WO2011116501A1 - 恒河猴自身免疫性1型糖尿病模型的建立 - Google Patents
恒河猴自身免疫性1型糖尿病模型的建立 Download PDFInfo
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- WO2011116501A1 WO2011116501A1 PCT/CN2010/000378 CN2010000378W WO2011116501A1 WO 2011116501 A1 WO2011116501 A1 WO 2011116501A1 CN 2010000378 W CN2010000378 W CN 2010000378W WO 2011116501 A1 WO2011116501 A1 WO 2011116501A1
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- diabetes
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/106—Primate
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
Definitions
- the invention relates to a method for preparing a rhesus monkey autoimmune type 1 diabetes model. Background technique
- autoimmune type 1 diabetes The cause of autoimmune type 1 diabetes is unknown, and there is no effective preventive measure so far.
- conventional insulin therapy can regulate blood glucose levels to a certain extent, long-term use does not effectively control the occurrence of diabetic complications, and some patients may also have hypoglycemia shock.
- How to prevent the autoimmune system from attacking islet ⁇ cells and effective ⁇ cell transplantation therapy is a key issue in diabetes research.
- the most commonly used model animals for diabetes research today are rodents. Because the rodents are far removed from the human race in terms of evolutionary relationship, the experimental data obtained from the study is of little significance for clinical guidance.
- the anatomical characteristics of rhesus monkeys, the physiological functions of various systems, and the responsiveness to diseases and therapeutic drugs are very similar to those of humans. They are sensitive animals for immunologically relevant treatment, stem cell transplantation, and preclinical efficacy and safety evaluation of xenotransplantation.
- the use of rhesus monkey diabetes model to evaluate new diabetes treatment drugs, study the immune rejection mechanism and transplantation tolerance induction of islet stem cells and xenogenic islet transplantation, is an internationally recognized preclinical experimental system, and has important guidance for the clinical application of these therapeutic programs in the future. significance. Therefore, the establishment of non-human primate diabetes model has become an urgent need for diabetic islet (dry) cell transplantation therapy research and preclinical drug evaluation.
- Currently used animal models of diabetes include experimental diabetes animal models and spontaneous diabetes animal models. Spontaneous models have higher application value, but because of the low incidence rate and high price, the feeding and breeding conditions are strict, and they cannot be widely used.
- the experimental model is widely used.
- Commonly used induction methods include pancreatectomy, chemical drug induction, viral infection, antagonistic insulin factor, food induction and fattening. Among them, the chemical drug induction method is highly praised by many researchers because of its simple operation and high feasibility. In these methods, streptozotocin is relatively less toxic to the body tissues, and the animal survival rate is high. A number of methods for preparing diabetic animal models.
- Streptozotocin is a commonly used inducing agent in the preparation of diabetes models. It selectively destroys islet ⁇ cells in certain species of animals, and many animals produce diabetes.
- the most commonly used rat model The commonly used induction methods are as follows: the rats were fasted for 12 hours, and the STZ was intraperitoneally injected 60 mg/g body weight once a day for 2 times to successfully prepare a rat model of type 1 diabetes, and the model had hyperglycemia and body weight. The characteristics of alleviating, drinking more, eating more urine are consistent with clinical type 1 diabetes; but in this experiment, if the model is only injected intraperitoneally with STZ-time and given high-calorie feed for 12 weeks, it can make type 2 diabetic animals.
- Models, and models prepared according to this method have the characteristics of overweight, impaired glucose tolerance, elevated blood lipids, elevated serum insulin, and decreased insulin receptor binding with insulin resistance, similar to the clinical features of patients with type 2 diabetes.
- Type 1 diabetes and type 2 diabetes animal models Preparation may be related to the dose of STZ injection: When a large dose (usually 120 mg/Kg) is injected, it can cause a type 1 diabetes model due to the direct destruction of islet ⁇ cells. When a small amount of STZ is injected, The function of destroying a part of islet ⁇ cells, causing peripheral tissues to be insensitive to insulin and feeding with high-calorie diet, the combination of the two induces pathological and physiological changes close to the animal model of human type 2 diabetes. Therefore, the amount of streptozotocin STZ is directly determined by the type of diabetic animal model prepared.
- STZ preparation of rhesus monkey diabetes model also related literature reports, such as: Yan Dexuan et al, the effect of different doses of streptozotocin on some common physiological indicators of rhesus monkeys, Chinese Journal of Experimental Animals, March 2003, Vol. 11 No. 1 In the period, the effects of streptozotocin (STZ) on physiological indexes such as feeding, drinking, urinating, body weight, blood sugar and urine sugar of rhesus monkeys were reported, and data were accumulated for establishing animal models of diabetes.
- STZ streptozotocin
- the dose and method of STZ are as follows: low dose is 30 mg/Kg, interval is 15 days, repeated injections are 1-2 times; medium dose is 45 mg/kg, STZ is injected once; high dose is 60 mg/kg, injection 1 Times.
