Construction method and application of type I diabetes animal model

Abstract

The invention provides a construction method and application of a type I diabetes animal model, and the construction method comprises the following steps: selecting 3-4 week old young rats, and feeding the selected young rats with a high-fat and high-sugar feed for 3-4 weeks; after the young rats are subjected to fasting for 6-12 h, streptozotocin slow-release liquid is injected into the young rats subjected to fasting; the blood glucose of the young rats is monitored once every day, and when the blood glucose concentration exceeds an index, it is determined that the type 1 diabetes mellitus animal model is established. According to the scheme, after the streptozotocin slow-release liquid is prepared and injected into the young rats, the in-vivo release speed of streptozotocin can be delayed, pancreatic islet inflammation of the young rats can be slowly induced, induction can be completed through one-time injection, the injection frequency of the young rats is reduced, the survival rate of the young rats is increased, and the survival rate of the young rats is increased. The method can be applied to experimental research on the aspect of type I diabetes of children, and a research model is provided for clinical research on the type I diabetes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal models of diseases, and particularly relates to a method for constructing an animal model of long-term induced type I diabetes and application of the method in an animal model of type I diabetes in young mice. BACKGROUND

[0002] Type 1 diabetes mellitus (T1DM), also called insulin-dependent diabetes mellitus (IDDM) or juvenile diabetes, is an autoimmune disease that occurs most often in children and young adults, but can occur at any age. The fundamental mechanism is that the beta cells of the islets of Langerhans responsible for producing insulin are mistakenly destroyed by the autoimmune system, so that the beta cells cannot produce enough insulin (or do not produce insulin) to reduce blood sugar, so that the body is in a long-term hyperglycemic state. The onset is relatively acute, the clinical symptoms are obvious and severe, and the typical symptoms of type I diabetes include frequent urination, thirst, dry mouth, itchy or dry skin, increased appetite, weight loss, etc. At present, type 1 diabetes mellitus cannot be cured, and early and lifelong use of insulin is the treatment principle for all type I diabetes patients.

[0003] Current data suggest that it is urgent to prevent and treat diabetes, and it is impossible to ignore scientific research on animal models for verifying the efficacy of drugs for curing or alleviating the progression of diabetes.

[0004] At present, it has been found that a variety of compounds can induce diabetes in animal models, and the two most commonly used compounds are Streptozotocin (STZ) and Alloxan (ALX), of which STZ is the most commonly used. Streptozotocin, also known as STZ, Streptozotocin, and Streptozocin, has the chemical name 2-deoxy-2-[[ (methyl nitrosyl amino) carbonyl]-amino]-D-glucopyranose, which is an organic compound with the chemical formula C8H 15 N3O7, is a light yellow crystalline powder, soluble in water, low-carbon alcohol and ketone. In animal experiments, STZ can damage the beta cells of the islets of Langerhans, and pharmacological studies have shown that the drug is rapidly metabolized after intravenous administration. The drug is not detected in the plasma 3 hours after administration, the metabolism of STZ is three-phase, and the terminal half-life is 40h, and 10%-20% is excreted with urine within 24h.

[0005] At present, when a mouse or rat model of type I diabetes is induced by streptozotocin, the survival period of the animal is short, and the survival period of the mouse or rat after the modeling is successful is generally within 3 weeks, and the success rate of the type I diabetes model of the young mouse is lower, and the young mouse is easy to die. However, the establishment of the type I diabetes model of the young mouse has important significance for the clinical research and treatment of children T1DM. SUMMARY

[0006] The main purpose of the present application is to provide a type I diabetes animal model construction method, evaluation method and application, which aims to solve the technical problems of low success rate and high mortality of young mice in the existing streptozotocin-induced type I diabetes of young mice.

[0007] In order to achieve the above-mentioned purpose, the present application provides a type I diabetes animal model construction method, evaluation method and application, the construction method comprising the following steps: S10, selecting 3-4 week old young mice, feeding with high-fat and high-sugar feed for 3-4 weeks; S20, after fasting the young mice for 6-12 hours, injecting streptozotocin sustained-release solution into the fasting young mice; S30, monitoring the blood glucose of the young mice once a day, and determining that the type I diabetes animal model is established when the blood glucose concentration exceeds the index.

