighth of the chromosomal genetic material from the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal lineage.

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

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

[0019] Step 6. Select female F4 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -), and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F5 SD / Zucker rats. F5 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F5 SD / Zucker rats carry one-thirty-second of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line.

[0020] Alternatively, female rats from the F4 generation heterozygous (fa+ / -) are selected, namely female Wistar / Zucker (fa+ / -) rats, and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F5 generation Wistar / Zucker rats. F5 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F5 generation Wistar / Zucker rats carry one thirty-second of the chromosomal genetic material of the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal lineage.

[0021] Step 7. Select female F5 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -), and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F6 SD / Zucker rats. F6 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F6 SD / Zucker rats carry one-sixty-fourth of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line.

[0022] Alternatively, female rats from the F5 generation heterozygous (fa+ / -) are selected, namely female Wistar / Zucker (fa+ / -) rats, and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F6 generation Wistar / Zucker rats. F6 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F6 generation Wistar / Zucker rats carry one-sixty-fourth of the chromosomal genetic material of the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal lineage.

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

[0024] Alternatively, female rats from the F6 generation heterozygous (fa+ / -) are selected and hybridized with male rats from the F6 generation heterozygous (fa+ / -) to obtain Wistar-Zucker recombinant inbred rats;

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

[0026] Or the Wistar-Zucker recombinant inbred rats obtained in step 8 have three genotypes, namely wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). Among them, Wistar / Zucker rats with the homozygous genotype (fa- / -) cannot express functional leptin receptors, that is, Wistar / Zucker recombinant inbred type 2 diabetic rats.

[0027] And the application of the method for constructing a recombinant inbred type 2 diabetes rat model in constructing a diabetic rat model.

[0028] Moreover, the recombinant inbred type 2 diabetic rats constructed were fed with ordinary feed and developed hyperglycemia at 8-10 weeks of age, with their non-fasting blood glucose levels ranging from 200 to 500 mg / dL at 8-10 weeks of age and from 400 to 600 mg / dL after 10 weeks. The main nutrients of the ordinary feed 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 the existing technology, the beneficial effects of this technical solution are:

[0030] A method for constructing a type 2 diabetic rat model is provided, and SD / Zucker recombinant inbred type 2 diabetic rats and Wistar / Zucker recombinant inbred type 2 diabetic rats are successfully constructed. The offspring of these two rat models are stable in reproduction and can spontaneously develop diabetes when fed with ordinary feed. They can be used for research on the biology, pathogenesis, and intervention methods of type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the construction of the SD / Zucker recombinant inbred strain type 2 diabetes rat model;

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

[0033] Figure 3 This is a schematic diagram of the weight changes of SD / Zucker recombinant inbred type 2 diabetic rats during growth;

[0034] Figure 4 This is a comparison photo of heterozygous (fa+ / -) SD / Zucker rats and homozygous (fa- / -) SD / Zucker rats in the 11th week of gestation;

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

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

[0037] Figure 7 This is a schematic diagram of blood glucose changes in Wistar / Zucker recombinant inbred type 2 diabetic rats under free feeding conditions;

[0038] Figure 8 This is a schematic diagram of the weight changes of Wistar / Zucker recombinant inbred type 2 diabetic rats during growth;

[0039] Figure 9 This is a comparison photo of heterozygous (fa+ / -) Wistar / Zucker rats and homozygous (fa- / -) Wistar / Zucker rats in the Wista Zucker recombinant inbred strain at week 11.

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

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

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

[0043] The principle behind constructing a recombinant inbred type 2 diabetes rat model in this invention is explained as follows: SD rats (or Wistar rats) have a stable genetic background, good health, and strong reproductive capacity. Zucker rats are ideal models for studying obesity and type 2 diabetes. Furthermore, their mitochondrial genetic material is maternally derived, and alterations in mitochondrial function are associated with many metabolic diseases. Crossing female SD (or Wistar) rats with male Zucker rats produces offspring with SD (or Wistar) mitochondria and a mix of Zucker and SD chromosomal genetic material. Crossing these two strains results in offspring inheriting half of their chromosomal genetic material from each parent. By repeatedly backcrossing heterozygous female offspring with their paternal parent, the maternal chromosomal genetic material is diluted (by the sixth generation, the maternal chromosomal genetic material is one-sixty-fourth, minimizing the impact of the maternal genetic component on the offspring) while retaining the maternal mitochondrial genetic material. This allows the leptin receptor gene mutation to be carried in strains other than Zucker rats, enabling the study of the contribution of mitochondrial genetic material to type 2 diabetes. Studies in this strain have also revealed the following: 1. The leptin receptor gene in SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) retains the mutation in a new mitochondrial context. 2. SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) develop diabetes when fed a standard rat maintenance diet. 3. SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats) exhibit severe hyperglycemia in adulthood and fail to gain weight. 4. Leptin receptor mutations in SD / Zucker (fa- / -) rats (or Wistar / Zucker (fa- / -) rats lead to polydipsia and polyphagia, as well as the development of diabetes. Therefore, they are an ideal animal model for studying overnutrition-induced diabetes.

