CN112933211A - Construction method of new animal model for type 1 diabetes - Google Patents
Construction method of new animal model for type 1 diabetes Download PDFInfo
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- CN112933211A CN112933211A CN201911373695.7A CN201911373695A CN112933211A CN 112933211 A CN112933211 A CN 112933211A CN 201911373695 A CN201911373695 A CN 201911373695A CN 112933211 A CN112933211 A CN 112933211A
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Abstract
The invention provides a method for constructing a new animal model of type 1 diabetes, which constructs the type 1 diabetes model by inducing islet cell injury and conditionally knocking out regulatory T cells, solves the problem of difference between the common NOD mouse diabetes model and the clinical symptoms of human diabetes, and constructs a new animal model which is closer to the morbidity state of human type 1 diabetes. The mouse diabetes model provided by the invention is simple and convenient to construct, has higher consistency with the clinical characteristics of human type 1 diabetes, and has stronger practical application value.
Description
Technical Field
The invention belongs to the field of preparation of diabetes animal models, and particularly relates to a construction method of a new type-1 diabetes animal model.
Background
Under the combined action of the environment and the susceptible gene, the organism of the type 1 diabetes patient has obvious immunological abnormality. The abnormal number or function of regulatory T cells that control immune balance leads to the development of inflammation in the body. Early CD8+After the T cells recognize MHC-I on the surface of the islet beta cells, the islet beta cells begin to be killed, and islet antigens are released. Dendritic cells, macrophages and their precursor monocytes present antigens to T cells in the pancreatic lymph nodes. In this process, an autoimmune response occurs because either islet antigen misfolds in the endoplasmic reticulum to form a new antigen, or T cells escape the negative selection in the thymus, resulting in activation of T cells that receive self islet antigen.
Among the most commonly used chemicals for inducing acute type 1 diabetes in chemical-induced animal models are Alloxan (ALX) and Streptozotocin (STZ), which are glucose analogs that have been previously accumulated in pancreatic islet beta cells by GLUT2 glucose transporters. In the presence of thiols in the cell, alloxan causes beta cell death by the reaction product hydroxyl radical, while preventing insulin secretion by inhibiting glucokinase. Streptozotocin can directly alkylate beta cell DNA or damage mitochondrial DNA to inhibit the mitochondrial metabolism of beta cells, thereby preventing insulin secretion. However, alloxan may cause damage to other organs in addition to the islets. Unlike type 1 diabetes, which is highly prevalent in younger children, older animals are more difficult to be sensitive to alloxan toxicity. Alloxan is unstable and has a short half-life of 1.5 min in phosphate buffer at pH7.4 at 37 deg.C, and can be rapidly decomposed in aqueous solution to obtain non-diabetic uric acid. Studies have shown that other conversion products of either aluric acid or alloxan can damage the liver tissue of animals. Unstable alloxan is more likely to damage other organs. In view of these disadvantages, alloxan is gradually replaced by streptozotocin.
Streptozotocin induces a diabetic model with pronounced insulitis. Multiple small doses of streptozotocin can promote local lymphocyte proliferation of transgenic mice and enhance the expression of a T cell costimulatory molecule CD80 on the surface of beta cells. Recent studies also indicate that high doses of streptozotocin cause islet inflammation due to phagocytic necrosis of macrophages without provoking adaptive immunity at all. The mechanism of immune cells in the streptozotocin-induced diabetes model is still unclear at present.
Injection of streptozotocin at high doses (150mg/kg) into mice induced acute diabetes without any complications. Unlike human type 1 diabetes, this acute form of diabetes is not immune-mediated, and high doses of streptozotocin directly toxic to beta cells result in rapid and severe toxic diabetes. Even if the required diabetes model can be quickly obtained by adjusting the dosage of the streptozotocin, the streptozotocin-induced model is unstable and is easily influenced by external factors such as the type of an experimental animal, the food intake amount, the initial weight of the experimental animal and the like.
For example: chinese patent 201910064657.7 discloses a method for constructing animal model of type I diabetes, wherein C57BL/6 mouse is selected for first tail vein injection, and then 50mg/kg BW streptozotocin reagent is injected into abdominal cavity for 5 days continuously.
For example: chinese patent 201610741829.6 discloses a method for establishing a type 1 diabetes animal model and application thereof, wherein a stable cynomolgus monkey type 1 diabetes model is established by using a 100mg/kg streptozotocin induction method, and type 1 diabetes can be completely induced and formed without causing complications. The invention not only evaluates the blood sugar index, but also evaluates the immunity level, but the cost of the animal cynomolgus monkey selected by the model is higher in practical application, so that an animal model with wider application is needed.
