WO2014202024A1 - Method for preparation of rhesus monkey diabetic nephropathy model - Google Patents

Method for preparation of rhesus monkey diabetic nephropathy model Download PDF

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Publication number
WO2014202024A1
WO2014202024A1 PCT/CN2014/080414 CN2014080414W WO2014202024A1 WO 2014202024 A1 WO2014202024 A1 WO 2014202024A1 CN 2014080414 W CN2014080414 W CN 2014080414W WO 2014202024 A1 WO2014202024 A1 WO 2014202024A1
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diabetic nephropathy
rhesus monkey
monkey
feed
rhesus
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PCT/CN2014/080414
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French (fr)
Chinese (zh)
Inventor
陆燕蓉
程惊秋
陈又南
刘敬平
刘芳
杨立川
王莉
李宏霞
张�杰
王丹
杨光
钟治晖
廖光能
李兰
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四川大学华西医院
四川康城生物科技有限公司
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Publication of WO2014202024A1 publication Critical patent/WO2014202024A1/en

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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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/02—Breeding vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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 diabetic nephropathy model.
  • Diabetes Mellitus DM
  • DN Diabetic Nephropathy
  • DN is one of the most serious chronic complications in diabetic patients and the leading cause of death in diabetic patients. Once DN patients have significant clinical symptoms, there are fewer drugs to choose from, and the drug treatment is less effective. The condition often progresses progressively until the end of renal failure, which brings huge economic burden to patients and families. Therefore, in-depth study of the pathogenesis of DN and early diagnosis methods, on this basis to explore effective measures to delay or reverse the disease process, is currently a hot and difficult issue in medical research.
  • the animal model is the basis for studying human major diseases. Establishing a DN animal model that is highly similar to humans will achieve a multiplier effect in studying its pathogenesis, early diagnosis and intervention therapy. As a close relative of human beings, non-human primates are about 96% homologous in genetics. Their morphological, physiological and biochemical metabolisms are very similar to humans. A non-human primate DN model was established. The occurrence and development of DN, early diagnosis and research and development of new drugs have important scientific significance and economic value.
  • the preparation method of the rhesus monkey diabetic nephropathy model of the present invention comprises the following steps:
  • the preparation method of the rhesus monkey diabetic nephropathy model of the present invention comprises the following steps:
  • the monkey After the blood glucose concentration is raised to ll.lmmol/1, feed the high-fat diet, feed twice a day, feed 0.3-0.4kg/time. Only, and apply insulin before the meal to make Ganges
  • the monkey has a blood glucose concentration of 10-20 mmol/L, and the feed comprises the following raw materials by weight: 80 parts of standard monkey feed, 15 parts of animal fat, and 5 parts of sugar.
  • Standard monkey feed refers to the monkey compound feed specified in the National Standard of the Chinese Republic of China GB14924.8-200O.
  • Animal fat refers to fat derived from animals, such as butter, sheep oil, and lard.
  • the administration method is intravenous injection.
  • the administration method is subcutaneous injection.
  • the animal fat is lard; and the sugar is sucrose.
  • the feed of the rhesus monkey diabetic nephropathy model of the present invention comprises the following raw materials by weight ratio: 80 parts of standard monkey feed, 15 parts of animal fat, and 5 parts of sugar.
  • the animal fat is lard; the sugar is sucrose.
  • the rhesus monkey diabetic nephropathy model prepared by the foregoing method of the present invention and its use in screening for a medicament for treating rhesus diabetes mellitus nephropathy.
  • the invention selects a method for treating a drug for a rhesus monkey diabetic nephropathy model, which comprises the following steps: a. establishing a rhesus monkey diabetic nephropathy model according to the foregoing method;
  • the modeling method of the invention can induce the clinical symptoms of diabetic kidney disease such as kidney damage and microalbuminuria in rhesus monkey, and the administration method is simple and reproducible.
  • Figure 3 shows the changes in fibrosis genes
  • TNF-a antibody abeam
  • CTGF antibody abeam
  • CD68(KP1) antibody abeam
  • Blood glucose detector Luo Kang full vital blood glucose detector, Luo Kang full vitality blood glucose test strip Automatic blood biochemical analyzer (Switzerland Roche COB AS Integra 400 Plus, No. MEB-02-01 )
  • Rhesus monkeys are raised in the General Animal Room of the National Chengdu Chinese Medicine Safety Evaluation Center (Experimental Animal House) License number: SYXK (chuan) 2003— 030), each of the rhesus monkeys are caged in stainless steel squirrel cages, and the feeding conditions are in line with the national standard GB14925-2001, room temperature 21 ⁇ 5°C (day temperature difference ⁇ 4°0, relative Humidity 55+15%, 12 hours of light and dark alternate, the number of air changes 8 ⁇ 10 times / hour.
  • the group consisted of the normal group (3 rats), experimental group A (after STZ diabetes model, subcutaneous injection of insulin 15 minutes before meals every day, control blood glucose ⁇ 1011111101/1, 3), experimental group B (STZ diabetes model, Intravenous injection of insulin 15 minutes before the meal every day, control blood glucose at 10-20mmol / l, 5), experimental group C (STZ diabetes model, by subcutaneous injection of insulin 15 minutes before meals every day, control blood sugar at 10-20mmol / l, and fed high-fat diet, 3).
  • Experimental group C High fat diet: 15% refined lard (commercially available), 5% sucrose (commercially available), 80% monkey standard monkey feed.
  • Feeding method Feed 2 times a day, feeding 0.3-0.413 ⁇ 4/time. Only.
  • Animals' body weight, blood pressure and food intake were measured by observing animal appearance signs (including animal hair, eyes and mucous membranes), behavioral activities, mental status, glandular secretion, respiration, diet, excretion, and local injection.
  • the animals were fasted for more than 12 hours, and after intravenous infusion of 100 ml of normal saline, the prepared STZ (80 mg/kg) was injected.
  • FBG fasting blood glucose
  • the animals were fasted for more than 12 hours, and after intravenous infusion of 100 ml of normal saline, the prepared STZ (80 mg/kg) was injected.
  • the rats were fed with regular monkey feed, fed twice a day, and the feeding amount was 0.3-0.413 ⁇ 4/time _ only; the fasting blood glucose (FBG) of the animals was detected, and the FBG was higher than ll.l mmol/1 for 2 consecutive days.
  • Insulin was injected subcutaneously 15 minutes before the meal to control the blood sugar concentration.
  • the high-fat diet was fed and fed twice a day.
  • the feeding amount was 0.3-0.413 ⁇ 4/time.
  • Detection indicators red blood cell count (RBC, capacitance method), hemoglobin (HGB, HiCN method), red blood cell volume (HCT, capacitance integration method), mean red blood cell volume (MCV, calculated), mean red blood cell hemoglobin (MCH, calculated), mean red blood cells Hemoglobin concentration (MCHC, calculated), reticulocyte count (RET, fluorescent staining), white blood cell count (WBC, laser method) and classification (neutrophil NEU, lymphocyte LYM, monocyte MONO, eosinophils) EOS, basophilic BASO, laser method, platelet count (PLT, capacitance method), reticulocyte (RET, flow + fluorescence staining), prothrombin time (PT, chromogenic substrate method), Activate partial thrombin time ( ⁇ , chromogenic substrate method).