[0044] Taking the commonly used ZDF rat model of type 2 diabetes as an example, research data shows that ZDF rats typically require Purina Rodent LabDiet 5008 to develop hyperglycemia, and their non-fasting blood glucose levels range from 200 to 400 mg / dL at 8 weeks of age and from 400 to 600 mg / dL at 12 weeks of age. The present invention, after constructing a recombinant inbred type 2 diabetic rat model, can induce diabetes by feeding these rats a maintenance diet, with blood glucose levels ranging from 200 to 500 mg / dL at 8-10 weeks of age and reaching 400 to 600 mg / dL after 10 weeks. A comparison of the specific diets is shown in Table 1.

[0045] Table 1: Comparison of the main nutrients of Purina Rodent LabDiet 5008 feed used for feeding ZDF rats and the ordinary rat maintenance feed used in the present invention

[0046] Main nutrients Animal feed formulation Animal feed formulation Feed name Purina Rodent LabDiet5008 (for feeding Zuckerdiabetic fatty rats) Ordinary rat maintenance feed (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 by the present 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 formula contains higher proportions of the major nutrients, including crude protein, crude fiber, crude fat, and carbohydrates, than conventional rat maintenance diets used to feed the SD / Zucker and Wistar / Zucker recombinant inbred type 2 diabetic rats constructed according to the present invention. This indicates that the SD / Zucker and Wistar / Zucker recombinant inbred type 2 diabetic rats constructed according to the present invention are more susceptible to developing type 2 diabetes at lower dietary nutrient intakes than traditional ZDF rats, making them ideal animal models for studying overnutrition-induced type 2 diabetes and its complications. This is due to a homozygous mutation in the leptin receptor, which causes the SD / Zucker and Wistar / Zucker recombinant inbred type 2 diabetic rats to spontaneously consume more food and drink more, which is more consistent with the real-world scenario of overnutrition and disease development in humans. This natural characteristic also avoids the harm to the rats themselves caused by other treatment methods such as gavage, intraperitoneal injection and subcutaneous injection during the study of food, medicine and nutritional substance intervention on animals.

[0048] On the other hand, mitochondria are important targets for metabolic diseases. This diabetic rat model retains the mitochondrial genetic material carried by the maternal line (SD or Wistar rats) by backcrossing the offspring with the sire, 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, expands the research field of animal models of metabolic diseases, and enriches the innovation points of Zucker rats as model animal research.

[0049] Example 1

[0050] As an example, the parent female rats are SD (fa+ / +) and the male rats are Zucker (fa+ / -), Figure 1 This is a schematic diagram of the construction of the SD / Zucker recombinant inbred rat model of type 2 diabetes. The construction method is as follows:

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

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

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

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

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

[0056] Step 6. Select female F4 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -), and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F5 SD / Zucker rats. F5 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F5 SD / Zucker rats carry one-thirty-second of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line.

[0057] Step 7. Select female F5 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -), and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F6 SD / Zucker rats. F6 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F6 SD / Zucker rats carry one-sixty-fourth of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line.

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

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

[0060] like Figure 2As shown in the figure, when fed with a normal rat maintenance diet, the blood sugar of lean rats remained at normal levels (blood sugar ≤ 200 mg / dL when fed freely) during the growth process after weaning, and the blood sugar fluctuations were small; the blood sugar of obese rats was higher than normal levels when fed freely from the 8th to 9th week, and the rats were in a diabetic state. Figure 3 As shown in the figure, when fed with normal rat maintenance feed, the weight of obese rats increased rapidly from 3 to 15 weeks, and almost stopped increasing after 15 weeks; while the weight of lean rats increased steadily from 2 to 22 weeks, and the weight of obese rats and lean rats was comparable at week 22. Figure 4 The following is a comparison of lean rats (left) and obese rats (right) at week 11. Figure 5 As shown in the figure, when normal rats were fed with maintenance diet, the serum total cholesterol content of lean rats and obese rats changed at 3, 5, 8, and 11 weeks. The serum total cholesterol content of obese rats was significantly higher than that of lean rats during the same period. The serum total cholesterol content of obese rats gradually increased with the development of diabetes, while that of lean rats remained almost stable. Figure 6 As shown in the figure, when normal rats were fed with maintenance diet, the total triglyceride content in the serum of lean rats and obese rats changed at 3 weeks, 5 weeks, 8 weeks and 11 weeks. The total cholesterol content in the serum of obese rats was significantly higher than that of lean rats during the same period. The triglyceride content in the serum of obese rats showed a gradual increasing trend with the development of diabetes, while that of lean rats remained almost stable.