NOD (non-obese diabetic) mice are a commonly used animal model for type 1 diabetes, because the abnormal structure of the thymus results in the inability of thymocytes to secrete autoantigens, so that T cells cannot be selected negatively during development, and these autoreactive T cells escape to the periphery to cause disease. Its onset is characterized by polyuria, polydipsia, and diabetic hyperglycemia accompanied by emaciation. Islet inflammation occurred in female mice at 2-4 weeks, and in males at 5-7 weeks later. In a sterile environment, the incidence rate of 30-week-old female mice is more than 90%, while that of male mice is 50% -80%. NOD mice develop early hyperglycemia and diabetes and survive for weeks without insulin treatment. While NOD is now used as a common animal model for type 1 diabetes to study the progression and treatment of type 1 diabetes in humans, it differs from type 1 diabetes in humans in many ways. Histologically, large inflammatory cell infiltrates were observed in the center of islets of diseased NOD mice while only a small number of leukocytes were detected in islets of type 1 diabetic patients. Unlike clinical patients with type 1 diabetes, the background of NOD onset is mainly CD4+T cell mediated, and long-term onset NOD mice do not develop ketonuria.
Although more and more animal models of type 1 diabetes are developed for the research on the pathogenesis of type 1 diabetes, the clinical characteristics of the current animal models and the human type 1 diabetes are difficult to be highly consistent, and particularly, the widely used NOD mice are widely applied to the pharmacodynamic evaluation of preclinical treatment drugs due to the characteristics of natural pathogenesis. Unfortunately, therapeutic agents that are effective in mice do not have significant effects in humans, even accelerating disease progression. These failures have led researchers to continually think whether the pathogenesis of NOD mice varies greatly from human to human. Therefore, there is an urgent need to develop a new animal model that more closely approximates the state of onset of type 1 diabetes in humans.
Disclosure of Invention
In order to solve the problems, the invention provides a construction method of a new animal model of type 1 diabetes.
The construction method provided by the invention aims at inducing islet cell injury and combining with conditional knockout Regulatory T cells (Tregs) of an original animal to induce type 1 diabetes. The specific principle is that streptozotocin is utilized to induce islet beta cell injury to generate inflammation, and meanwhile, regulatory T cells are knocked out to cause autoreactive T cells to further attack islet beta cells, so that the same pathogenesis as human type 1 diabetes is caused.
The invention completes the construction of a new animal model of type 1 diabetes by inducing islet cell injury and conditionally knocking out regulatory T cells.
Specifically, the original animal includes but is not limited to an original mouse.
More specifically, the original mice include but are not limited to BALB/C mice, C57BL/6 mice.
More specifically, the original mice include but are not limited to Foxp3-DTR-eGFP mice.
Specifically, the induced islet cell damage includes, but is not limited to, injection of an islet cell damage inducing agent.
Further specifically, the islet cell injury inducing agent includes, but is not limited to, alloxan and/or streptozotocin. Further preferred is streptozotocin.
Further specifically, the injection amount of the streptozotocin is 20-200mg/kg body weight; preferably 40-80mg/kg body weight; more preferably 50mg/kg body weight.
Further specifically, the injection amount of the alloxan is 50-500mg/kg body weight; preferably 80-250mg/kg body weight; more preferably 100-200mg/kg body weight.
In some embodiments, the islet cell injury inducing agent is injected at the above dose for a period of 1 to 5 consecutive days, once a day; preferably for 3-4 days, once daily; further preferably for 3 consecutive days, once daily.
Specifically, the conditional knockout regulatory T cells include, but are not limited to, Diphtheria Toxin (DT) injection.
Preferably, the diphtheria toxin is injected in an amount of 0.001-0.1mg/kg body weight; preferably 0.005-0.05mg/kg body weight; further preferably 0.0125mg/kg of body weight.
In a further embodiment, the injection method may be intraperitoneal injection. Other injection methods may be used to achieve equivalent results.
Specifically, the islet cell injury induction and conditional knock-out of regulatory T cells can be performed simultaneously or sequentially.
More specifically, the injection of streptozotocin and the injection of diphtheria toxin can be performed synchronously or sequentially.
In some embodiments, the injection of streptozotocin is performed prior to the injection of diphtheria toxin.
As some specific preferred embodiments, the construction method comprises the following steps:
(1) preparing an original animal;
(2) preparing a medicament:
preparing a streptozotocin solution,
preparing diphtheria toxin solution;
(3) medicine injection:
the original animal was given a solution of streptozotocin of 40-80mg streptozotocin/kg body weight for intraperitoneal injection and a solution of diphtheria toxin of 0.001-0.1mg diphtheria toxin/kg body weight for injection.
As some recommended construction methods, the original animal preparation includes routine rearing of the original animal in a Specific Pathogen Free (SPF) environment for 1-3 weeks.
As some recommended construction methods, the concentration of streptozotocin solution may be 10 mg/mL.
As some recommended construction methods, the buffer system of streptozotocin solution can be citrate buffer, acetate solution, PBS solution, PB solution or physiological saline solution.
As some recommended construction methods, the concentration of diphtheria toxin solution may be 2.5. mu.g/mL.
As some recommended construction methods, the buffer system of diphtheria toxin solution may be phosphate buffer, water for injection, PBS solution, PB solution, or physiological saline solution.
As some recommended construction methods, the injection is performed by intraperitoneal injection.