  • RBC red blood cell count
  • HGB hemoglobin
  • HCT red blood cell volume
  • MCV mean red blood cell volume
  • MH mean red blood cell hemoglobin
  • Aspartate aminotransferase (AST, IFCC P-5'-P method), alanine aminotransferase (ALT, IFCC P-5'-P method), ⁇ -glutamyltransferase (GGT, L) - ⁇ -glutamyl-3-carboxy-p-nitroaniline method), creatine phosphokinase (CK, HK-G6PD method), alkaline phosphatase (ALP, 4-NPP method), lactate dehydrogenase (LDH) , IFCC method), urea nitrogen (Urea, Urcase-GLDH-Kinetic method), total protein (TP, Biuret method), albumin (ALB, BCG method), blood glucose (GLU, GLK-G6PDH method), total bilirubin (TBIL, Diazo method), creatinine (Crea, JAFF method), total cholesterol (CHOL, CHOD-P method), triglyceride (TG, GPO-PAP method), sodium ion concentration (N
  • Detection time Test each time for STZ 1, 4, and every week, and then every 1 ⁇ 2 months.
  • Blood samples were collected from the saphenous vein of the hind legs of rhesus monkeys. Hematological examination blood is anticoagulated with EDTA, and blood clotting time is anticoagulated with sodium citrate.
  • FBG Fasting blood glucose
  • PBG postprandial blood glucose
  • Detection time FBG (9 am, 12 h after fasting) and PBG (2 h after eating) were tested twice a week.
  • Test method Take the finger or toe peripheral blood and test with Roche blood glucose meter.
  • Radioimmunoassay (RIA) kit radioimmunoassay (RIA) kit, enzyme-linked immunosorbent assay (ELISA)
  • 2.7 insulin therapy Fasting and postprandial blood glucose monitoring, adjusting the type, dosage form and dosage of insulin injected subcutaneously in diabetic monkeys according to blood glucose levels. 24-hour blood glucose monitoring to observe the treatment effect, determine the optimal program. Regular blood tests and biochemical tests are performed.
  • Detection time before administration and at the end of the observation period after administration.
  • Determination method Rhesus monkey was anesthetized with ketamine hydrochloride (intramuscular injection, 15 mg/kg), Medea dilated (eye drops, 1 drop/eye), and examined by binocular indirect ophthalmoscopy. All imaging evaluations were performed. Under general anesthesia (animal anesthesia method reference rhesus surgery anesthesia SOP). Use the ophthalmoscope to observe the shape, size, color and sharpness of the optic nerve head. There is no edema, hemorrhage, exudation and pigmentation disorder in the macula. There are no edema, exudation, hemorrhage, exfoliation and new blood vessels in the retina.
  • RNA latter tissue extraction RNA was reverse transcribed and the expression of smad2, smad3, smad4, smad7, actin and NF-Kb genes was detected by real-time PCR.
  • Rhesus monkeys were placed in metabolic cages to collect morning urine and 24-hour urine to detect urine routine, urinary albumin and creatinine.
  • the blood glucose of each experimental group was kept within a predetermined range.
  • the early renal vascular bed resistance increases, so that the blood flow in the kidney changes.
  • the elasticity of the arteriosclerotic blood vessel wall decreases, the lumen narrows, the peripheral vascular resistance increases, and the renal blood flow perfusion decreases.
  • the renal blood flow was in a low-flow, high-resistance state. Among them, the pathological changes of group B and group C were very obvious, and group C was the most obvious.
  • Renal angiography showed that the blood perfusion coefficient AUC and the flow dynamics resistance index RI of the experimental group B and C increased significantly, the minimum flow velocity EDV decreased at the end of diastole (P ⁇ 0.05), the elasticity of the arteriosclerotic vessel wall decreased, and the lumen Stenosis, peripheral vascular resistance is increased, renal blood perfusion is reduced, and renal blood flow is in a low-flow, high-resistance state.
  • the present invention allows diabetic rhesus monkeys to have a blood glucose concentration ranging from 10 to 20 mmol/L for a long time.
  • Typical clinical symptoms of diabetic nephropathy such as renal cell inflammatory cell infiltration, mesangial cell hyperplasia, basement membrane thickening, collagen and glycogen deposition, kidney damage, microalbuminuria, etc., indicating that the rhesus monkey diabetic nephropathy model of the present invention success.
  • the modeling method of the present invention induces the clinical symptoms of diabetic nephropathy in rhesus monkeys by long-term control of blood glucose.
  • the combination of long-term glycemic control and high-fat intake can also induce the clinical manifestation of diabetic nephropathy in rhesus monkeys. Symptoms, and the symptoms are more significant, indicating that the two methods of the present invention can effectively establish a rhesus monkey diabetic nephropathy model.
  • the modeling method of the invention effectively establishes a rhesus monkey diabetic nephropathy model, which can be used for rhesus monkey diabetes

Abstract

Disclosed is a method for preparation of a rhesus monkey diabetic nephropathy (DN) model, comprising the following steps: (1) administering streptozocin with the dosage of 80-100 mg/Kg to a rhesus monkey; (2) when the blood glucose concentration is increased to 11.1 mmol/l, administering insulin before a meal, such that the blood glucose concentration of the rhesus monkey is 10-20 mmol/L. The present invention employs a blood glucose control method on a rhesus monkey having diabetes mellitus (DM), and successfully induces a rhesus monkey DN model.

Description

说 明 书  Description
一种恒河猴糖尿病肾病模型的制备方法 技术领域  Method for preparing rhesus monkey diabetic nephropathy model
本发明涉及恒河猴糖尿病肾病模型的制备方法。  The invention relates to a method for preparing a rhesus monkey diabetic nephropathy model.
背景技术 Background technique
糖尿病 （Diabetes Mellitus, DM) 的发病率呈快速增长的趋势。 据报道 我国现约有 9200万 DM和 1.48亿 DM前期患者， 成为继美国之后的第二个 糖尿病发病大国。 随着病情的逐渐发展加重， 这些患者中约有 30-40%的 1 型糖尿病患者和约 20%的 2型糖尿病患者伴随严重的肾功能损害一糖尿病 肾病 (Diabetic Nephropathy, DN)。  The incidence of diabetes (Diabetes Mellitus, DM) is increasing rapidly. According to reports, there are about 92 million DM and 148 million DM pre-patients in China, making it the second largest country for diabetes after the United States. As the disease progresses, about 30-40% of patients with type 1 diabetes and about 20% of type 2 diabetes suffer from severe renal impairment, Diabetic Nephropathy (DN).
DN 是糖尿病患者最严重的慢性并发症之一， 也是糖尿病患者最主要的 死亡原因。 一旦 DN患者出现了显著的临床症状， 此时可选择的药物较少， 且药物的治疗效果较差， 病情往往呈进行性发展直至末期肾衰， 给患者家庭 和社会带来巨大的经济负担。因此深入研究 DN的发病机制及早期诊断方法， 在此基础上探索延缓或逆转疾病进程的有效措施， 是目前医学研究的热点和 难点问题。  DN is one of the most serious chronic complications in diabetic patients and the leading cause of death in diabetic patients. Once DN patients have significant clinical symptoms, there are fewer drugs to choose from, and the drug treatment is less effective. The condition often progresses progressively until the end of renal failure, which brings huge economic burden to patients and families. Therefore, in-depth study of the pathogenesis of DN and early diagnosis methods, on this basis to explore effective measures to delay or reverse the disease process, is currently a hot and difficult issue in medical research.
动物模型是研究人类重大疾病的基础， 建立与人类高度近似的 DN动物 模型， 将会在研究其发病机理、 早期诊断和干预治疗等方面取得事半功倍的 效果。 非人灵长类动物作为人类的近亲， 在遗传基因方面约 96%以上同源， 其形态结构、 生理机能和生化代谢均同人类非常相似， 建立一种非人灵长类 动物 DN模型对研究 DN的发生发展、 早期诊断和新药研发等具有重要的科 学意义和经济价值。  The animal model is the basis for studying human major diseases. Establishing a DN animal model that is highly similar to humans will achieve a multiplier effect in studying its pathogenesis, early diagnosis and intervention therapy. As a close relative of human beings, non-human primates are about 96% homologous in genetics. Their morphological, physiological and biochemical metabolisms are very similar to humans. A non-human primate DN model was established. The occurrence and development of DN, early diagnosis and research and development of new drugs have important scientific significance and economic value.