- 7 rhesus monkeys showed symptoms of "three more and one less" similar to human diabetes at different times and degrees, and the use of different doses of insulin in diabetic monkeys can alleviate the symptoms.
- the symptoms of the animals were more obvious, while in the low dose group, the body weight decreased rapidly after a short period of time.
- the blood glucose and urine sugar in the experimental group increased in different degrees, and the animals in the middle and high dose groups changed greatly.
- STZ in the middle and high dose groups can induce an acute animal model similar to human diabetes, and the use of insulin therapy or low-dose STZ can prolong the disease course of the animal model, which is conducive to its concurrent Study of the disease.
- Biochemical changes in rhesus monkey during the first days after streptozotocin administration are indicated of selective beta cell destruction/Takimoto G, Jones C, Lands W, Bauman A, Jeffrey J, Jonasson 0.//Metabolism. 1988 Apr; 37(4): 364-70. 22 rhesus monkeys were selected and intravenously injected with STZ (45 to 55 mg/Kg).
- Autoimmune diabetes can be divided into Diabetes in children and adolescents and latent autoimmune diabetes in adults (LAD A), which are subtypes of type 1 diabetes.
- LAD A latent autoimmune diabetes in adults
- the characteristics are: 1. Adolescent age at onset Less than 15 years old, LADA can be independent of insulin for half a year after the age of 15 years, no ketosis occurs; 2. Most of the disease is non-obese; 3. In vivo islet B cell antibodies (ICA, GAD and insulin itself) Antibodies, etc.) are often persistently positive; 4. Susceptible genes with type 1 diabetes (eg HLA-DR3, HLA-DR4, BW54 and DQ-131-57-Non-Asp, etc.); 5.
- LADA LADA- Diagnosed early insulin therapy to protect residual beta cells.
- European and American data 4 reports that LADA accounts for about 10% to 15% of type 2 diabetes, and is reported to be up to 50% in non-obese patients with type 2 diabetes; there is a report that the positive rate of GAD-Ab in patients with type-2 diabetes is 14.2.
- This type of cause is insulin deficiency caused by autoimmune destruction of islet beta cells. Markers of autoimmune processes include lymphocyte infiltration of islets and islet cell autoantibodies, Detection of islet cell antigen autoantibodies and insulin autoantibodies.
- the technical solution of the present invention is to provide a use of a low dose of streptozotocin for the preparation of an animal model for screening for the treatment of autoimmune type 1 diabetes.
- Another technical solution of the present invention provides for the establishment of a rhesus monkey autoimmune type 1 diabetes model.
- the present invention provides the use of low doses of streptozotocin for the preparation of an animal model for screening for the treatment of autoimmune type 1 diabetes, wherein the streptozotocin is administered at a dose of 15-30 mg/kg/time.
- the administration method and dosage of streptozotocin are: intravenous administration of small doses of streptozotocin for 5 consecutive days: 15-30 mg/Kg each time; re-administration on the 7th and 14th days after the last administration. Further preferably, the streptozotocin is administered at a dose of 20-25 mg/Kg.
- the invention also provides a method for preparing an animal model of autoimmune type I diabetes, which is to apply streptozotocin to a primate, and the method and dosage of streptozotocin are: intravenous administration, 15- 30 mg/Kg/time.
- the primate is a rhesus monkey.
- the method and dosage of the streptozotocin are: intravenous administration of a small dose of streptozotocin for 15 consecutive days: 15-30 mg/Kg each time; and re-administration on the 7th and 14th days after the last administration.
- the invention also provides an animal model of autoimmune type 1 diabetes prepared by the method.
- the invention also provides the use of the animal model for screening for a medicament for the treatment of autoimmune type 1 diabetes.
- the present invention also provides a method of screening for a medicament for treating autoimmune type 1 diabetes, comprising the steps of:
- streptozotocin is administered at a dose of 15-30 mg / Kg / time;
- step b applying the candidate to the primate described in step a;
- the primate in step a is a rhesus monkey.
- the method and dosage of streptozotocin were: intravenous administration of small doses of streptozotocin for 5 consecutive days: 15-30 mg/Kg each time; re-administration on days 7 and 14 after the last administration.
- the evaluation indicators described in step c are mainly: the onset time of animal diabetes; the incidence rate and the degree of damage of ⁇ cells.
- the animal model of the present invention can be used for the evaluation of new biotech drugs and the evaluation of stem cell transplantation therapy techniques which cannot be performed in a rodent model.
- Figure 1 Results of serum anti-islet cell antibody test (wherein, Figure 1A is an anti-insulin antibody positive control, Figure 1B is a negative control, Figure 1 C is an animal model of the invention (No. 05539) serum anti-islet cell antibody test results; Figure 1D For the animal model of the present invention (No. 05572), the serum anti-islet cell antibody test results), the scale is 50 ⁇ m.