[0008] Optionally, in the step S20, the preparation method of the streptozotocin sustained-release solution comprises the following steps: adding polylactic acid-glycolic acid copolymer and streptozotocin into an organic solvent to mix uniformly to obtain an oil phase, dissolving an emulsifier into injection water to form an aqueous phase; mixing the oil phase with the aqueous phase to prepare an oil-in-water emulsion; removing the organic solvent in the oil-in-water emulsion by volatilization to obtain the streptozotocin sustained-release solution.

[0009] Optionally, the organic solvent is acetone.

[0010] Optionally, the mass ratio of streptozotocin to polylactic acid-glycolic acid copolymer is 1:2.5.

[0011] Optionally, the emulsifier is polyvinyl alcohol.

[0012] Optionally, the step S20 further comprises, after 2 hours of injection, gavage of the young mice with a glucose aqueous solution.

[0013] Optionally, the mouse is a C57BL / 6J mouse, and the rat is an SD rat.

[0014] Optionally, in the step S10, the young mouse is a young C57BL / 6J mouse, and the young rat is a young SD rat.

[0015] Optionally, in the step S20, the injection amount of STZ for the young mouse is 180-220 mg / kg.

[0016] Optionally, in the step S20, the injection amount of STZ for the young rat is 60-70 mg / kg.

[0017] Optionally, in the step S30, the index is that the blood glucose concentration is greater than or equal to 15 mmol / L for three consecutive days.

[0018] The application further provides an application of the construction method to a young mouse animal model of type I diabetes.

[0019] In the technical scheme, the STZ sustained-release solution is prepared, and then injected into the young mouse, so that the release speed of STZ in the body is delayed, the inflammation of the islet of the young mouse is induced slowly, the induction is completed by one injection, the injection frequency of the young mouse is reduced, the survival rate of the young mouse is prolonged, and the application can be used for experimental research on type I diabetes of children, and a research model is provided for clinical research on type I diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical scheme in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only show some embodiments of the application, and for those skilled in the art, other drawings can be obtained without creative labor based on the drawings shown.

[0021] Figure 1 A flowchart of an embodiment of the construction method of the atopic dermatitis animal model provided by the application is shown. Figure 2 The change of fasting blood glucose of the C57BL / 6J mouse after STZ induction in the embodiment provided by the application is shown. Figure 3 The change of fasting body weight of the C57BL / 6J mouse after STZ induction in the embodiment provided by the application is shown. Figure 4 The change of fasting blood glucose of the SD rat after STZ induction in the embodiment provided by the application is shown. Figure 5 The change of fasting body weight of the SD rat after STZ induction in the embodiment provided by the application is shown.

[0022] The objectives, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments.

[0024] It should be noted that, in the embodiments, the specific conditions not mentioned are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments used are not mentioned the manufacturers, which are all the conventional products that can be purchased in the market. In addition, the meaning of "and / or" appearing in the whole text includes three parallel solutions, for example, "A and / or B" includes the solution of A, or the solution of B, or the solution of A and B. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that the technical solutions can be realized by the ordinary skilled in the art. When the combination of the technical solutions appears contradictory or unachievable, it should be considered that the combination of the technical solutions does not exist, and is not within the protection scope of the present application. Based on the embodiments in the present application, all the other embodiments obtained by the ordinary skilled in the art without making creative efforts are within the protection scope of the present application.

[0025] At present, when the mouse or rat model of type I diabetes is induced by streptozotocin, the survival period of the animal is short, and the survival period of the mouse or rat after the modeling is successful is generally within 3 weeks. The success rate of the type I diabetes model of the young mouse is lower, and the young mouse is easy to die.

[0026] In view of this, the present application provides a method for constructing a type I diabetes animal model and application, which is mainly applied to the modeling of young mice, and combines the advantages of the existing methods. Figure 1 The flowchart of the method for constructing a type I diabetes animal model according to an embodiment is shown in the figure, and the method comprises the following steps.