发明内容 Summary of the invention
本发明的目的在于提供恒河猴糖尿病肾病模型的制备方法。  It is an object of the present invention to provide a method for preparing a rhesus monkey diabetic nephropathy model.
本发明恒河猴糖尿病肾病模型的制备方法， 包括如下步骤：  The preparation method of the rhesus monkey diabetic nephropathy model of the present invention comprises the following steps:
( 1 ) 将剂量为 80~100mg/Kg的链脲菌素施用于恒河猴；  (1) administering streptozotocin at a dose of 80-100 mg/Kg to rhesus monkeys;
(2) 血糖浓度升至 ll.lmmol/1后， 餐前施用胰岛素， 使恒河猴血糖浓 度为 10-20mmol/L。  (2) After the blood glucose concentration rises to ll.lmmol/1, insulin is administered before the meal to make the rhesus monkey blood sugar concentration 10-20mmol/L.
本发明恒河猴糖尿病肾病模型的制备方法， 包括如下步骤：  The preparation method of the rhesus monkey diabetic nephropathy model of the present invention comprises the following steps:
( 1 ) 将剂量为 80~100mg/Kg的链脲菌素施用于恒河猴；  (1) administering streptozotocin at a dose of 80-100 mg/Kg to rhesus monkeys;
(2) 血糖浓度升高至 ll.lmmol/1后， 饲喂高脂饲料， 每天饲喂 2次， 饲喂量为 0.3-0.4kg/次.只， 并在餐前施用胰岛素， 使恒河猴血糖浓度为 10-20mmol/L, 所述饲料包含如下重量配比的原料： 标准猴饲料 80份、 动物 油脂 15份、 糖 5份。 标准猴饲料， 是指《中华人名共和国国家标准 GB14924.8-200O规定的 猴配合饲料。 (2) After the blood glucose concentration is raised to ll.lmmol/1, feed the high-fat diet, feed twice a day, feed 0.3-0.4kg/time. Only, and apply insulin before the meal to make Ganges The monkey has a blood glucose concentration of 10-20 mmol/L, and the feed comprises the following raw materials by weight: 80 parts of standard monkey feed, 15 parts of animal fat, and 5 parts of sugar. Standard monkey feed refers to the monkey compound feed specified in the National Standard of the Chinese Republic of China GB14924.8-200O.
动物油脂， 是指来源于动物的脂肪， 如， 牛油、 羊油、 猪油。  Animal fat refers to fat derived from animals, such as butter, sheep oil, and lard.
步骤 （1 ) 中， 所述的施用方式是静脉注射。  In the step (1), the administration method is intravenous injection.
步骤 （2) 中， 所述的施用方式是皮下注射。  In the step (2), the administration method is subcutaneous injection.
步骤 （2) 中， 所述动物油脂是猪油； 所述糖是蔗糖。  In the step (2), the animal fat is lard; and the sugar is sucrose.
本发明恒河猴糖尿病肾病模型的饲料， 它包含如下重量配比的原料： 标 准猴饲料 80份、 动物油脂 15份、 糖 5份。  The feed of the rhesus monkey diabetic nephropathy model of the present invention comprises the following raw materials by weight ratio: 80 parts of standard monkey feed, 15 parts of animal fat, and 5 parts of sugar.
所述动物油脂是猪油； 所述糖是蔗糖。  The animal fat is lard; the sugar is sucrose.
本发明前述方法制备的恒河猴糖尿病肾病模型及其在筛选治疗恒河猴糖 尿病肾病的药物中的用途。  The rhesus monkey diabetic nephropathy model prepared by the foregoing method of the present invention and its use in screening for a medicament for treating rhesus diabetes mellitus nephropathy.
本发明筛选治疗恒河猴糖尿病肾病模型的药物的方法，它包括如下步骤： a、 按照前述方法， 建立恒河猴糖尿病肾病模型；  The invention selects a method for treating a drug for a rhesus monkey diabetic nephropathy model, which comprises the following steps: a. establishing a rhesus monkey diabetic nephropathy model according to the foregoing method;
b、 将候选药物施用于动物模型；  b. administering the candidate drug to the animal model;
c、 用动物模型评价潜在的治疗恒河猴糖尿病肾病的药物。  c. Evaluation of potential drugs for the treatment of rhesus diabetic nephropathy using animal models.
本发明造模方法可以诱导恒河猴出现肾脏损伤、 微量蛋白尿等糖尿病肾 病的临床症状， 给药方法简单， 可重复性强。  The modeling method of the invention can induce the clinical symptoms of diabetic kidney disease such as kidney damage and microalbuminuria in rhesus monkey, and the administration method is simple and reproducible.
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段， 在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、 替换或变更。  It is apparent that various other modifications, substitutions and changes can be made in the form of the above-described embodiments of the present invention without departing from the spirit and scope of the invention.
以下通过实施例形式的具体实施方式， 对本发明的上述内容再作进一步 的详细说明。 但不应将此理解为本发明上述主题的范围仅限于以下的实例。 凡基于本发明上述内容所实现的技术均属于本发明的范围。  The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following examples. Any technique implemented based on the above description of the present invention is within the scope of the present invention.
附图说明  DRAWINGS
图 1 血糖控制情况；  Figure 1 Blood sugar control;
图 2 肾功能检测结果；  Figure 2 results of renal function tests;
图 3 致纤维化基因改变情况；  Figure 3 shows the changes in fibrosis genes;
图 5 肾血流动力学参数和血流灌注系数；  Figure 5 Renal hemodynamic parameters and blood perfusion coefficient;
图 6 肾脏 B超检查的血流分布和频谱；  Figure 6 Blood flow distribution and spectrum of kidney B-ultrasound;
图 7 肾组织病理改变；  Figure 7 Pathological changes in kidney tissue;
图 8 肾组织免疫荧光。  Figure 8 Immunofluorescence of kidney tissue.
具体实施方式  detailed description
实施例 1 本发明恒河猴糖尿病肾病征模型的制备  Example 1 Preparation of Diabetic Nephropathy Model of Rhesus Monkey of the Present Invention
1实验鼎和仪器  1 experimental tripod and instrument
1.1 供试品基本信息 IL-Ιβ抗体 - abeam 1.1 Basic information of the test article IL-Ιβ antibody - abeam
TNF-a抗体： abeam  TNF-a antibody: abeam
TGF-βΙ 抗体： Bioworld  TGF-βΙ Antibody: Bioworld
CTGF抗体： abeam  CTGF antibody: abeam
Collagen IV抗体： abeam  Collagen IV Antibody: abeam
Fibronection 抗体： Bioworld  Fibronection antibody: Bioworld
MCP-1抗体： ebioscience  MCP-1 antibody: ebioscience
CD68(KP1) 抗体： abeam  CD68(KP1) antibody: abeam
eNOS 抗体: abeam  eNOS antibody: abeam
动物组织总 RNA提取试剂盒： 天根  Animal Tissue Total RNA Extraction Kit: Tiangen
iScriptTM cDNA合成试剂盒： BIO-RAD iScript TM cDNA Synthesis Kit: BIO-RAD
iQTM SYBR® Green Supermix ： BIO-RAD iQ TM SYBR® Green Supermix : BIO-RAD
1.2 试验所需主要仪器、 器械  1.2 Main instruments and instruments required for testing
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 全自动血液生化分析仪 （瑞士 Roche COB AS Integra 400 Plus, 编号 MEB-02-01 )  Blood glucose detector: Luo Kang full vital blood glucose detector, Luo Kang full vitality blood glucose test strip Automatic blood biochemical analyzer (Switzerland Roche COB AS Integra 400 Plus, No. MEB-02-01 )
BIO-RAD CFX96 荧光定量 PCR仪  BIO-RAD CFX96 Fluorescence Quantitative PCR System
BIO-RAD S200 PCR仪  BIO-RAD S200 Cycler
LEICA DM4000B 荧光显微镜  LEICA DM4000B fluorescence microscope
Olympus BX51 光学显微镜  Olympus BX51 Optical Microscope
穿剌针， 穿剌枪： BARD  Wear a needle, wear a rifle: BARD
彩超；Iu22 philips  Color ultrasound; Iu22 philips
眼底荧光造影： Topcon TRC. 50DX (日本拓普康公司生产）  Fundus fluorescein: Topcon TRC. 50DX (produced by Japan Topcon)
1.3 实验动物  1.3 Experimental animals
1.3.1 实验***  1.3.1 Experimental system
恒河猴 14只，体重 3.5~5.5 Kg,拟由四川成都平安动物繁育研究基地（生 产许可证号： SCXK (川) 2004— 013 ) 引进。 入室前一周进行结核菌素、 寄生 虫、 沙门氏菌、 致贺氏菌检查。  14 rhesus monkeys, weighing 3.5~5.5 Kg, are proposed to be imported from Sichuan Chengdu Pingan Animal Breeding Research Base (production license number: SCXK (Chuan) 2004-013). Tuberculin, parasitic, Salmonella, and Hercules were examined one week before entering the room.