In some preferred embodiments, 50mg/kg body weight of streptozotocin is injected daily for 1-5 consecutive days; an additional injection of 0.0125mg/kg body weight of diphtheria toxin was made. Further preferably, the injection is performed for 3 consecutive days, and 50mg/kg body weight of streptozotocin is injected every day; an additional injection of 0.0125mg/kg body weight of diphtheria toxin was made.
According to the mouse type 1 diabetes model established by the method, compared with a diabetes animal model NOD/ShiLtJ mouse, the result shows that compared with the NOD/ShiLtJ mouse, the mouse type 1 diabetes model established by the method is not only similar to the symptoms of human type 1 diabetes in blood sugar change, but also has a T cell immune response specific to islet antigen in pancreatic tissues of the model after being successfully established through blood sugar change detection, a sugar tolerance test, serum insulin determination, C-peptide concentration determination, glycosylated hemoglobin (HbA1C) content determination, survival rate calculation, a pancreatic T cell infiltration experiment, an antigen specific T cell immune response experiment and beta-hydroxybutyric acid level determination causing ketoacidosis. Inflammatory cell infiltration can occur in the pancreas of a mouse model constructed by the method provided by the invention, the infiltrated inflammatory cells are mainly CD 8T cells, and a certain number of CD 4T cells exist. The T cells are islet associated antigen-specific T cells, consistent with the specific T cells found in humans, which is not present in NOD/ShiLtJ mice. In addition, the level of beta-hydroxybutyric acid in the mouse body constructed by the method provided by the invention is increased, and is consistent with the symptoms of type 1 diabetes mellitus ketoacidosis of human beings. In addition, the NOD/ShiLtJ mice show abnormal glucose metabolism in the early stage of disease occurrence in a glucose tolerance experiment, and the model constructed by the model construction method provided by the invention can avoid the deficiency. Compared with NOD/ShiLtJ mice, the model constructed by the model construction method provided by the invention is more similar to the symptoms of human type 1 diabetes.
The novel animal model constructed by the method provided by the invention is consistent with the pathogenesis of human type 1 diabetes, is autoimmune disease, and the pathogenesis symptoms are highly consistent with the pathogenesis symptoms and indexes of human type 1 diabetes. The animal model is convenient to prepare, high in morbidity, free of sex selectivity and controllable in morbidity cycle.
The invention also provides application of the mouse constructed by the model in the research related to type 1 diabetes.
Specifically, the applications include but are not limited to the research on the pathogenesis of diabetes, the evaluation of islet cell transplantation technology, the screening and evaluation of type 1 diabetes drugs, and the application in the evaluation of anti-hyperglycemia health-care foods.
Drawings
FIG. 1 is a schematic diagram of injection modes of various groups of mice in the initial establishment of a new animal model of type 1 diabetes mellitus and detection results of various indexes.
FIG. 2 is the results of blood glucose measurements in Foxp3-DTR-eGFP mice injected with different amounts of streptozotocin.
FIG. 3 is a comparison of blood glucose changes and incidence in a new model of type 1 diabetes mellitus and NOD/ShiLtJ mouse model.
FIG. 4 is a comparison of the results of the glucose tolerance test in a new model of type 1 diabetes mellitus and in a NOD/ShiLtJ mouse model.
FIG. 5 is a comparison of serum insulin and C-peptide concentrations in a new model of type 1 diabetes mellitus and NOD/ShiLtJ mouse model.
FIG. 6 shows the results of immunofluorescent staining of pancreatic tissues and the change in HbA1c content in peripheral blood of a novel model animal of type 1 diabetes and a model NOD/ShiLtJ mouse.
FIG. 7 shows the serum beta-hydroxybutyrate content changes of the new model of type 1 diabetes and NOD/ShiLtJ mouse model.
FIG. 8 is a survival curve for a new model of type 1 diabetes and a NOD/ShiLtJ mouse model.
FIG. 9 shows the results of immunofluorescent staining of pancreatic tissue in a novel model of type 1 diabetes and NOD/ShiLtJ mouse model and the results of cellular immunoreaction detected by flow cytometry.
FIG. 10 shows the results of islet antigen-specific T cell response experiments in a novel model of type 1 diabetes and a NOD/ShiLtJ mouse model.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Of the experimental animals in all examples, 6-8 weeks of BALB/c background Foxp3-DTR-eGFP mice were purchased from Jackson Lab, U.S.A., 6-8 weeks of BALB/c mice were purchased from Shanghai Jersey laboratory animals Co., Ltd, and 10 weeks of female non-organism diabetic ShiLtJ (NOD/ShiLtJ) mice were purchased from Beijing Wahuafukang Biotechnology Ltd. Mice were all housed under SPF ambient conditions. All purchased mice were left in an SPF environment for 1 week prior to the experiment.
The blood sugar measuring method comprises the following steps: the mouse orbit collected 10 μ L of blood, and the blood glucose concentration was measured and recorded using a glucometer (JPS-5 model kyo yi). The specific measurement method refers to the specification of the blood glucose meter.
The immunofluorescence staining method comprises the following steps: the whole pancreas tissue of the mice was fixed in 6mL of 4% paraformaldehyde. HE and immunofluorescence assays were performed. HE sections were observed under 40-fold microscope with a normal microscope. Immunofluorescent sections were observed under a confocal microscope oil microscope.