- Fig. 1 HE staining of monkey pancreatic tissue sections
- Fig. 2 is a normal monkey pancreas (scale is 50 ⁇ )
- Fig. 2 ⁇ is an animal model group pancreas of the invention: lymphocyte infiltration is observed around the islets (scale is 50 ⁇ );
- Fig. 2C is anti-CD3 Monoclonal antibody immunohistochemical staining showed mainly sputum cell infiltration
- Figure 3 shows the immunohistochemical results of pancreatic tissue sections of the animal model of the present invention (Fig. 3 ⁇ insulin expression. cell distribution; Fig. 3 ⁇ distribution of glucagon-expressing cells (scales are ⁇ ))
- Streptozotocin (STZ) purchased from Chengdu Yuyang High-Tech Development Co., Ltd.
- Solution A (0.1 mol L citric acid solution): Weigh 2.1 g of citric acid (C6H807'H20 FW: 210.14) in 100 ml of double distilled water.
- Solution B (0.1 M sodium citrate solution): Weigh 2.94 g of sodium citrate (C6H5Na307'2H20 FW: 294.10) in 100 ml of double distilled water.
- Streptozotocin solution Weigh STZ according to animal weight ratio, dissolve STZ at 1% concentration with the above mixed working solution of A and B, and mix and match with the solution. The solution requires 4°C configuration and storage. Try to be within 30 minutes. The injection is completed.
- High-frequency electric knife (H.F.ELECTROTOME): GD-350-P type, Shanghai Hutong Electronics Co., Ltd.
- Blood glucose detector Luo Kang full vitality blood glucose detector, Luo Kang full vitality blood glucose test strip
- the pancreatic function of the animals was tested by the intravenous glucose tolerance test (IVGTT) to understand the range of blood glucose fluctuations and to rule out the possibility of spontaneous diabetes in animals.
- IVGTT intravenous glucose tolerance test
- Blood samples were taken at the 1st, 3rd, 5th, 10th, and 30th minute after the time of recording, and placed in the red blood collection tube to measure the insulin level; at 0, 1, 3, 5, 10, 30, respectively. Blood glucose is measured at 60, 120 min;
- Rhesus serum islet cell antibody detection (immunohistochemistry): After using normal monkey pancreatic tissue sections, respectively, after reacting with test group monkey serum, normal monkey serum (negative control) and anti-insulin antibody (positive control), Then combined with HRP drunken secondary antibody, after adding the substrate to color, the coloration of islet cells was observed under the microscope, and the presence or absence of anti-islet antibody in the serum was determined.
- Rhesus serum insulin antibody test (enzyme-linked immunosorbent assay): The insulin antibody test kit of Beijing North Institute of Biotechnology was used to analyze the presence or absence of insulin antibodies in animal serum according to the procedures required by the operating instructions.
- the skin preparation area is: below the bilateral nipple connection, above the groin, before the anterior line;
- Pancreatic biopsy tissue acquisition bluntly stripped 1 cm from the pancreatic body and duodenum, and the pancreatic tissue (about 0.5 g) was taken after ligation of the blood vessel with the 1st silk thread; 10) Liver biopsy tissue acquisition: No. 1 silk thread ligation of the edge of the liver, do wedge-shaped cutting to obtain tissue about 1 g;
- Kidney biopsy tissue acquisition No. 1 silk ligation of the kidney edge, wedge cutting to obtain tissue about 0.3 g;
- the fasting blood glucose of rhesus monkeys fluctuated between 3.277.6 mmol/L after intravenous injection of STZ.
- the fasting blood glucose of animals continued to be higher than 11.1 mmol/L, and the serum C-peptide was lower than 0.3 nM.
- Serum anti-islet fine See Figure 1C, ID) and anti-insulin antibody test positive; animal pancreatic tissue pathological biopsy showed lymphocytic infiltration around the islets (Fig. 2B), anti-CD3 monoclonal antibody immunohistochemical staining showed mainly T cell infiltration (Fig. 2C); Only a small amount of insulin-positive cells remained in the islet tissue (Fig. 3A), and more than 90% of the glucagon-positive cells (Fig. 3B) were judged to be successful in modeling the autoimmune type 1 diabetes model.
- test drug cyclosporine (immunosuppressive drugs, can reduce the incidence of autoimmune diabetes in mice);
- Test methods The intervention effect of cyclosporine on the pathogenesis of autoimmune diabetes was divided into saline control group and cyclosporine intervention group: in the process of STZ-induced autoimmune diabetes, the intervention group was also daily. Cyclosporine (2.5 mg/Kg) was administered to compare the onset time of diabetes, the incidence of disease, and the degree of damage to ⁇ -cells in the blank control group and the intervention group.
- autoimmune type 1 diabetes is mainly treated with insulin injection.
- insulin is used in the rhesus monkey autoimmune type 1 diabetes model.
- the specific method is as follows:
- the autoimmune type 1 diabetes of the present invention prepared by using the method of injecting different doses of different kinds of insulin twice a day (9:30 am & 17:30 pm) is applied.
- the animal model of the present invention is suitable for the treatment research of immune type 1 diabetes and the occurrence and treatment of diabetic complications, and can be used for screening drugs and treatment methods for treating autoimmune type 1 diabetes.