1.4 动物饲养管理  1.4 Animal feeding management
1.4.1 检疫 /环境适应  1.4.1 Quarantine / Environmental Adaptation
入室恒河猴试验前先适应环境并检疫一周， 选择健康雄性动物作为受试 动物， 检疫内容： 是否与订购时要求的质量指标一致； 体温、 摄食量、 血生 化、 电解质； 动物一般状态； 动物体重是否达到试验要求体重的范围。  Enter the rhesus monkey to adapt to the environment and quarantine for one week before the test, select healthy male animals as the test animals, quarantine content: whether it is consistent with the quality indicators required at the time of ordering; body temperature, food intake, blood biochemistry, electrolytes; animal general state; animals Whether the weight reaches the range of weight required for the test.
1.4.2动物饲养  1.4.2 Animal breeding
恒河猴饲养于国家成都中药安全性评价中心普通动物房 （实验动物房使 用许可证号： SYXK (川） 2003— 030)、 恒河猴每只用不锈钢鼠笼分笼饲养， 饲养条件符合国标 GB14925-2001 , 室温 21±5°C (日温差≤4°0， 相对湿度 55+15 % , 12小时明暗交替， 换气次数 8〜10次 /小时。 Rhesus monkeys are raised in the General Animal Room of the National Chengdu Chinese Medicine Safety Evaluation Center (Experimental Animal House) License number: SYXK (chuan) 2003— 030), each of the rhesus monkeys are caged in stainless steel squirrel cages, and the feeding conditions are in line with the national standard GB14925-2001, room temperature 21±5°C (day temperature difference ≤4°0, relative Humidity 55+15%, 12 hours of light and dark alternate, the number of air changes 8~10 times / hour.
1.4.3 动物标识方法  1.4.3 Animal identification method
恒河猴由胸牌及笼上标签共同标记  Rhesus monkeys are marked by badges and cage tags
1.5 实验分组  1.5 Experimental grouping
分组包括正常组 （3只） ，实验组 A (STZ糖尿病造模后， 通过每天餐前 15分钟皮下注射胰岛素， 控制血糖<1011111101/1， 3只）， 实验组 B (STZ糖尿 病造模后，通过每天餐前 15分钟皮下注射胰岛素，控制血糖于 10-20mmol/l， 5只）， 实验组 C (STZ糖尿病造模后， 通过每天餐前 15分钟皮下注射胰岛 素， 控制血糖于 10-20mmol/l， 并饲喂高脂饲料， 3只）。  The group consisted of the normal group (3 rats), experimental group A (after STZ diabetes model, subcutaneous injection of insulin 15 minutes before meals every day, control blood glucose <1011111101/1, 3), experimental group B (STZ diabetes model, Intravenous injection of insulin 15 minutes before the meal every day, control blood glucose at 10-20mmol / l, 5), experimental group C (STZ diabetes model, by subcutaneous injection of insulin 15 minutes before meals every day, control blood sugar at 10-20mmol / l, and fed high-fat diet, 3).
饲料配方：  Feed formula:
实验组 A、 B: 猴标准猴饲料一商业化的猴颗粒饲料 （购自四川省医 学科学院动物研究所， 配方与 《中华人名共和国国家标准 GB 14924.8-2001》 规定的猴配合饲料相同）。  Experimental group A, B: Monkey standard monkey feed-commercial monkey pellet feed (purchased from the Institute of Zoology, Sichuan Academy of Medical Sciences, the formula is the same as the monkey compound feed specified in the National Standard GB 14924.8-2001 of the Chinese Republic of China).
实验组 C: 高脂饲料： 15%精炼猪油 （市售）， 5%蔗糖 （市售）， 80%猴 标准猴饲料。  Experimental group C: High fat diet: 15% refined lard (commercially available), 5% sucrose (commercially available), 80% monkey standard monkey feed.
饲喂方式： 每天饲喂 2次， 饲喂量为 0.3-0.41¾/次.只。  Feeding method: Feed 2 times a day, feeding 0.3-0.413⁄4/time. Only.
2实验方法  2 experimental methods
2.1动物一般状况观察  2.1 General observation of animals
观察动物外观体征（包括动物的毛发、眼和粘膜）、行为活动、精神状况、 腺体分泌、 呼吸、 饮食、 ***物、 注射局部等情况， 检测动物体重、 血压及 摄食量。  Animals' body weight, blood pressure and food intake were measured by observing animal appearance signs (including animal hair, eyes and mucous membranes), behavioral activities, mental status, glandular secretion, respiration, diet, excretion, and local injection.
2.2造模方法  2.2 Modeling method
实验组 A、 B:  Experimental group A, B:
给药前动物禁食禁饮 12h以上， 静脉输注生理盐水 100 ml扩容后， 再推 注配制好的 STZ (80 mg/kg);  Before the administration, the animals were fasted for more than 12 hours, and after intravenous infusion of 100 ml of normal saline, the prepared STZ (80 mg/kg) was injected.
给予常规猴饲料喂养， 每天饲喂 2次， 饲喂量为 0.3-0.41¾/次_只； 检测动物空腹血糖 （FBG)， FBG连续 2天高于 ll.l mmol/1后， 通过每 天餐前 15分钟皮下注射胰岛素， 控制血糖浓度。  Feeding to regular monkey feed, feeding twice a day, feeding 0.3-0.413⁄4/time _ only; detecting fasting blood glucose (FBG) in animals, FBG is higher than ll.l mmol/1 for 2 consecutive days, through daily meal Insulin was injected subcutaneously in the first 15 minutes to control blood glucose levels.
实验组 C:  Experimental group C:
给药前动物禁食禁饮 12h以上， 静脉输注生理盐水 100 ml扩容后， 再推 注配制好的 STZ (80 mg/kg);  Before the administration, the animals were fasted for more than 12 hours, and after intravenous infusion of 100 ml of normal saline, the prepared STZ (80 mg/kg) was injected.
给予常规猴饲料喂养， 每天饲喂 2次， 饲喂量为 0.3-0.41¾/次_只； 检测动物空腹血糖 （FBG)， FBG连续 2天高于 ll.l mmol/1后， 通过每 天餐前 15分钟皮下注射胰岛素， 控制血糖浓度， 同时给与高脂饲料喂养， 每 天饲喂 2次， 饲喂量为 0.3-0.41¾/次_只。 The rats were fed with regular monkey feed, fed twice a day, and the feeding amount was 0.3-0.413⁄4/time _ only; the fasting blood glucose (FBG) of the animals was detected, and the FBG was higher than ll.l mmol/1 for 2 consecutive days. Insulin was injected subcutaneously 15 minutes before the meal to control the blood sugar concentration. At the same time, the high-fat diet was fed and fed twice a day. The feeding amount was 0.3-0.413⁄4/time.