Example 11 construction method of mouse model for diabetes mellitus
Preparing and injecting a streptozotocin solution: streptozotocin powder was weighed out of the dark and prepared into a solution having a streptozotocin concentration of 10mg/mL using a 100mM citric acid-trisodium citrate buffer solution having a pH of 4.5. Each 100. mu.L intraperitoneal injection was performed 5 minutes into the mice from the start of lysis (i.e., a streptozotocin dose of 50mg/kg body weight).
Preparation and injection of diphtheria toxin solution: mu.g of diphtheria toxin was dissolved in 100. mu.L of PBS buffer (pH7.4) at 100mM, and 100. mu.L of diphtheria toxin was intraperitoneally injected to each mouse (i.e., a diphtheria toxin dose of 0.0125mg/kg body weight).
The construction process comprises the following steps:
(1) streptozotocin injection
Foxp3-DTR-eGFP mice with a BALB/c background weighing approximately 20 grams were selected at 6-8 weeks. On the day of diphtheria toxin injection, the day is 0, the day before the injection is-1 day, and the day after the injection is +1 day; and so on.
Grouping mice:
and DT group: diphtheria toxin solution was injected on day 0 at a dose of 0.0125mg/kg body weight of diphtheria toxin.
1 × STZ group: streptozotocin solution at a dose of 50mg/kg body weight was injected on day-1 without any drug injection on day 0.
2 × STZ group: day-2, -1 day daily injection of streptozotocin solution at a dose of 50mg/kg body weight, no drug injection on day 0.
3 × STZ group: day-3, -2, -1 daily injection of streptozotocin solution at a dose of 50mg/kg body weight, day 0 without any drug injection.
5 × STZ group: day-5, -4, -3, -2, -1, a streptozotocin solution with a streptozotocin dose of 50mg/kg body weight was injected daily, and no drug injection was performed on day 0.
1 × STZ + DT group: the day-1 was injected with a streptozotocin solution at a dose of 50mg/kg body weight, and the day-0 was injected with a diphtheria toxin solution at a dose of 0.0125mg/kg body weight.
2 × STZ + DT group: day-2 and day-1, a solution of streptozotocin at a dose of 50mg/kg body weight per day, and a solution of diphtheria toxin at a dose of 0.0125mg/kg body weight per day 0 were injected.
3 × STZ + DT group: day-3, -2, -1, a daily injection of a streptozotocin solution at a dose of 50mg/kg body weight of streptozotocin, and day 0 injection of a diphtheria toxin solution at a dose of 0.0125mg/kg body weight of diphtheria toxin.
5 × STZ + DT group: day-5, -4, -3, -2, -1, a streptozotocin solution with 50mg/kg body weight of streptozotocin is injected every day, and day 0 a diphtheria toxin solution with 0.0125mg/kg body weight of diphtheria toxin is injected.
PBS group: day-5, -4, -3, -2, -1, and 0 days, PBS buffer with pH of 7.4 and 100mM concentration was injected daily.
Experimental example 11 preliminary establishment of novel animal model for diabetes mellitus
Detection of relevant index was performed for each group of mice in example 1
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 1:
FIG. 1-A shows the injection pattern and blood glucose monitoring pattern of mice in the 1 XSTZ group, 2 XSTZ group, 3 XSTZ group, and 5 XSTZ group.
FIG. 1-B shows the blood glucose changes in the PBS group, 1 XSTZ group, 2 XSTZ group, 3 XSTZ group, and 5 XSTZ group mice.
FIG. 1-C shows local immunofluorescence staining of islets in PBS and 5 XSTZ mice. Islets of mice were removed on day +6 of the end of injection and stained with insulin (green), CD3 (red) and DAPI (blue).
FIG. 1-D shows the change in the ratio of Treg cells in peripheral blood of mice in PBS and DT groups. The peripheral blood Treg cell ratio of the mice was measured on days 0, +1, +6, +12, respectively.
FIG. 1-E shows the blood glucose changes of the PBS and DT mice.
FIG. 1-F shows local immunofluorescence staining of islets in PBS and DT mice. Islets of mice were removed on day +6 of the end of injection and stained with insulin (green), CD3 (red) and DAPI (blue). Scale bar 25 μm.
FIG. 1-G shows the injection pattern and blood glucose monitoring pattern of 5 XSTZ + DT mice.
FIG. 1-H shows the blood glucose changes of the PBS group and the 5 XSTZ + DT group mice.
FIG. 1-I shows local immunofluorescence staining of islets in PBS and 5 XSTZ + DT mice. Islets of mice were removed on day +6 of the end of injection and stained with insulin (green), CD3 (red) and DAPI (blue). Scale bar 25 μm.