- the animal model of autoimmune type 1 diabetes prepared by the present invention is successfully modeled and can be used for the evaluation of new biotech drugs in which rodent models cannot be completed.
- the present invention provides the use of a low dose of streptozotocin for the preparation of an animal model for screening for an autoimmune type 1 diabetes drug, wherein the administration method and dosage of streptozotocin are: multiple consecutive veins Give, 15-30 mg / Kg / time.
- the animal model of the present invention can be used for evaluation of new biotechnological drugs and evaluation of stem cell transplantation therapeutic techniques which cannot be completed in a rodent model, and is suitable for industrial applications.
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Description
恒河猴自身免疫性 1型糖尿病模型的建立 技术领域
本发明涉及一种恒河猴自身免疫性 1型糖尿病模型的制备方法。 背景技术
自身免疫性 1型糖尿病的发病病因不明， 至今没有有效的预防办法。 常 用的胰岛素治疗尽管可以在一定程度上调节血糖水平， 但长期使用并不能有 效地控制糖尿病并发症的发生， 而且部分患者还有可能发生低血糖休克。 如 何阻止自身免疫系统对胰岛 β细胞的攻击和进行有效的 β细胞移植治疗， 成 为糖尿病研究中的关键问题。
目前糖尿病研究最常用模型动物是啮齿类。由于啮齿类在进化关系上与人 类相距甚远， 以其为研究对象所获得的实验数据对临床指导意义不大。 恒河猴 的解剖特点、 各系统生理功能及对疾病和治疗药物的反应性与人类极为相近， 是进行免疫学相关治疗和干细胞移植、异种移植临床前有效性和安全性评价的 敏感动物。 利用恒河猴糖尿病模型评价新型糖尿病治疗药物，研究胰岛干细胞 和异种胰岛移植的免疫排斥机理和移植耐受诱导，是国际公认的临床前实验体 系，对未来这些治疗方案的临床应用具有重要的指导意义。 因此建立非人灵长 类糖尿病模型成为糖尿病胰岛（干）细胞移植治疗研究和临床前药物评价的迫 切需要。
现在常用的糖尿病动物模型有实验性糖尿病动物模型和自发性糖尿病动 物模型。 自发性模型应用价值较高， 但因发病率低， 价格昂贵， 饲养、 繁殖条 件要求严格， 而不能得到广泛应用。 实验性模型则应用比较广泛， 常用的诱导 方法有胰腺切除术、 化学药物诱发、 病毒感染、 拮抗胰岛素因子、 食物诱发及 催肥等。 其中化学药物诱导方法因为操作简单、 可行性高， 而得到众多研究人 员的推崇； 而在这些方法中链脲佐菌素对机体组织毒性相对较小， 动物存活率 高， 是目前国内外使用较多的制备糖尿病动物模型的方法。
链脲菌素（ streptozotocin, STZ )是制备糖尿病模型中比较常用的诱导试剂， 对一定种属动物的胰岛 β细胞有选择性破坏作用， 而使许多动物产生糖尿病， 最常用的是大鼠模型。一般常用的诱导方法如下：将大鼠禁食 12h,按 60 mg/ g 体重腹腔注射 STZ， 每日 1次， 连续 2次， 成功制备 1型糖尿病大鼠模型， 并 且该模型具有高血糖、 体重减轻、 多饮多食多尿的特点， 与临床 1型糖尿病吻 合； 但在此实验中， 若造模组只腹腔注射 STZ—次， 并给予高热量饲料饲养 12周， 则可制 2型糖尿病动物模型， 且按该法制备出的模型具有超重、 糖耐 量减低、血脂升高、血清胰岛素升高及胰岛素受体结合力降低伴胰岛素抵抗的 特点， 类似 2型糖尿病病人的临床特征。 1型糖尿病与 2型糖尿病动物模型的