[0027] In step S10, a 3-4-week-old young mouse is selected, and then the young mouse is fed with high-fat and high-sugar feed for 3-4 weeks.

[0028] In this embodiment, the 3-4-week-old C57BL / 6 mouse or SD rat is selected as the young mouse, the mouse at this stage is weaned for a short time, which is equivalent to 1-3 years old of human, the initial weight of the C57BL / 6 mouse is controlled to be 15-20 g, and the initial weight of the SD rat is controlled to be 60-80 g. The modeling is started from the young mouse at this age until the modeling is successful, and the age of the surviving young mouse is controlled to be about 10 weeks, which is equivalent to 6-8 years old of human, and is very suitable for the clinical research and treatment of children and adolescents T1DM.

[0029] Step S20, after the young mice are fasted for 6-12 hours, the streptozotocin slow-release solution is injected into the fasted young mice.

[0030] In this embodiment, the slow-release material is selected from PLGA copolymer or PLGA-PEG block copolymer, both of which have certain hydrophilicity. The PLGA-PEG block copolymer contains hydrophilic PEG (polyethylene glycol) and hydrophobic PLGA (poly lactic acid-glycolic acid) block, and the hydrophilicity is further enhanced by grafting. Both of the materials have biodegradability and biocompatibility, and the slow-release effect and biocompatibility are suitable for injection in young mice.

[0031] The molecular structure of streptozotocin is as follows. It has a large number of hydroxyl groups, and the slow-release material can form hydrogen bonds with the hydrophilic groups, so that the slow-release microspheres after encapsulation are more stable.

[0032] .

[0033] The preparation method of the streptozotocin slow-release solution comprises the following steps.

[0034] Step S21, the polylactic acid-glycolic acid copolymer and streptozotocin are added to the organic solvent to mix uniformly to obtain an oil phase.

[0035] In specific implementation, if the hydrophilicity of the slow-release material is too high, the release rate of streptozotocin will be too fast, the pancreatic beta cells of the young mice will be damaged quickly, and the mortality of the young mice will be high. Therefore, the PLGA copolymer with lower hydrophilicity is selected. The mass ratio of streptozotocin to polylactic acid-glycolic acid copolymer is controlled at 1: (2-3.5), that is, a good encapsulation effect can be achieved. In this embodiment, the mass ratio is preferably 1:2.5, and the encapsulation rate is best observed by electron microscopy.

[0036] The ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50. The higher the proportion of glycolic acid units, the stronger the hydrophilicity of the material, and the faster the degradation. However, considering the influence of the release rate on the mortality of young mice, it is not appropriate to use a slow-release material with a high proportion of glycolic acid units. In this embodiment, the ratio of 50:50 is preferred.

[0037] The organic solvent is acetone which can dissolve both PLGA copolymer and streptozotocin.

[0038] Step S22, the emulsifier is dissolved in the injection water to form an aqueous phase.

[0039] In specific implementation, the emulsifier is preferably polyvinyl alcohol, which is easily soluble in water and can effectively stabilize the oil-water two phases in the emulsion to prevent layering. Step S23, mixing the oil phase and the water phase to form an oil-in-water emulsion.

[0040] In practice, the oil phase and the water phase are mixed at the same temperature.

[0041] Step S24, removing the organic solvent in the oil-in-water emulsion by volatilization to obtain the streptozotocin sustained-release solution.

[0042] In practice, the emulsion is stirred by a stirrer, the acetone in the oil phase is volatilized by sufficient air contact, and finally the streptozotocin sustained-release solution is obtained. During the volatilization process, the injection water can be supplemented to prevent water volatilization.

[0043] When the young mice are injected, the injection amount of streptozotocin for young mice is controlled to be 180-220 mg / kg, and the injection amount of streptozotocin for young rats is controlled to be 60-70 mg / kg, so that the young mouse model of type I diabetes can be modeled efficiently while reducing the mortality of young mice.

[0044] After step S20, the young mice are also given a glucose aqueous solution by gavage 2 hours after injection to prevent the death of the young mice due to stress and other phenomena caused by discomfort after injection.