2.3血液学检查  2.3 Hematology examination
检测指标： 红细胞计数 （RBC， 电容法）、 血红蛋白 （HGB， HiCN法）、 红细胞容积 （HCT， 电容积分法）、 平均红细胞容积 （MCV， 计算）、 平均红 细胞血红蛋白 （MCH， 计算）、 平均红细胞血红蛋白浓度 （MCHC, 计算）、 网织红细胞计数 （RET, 荧光染色法）、 白细胞计数 （WBC， 激光法） 及分 类（中性粒细胞 NEU、淋巴细胞 LYM、单核细胞 MONO、嗜酸性粒细胞 EOS、 嗜碱性粒细胞 BASO， 激光法）、 血小板计数 （PLT， 电容法）、 网织红细胞 (RET, 流氏 +荧光染色法）、 凝血酶原时间 （PT， 发色底物法）、 活化部分 凝血酶时间（ΑΡΤΤ,发色底物法）。天门冬氨酸氨基转移酶（AST, IFCC P-5'-P 法）、 丙氨酸氨基转移酶 （ALT, IFCC P-5'-P法）、 γ-谷氨酰转移酶 （GGT， L-γ-谷氨酰 -3-羧基 -对硝基苯胺法）、 肌酸磷酸激酶 （CK， HK-G6PD法）、 碱 性磷酸酶（ALP， 4-NPP法）、 乳酸脱氢酶（LDH， IFCC法）、 尿素氮（Urea, Urcase-GLDH-Kinetic法）、 总蛋白 （TP， Biuret法）、 白蛋白 （ALB， BCG 法）、血糖（GLU， GLK-G6PDH法）、总胆红素（TBIL， Diazo法）、肌酐（Crea， JAFF法）、 总胆固醇（CHOL， CHOD-P法）、甘油三酯（TG， GPO-PAP法）、 钠离子浓度（Na+， 离子选择电极法）、 钾离子浓度（K+， 离子选择电极法）、 氯离子浓度 （Cl-， 离子选择电极法） 等。  Detection indicators: red blood cell count (RBC, capacitance method), hemoglobin (HGB, HiCN method), red blood cell volume (HCT, capacitance integration method), mean red blood cell volume (MCV, calculated), mean red blood cell hemoglobin (MCH, calculated), mean red blood cells Hemoglobin concentration (MCHC, calculated), reticulocyte count (RET, fluorescent staining), white blood cell count (WBC, laser method) and classification (neutrophil NEU, lymphocyte LYM, monocyte MONO, eosinophils) EOS, basophilic BASO, laser method, platelet count (PLT, capacitance method), reticulocyte (RET, flow + fluorescence staining), prothrombin time (PT, chromogenic substrate method), Activate partial thrombin time (ΑΡΤΤ, chromogenic substrate method). Aspartate aminotransferase (AST, IFCC P-5'-P method), alanine aminotransferase (ALT, IFCC P-5'-P method), γ-glutamyltransferase (GGT, L) -γ-glutamyl-3-carboxy-p-nitroaniline method), creatine phosphokinase (CK, HK-G6PD method), alkaline phosphatase (ALP, 4-NPP method), lactate dehydrogenase (LDH) , IFCC method), urea nitrogen (Urea, Urcase-GLDH-Kinetic method), total protein (TP, Biuret method), albumin (ALB, BCG method), blood glucose (GLU, GLK-G6PDH method), total bilirubin (TBIL, Diazo method), creatinine (Crea, JAFF method), total cholesterol (CHOL, CHOD-P method), triglyceride (TG, GPO-PAP method), sodium ion concentration (Na+, ion selective electrode method), Potassium ion concentration (K+, ion selective electrode method), chloride ion concentration (Cl-, ion selective electrode method), and the like.
检测时间： 给 STZ第 1， 4， 周各测一次， 以后每 1~2月测一次。  Detection time: Test each time for STZ 1, 4, and every week, and then every 1~2 months.
方法： 自恒河猴后腿大隐静脉采血检测。血液学检查用血以 EDTA抗凝， 凝血时间用血以枸橼酸钠抗凝。  Methods: Blood samples were collected from the saphenous vein of the hind legs of rhesus monkeys. Hematological examination blood is anticoagulated with EDTA, and blood clotting time is anticoagulated with sodium citrate.
2.4 空腹血糖（Fasting blood glucose, FBG)及餐后血糖（Postprandial blood glucose, PBG) 检测  2.4 Fasting blood glucose (FBG) and postprandial blood glucose (PBG) detection
检测时间： 每周 2次检测 FBG (上午 9点， 空腹 12h后）及 PBG (进食 后 2h)。  Detection time: FBG (9 am, 12 h after fasting) and PBG (2 h after eating) were tested twice a week.
检测方法： 取手指或脚趾末梢血， 用罗氏血糖仪检测。  Test method: Take the finger or toe peripheral blood and test with Roche blood glucose meter.
2.5静脉葡萄糖耐量实验 （IVGTT)  2.5 intravenous glucose tolerance test (IVGTT)
静脉给予 50%的葡萄糖注射液 （0.5 g/kg体重）， 在推注后 0， 1， 3， 5， 10， 30， 60和 120分钟测定血糖水平， 同时检查葡萄糖注射后 0， 1， 3， 5， 30分钟血样中的胰岛素水平 （ RIA or ELISA )  50% glucose injection (0.5 g/kg body weight) was administered intravenously, blood glucose levels were measured at 0, 1, 3, 5, 10, 30, 60 and 120 minutes after the bolus, and 0, 1, 3 after glucose injection were examined. , 5, 30 minutes of insulin levels in blood samples (RIA or ELISA)
2.6 空腹糖化血红蛋白、 血清胰岛素及 C肽 (C-peptide， C-P) 水平检测 检测时间： 每隔 4周取血清检测。  2.6 Fasting glycated hemoglobin, serum insulin and C-peptide (C-peptide, C-P) levels Detection time: Serum was taken every 4 weeks.
检测方法： 放射免疫法 (RIA) 试剂盒，酶联免疫检测法 （ELISA) Detection method: Radioimmunoassay (RIA) kit, enzyme-linked immunosorbent assay (ELISA)
2.7 胰岛素治疗 空腹和餐后血糖监控， 根据血糖水平调整糖尿病猴皮下注射胰岛素的种 类、 剂型及用量。 24小时血糖监控观察治疗效果， 确定最优方案。 定期行血 常规及生化检查。 2.7 insulin therapy Fasting and postprandial blood glucose monitoring, adjusting the type, dosage form and dosage of insulin injected subcutaneously in diabetic monkeys according to blood glucose levels. 24-hour blood glucose monitoring to observe the treatment effect, determine the optimal program. Regular blood tests and biochemical tests are performed.
2.8眼底检查  2.8 fundus examination
检测时间： 给药前及给药后观察期结束时。  Detection time: before administration and at the end of the observation period after administration.