制备可能与 STZ注射的剂量有关系： 大剂量（常为 120 mg/Kg )注射时， 由于 直接引起胰岛 β细胞的广泛破坏，可造成 1型糖尿病模型； 而注射较少量 STZ 时， 由于只是破坏一部分胰岛 β细胞的功能， 造成外周组织对胰岛素不敏感， 同时给予高热量饲料喂养， 两者结合便诱导出病理、 生理改变都接近于人类 2 型糖尿病的动物模型。 因此， 链脲菌素 STZ的用量的多少， 直接决定了制备 的糖尿病动物模型的类型。
STZ制备恒河猴糖尿病模型， 也有相关文献报道， 如： 匡德宣 等， 不同 剂量链脲菌素对恒河猴某些常规生理指标的影响， 中国实验动物学报， 2003 年 3月第 11卷第 1期，报道了研究链脲菌素 (STZ)对恒河猴摄食、饮水、排尿、 体重、血糖及尿糖等生理指标的影响，为建立糖尿病动物模型积累资料。方法： 通过静脉给 7只恒河猴注射不同剂量的 STZ。 STZ的使用剂量和方法为： 低剂 量为 30 mg/Kg, 中间间隔 15天， 重复注射 1-2次； 中剂量为 45 mg/Kg, 注射 STZ1次； 高剂量为 60 mg/Kg， 注射 1次。 试验结果使用不同剂量 STZ后， 7 只恒河猴均在不同时间、 不同程度出现与人糖尿病相类似的 "三多一少"症状， 同时对糖尿病猴使用不同剂量胰岛素可使其症状减轻。尤其是中、 高剂量组的 动物症状较为明显， 而低剂量组体重有一个短时间的增加后又迅速下降。 实验 组中血糖和尿糖均出现不同程度的升高，以中、高 2个剂量组的动物变化较大。 因此， 采用中、 高剂量组 （45-60 mg/Kg ) 的 STZ可诱导类似人类糖尿病的急 性动物模型， 而使用胰岛素治疗或低剂量 STZ可使该动物模型疾病病程延长， 有利于进行其并发症的研究。 Biochemical changes in rhesus monkey during the first days after streptozotocin administration are indicative of selective beta cell destruction/Takimoto G, Jones C, Lands W, Bauman A, Jeffrey J, Jonasson 0.//Metabolism. 1988 Apr; 37(4): 364-70.选用 22只恒河猴，静脉注射 STZ(45 to 55 mg/Kg), 约半数的恒河猴在注射后的 5天内诱生胰岛素依赖性糖尿病， 剩 余实验恒河猴中的 4只在给药后 6个月内未产生胰岛素依赖性糖尿病，被认为 诱生了非胰岛素依赖型糖尿病， 其余 8只恒河猴未对外源性胰岛素产生需求。
自身免疫性糖尿病可以分为青少年型 （ Diabetes in children and adolescents ) 和成年隐匿性 ( latent autoimmune diabetes in adults, LAD A ), 属于 1型糖尿病的亚型， 其特点为： 1. 青少年型起病年龄小于 15岁， LADA 可以在 15岁以后的任何年龄段， 发病半年内不依赖胰岛素， 无酮症发生； 2. 发病时多为非肥胖； 3. 体内胰岛 B细胞抗体（ICA、 GAD和胰岛素自身抗 体等）常持续阳性； 4. 具有 1型糖尿病的易感基因（如 HLA-DR3、 HLA-DR4、 BW54及 DQ-131-57-Non-Asp等）； 5. 常伴有曱状腺和胃壁细胞等器官特异 性抗体阳性。 LADA—经诊断应早期采用胰岛素治疗以保护残存的 β细胞。 欧美人资料 4艮告 LADA约占 2型糖尿病 10% ~ 15%, 在非肥胖的 2型糖尿病 患者中 ^"报道高达 50%; 有文献报告 -2型糖尿病患者 GAD-Ab的阳性率达 14.2 %。 这一类型的病因是由胰岛 β细胞的自身免疫性破坏而引起的胰岛素 缺乏。自身免疫过程的标记物包括胰岛的淋巴细胞浸润和胰岛细胞自身抗体、
胰岛细胞抗原自身抗体以及胰岛素自身抗体的检测。
目前文献报道的是中、 高剂量组（ 45-60 mg/Kg ) STZ诱导胰岛素依赖性 糖尿病的恒河猴动物模型， 没有将低剂量 STZ (小于 30 mg/Kg )用于制备自 身免疫性 1型糖尿病灵长类动物模型的相关报道。
发明内容
本发明的技术方案是提供了低剂量的链脲菌素在制备用于筛选治疗自身 免疫性 1型糖尿病的动物模型中的用途。 本发明的另一技术方案是提供了恒 河猴自身免疫性 1型糖尿病模型的建立。
本发明提供了低剂量的链脲菌素在制备用于筛选治疗自身免疫性 1型糖 尿病的动物模型中的用途， 其中链脲菌素的施用剂量为： 15-30 mg/Kg/次。