[0045] Step S30, monitoring the blood glucose of the young mice once a day, and determining that the type I diabetes animal model is established when the blood glucose concentration exceeds the index.

[0046] In practice, the index is that the measured blood glucose concentration is ≥15 mmol / L for 3 consecutive days.

[0047] The application also provides an application of the construction method to a young mouse model of type I diabetes, which mainly constructs a young mouse model of type I diabetes with high survival rate and high standard by injecting the streptozotocin sustained-release solution.

[0048] In the technical scheme of the application, after the young mice are injected with the streptozotocin sustained-release solution, a postoperative care plan is also used for the young mice, the survival cycle of the model animals is prolonged by supplementing glucose, vitamins or antibiotics, etc. after the operation. After the modeling is successful, the young mice are fed with low blood glucose generation index value feed to maintain the long-term stability of the model. At the same time, a double-dimension evaluation system is used to combine the blood glucose level and the complication evaluation to comprehensively evaluate the effectiveness of the model.

[0049] The technical scheme of the application will be further described in detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the application and not to limit the application. The mice or rats used in the embodiments are bred by the inventors, and the reagents and materials are purchased on the market. Example 1

[0050] I. Preparation of experimental materials Animals: 8 three-week-old male C57BL / 6 mice, 8 three-week-old male SD rats.

[0051] Reagents and consumables: high-fat high-sugar feed, STZ, polylactic acid-glycolic acid copolymer, polyvinyl alcohol, injection water, acetone, sucrose water.

[0052] Preparation of STZ sustained-release solution (streptozotocin sustained-release solution): 250 mg of polylactic acid-glycolic acid copolymer (50:50, weight average molecular weight 20000 Da, functional group capped with carboxylic acid) and 100 mg of streptozotocin were ultrasonically dissolved in 5 mL of acetone to form an oil phase, the oil phase was poured into 20 mL of 1% (w / v) polyvinyl alcohol aqueous solution (0.2 g of polyvinyl alcohol and 20 mL of injection water were heated and stirred to dissolve), a small emulsifier was used to shear at 2000 rpm for 60 s to form an oil-in-water emulsion, then a magnetic stirrer was used to stir at a speed of 250 rpm for 3 h, after the acetone in the emulsion was completely volatilized, 0.5% (w / v) of STZ sustained-release solution 20 mL was obtained.

[0053] Preparation of STZ solution: 2.1 g of citric acid was added to 100 mL of injection water to prepare a citric acid solution, 2.94 g of sodium citrate was added to 100 mL of double distilled water to prepare a sodium citrate solution. Mix the citric acid solution and the sodium citrate solution in a volume ratio of 1:1, and measure the pH value with a pH meter, adjust to 4.5, and STZ citric acid buffer is obtained. When injected, dissolve STZ in the citric acid buffer to obtain 0.5% (w / v) STZ solution.

[0054] Preparation of glucose aqueous solution: 40 g of glucose was added to 100 mL of injection water to obtain 40% (w / v) glucose aqueous solution.

[0055] Preparation of sucrose aqueous solution: 10 g of sucrose was added to 100 mL of injection water to obtain 10% (w / v) sucrose aqueous solution. Example 2

[0056] Eight 3-week-old C57BL / 6 mice were randomly divided into control group 1, control group 2 and experimental group 1, experimental group 2, respectively, and fed with high-fat high-sugar feed for 3 weeks. After fasting and water deprivation for 10 h, the fasting body weight and blood glucose of the mice were measured and recorded, and then 200 mg / kg of STZ sustained-release solution was injected into the tail vein of the mice in experimental group 1, and 200 mg / kg of STZ solution was injected into the tail vein of the mice in experimental group 2. The mice in control group 1 and control group 2 were injected with the same amount of injection water and citric acid buffer, respectively. Two hours after injection, 20 mL / kg of glucose aqueous solution was administered to the pups by gavage, and the required injection and gavage amounts of the mice in experimental group 1, experimental group 2, control group 1 and control group 2 were calculated based on the fasting body weight.