And (4) analyzing results: damage to local islets or disruption of immune balance alone cannot cause hyperglycemia in which T cells participate (fig. 1-A, B, C, D, E, F). Combining streptozotocin with diphtheria toxin injection, mice after 5 × STZ + DT were found to cause hyperglycemia on day 3 after diphtheria toxin injection and reached the highest level on day 6, and immunofluorescence showed that islets had CD3 at this time+T cell infiltration including CD4+And CD8+T cells (FIGS. 1-G, H, I). The results show that only streptozotocin damaged islets combined with DT-knocked out Tregs can establish a T cell-mediated type 1 diabetes model.
Experimental example 21 New animal model for diabetes mellitus streptozotocin dose screening
Detection of relevant index was performed for each group of mice in example 1
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 2:
FIG. 2-A shows the injection pattern and blood glucose monitoring pattern of mice in the 1 XSTZ + DT, 2 XSTZ + DT, 3 XSTZ + DT, and 5 XSTZ + DT groups.
FIG. 2-B shows the blood glucose changes in the PBS group, 1 XSTZ + DT group, 2 XSTZ + DT group, 3 XSTZ + DT group, and 5 XSTZ + DT group mice.
FIG. 2-C shows the survival percentage of mice in PBS group, 1 XSTZ + DT group, 2 XSTZ + DT group, 3 XSTZ + DT group, and 5 XSTZ + DT group.
The 5 × STZ + DT group was able to cause acute hyperglycemia in mice on day 3, while the 3 × STZ + DT group caused persistent hyperglycemia in mice around 2 weeks with an incidence rate close to that of 5 × STZ + DT, while the 2 × STZ + DT group and 1 × STZ + DT group had little hyperglycemia. The injection modes of the 1 × STZ + DT group, the 2 × STZ + DT group, the 3 × STZ + DT group and the 5 × STZ + DT group can cause the hyperglycemia of mice to a certain extent, so that the injection modes can be used for establishing a type 1 diabetes animal model; however, the 3 XSTZ + DT group is preferably constructed by a method for establishing a type 1 diabetes animal model.
Experimental example 31 comparison of New animal model of diabetes mellitus with blood glucose Change in NOD mice
To verify that 3 × STZ + DT was able to successfully establish a new type 1 diabetes model, the success of the animal model was evaluated by examining clinical symptoms of type 1 diabetes, and compared to NOD/ShiLtJ mice, a spontaneous animal model.
The relevant index test was performed for each group of mice in example 1.
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 3:
FIG. 3-A shows the injection pattern and blood glucose monitoring pattern of 3 XSTZ + DT mice, and blood glucose monitoring pattern of NOD/ShiLtJ mice.
FIG. 3-B shows the blood glucose changes in the PBS, DT, 3 XSTZ + DT mice.
FIG. 3-C shows the incidence of mice in PBS, DT, 3 XSTZ, and 3 XSTZ + DT groups.
FIG. 3-D is the blood glucose changes in NOD/ShiLtJ mice.
FIG. 3-E is the incidence of NOD/ShiLtJ mice.
The blood sugar result shows that hyperglycemia occurs at 9 days after 3 XSTZ + DT mouse diphtheria toxin injection, the incidence rate reaches more than 80% after 2 weeks, and the mice are not hyperglycemic due to Treg cell knockout only by injecting 3 XSTZ. NOD/ShiLtJ mice develop hyperglycemia sequentially from week 12 with 75% incidence at week 27. The above results indicate that both the 3 XSTZ + DT and NOD/ShiLtJ models produce hyperglycemia.
Experimental example 41 comparison of New animal model of diabetes mellitus with oral glucose tolerance (OGTT) in NOD/ShiLtJ mice
The exogenous glucose utilization capacity of the patient is clinically tested through 2-hour oral glucose tolerance, so that the pancreatic islet beta cell function of the patient is evaluated. Oral glucose tolerance test of mice 2g/kg glucose was administered after fasting for 6 hours, and blood glucose was measured at 30min, 60min, 90min and 120min, respectively.
Oral glucose tolerance experiments were performed on each group of mice in example 1.
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 4:
FIG. 4-A shows the injection pattern and the glucose administration pattern of 3 XSTZ + DT mice, and the glucose administration pattern of NOD/ShiLtJ mice.
FIG. 4-B shows the blood glucose changes in the PBS, DT, 3 XSTZ + DT mice.
FIG. 4-C is the blood glucose changes in NOD/ShiLtJ mice.
The blood glucose reached the highest level after 30 minutes of oral glucose administration in the control group, returned to the initial level after 60 minutes, with the overall blood glucose excursion in the normoglycemic range (<16.7mmol/L), whereas the blood glucose level in the 3 XSTZ + DT group was above 16.7 mmol/L30 minutes after drinking high glucose and remained hyperglycemic after 120 minutes. It was shown that the 3 XSTZ + DT group of mice failed to normally utilize exogenous glucose. In NOD/ShiLtJ mice, pre-morbid (blood glucose at 150-. This result indicates that NOD/ShiLtJ mice had abnormal carbohydrate metabolism in the pre-morbid stage.