其中， 链脲菌素的施用方法和剂量为： 连续 5天静脉给予小剂量链脲菌 素： 每次 15-30 mg/Kg; 末次给药后第 7和 14天再次给药。 进一步优选地， 链脲菌素的施用剂量为 20-25 mg/Kg。
本发明还提供了一种制备自身免疫性 I型糖尿病的动物模型的方法， 它 是将链脲菌素施用于灵长类动物，链脲菌素的施用方法和剂量为：静脉给予， 15- 30 mg/Kg/次。
其中， 所述灵长类动物为恒河猴。
其中， 所述的链脲菌素的施用方法和剂量为： 连续 5天静脉给予小剂量 链脲菌素： 每次 15-30 mg/Kg; 末次给药后第 7和 14天再次给药。
本发明还提供了该方法制备得到的自身免疫性 1型糖尿病动物模型。 本发明还提供了该动物模型在筛选治疗自身免疫性 1型糖尿病的药物中 的应用。
本发明还提供了一种筛选治疗自身免疫性 1型糖尿病的药物的方法， 包 括如下步骤：
a、 它是将链脲菌素施用于灵长类动物， 链脲菌素的施用剂量为： 15-30 mg/Kg/次；
b、 将候选物施用于 a步骤所述的灵长类动物；
c、用自身免疫性 1型糖尿病动物模型评价潜在的治疗自身免疫性 1型糖 尿病的药物。
其中， a步骤所述灵长类动物为恒河猴。 链脲菌素的施用方法和剂量为： 连续 5天静脉给予小剂量链脲菌素： 每次 15-30 mg/Kg; 末次给药后第 7和 14天再次给药。
其中， c 步骤所述的评价指标主要有： 动物糖尿病的发病时间； 发病率 和 β细胞的损伤的程度。
本发明动物模型可以用于啮齿类动物模型无法完成的生^技术新药的评 价和干细胞移植治疗技术的评价。
以下通过具体实施方式对本发明作进一步的详细描述， 但并不限制本发 明， 本领域技术人员可以根据本发明作出各种改变和变形， 只要不脱离本发 明的精神， 均应属于本发明所附权利要求的范围。
附图说明
图 1 血清抗胰岛细胞抗体检测结果（其中， 图 1A为抗胰岛素抗体阳性 对照、 图 1B为阴性对照、 图 1 C为本发明动物模型 （编号： 05539 )血清抗 胰岛细胞抗体检测结果； 图 1D为本发明动物模型 （编号： 05572 )血清抗胰 岛细胞抗体检测结果）， 标尺为 50μηι。
图 1 猴胰腺组织切片 HE染色 （图 2Α为正常猴胰腺（标尺为 50μπι ); 图 2Β为本发明动物模型组胰腺： 在胰岛周围观察到淋巴细胞浸润 （标尺为 50μηι )； 图 2C为抗 CD3单抗免疫组化染色显示主要为 Τ细胞浸润）
图 3 本发明动物模型胰腺组织切片的免疫组化结果 （图 3Α胰岛素表达. 细胞的分布； 图 3Β胰高血糖素表达细胞的分布 （标尺均为 ΙΟΟμπι ) ) 发明的具体实施方式
实施例 1 本发明动物模型的制备
1、 材料与方法：
1.1 实验材料
2-3岁雌性和雄性恒河猴 6只， 无 Β疱疹病毒、 猴逆转录病毒、 猴白血 病病毒和免疫缺陷病毒感染； 购自成都平安动物繁育基地。
1.2 主要试剂
链脲菌素 （ STZ )： 购自成都宇洋高科技发展有限公司
其他药品和耗材均购自华西医院利康药房
1.3 主要溶液的配制
链脲菌素溶液的配制：
0.1 mol/L枸橼酸钠緩沖液配制：
A液（0.1mol L枸橼酸溶液）： 称取 2.1 g枸櫞酸（ C6H807'H20 FW: 210.14 )溶于 100 ml双蒸水中。 B液（ 0.1 M枸橼酸钠溶液）： 称取 2.94 g枸 橼酸钠 （ C6H5Na307'2H20 FW: 294.10 )溶于 100 ml双蒸水中。
工作液： 用时取 28 ml A液加 22 ml B液（按照 1.32:1的比例）， 双蒸水 稀释到 100 ml，调 pH=4.5。
替换页 (细则第 26奈)
链脲菌素溶液： 按动物重量比称取 STZ， 用上述 A、 B混合工作液以 1% 的浓度溶解 STZ， 随用随配， 该液要求 4°C配置、 保存， 尽量在 30分钟内注 射完毕。
1.4 主要仪器
1) 麻醉机： Excel 210 SE， Datex-Ohmeda
2) 呼吸机配心电监护仪： M/206B, Philips公司
3) 高频电刀（H.F.ELECTROTOME): GD-350-P 型， 上海沪通电子有限公 司
4) 血糖检测仪： 罗康全活力型血糖检测仪， 罗康全活力型血糖试纸
5) 全波长分光光度计
1.5 实验方法
1.5.1 恒河猴静脉葡萄糖耐受实验
通过静脉葡萄糖耐受实验 ( The intravenous glucose tolerance test, IVGTT ) 测试动物胰腺功能， 了解血糖波动范围， 排除动物患有自发性糖尿病的可能。
1) 动物禁食 10-12 h， 不禁水；
2) 根据动物购买时体重 , 按 15 mg/Kg肌肉注射氯氨酮 （ 50 mg/ml );