[0057] On the second day of the experiment, the injection of STZ slow-release solution was replaced with the injection of 10% (w / v) sucrose aqueous solution: On the third day of the experiment, the injection of 10% (w / v) sucrose aqueous solution was replaced with the injection of normal water.

[0058] On the fourth to the fortieth day of the experiment, the normal diet was restored, and the vital signs and survival status of the mice were observed at any time, and vitamins and antibiotics were appropriately supplemented.

[0059] Blood glucose detection: On the first to the fortieth day of the experiment, all the mice were fasted for 6 hours, and then the blood glucose was measured through a tail vein blood sample, wherein the mice injected with the STZ slow-release solution began to have severe diabetes on the tenth day; the mice injected with the STZ solution died on the ninth and tenth days, respectively.

[0060] Experimental results: combined with the blood glucose change graph and the body weight change graph of Figure 2 and Figure 3 , wherein the blood glucose concentration and the body weight are the average values of the same group, and the control group 1, the control group 2, and the experimental group 1 and the experimental group 2 correspond to Vehicle 1, Vehicle 2, STZ-slow-treatment, and STZ-treatment, respectively.

[0061] Compared with the control group 1, the blood glucose value of the mice in the experimental group 1 was significantly increased, and the blood glucose concentration was > 15 mmol / L within 72 hours from the sixth day, and at the same time, compared with the control group 1, the body weight value of the mice grew slowly during the growth period, indicating that a single high-dose STZ has direct toxicity to pancreatic beta cells and can rapidly cause diabetes and induce type I diabetes in mice. The general signs of the mice in the experimental group 1 and the control group 1 were observed, and the mice in the experimental group 1 showed the typical symptoms of diabetes, such as polyuria, polydipsia, increased appetite, and weight loss.

[0062] Compared with the control group 2, the blood glucose value of the mice in the experimental group 2 was significantly increased, and the blood glucose concentration was > 15 mmol / L within 72 hours from the fifth day, but the mice began to die successively from the tenth day.

[0063] Moreover, compared with the experimental group 1, the mice injected with the STZ solution without the slow-release coating in the experimental group 2 all died in the early modeling period, and the mice injected with the STZ solution with the slow-release coating in the experimental group 1 survived for more than 40 days, so it can be seen that the STZ slow-release solution with the slow-release coating in this embodiment can effectively prolong the survival rate of the young mice. Example 3

[0064] 8 three-week-old SD rats were randomly divided into control group 3, control group 4 and experimental group 3, experimental group 4, and fed with high-fat high-sugar feed for 3 weeks, and then fasted and watered for 8 hours. The fasting body weight and blood glucose of the rats were weighed and recorded, and then the rats in experimental group 3 were injected with STZ slow-release solution and the rats in experimental group 4 were injected with STZ solution, and the rats in control group 3 and control group 4 were injected with the same amount of injection water and citric acid buffer solution, respectively. 2 hours after injection, the pups were given glucose solution by gavage, and the rats in experimental group 3 and experimental group 4 were given injection and gavage according to the fasting body weight of the rats.

[0065] On the second day of the test, the injection of STZ slow-release solution was replaced by injection of 10% (w / v) sucrose solution: On the third day of the test, the injection of 10% (w / v) sucrose solution was replaced by injection of ordinary water.

[0066] From the 4th to the 40th day of the test, the normal diet was restored, and the vital signs and survival status of the rats were observed at any time, and vitamins and antibiotics were appropriately supplemented.

[0067] Blood glucose detection: from the 1st to the 40th day of the test, all rats were fasted for 6 hours, and then blood glucose was measured by tail vein blood sample, and rats injected with STZ slow-release solution developed severe diabetes starting on the 10th day; rats injected with STZ solution died on the 9th and 10th days, respectively.

[0068] Experimental results: combined with the blood glucose change chart and body weight change chart of Figure 4 and Figure 5 , the blood glucose concentration is the average value of the same group, and control group 3, control group 4 and experimental group 3, experimental group 4 correspond to Vehicle1, Vehicle2, STZ-slow-treatment and STZ-treatment, respectively.