Example 51 comparison of New animal models of diabetes mellitus with serum insulin and C-peptide concentrations in NOD/ShiLtJ mice
Serum insulin and C-peptide levels in type 1 diabetic patients are common diagnostic indicators of clinical response to insulin secretion from islet beta cells. To further verify the function of islet beta cells, fasting serum from mice was collected and serum insulin and C-peptide levels were measured. The mice need to be cut off food for 6 hours and then blood is collected after fasting serum.
The relevant index test was performed for each group of mice in example 1.
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 5:
FIG. 5-A shows the injection pattern and serum collection pattern for 3 XSTZ + DT group mice, and the serum collection pattern for NOD/ShiLtJ mice.
FIG. 5-B shows the change in serum insulin in the PBS, DT, 3 XSTZ + DT mice.
FIG. 5-C is the change in serum insulin in NOD/ShiLtJ mice.
FIG. 5-D shows the change in serum C-peptide content in the PBS, DT, 3 XSTZ + DT mice.
FIG. 5-E shows the change in serum C-peptide content of NOD/ShiLtJ mice.
The results show that serum insulin in mice in the 3 × STZ + DT group gradually decreased with increasing blood glucose, significantly lower than in the 3 × STZ and DT groups after 2 weeks. Serum insulin was also measured in NOD/ShiLtJ mice after onset, and gradually decreased significantly below that in healthy NOD/ShiLtJ mice over the course of the disease, and almost no expression of serum insulin was detected 2 weeks after onset. The serum C-peptide data show a compensatory increase in serum C-peptide levels in mice in the 3 XSTZ + DT group, at around day 7 after diphtheria toxin injection, followed by a sustained decrease significantly lower than in the control group. In parallel, serum C-peptide was examined in diseased NOD/ShiLtJ mice and it was also found that with the development of hyperglycemia, C-peptide gradually decreased and was significantly lower than in healthy NOD/ShiLtJ mice.
To further demonstrate that hyperglycemia due to streptozotocin in combination with diphtheria toxin was caused by β cell injury, pancreatic tissue from mice was taken on day 9 for immunofluorescent staining.
FIG. 5-F shows immunofluorescent staining results of pancreatic tissues of PBS, DT, 3 XSTZ + DT and NOD/ShiLtJ mice.
Insulin-secreting beta cells were seen in the pancreas of control mice, whereas almost no insulin-secreting cells were seen in 3 × STZ + DT mice, and an increase in glucagon-expressing cells was observed in the islets of 3 × STZ + DT and 3 × STZ mice. Meanwhile, NOD/ShiLtJ mice are observed, and the fact that cells for expressing insulin are obvious in pancreatic tissues of 2-week-old NOD/ShiLtJ mice is found, as the mice grow to be 10 weeks old, the mice still have obvious insulin secretion, compact cell nuclei are arranged around the pancreatic islets, the existence of inflammatory cells is indicated, and after the mice are hyperglycemic (blood sugar is more than 16.7mmol/L) in 17 weeks, almost no cells for expressing insulin exist in the pancreatic islets, and the number of cells for expressing glucagon is increased. The above results demonstrate that the function of β cells to secrete insulin is impaired after the onset of 3 × STZ + DT mice and NOD/ShiLtJ mice, and is accompanied by the disappearance of insulin-expressing β cell masses.
Experimental example comparison of HbA1c content in New animal model of type 61 diabetes mellitus and peripheral blood of NOD/ShiLtJ mice
High concentrations of glucose in the blood of a patient can combine with hemoglobin to form stable glycosylated hemoglobin (HbA1 c). HbA1c disappears with 3-4 months of red blood cells renewal, so this indicator reflects the ability of a diabetic to control blood glucose for about 3 months.
The relevant index test was performed for each group of mice in example 1.
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 6:
FIG. 6-A shows the injection pattern and peripheral blood collection pattern of 3 XSTZ + DT mice, and peripheral blood collection pattern of NOD/ShiLtJ mice.
FIG. 6-B shows changes in peripheral blood glycosylated hemoglobin of mice in PBS group, DT group, 3 XSTZ group, and 3 XSTZ + DT group.
FIG. 6-C is the change in peripheral blood glycosylated hemoglobin of NOD/ShiLtJ mice.
And (4) analyzing results: the detection result shows that the glycosylated hemoglobin level in the blood of the 3 XSTZ + DT mice is obviously increased compared with the control mice. Peripheral blood glycosylated hemoglobin of NOD/ShiLtJ mice was also tested and compared with healthy NOD/ShiLtJ mice, the results showed that glycosylated hemoglobin was significantly higher in NOD/ShiLtJ mice 1-8 weeks after onset than in the control group. And peaked at week 4 and declined slightly at week 8. The above results indicate that both 3 XSTZ + DT and NOD/ShiLtJ mice lose glycemic control after disease.
Experimental example 71 comparison of New animal model of type 71 diabetes mellitus and serum beta-hydroxybutyrate content of NOD/ShiLtJ mice
The long-term glucose metabolism blockage of patients promotes the liver to start metabolizing fat, and ketone bodies are formed to enter the circulation to cause ketotic acid (DKA) poisoning. About 80% of the ketone bodies are beta-hydroxybutyrate (beta-HB), so the content of beta-hydroxybutyrate in mouse serum is detected after the hyperglycemia is stabilized, namely 5 th week to judge whether the mouse model with type 1 diabetes is accompanied with ketoacidosis complications.