3) 待动物麻醉后， 准确称量体重， 并记录；
4) 将恒河猴上下肢固定于手术台上， 下肢小腿后侧备皮暴露大隐静脉， 碘 氟消毒；
5) 20 G留滞针穿刺大隐静脉， 5ml注射器抽血 6 ml (分别做生化、血常规、 血糖和空腹胰岛素， 并标记为 0分钟数值）；
6) 立即用 5 ml注射器静脉推注 50 %葡萄糖高渗溶液 0.5 g Kg(在 30 sec内 推完），并开始记时，再注射生理盐水 5 ml, 向留滞针内推肝素（ 250 U/ml ) 1 ml;
7) 分别于记时后的第 1 , 3, 5， 10, 30 min时采血 1.5 ml, 放入红头采血 管， 测定胰岛素水平； 同时分别于第 0, 1 , 3 , 5, 10， 30, 60, 120 min 时测定血糖；
8) 检测结束， 将动物放回饲养笼中， 观察动物状况至苏醒。
1.5.2 小剂量 STZ诱导造糖尿病模型
连续 5天静脉给予小剂量 STZ: 每次 15-30 mg/Kg; 末次给药后第 7和 14 天再次给药； 每周观察空腹血糖一次， 以连续 2 天空腹血糖高于 11.1 mmol/L, C-肽水平低于 0.3 nM, 抗胰岛细胞抗体和抗胰岛素抗体阳性为建模 成功。
以下为两组实验的动物给药方案:
6只恒河猴按本发明方法造模， 有 4只造模成功。
1.5.3 恒河猴血清胰岛素测定 (放射免疫分析法)： 使用北京北方生物技术 研究所胰岛素检测试剂盒， 按照操作说明书要求程序， 分析动物血清中胰岛 素含量。
1.5.4 恒河猴血清胰岛细胞抗体检测 （免疫组织化学法）： 使用正常猴胰 腺组织切片， 分别与试验组猴血清、 正常猴血清（阴性对照）和抗胰岛素抗 体（阳性对照）反应后， 再与 HRP醉标二抗结合， 加入底物显色后， 显微镜 下观察胰岛细胞着色情况， 判断血清中有无抗胰岛细胞抗体存在。
1.5.5 恒河猴血清胰岛素抗体检测 （酶联免疫分析法）： 使用北京北方生 物技术研究所胰岛素抗体检测试剂盒， 按照操作说明书要求程序， 分析动物 血清中有无胰岛素抗体。
1.5.6 胰腺、 肝、 肾活检手术
1) 动物麻醉： 15 mg/Kg氯氨酮（ 50 mg/ml )基础麻醉， 麻醉后称动物体重；
2) 动物固定于手术台上， 仰卧位， 双下肢备皮， 建立静脉通道；
3) 手术区域皮肤准备， 备皮区域为： 双侧乳头连线以下、 腹股沟以上、 腋 前线以前；
4) 通过静脉通道， 进行静脉全麻， 气管插管， 术中全程使用呼吸机、 麻醉 机， 同时进行心电监护。 监测氧饱和度、 呼吸及体温； 建立股动脉通道， 测动脉血压；
5) 手术人员洗手消毒， 手术区域消毒： 艾力克纱布对以手术切口 （上腹正 中切口） 为中心向周围至少 15 cm皮肤无遗漏地涂擦。 待艾力克涂液自 然干燥后， 再用 70 %酒精棉球将艾力克擦去；
6) 铺无菌巾单和无菌大单和大洞单， 无菌大单上端应盖过麻醉架， 下端盖 过动物足部。 大洞单洞口要对准手术野皮肤；
7) 上腹正中切口， 切口前用 70 %酒精再次消毒切口皮肤， 依次切开皮肤、 皮下脂肪、 肌层、 腹膜进入腹腔；
8) 胰腺探查， 切开胃结肠韧带， 进入小网膜腔；
9) 胰腺活检组织获取： 从胰体与十二指肠部分钝性剥离 1 cm, 1号丝线结 扎血管后切取胰腺组织 （约 0.5 g )；
10) 肝脏活检组织获取： 1号丝线结扎肝叶边缘，做楔形切割获取组织约 1 g;
11) 肾脏活检组织获取： 1号丝线结扎肾边缘，做楔形切割获取组织约 0.3 g;
12) 清洗腹腔， 按层缝合腹壁切口；
13) 手术苏醒及术后护理： 动物术后禁食 1 天。 手术苏醒后 24 小时内肌肉 注射曲马多镇痛治疗， 头孢噻肟钠抗感染治疗 5天；
14) 术后检测动物血糖， 7天后拆线， 生化血常规检查；
2. 实验结果
恒河猴静脉注射 STZ后空腹血糖在 3.2-17.6 mmol/L之间波动， 动物的 空腹血糖持续高于 11.1 mmol/L， 血清 C-肽检测低于 0.3 nM， 血清抗胰岛细. - 胞抗体（见图 1C， ID )和抗胰岛素抗体检测阳性； 动物胰腺组织病理活检 显示胰岛周围有淋巴细胞浸润（图 2B ), 抗 CD3单抗免疫组化染色显示主要 为 T细胞浸润（图 2C );胰岛组织中仅有极少量胰岛素表达阳性细胞残存（图 3A ), 90%以上为胰高血糖素表达阳性细胞（图 3B ), 判断为自身免疫性 1 型糖尿病模型建模成功。 实施例 2 本发明动物模型的药物干预试验
一、 受试药物： 环孢霉素 （免疫抑制药物， 可降低小鼠自身免疫性糖尿 病的发生率）；