测定方法： 恒河猴经盐酸***麻醉（肌肉注射， 15 mg/kg)， 美多丽散 瞳 （滴眼， 1滴 /眼）， 以双目间接检眼镜进行检查， 所有影像学评估均在全 麻状态下进行 （动物麻醉方法参考恒河猴手术麻醉 SOP)。 用眼底镜观察视 神经***的形状、 大小、 色泽， 边缘是否清晰； 黄斑部有无水肿、 出血、 渗 出及色素紊乱； 视网膜有无水肿、 渗出、 出血、 剥离及新生血管等。  Determination method: Rhesus monkey was anesthetized with ketamine hydrochloride (intramuscular injection, 15 mg/kg), Medea dilated (eye drops, 1 drop/eye), and examined by binocular indirect ophthalmoscopy. All imaging evaluations were performed. Under general anesthesia (animal anesthesia method reference rhesus surgery anesthesia SOP). Use the ophthalmoscope to observe the shape, size, color and sharpness of the optic nerve head. There is no edema, hemorrhage, exudation and pigmentation disorder in the macula. There are no edema, exudation, hemorrhage, exfoliation and new blood vessels in the retina.
2.9 肾 B超和肾血管造影  2.9 Kidney B ultrasound and renal angiography
彩超观察肾脏形态和血流灌注分析血流动力学指标和血流灌注系数。 造 影剂 SonoVue (Bracco SpA, Milan, Italy) 通过大隐静脉注射 lml (5mg/ml)， 然后以 2ml生理盐水冲洗。 之后再做左肾， 中间间隔不小于 10分钟， 中途 加以 flash***。  Color Doppler ultrasound observation of renal morphology and blood perfusion analysis of hemodynamic parameters and blood perfusion coefficient. The agent SonoVue (Bracco SpA, Milan, Italy) was injected with lml (5 mg/ml) through the saphenous vein and then rinsed with 2 ml of saline. Then do the left kidney again, with an interval of no less than 10 minutes in the middle, and flash blasting midway.
2. 10 B超引导下肾穿剌活检术  2. 10 B-guided renal biopsy
恒河猴肌注***和安泰麻醉后去俯卧位， 备皮消毒后铺巾， 穿剌点选 择左肾上极或下极， 避开血管和肾盂。 BARD肾穿剌针穿剌抽吸术， 分别放 入 10% 多聚甲醛和 RNA latter的 EP管保存。所有固定组织进行 HE、Masson、 PAS常规染色， 免疫荧光染色 （CTGF、 eNOS、 IL-Ιβ, IV型胶原、 纤连蛋 白 FN、 TGF-β, MCP- TNF-a、 CD68) 于显微镜下观察病理组织学变化。 RNA latter保存组织提取 RNA逆转录后用荧光定量 PCR检测 smad2、smad3、 smad4、 smad7、 actin、 NF-Kb基因表达。  Rhesus monkeys were injected with ketamine and Antai anesthesia and then placed in a prone position. After skin preparation, the towel was placed and the upper or lower pole of the left kidney was selected to avoid the blood vessels and renal pelvis. BARD renal percutaneous needle aspiration was performed by placing a 10% paraformaldehyde and RNA latter EP tube. All fixed tissues were routinely stained with HE, Masson, and PAS, and immunofluorescence staining (CTGF, eNOS, IL-Ιβ, type IV collagen, fibronectin FN, TGF-β, MCP-TNF-a, CD68) was observed under a microscope. Histological changes. RNA latter tissue extraction RNA was reverse transcribed and the expression of smad2, smad3, smad4, smad7, actin and NF-Kb genes was detected by real-time PCR.
2.11液相芯片检测  2.11 liquid phase chip detection
取不同病程的血浆，检测因子（IL-1、 IL-6、 MCP-1、 TNF-a、 IL-17、 IL-18) 2.12尿液检测  Take plasma from different stages of disease, detection factors (IL-1, IL-6, MCP-1, TNF-a, IL-17, IL-18) 2.12 Urine test
恒河猴放入代谢笼中， 收集晨尿和 24小时尿， 检测尿常规， 尿白蛋白和 肌酐。  Rhesus monkeys were placed in metabolic cages to collect morning urine and 24-hour urine to detect urine routine, urinary albumin and creatinine.
3实验结果  3 experimental results
3.1血糖控制情况  3.1 blood sugar control situation
如图 1所示， 各实验组的血糖均保持在预先规定的范围内。  As shown in Fig. 1, the blood glucose of each experimental group was kept within a predetermined range.
3.2肾功能检测  3.2 renal function test
如图 2所示， 尿液检查示实验组 B和 C在第 36个月开始出现了微量蛋 白尿 （MAU)， 并且， 随着病程推移有增加趋势。 血生化显示 42个月 ABC 三组血肌酐和尿素氮和正常组相比有明显差异。 3.3肾脏 B超检查的血流分布和频谱 As shown in Figure 2, urine tests showed that experimental groups B and C began to show microalbuminuria (MAU) at the 36th month, and there was an increasing trend as the disease progressed. Blood biochemistry showed a significant difference in serum creatinine and urea nitrogen between the 42-month ABC group and the normal group. 3.3 Blood flow distribution and spectrum of renal B-ultrasound
如图 5所示， 早期肾血管床阻力增加， 从而使肾内血流发生改变， 随着 病情加重， 动脉硬化的血管壁弹性减低， 管腔狭窄， 外周血管阻力增高， 肾 血流灌注减少， 使肾血流呈低流， 高阻状态， 其中， B组和 C组的的病理改 变非常明显， C组最为明显。  As shown in Fig. 5, the early renal vascular bed resistance increases, so that the blood flow in the kidney changes. As the condition worsens, the elasticity of the arteriosclerotic blood vessel wall decreases, the lumen narrows, the peripheral vascular resistance increases, and the renal blood flow perfusion decreases. The renal blood flow was in a low-flow, high-resistance state. Among them, the pathological changes of group B and group C were very obvious, and group C was the most obvious.
3.4肾血流动力学参数和血流灌注系数  3.4 renal hemodynamic parameters and blood perfusion coefficient
如图 4所示， 与对照组相比， ABC三组血流动力学参数阻力指数 （RI) 增加， 舒张末期最低流速 （EDV) 降低， 血流灌注系数曲线下面积 （AUC) 降低， C组与对照组的差异最大， B组其次， A组差异最小。  As shown in Figure 4, compared with the control group, the ABC three groups of hemodynamic parameters resistance index (RI) increased, the end-diastolic minimum flow rate (EDV) decreased, the area under the perfusion coefficient curve (AUC) decreased, group C The difference was the largest with the control group, followed by the B group, and the A group had the smallest difference.
3.5肾组织病理改变  3.5 renal tissue pathological changes
如图 6所示， 与正常组相比 A组无明显改变， BC组炎性细胞侵润、 系 膜细胞增生、 基底膜增厚、 胶原和糖原的沉积, C组更加显著。  As shown in Fig. 6, there was no significant change in group A compared with the normal group. Inflammatory cell invasion, mesangial cell proliferation, basement membrane thickening, collagen and glycogen deposition were more prominent in group C.
3.6肾组织免疫荧光  3.6 renal tissue immunofluorescence
如图 7所示， 肾组织免疫荧光显示实验组 B C与正常组和实验 A组相比 CTGF、 eNOS、 IL-1、 IV型胶原、 纤连蛋白 (FN)、 TGF-β表达增加， 并且在 实验组 C更加显著， 同时实验组 C还出现 MCP1和 TNF-a表达。  As shown in Figure 7, immunofluorescence of renal tissue showed that the expression of CTGF, eNOS, IL-1, type IV collagen, fibronectin (FN), and TGF-β was increased in the experimental group BC compared with the normal group and the experimental group A, and Experimental group C was more significant, and MCP1 and TNF-a expression also appeared in experimental group C.
3.7致纤维化基因改变  3.7 Fibrosis gene change
如图 3所示， Real-time PCR显示实验组 B和 C在第 24个月开始出现 Smad2和 Smad3 mRNA表达，实验组 C更显著，并且在第 42个月出现 Smad7 和 NF-Kb mRNA表达增加。  As shown in Figure 3, Real-time PCR showed that Smad2 and Smad3 mRNA expression began to appear in the experimental group B and C at the 24th month, the experimental group C was more significant, and the Smad7 and NF-Kb mRNA expression increased at the 42nd month. .