The relevant index test was performed for each group of mice in example 1.
The schematic diagram of the injection mode and the detection results of each index are shown in fig. 7:
FIG. 7-A shows the injection pattern and peripheral blood collection pattern of 3 XSTZ + DT mice, and peripheral blood collection pattern of NOD/ShiLtJ mice.
FIG. 7-B shows the change in peripheral blood β -hydroxybutyrate in PBS, DT, 3 XSTZ + DT mice.
FIG. 7-C is the change in peripheral blood β -hydroxybutyrate of NOD/ShiLtJ mice.
And (4) analyzing results: 3 XSTZ + DT mice are significantly higher than the control group by respectively injecting 3 XSTZ, DT, PBS and healthy NOD/ShiLtJ mice as the control group, which shows that the 3 XSTZ + DT mice have ketoacidosis. In contrast, the serum levels of β -hydroxybutyrate were not significantly different from healthy NOD/ShiLtJ mice at any time after the NOD/ShiLtJ mice developed hyperglycemia, indicating that the onset of NOD/ShiLtJ mice did not produce ketotic acid complications. The 3 XSTZ + DT mouse model is shown to be more similar to the onset symptoms of the human type 1 diabetes in the aspect of ketotic acid complications.
Experimental example 81 comparison of survival curves of New animal models of diabetes mellitus and NOD/ShiLtJ mice
Patients with type 1 diabetes, once diagnosed, require exogenous insulin to maintain blood glucose balance, but without any treatment, patients develop hyperglycemia, wasting, dehydration of ketonic acid and ultimately death. Suitable animal models of type 1 diabetes should also meet this profile.
For each group of mice in example 1, after the mouse model was successfully prepared, the mice were normally diet and measured for body weight, the mice were sacrificed humanely when the body weight of the mice was 20% of the initial body weight and the mice were calculated to be in a dead state, and the survival of the PBS, DT, 3 × STZ and 3 × STZ + DT and NOD/ShiLtJ mice was recorded and each group of death curves was plotted on day 0 from the onset of the mice.
FIG. 8 shows that 3 XSTZ + DT group mice and NOD/ShiLtJ mice died as disease progressed without any drug treatment.
EXAMPLE 91 novel animal model of diabetes mellitus and infiltration of pancreatic T cells in NOD/ShiLtJ mice
In the PBS, DT, 3 XSTZ and 3 XSTZ + DT mice of example 1, islets were removed on day 9 for HE staining, and islet cells were isolated for flow staining. At the same time, pancreata of NOD/ShiLtJ mice aged 2 weeks, 10 weeks and 17 weeks were removed for HE staining, and islet cells were isolated from NOD/ShiLtJ mice aged 12 weeks for flow staining.
The results are shown in FIG. 9:
FIG. 9-A shows the results of day 9 islet HE staining in PBS, DT, 3 XSTZ + DT mice.
FIG. 9-B is the results of HE staining of islets of NOD/ShiLtJ mice at 2 weeks, 10 weeks and 17 weeks of age.
FIG. 9-C shows the results of day 9 pancreatic cell flow staining of mice in PBS, DT, 3 XSTZ + DT groups.
FIG. 9-D is the results of flow staining of pancreas cells of 12 week old NOD/ShiLtJ mice.
And (4) analyzing results: HE pathological section results showed that the islets in the control group were morphologically intact, while the islets in the 3 × STZ + DT group shrank and there was infiltration of inflammatory cells around them. HE staining of NOD/ShiLtJ mice showed intact islet morphology in 2-week NOD/ShiLtJ mice, significant inflammatory cell infiltration in islets after 10 weeks, and islet atrophy after 17 weeks. Flow cytometry results further demonstrated CD8 infiltration in the pancreas of 3 × STZ + DT group mice+The number of T cells is significantly higher than CD4+T cells, except NOD/ShiLtJ mice local CD4+T number significantly higher than CD8+T cells. The above results indicate that the 3 × STZ + DT model is involved in T cells and is expressed as CD8+T cells arePredominant islet infiltration, whereas NOD is CD4+T is the main.
Experimental example 101 novel animal model of diabetes mellitus and antigen-specific T cell immune response in NOD/ShiLtJ mice
For each group of mice in example 1, day 6 pancreata of 3 XSTZ + DT mice and week 12 NOD mice were removed and then treated with Ficoll to isolate lymphocytes, 10. mu.g/mL of antigenic peptide (Table 1) was added for stimulation for 6 hours, 10. mu.g/mL of irrelevant antigen OVA polypeptide was added to the control group, PMA (0.1. mu.g/mL) and IONO (1. mu.g/mL) were added to the positive control, and anti-CD28 (0.1. mu.g/mL) and BFA were added to the system for stimulation. 6 hours post-flow detection of 3 XSTZ + DT and NOD mice CD4+、CD8+、IFN-γ+Secretion and in vivo CTL.