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

[0062] Example 2

[0063] When the parent 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 in the figure, when fed with a normal rat maintenance diet, the blood sugar of lean rats remained at normal levels (blood sugar ≤ 200 mg / dL when fed freely) during the growth process after weaning, and the blood sugar fluctuations were small; the blood sugar of obese rats was higher than normal levels when fed freely from the 7th to the 9th week, and the rats were in a diabetic state. Figure 8 As shown in the figure, when fed with normal rat maintenance feed, the weight of obese rats increased rapidly from 3 to 15 weeks and hardly increased after 15 weeks; while the weight of lean rats increased steadily from 2 to 21 weeks. Figure 9 The following is a comparison of lean rats (left) and obese rats (right) at week 11. Figure 10 As shown in the figure, when normal rats were fed with maintenance diet, the serum total cholesterol content of lean rats and obese rats changed at 3, 5, 8, and 11 weeks. The serum total cholesterol content of obese rats was significantly higher than that of lean rats during the same period. The serum total cholesterol content of obese rats gradually increased with the development of diabetes, while that of lean rats remained almost stable. Figure 11 As shown in the figure, when normal rats were fed with maintenance diet, the total triglyceride content in the serum of lean rats and obese rats changed at 3 weeks, 5 weeks, 8 weeks and 11 weeks. The total cholesterol content in the serum of obese rats was significantly higher than that of lean rats during the same period. The triglyceride content in the serum of obese rats showed a gradual increasing trend with the development of diabetes, while that of lean rats remained almost stable.

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

Claims

1. A method for constructing a recombinant inbred type 2 diabetes rat model, characterized by: Select SD (fa+ / +) or Wistar (fa+ / +) as the female parent rats, and select Zucker (fa+ / -) as the male parent rats; hybridize the female parent rats with the male parent rats to obtain the F1 generation, backcross the female heterozygotes (fa+ / -) of the F1 generation with the male parent rats to obtain the F2 generation, backcross the female heterozygotes (fa+ / -) of the F2 generation with the male parent rats to obtain the F3 generation, and backcross the female heterozygotes (fa+ / -) of the F3 generation with the male parent rats to obtain the F3 generation. The F4 generation is obtained by backcrossing female rats of the F4 generation heterozygous (fa+ / -) with male rats of the father's parent to obtain the F5 generation, the female rats of the F5 generation heterozygous (fa+ / -) are backcrossed with male rats of the father's parent to obtain the F6 generation, and then the female rats of the F6 generation heterozygous (fa+ / -) are hybridized with male rats of the F6 generation heterozygous (fa+ / -). The obtained SD / Zucker rats or Wistar / Zucker rats with a homozygous (fa- / -) genotype are recombinant inbred type 2 diabetic rats.