3.8眼科检查  3.8 eye examination
除一只 （05539)有双眼白内障未能检查外， 其余双外眼无异常， 结膜无 充血水肿， 角膜透明， 前房无异常， 瞳孔中度散大（均已滴用散瞳剂）。 晶体 透明， 玻璃体无浑浊。 眼底： 双眼豹纹状眼底， 视神经***色淡红，  Except for one (05539) binocular cataract failed to be examined, the other double external eyes were normal, the conjunctiva was free of congestion and edema, the cornea was transparent, the anterior chamber was not abnormal, and the pupil was moderately dilated (both of which had been drenched with a mydriatic agent). The crystal is transparent and the vitreous is turbid. Fundus: Eyes of the leopard-like fundus, the optic nerve head is reddish,
C/D=0.3-0.7,A:V=1:2,可见筛孔和血管波动。 未见出血、 渗出、 微血管瘤及水 肿， 黄斑中心凹反光清。 综上， 实验组 A未出现糖尿病肾病的相关症状， 实验组 B和 C出现了 糖尿病肾病的典型临床症状： C/D = 0.3-0.7, A: V = 1: 2, seeing the mesh and blood vessel fluctuations. No bleeding, exudation, microangioma and edema were observed, and the fovea was concavely reflected. In summary, there were no symptoms associated with diabetic nephropathy in experimental group A. Typical clinical symptoms of diabetic nephropathy occurred in experimental groups B and C:
1、 实验组 B和 C在第 36个月开始出现了微量蛋白尿 （MAU) 并且随 着病程推移有增加趋势；  1. Experimental group B and C began to appear microalbuminuria (MAU) at the 36th month and increased with the course of the disease;
2、 肾组织 HE、 Masson、 PAS染色显示： 实验组 A无病变， 实验组 B和 C肾脏从第 24个月开始出现炎性细胞侵润、系膜细胞增生、基底膜增厚、胶 原和糖原的沉积， 并且发现实验组 C在第 42个月出现局部肾小球硬化。 同 时期肾组织免疫荧光显示实验组 B C于正常组和实验 A组相比 CTGF、 eNOS、 IL-1、 IV型胶原、 纤连蛋白 (FN)、 TGF-β表达增加， 并且在实验组 C 更加显著， 同时实验组 C还出现 MCP1和 TNFa表达； 2. HE, Masson, and PAS staining of renal tissue showed that there was no lesion in experimental group A. Inflammatory cell infiltration, mesangial cell hyperplasia, basement membrane thickening, collagen and sugar appeared in the experimental group B and C kidneys from the 24th month. The original deposition, and found that experimental group C showed local glomerular sclerosis at the 42nd month. Simultaneous renal tissue immunofluorescence showed that the experimental group BC was compared with CTGF in the normal group and the experimental group A. The expression of eNOS, IL-1, type IV collagen, fibronectin (FN) and TGF-β was increased, and it was more significant in experimental group C. At the same time, MCP1 and TNFa expression also appeared in experimental group C;
3、 Real-time PCR显示： 实验组 B和 C在第 24个月开始出现 Smad2和 Smad3 mRNA表达，实验组 C更显著，并且在第 42个月出现 Smad7和 NF-Kb mRNA表达增加；  3. Real-time PCR showed that Smad2 and Smad3 mRNA expression appeared in the experimental group B and C at the 24th month, the experimental group C was more significant, and the Smad7 and NF-Kb mRNA expression increased at the 42nd month;
4、 肾血管造影显示， 实验组 B和 C的血流灌注系数 AUC 和流动力学 参数阻力指数 RI显著增加， 舒张末期最低流速 EDV降低（P<0.05 )， 动脉硬 化的血管壁弹性减低， 管腔狭窄， 外周血管阻力增高， 肾血流灌注减少， 使 肾血流呈低流、 高阻状态； 本发明通过长期控制糖尿病恒河猴的血糖浓度范围为 10-20mmol/L， 使 得糖尿病恒河猴出现肾组织炎性细胞侵润、 系膜细胞增生、 基底膜增厚、 胶 原和糖原的沉积、 肾脏损伤、 微量蛋白尿等糖尿病肾病的典型临床症状， 说 明本发明恒河猴糖尿病肾病造模成功。  4. Renal angiography showed that the blood perfusion coefficient AUC and the flow dynamics resistance index RI of the experimental group B and C increased significantly, the minimum flow velocity EDV decreased at the end of diastole (P<0.05), the elasticity of the arteriosclerotic vessel wall decreased, and the lumen Stenosis, peripheral vascular resistance is increased, renal blood perfusion is reduced, and renal blood flow is in a low-flow, high-resistance state. The present invention allows diabetic rhesus monkeys to have a blood glucose concentration ranging from 10 to 20 mmol/L for a long time. Typical clinical symptoms of diabetic nephropathy such as renal cell inflammatory cell infiltration, mesangial cell hyperplasia, basement membrane thickening, collagen and glycogen deposition, kidney damage, microalbuminuria, etc., indicating that the rhesus monkey diabetic nephropathy model of the present invention success.
本发明通过长期控制糖尿病恒河猴的血糖浓度范围为 10-20mmol/L， 结 合长期高脂肪摄入的方式， 使得糖尿病恒河猴的各种糖尿病肾病典型症状比 单独控制血糖的方式更为显著， 说明二者联合处理的方式更优。 实施例 2 用本发明模型筛选治疗糖尿病肾病的药物  The long-term control of diabetic rhesus monkey blood glucose concentration range of 10-20mmol / L, combined with long-term high fat intake, the typical symptoms of diabetic rhesus monkey diabetes is more significant than the method of controlling blood glucose alone , indicating that the two methods of joint processing are better. Example 2 Screening for the treatment of diabetic nephropathy using the model of the present invention
a、 按照实施例 1方法建立的恒河猴糖尿病肾病模型；  a. Rhesus monkey diabetic nephropathy model established according to the method of Example 1;
b、 将候选药物施用于动物模型；  b. administering the candidate drug to the animal model;
c、观察候选药物对代谢综合征的各种指标的影响情况，评价潜在的治疗 糖尿病肾病疾病的药物。 综上， 本发明造模方法通过长期控制血糖的方式， 诱导恒河猴出现糖尿 病肾病的临床症状， 另外， 长期血糖控制与高脂摄入联合的方式也可以诱导 恒河猴出现糖尿病肾病的临床症状， 并且症状更为显著， 说明本发明两种方 式均可以有效建立恒河猴糖尿病肾病模型。 工业应用性  c. Observe the effects of drug candidates on various indicators of metabolic syndrome, and evaluate potential drugs for the treatment of diabetic nephropathy. In summary, the modeling method of the present invention induces the clinical symptoms of diabetic nephropathy in rhesus monkeys by long-term control of blood glucose. In addition, the combination of long-term glycemic control and high-fat intake can also induce the clinical manifestation of diabetic nephropathy in rhesus monkeys. Symptoms, and the symptoms are more significant, indicating that the two methods of the present invention can effectively establish a rhesus monkey diabetic nephropathy model. Industrial applicability
本发明造模方法有效建立恒河猴糖尿病肾病模型， 可用于恒河猴糖尿病  The modeling method of the invention effectively establishes a rhesus monkey diabetic nephropathy model, which can be used for rhesus monkey diabetes

Claims

权 利 要 求 书 claims
1、一种恒河猴糖尿病肾病模型的制备方法，其特征在于：包括如下步骤:1. A method for preparing a rhesus monkey diabetic nephropathy model, which is characterized by comprising the following steps:
( 1 ) 将剂量为 80~100mg/Kg的链脲菌素施用于恒河猴； (1) Administer streptozotocin at a dose of 80~100mg/Kg to rhesus monkeys;
( 2) 血糖浓度升至 l l.lmmol/1后， 餐前施用胰岛素， 使恒河猴血糖浓 度为 10-20mmol/L。 (2) After the blood sugar concentration rises to l l.lmmol/1, administer insulin before meals to make the blood sugar concentration of the rhesus monkey 10-20mmol/L.