TABLE 11 type diabetes islet antigen polypeptides
Spleen cells of wild type mice were separately incubated with GAD65114-122And stained with 5. mu.M efluor670, control cells were incubated with OVA257-264Polypeptides were stained with 0.5 μ M efluor670, high-stained and low-stained cells 1: a ratio of 1 tail vein was injected into mice, and after 4 hours, efluor670 cells in the spleen were detected to calculate the degree of specific lysis.
The results are shown in FIG. 10:
(1) stimulation by InsC17-A1 antigen in the 3 XSTZ + DT model compared to OVA323-339Stimulation, CD4+T cells significantly upregulate IFN- γ expression, while IA-2841-860CD4 under stimulation+T cells expressing IFN-gamma and OVA323-339There were no significant differences in stimulation (a and B in fig. 10). However CD4 in 12-week NOD mice+T cells express IFN-gamma in InsC17-A1Or IA-2841-860Comparative OVA under stimulation323-339There were no significant differences in stimulation (a and B in fig. 10).
(2) CD8 of 3 XSTZ + DT+T cells are in GAD65114-122Or IAPP (prepro)5-13Under stimulation, in comparison with OVA257-263Stimulating, remarkably expressing more IFN-gamma, and taking GAD65114-122Mainly (C and D in fig. 10). The in vivo CTL assay also demonstrated 3 XSTZ + DT mouse CD8+T is GAD65114-122Antigen-specific (E in fig. 10). In contrast, NOD mouse CD8+T cells are in GAD65114-122Or IAPP (prepro)5-13No IFN-. gamma.secretion was evident upon stimulation (C and D in FIG. 10). Islet antigen-specific T cell immune responses were present in pancreatic tissue from the 3 × STZ + DT model, but were not detectable in the pancreas of classical NOD mice.
Claims (10)
1. A method for constructing a new animal model of type 1 diabetes is characterized in that the method for constructing the model of type 1 diabetes is implemented by inducing islet cell injury and combining conditional knockout of regulatory T cells.
2. The method of claim 1, wherein the step of inducing islet cell injury and the conditional knock-out of regulatory T cells is performed simultaneously or sequentially.
3. The method of claim 1, wherein the method for inducing islet cell damage is injecting an islet cell damage inducing agent; the conditional knockout method of regulatory T cells is injection of diphtheria toxin.
4. The method according to claim 3, wherein the islet cell injury-inducing agent is one or more of alloxan and streptozotocin.
5. The method according to claim 3, wherein the diphtheria toxin is injected in an amount of 0.001-0.1mg/kg body weight.
6. The method for constructing a recombinant streptozotocin vector as claimed in claim 4, wherein the injection amount of streptozotocin is 20-200mg/kg body weight.
7. The method for constructing a bioactive peptide according to claim 4, wherein the injection amount of the alloxan is 50 to 500mg/kg body weight.
8. The application of the new animal model of type 1 diabetes mellitus constructed by the construction method of claims 1-7 in the diabetes related research includes the research on the pathogenesis of diabetes mellitus, the evaluation of islet cell transplantation technology, the screening and evaluation of novel type 1 diabetes mellitus drugs and the evaluation of anti-hyperglycemia health-care food.
9. The construction method according to claims 1-7, characterized in that it comprises the steps of:
(1) preparing an original animal;
(2) preparing a medicament: preparing a streptozotocin solution; preparing diphtheria toxin solution;
(3) and (5) injecting the medicine.
10. The construction method according to claim 9, characterized in that it comprises the steps of:
(1) preparation of original animals: foxp3-DTR-eGFP mice were routinely bred for 1-3 weeks in a pathogen-free environment.
(2) Preparing a medicament: preparing 10mg/mL streptozotocin solution; preparing 2.5 mu g/mL diphtheria toxin solution;
(3) medicine injection: injecting 50mg/kg body weight of streptozotocin every day for 3 consecutive days; an additional injection of 0.0125mg/kg body weight of diphtheria toxin was made.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114324860A (en) * | 2021-12-07 | 2022-04-12 | 南京鼓楼医院 | Assessment method for correlation between plasmablasts and pancreatic islet immune injury |
| CN115299406A (en) * | 2022-08-19 | 2022-11-08 | 中南大学湘雅二医院 | Animal model construction method and method for ablating fat cells |
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Non-Patent Citations (2)
| Title |
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| NICOLAS DAMOND等: "Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist", 《BIOCHEMISTRY AND CHEMICAL BIOLOGY》 * |
| 郭波等: "人脐带间充质干细胞联合免疫干预治疗1型糖尿病小鼠的实验研究", 《中国组织工程研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114324860A (en) * | 2021-12-07 | 2022-04-12 | 南京鼓楼医院 | Assessment method for correlation between plasmablasts and pancreatic islet immune injury |
| CN115299406A (en) * | 2022-08-19 | 2022-11-08 | 中南大学湘雅二医院 | Animal model construction method and method for ablating fat cells |
| CN115299406B (en) * | 2022-08-19 | 2023-06-16 | 中南大学湘雅二医院 | Animal model construction method and method for ablating adipocytes |
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