2. The method for constructing a recombinant inbred type 2 diabetes 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: Use female SD (fa+ / +) rats and male Zucker (fa+ / -) rats as parents to crossbreed and obtain F1 generation SD / Zucker rats. F1 generation SD / Zucker rats have only two genotypes: wild type (fa+ / +) and heterozygous (fa+ / -). The resulting F1 generation SD / Zucker rats carry half of the chromosomal genetic material of the maternal SD (fa+ / +) rats, and all of the mitochondrial genetic material of the female rats comes from the maternal parent. Alternatively, female Wistar (fa+ / +) rats and male Zucker (fa+ / -) rats are used as parents to crossbreed and obtain F1 generation Wistar / Zucker rats. F1 generation Wistar / Zucker rats have only two genotypes: wild type (fa+ / +) and heterozygous (fa+ / -). The resulting F1 generation Wistar / Zucker rats carry half of the chromosomal genetic material of the maternal Wistar (fa+ / +) rats, and all of the mitochondrial genetic material of the female rats comes from the maternal parent. Step 3. Select female F1 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F2 SD / Zucker rats. F2 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F2 SD / Zucker rats carry one-quarter of the chromosomal genetic material from the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material in the female rats comes from the maternal line. Alternatively, female rats from the F1 generation heterozygous (fa+ / -), namely female Wistar / Zucker (fa+ / -), are selected and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F2 generation Wistar / Zucker rats. 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+ / +) rat, and all of the mitochondrial genetic material in the female rats comes from the maternal lineage. Step 4. Select female F2 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F3 SD / Zucker rats. F3 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F3 SD / Zucker rats carry one-eighth of the chromosomal genetic material from the maternal SD (fa+ / +) rats, and all of the mitochondrial genetic material of the female rats comes from the maternal lineage. Alternatively, female rats from the F2 generation heterozygous (fa+ / -), namely female Wistar / Zucker (fa+ / -), are selected and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F3 generation Wistar / Zucker rats. F3 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F3 generation Wistar / Zucker rats carry one-eighth of the chromosomal genetic material from the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal lineage. Step 5. Select female F3 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -) rats, and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F4 SD / Zucker rats. F4 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F4 SD / Zucker rats carry one-sixteenth of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line. Alternatively, female rats from the F3 generation heterozygous (fa+ / -) are selected, namely female Wistar / Zucker (fa+ / -) rats, and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F4 generation Wistar / Zucker rats. F4 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F4 generation Wistar / Zucker rats carry one-sixteenth of the chromosomal genetic material of the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal line. Step 6. Select female F4 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -), and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F5 SD / Zucker rats. F5 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F5 SD / Zucker rats carry one-thirty-second of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line. Alternatively, female rats from the F4 generation heterozygous (fa+ / -) are selected, namely female Wistar / Zucker (fa+ / -) rats, and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F5 generation Wistar / Zucker rats. F5 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F5 generation Wistar / Zucker rats carry one thirty-second of the chromosomal genetic material of the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal lineage. Step 7. Select female F5 heterozygous (fa+ / -) rats, namely female SD / Zucker (fa+ / -), and backcross them with male Zucker (fa+ / -) rats from the parental line to obtain F6 SD / Zucker rats. F6 SD / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F6 SD / Zucker rats carry one-sixty-fourth of the chromosomal genetic material of the maternal SD (fa+ / +) rat, and all of the mitochondrial genetic material of the female rats comes from the maternal line. Alternatively, female rats from the F5 generation heterozygous (fa+ / -) are selected, namely female Wistar / Zucker (fa+ / -) rats, and backcrossed with male Zucker (fa+ / -) rats from the parental line to obtain F6 generation Wistar / Zucker rats. F6 generation Wistar / Zucker rats have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). The resulting F6 generation Wistar / Zucker rats carry one-sixty-fourth of the chromosomal genetic material of the maternal Wistar (fa+ / +) rat, and all of the female rats' mitochondrial genetic material comes from the maternal lineage. Step 8. Screen out female rats from the F6 generation heterozygous (fa+ / -) and cross them with male rats from the F6 generation heterozygous (fa+ / -) to obtain SD-Zucker recombinant inbred strain rats; Alternatively, female rats from the F6 generation heterozygous (fa+ / -) are selected and hybridized with male rats from the F6 generation heterozygous (fa+ / -) to obtain Wistar-Zucker recombinant inbred rats; Step 9. The SD-Zucker recombinant inbred rats obtained in Step 8 have three genotypes: wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). SD / Zucker rats with the homozygous (fa- / -) genotype cannot express functional leptin receptors and are SD / Zucker recombinant inbred type 2 diabetic rats. Or the Wistar-Zucker recombinant inbred rats obtained in step 8 have three genotypes, namely wild type (fa+ / +), heterozygous (fa+ / -), and homozygous (fa- / -). Among them, Wistar / Zucker rats with the homozygous genotype (fa- / -) cannot express functional leptin receptors, that is, Wistar / Zucker recombinant inbred type 2 diabetic rats.

3. Use of the method for constructing a recombinant inbred type 2 diabetes rat model according to claim 1 or 2 in constructing a type 2 diabetes rat model.

4. The use according to claim 3, characterized in that: The constructed recombinant inbred type 2 diabetic rats were fed with ordinary feed and developed hyperglycemia at 8-10 weeks of age, with non-fasting blood glucose levels ranging from 200 to 500 mg / dL at 8-10 weeks of age and from 400 to 600 mg / dL after 10 weeks. The main nutrients of the ordinary feed 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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