2、一种恒河猴糖尿病肾病模型的制备方法，其特征在于：包括如下步骤: 2. A method for preparing a rhesus monkey diabetic nephropathy model, which is characterized by comprising the following steps:
( 1 ) 将剂量为 80~100mg/Kg的链脲菌素施用于恒河猴； (1) Administer streptozotocin at a dose of 80~100mg/Kg to rhesus monkeys;
( 2) 血糖浓度升高至 ll.lmmol/1后， 饲喂高脂饲料， 每天饲喂 2次， 饲喂量为 0.3-0.4kg/次.只， 并在餐前施用胰岛素， 使恒河猴血糖浓度为 10-20mmol/L, 所述饲料包含如下重量配比的原料： 标准猴饲料 80份、 动物 油脂 15份、 糖 5份。 (2) After the blood sugar concentration rises to 11.1 mmol/1, feed high-fat feed twice a day, the feeding amount is 0.3-0.4kg/time, and administer insulin before meals to make the Ganges The monkey blood sugar concentration is 10-20 mmol/L, and the feed contains raw materials in the following weight ratio: 80 parts of standard monkey feed, 15 parts of animal fat, and 5 parts of sugar.
3、 根据权利要求 1或 2所述的制备方法， 其特征在于： 步骤 （1 ) 中， 所述的施用方式是静脉注射。 3. The preparation method according to claim 1 or 2, characterized in that: in step (1), the administration method is intravenous injection.
4、 根据权利要求 1或 2所述的制备方法， 其特征在于： 步骤 （2) 中， 所述的施用方式是皮下注射。 4. The preparation method according to claim 1 or 2, characterized in that: in step (2), the administration method is subcutaneous injection.
5、 根据权利要求 2所述的制备方法， 其特征在于： 步骤 （2) 中， 所述 动物油脂是猪油； 所述糖是蔗糖。 5. The preparation method according to claim 2, characterized in that: in step (2), the animal fat is lard; and the sugar is sucrose.
6、一种建立恒河猴糖尿病肾病模型的饲料， 其特征在于： 它包含如下重 量配比的原料： 标准猴饲料 80份、 动物油脂 15份、 糖 5份。 6. A feed for establishing a rhesus monkey diabetic nephropathy model, characterized in that: it contains raw materials in the following weight ratio: 80 parts of standard monkey feed, 15 parts of animal fat, and 5 parts of sugar.
7、根据权利要求 6所述的饲料， 其特征在于： 所述动物油脂是猪油； 所 述糖是蔗糖。 7. The feed according to claim 6, characterized in that: the animal fat is lard; and the sugar is sucrose.
8、 权利要求 1~4任意一项所述方法制备的恒河猴糖尿病肾病模型。 8. The rhesus monkey diabetic nephropathy model prepared by the method of any one of claims 1 to 4.
9、权利要求 8所述恒河猴糖尿病肾病模型在筛选治疗恒河猴糖尿病肾病 的药物中的用途。 9. Use of the rhesus monkey diabetic nephropathy model described in claim 8 in screening drugs for treating rhesus monkey diabetic nephropathy.
10、 一种筛选治疗恒河猴糖尿病肾病模型的药物的方法， 其特征在于： 它包括如下步骤： 10. A method for screening drugs for treating rhesus monkey diabetic nephropathy model, characterized by: It includes the following steps:
a、 按照权利要求 1~4任意一项所述方法， 建立恒河猴糖尿病肾病模型； b、 将候选药物施用于动物模型； a. Establish a rhesus monkey diabetic nephropathy model according to the method described in any one of claims 1 to 4; b. Apply the candidate drug to the animal model;
c、 用动物模型评价潜在的治疗恒河猴糖尿病肾病的药物。 c. Use animal models to evaluate potential drugs for the treatment of diabetic nephropathy in rhesus monkeys.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9233204B2 (en) 2014-01-31 2016-01-12 Aseko, Inc. Insulin management
US9483619B2 (en) 2012-09-11 2016-11-01 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
US9486580B2 (en) 2014-01-31 2016-11-08 Aseko, Inc. Insulin management
US9886556B2 (en) 2015-08-20 2018-02-06 Aseko, Inc. Diabetes management therapy advisor
US9892234B2 (en) 2014-10-27 2018-02-13 Aseko, Inc. Subcutaneous outpatient management
US9897565B1 (en) 2012-09-11 2018-02-20 Aseko, Inc. System and method for optimizing insulin dosages for diabetic subjects
US11081226B2 (en) 2014-10-27 2021-08-03 Aseko, Inc. Method and controller for administering recommended insulin dosages to a patient

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103432572B (en) * 2013-06-21 2018-09-18 四川大学华西医院 A kind of preparation method of rhesus monkey diabetic nephropathy model
CN105726568B (en) * 2016-04-11 2019-01-29 陕西科技大学 A kind of construction method of diabetic nephropathy animal model
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005009119A1 (en) * 2003-07-23 2005-02-03 Mcw Research Foundation, Inc. Rat model of diabetic nephropathy
CN101766149A (en) * 2010-01-29 2010-07-07 东南大学 Preparation method of 2-type diabetic nephropathy model
CN103432572A (en) * 2013-06-21 2013-12-11 四川大学华西医院 Rhesus monkey diabetic nephropathy model preparation method

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1785433A (en) * 2004-12-09 2006-06-14 上海凯曼生物科技有限公司 Preparation method of Type II diabetes animal model
CN101095958A (en) * 2007-07-04 2008-01-02 山西医科大学第一医院 Method for preparing 2 type diabetes rat model
CN101637405A (en) * 2008-07-31 2010-02-03 四川大学华西医院 Method for preparing diabetic animal model
CN101804211B (en) * 2009-02-13 2012-12-05 四川大学华西医院 Establishment of rhesus autoimmune 1 type diabetes model

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005009119A1 (en) * 2003-07-23 2005-02-03 Mcw Research Foundation, Inc. Rat model of diabetic nephropathy
CN101766149A (en) * 2010-01-29 2010-07-07 东南大学 Preparation method of 2-type diabetic nephropathy model
CN103432572A (en) * 2013-06-21 2013-12-11 四川大学华西医院 Rhesus monkey diabetic nephropathy model preparation method

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US10629294B2 (en) 2012-09-11 2020-04-21 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
US10410740B2 (en) 2012-09-11 2019-09-10 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
US9773096B2 (en) 2012-09-11 2017-09-26 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
US9811638B2 (en) 2012-09-11 2017-11-07 Aseko, Inc. Means and method for improved glycemic control for diabetic patients
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US11468987B2 (en) 2014-01-31 2022-10-11 Aseko, Inc. Insulin management
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US9898585B2 (en) 2014-01-31 2018-02-20 Aseko, Inc. Method and system for insulin management
US11621074B2 (en) 2014-01-31 2023-04-04 Aseko, Inc. Insulin management
US9710611B2 (en) 2014-01-31 2017-07-18 Aseko, Inc. Insulin management
US10453568B2 (en) 2014-01-31 2019-10-22 Aseko, Inc. Method for managing administration of insulin
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US11783946B2 (en) 2014-01-31 2023-10-10 Aseko, Inc. Method and system for insulin bolus management
US10811133B2 (en) 2014-01-31 2020-10-20 Aseko, Inc. System for administering insulin boluses to a patient
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