二、 试验方法： 环孢霉素对自身免疫性糖尿病发病过程的干预作用 实验分为生理盐水空白对照组和环孢霉素干预组：在 STZ诱导自身免疫 性糖尿病的过程中， 干预组同时每天给予环孢霉素 （2.5 mg/Kg ), 比较空白 对照组和干预组动物糖尿病的发病时间、 发病^ ^和 β细胞的损伤的程度。
通过免疫抑制药物干预自身免疫性糖尿病的发病过程， 观察到动物模型 中 β细胞的损伤程度降低， 进一步证明本发明动物模型为自身免疫性 1型糖 尿病模型， 且采用本发明的造模方式可成功造模。 实施例 3 用治疗自身免疫性 1型糖尿病的药物验证自身免疫性 1型糖 尿病恒河猴动物模型并初步建立相关药物筛选平台
目前自身免疫性 1型糖尿病的治疗方法主要以注射胰岛素治疗， 本实验 将胰岛素用于恒河猴自身免疫性 1型糖尿病模型， 具体方式为：
1、根据恒河猴每天进食两餐的习惯，应用一天两次（ 9:30am & 17:30pm ) 注射不同剂量不同种类胰岛素的方法， 施用于实施例 1制备的本发明自身免 疫性 1型糖尿病模型： ①长效胰岛素： 万邦 （徐州） 产猪胰岛素 （精蛋白锌 胰岛素）和②短效胰岛素： 万邦 （徐州）产猪普通胰岛素的联合治疗；
2、 为了防止低血糖发生， 从小剂量 （0.4 U/Kg ) 开始皮下注射胰岛素；
3、监测空腹和餐后血糖水平， 根据血糖水平调整给药剂量， 最终可将动 物的空腹和餐后血糖控制在低于 10 mmol/L水平，糖化血红蛋白低于 6.5%;
试险结果： 胰岛素对自身免疫性 1型糖尿病具有较佳的治疗效果。
该实验证明， 本发明动物模型适用于身免疫性 1型糖尿病的治疗研究和 糖尿病并发症的发生和治疗研究， 可用于筛选治疗自身免疫性 1型糖尿病的 药物及治疗方法。 综上所述， 本发明制备的自身免疫性 1型糖尿病动物模型建模成功， 可 以用于啮齿类动物模型无法完' 的生物技术新药的评价 ^干细胞移植治 ^技 术的评价。 工业应用性 本发明提供了低剂量的链脲菌素在制备用于筛选治疗自身免疫性 1型糖 尿病药物的动物模型中的用途， 其中链脲菌素的施用方法和剂量为： 连续多 次静脉给予， 15-30 mg/Kg/次。 本发明动物模型可以用于啮齿类动物模型无 法完成的生物技术新药的评价和干细胞移植治疗技术的评价,适于工业应用。
Claims
1、低剂量的链脲菌素在制备用于筛选治疗自身免疫性 1型糖尿病药物的 动物模型中的用途， 其中链脲菌素的施用剂量为： 15-30 mg/Kg/次。
2、根据权利要求 1所述的用途，其特征在于： 链脲菌素的施用方法和剂 量为： 连续 5天静脉给予小剂量链脲菌素： 每次 15-30 mg/Kg; 末次给药后 第 7和 14天再次给药。
3、一种制备权利要求 1或 2所述的自身免疫性 1型糖尿病动物模型的方 法， 其特征在于： 它是将链脲菌素施用于灵长类动物， 链脲菌素的施用剂量 为： 15-30 mg/Kg/次。
4、 根据权利要求 3所述的自身免疫性 1型糖尿病动物模型的制备方法， 其特征在于： 所述灵长类动物为恒河猴。
5、根据权利要求 3或 4所述的自身免疫性 1型糖尿病动物模型的制备方 法， 其特征在于： 所述的链脲菌素的施用方法和剂量为： 连续 5天静脉给予 小剂量链脲菌素： 每次 15-30 mg/Kg; 末次给药后第 7和 14天再次给药。
6、权利要求 3-5任一一项所述的方法制备得到的自身免疫性 1型糖尿病 动物模型。
7、权利要求 6所述的动物模型在筛选治疗自身免疫性 1型糖尿病的药物 中的应用。
8、 一种筛选治疗自身免疫性 1型糖尿病的药物的方法， 包括如下步骤： a、 它是将链脲菌素施用于灵长类动物， 链脲菌素的施用剂量为： 15-30 mg/Kg/次；
b、 将候选物施用于 a步骤所述的灵长类动物；
c、用自身免疫性 1型糖尿病动物模型评价潜在的治疗自身免疫性 1型糖 展病的药物。
9、 根据权利要求 8所述的筛选药物的方法， 其特征在于： a步驟所述灵 长类动物为恒河猴。
10、 根据权利要求 8或 9所述的筛选药物的方法， 其特征在于： a步骤 所述的链脲菌素的施用方法和剂量为： 连续 5天静脉给予小剂量链脲菌素： 每次 15-30 mg/Kg; 末次给药后第 7和 14天再次给药。
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| CN111066728A (zh) * | 2020-01-02 | 2020-04-28 | 康美华大基因技术有限公司 | 一种高血糖动物模型的构建方法 |
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