[0069] Compared with control group 3, the blood glucose value of rats in experimental group 3 increased significantly, and the blood glucose concentration was >15mmol / L within 72 hours from the 7th day, and at the same time, compared with control group 3, the body weight value of the rats grew slowly during the growth period, indicating that a single high-dose STZ has direct toxicity to pancreatic beta cells and can rapidly cause diabetes and induce type I diabetes in rats. The general signs of rats in experimental group 3 and control group 4 were observed, and rats in experimental group 3 showed typical symptoms of diabetes such as polyuria, polydipsia, increased appetite, and weight loss.

[0070] Compared with control group 4, the blood glucose value of rats in experimental group 4 increased significantly, and the blood glucose concentration was >15mmol / L within 72 hours from the 5th day, but rats began to die successively from the 9th day.

[0071] And, compared with the experimental group 3, all the rats in the experimental group 4 injected with the STZ solution without the sustained-release coating died in the early stage of modeling, and the rats in the experimental group 3 injected with the STZ solution with the sustained-release coating survived for more than 40 days, so it can be seen that the STZ sustained-release solution with the sustained-release coating of the embodiment can effectively prolong the survival rate of the young rats.

[0072] The above results show that the STZ sustained-release solution with the coating can successfully establish T1DM in young mice or rats by once injection through the tail vein, and the model induced by this method has typical symptoms of diabetes, and is suitable for experimental research and treatment of T1DM in children and adolescents.

[0073] The technical features of the above embodiments can be combined in any manner. In order to make the description simple, all possible combinations of the technical features in the above embodiments are not described, but as long as the combinations of the technical features do not contradict, they should be considered as the scope of the description.

[0074] The above embodiments only express several implementation manners of the application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are all within the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A method for constructing an animal model of type I diabetes, characterized by, The construction method comprises the following steps: S10, selecting 3-4-week-old young mice, feeding with high-fat and high-sugar feed for 3-4 weeks; S20, after fasting the young mice for 6-12 hours, injecting streptozotocin slow-release solution into the fasted young mice; S30, monitoring the blood glucose of the young mice once a day, and determining that the type 1 diabetes animal model is established when the blood glucose concentration exceeds the index.

2. The method for constructing a type 1 diabetes animal model as described in claim 1, characterized in that, In the step S20, the preparation method of the streptozotocin slow-release solution comprises the following steps: adding polylactic acid-glycolic acid copolymer and streptozotocin into an organic solvent to mix uniformly to obtain an oil phase, dissolving an emulsifier into injection water to form an aqueous phase; mixing the oil phase with the aqueous phase to prepare an oil-in-water emulsion; removing the organic solvent in the oil-in-water emulsion by volatilization to obtain the streptozotocin slow-release solution.

3. The method for constructing an animal model of type I diabetes according to any one of claims 2, wherein The organic solvent is acetone.

4. The method for constructing an animal model of type I diabetes according to any one of claims 2, wherein The mass ratio of streptozotocin to polylactic acid-glycolic acid copolymer is 1:2.

5.

5. The method for constructing an animal model of type I diabetes according to any one of claims 2, wherein The emulsifier is polyvinyl alcohol.

6. The method for constructing a model of diabetes mellitus type I according to claim 1, wherein the non-human animal is a mouse. After the step S20, the young mice are given intragastrically glucose aqueous solution after 2 hours of injection.

7. The method for constructing a model of Type I diabetes mellitus according to claim 1, wherein the non-obese diabetic animal is a non-obese diabetic mouse. In the step S10, the young mice are young mice or young rats, the young mice are C57BL / 6J mice, and the young rats are SD rats.

8. The method for constructing an animal model of type I diabetes according to claim 7, wherein the non-human animal is a mouse. In the step S20, the streptozotocin injection amount of the young mice is 180-220 mg / kg, and the streptozotocin injection amount of the young rats is 60-70 mg / kg.

9. The method for constructing a model of non-obese type I diabetic animal according to claim 1, wherein the non-obese type I diabetic animal is a mouse. In the step S30, the index is that the measured blood glucose concentration is greater than or equal to 15 mmol / L for three consecutive days.

10. Application of the construction method according to any one of claims 1-9 to a type 1 diabetes young mouse animal model.

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