WO2019100602A1 - 一种溃疡性结肠炎动物模型的构建方法和应用 - Google Patents

一种溃疡性结肠炎动物模型的构建方法和应用 Download PDF

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WO2019100602A1
WO2019100602A1 PCT/CN2018/075705 CN2018075705W WO2019100602A1 WO 2019100602 A1 WO2019100602 A1 WO 2019100602A1 CN 2018075705 W CN2018075705 W CN 2018075705W WO 2019100602 A1 WO2019100602 A1 WO 2019100602A1
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dog
group
drug
control group
colon
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French (fr)
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赵昱
耿福能
徐在品
沈咏梅
刘衡
赵海荣
陈丽
张成桂
巫秀美
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大理大学
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    • 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
    • A01K67/02—Breeding vertebrates
    • 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
    • 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; 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
    • 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

Definitions

  • the invention relates to the field of experimental animal models, in particular to a method for constructing an animal model of ulcerative colitis and an application thereof.
  • Ulcerative Colitis is a chronic non-specific ulcerative colitis characterized by ulceration and chronic inflammation, which is a type of Inflammatory Bowel Disease (IBD).
  • IBD Inflammatory Bowel Disease
  • the etiology of UC is unknown.
  • the main clinical manifestations of gastrointestinal symptoms such as diarrhea, abdominal pain and mucus pus and bloody stools; the lesions involve the rectum and sigmoid colon, and can spread throughout the colon, mainly invading the mucosa and submucosa of the large intestine, showing a staged and diffuse distribution.
  • the UC attack period and remission period are repeated alternately, the course of disease is long, and the cure is difficult.
  • the recurrence rate after cure is high, and it has a certain correlation with the onset of colon cancer. It is poor after the treatment, so it is listed as a modern refractory disease by the World Health Organization. First, it has received widespread attention from the medical community. Establishing an animal model suitable for UC research with similar clinical symptoms, good reproducibility and simple operation not only provides a basis for studying disease patterns, but also provides suitable conditions for the development of new drugs for the treatment of UC.
  • UC disease research/pharmacodynamic evaluation experimental animal model is to induce intestinal ulcer lesions and inflammatory reactions by using chemical stimulation method, immunological method, and composite method for healthy experimental animals.
  • the method of chemical stimulation of acetic acid destroys the intestinal mucosal barrier structure of experimental animals, which in turn initiates inflammation, causes inflammatory mediators, pathological changes of the lesions, and intestinal inflammatory properties caused by abnormal arachidonic acid metabolism in humans.
  • Similar, simple preparation, low cost, high success rate, good repeatability, obvious symptoms can occur in a short period of time, and the UC induction method is relatively simple.
  • mice are also useful; however, rodents of rabbits and rodents are different in feeding habits, and their physiology and toxicology of the digestive system are different. The response to the disease is very different from that of humans and therefore cannot be directly promoted to the human body.
  • the UC animal model established by dogs is an adult German Shepherd or Beagle dog. These dogs are susceptible to external environmental factors during breeding. The large size of adult dogs leads to the artificial induction of UC. The program is difficult to standardize, which makes it difficult to effectively control the confounding factors associated with UC. Moreover, the individual's resistance is poor, and it is easy to induce other diseases or even death during the modeling process. Furthermore, in order to avoid other diseases or deaths in the German Shepherd or Beagle during the modeling process, the chemical agents that induce UC in the literature method have lower doses, and thus the degree of inflammation caused is different from that of human clinical manifestations. Large, unable to objectively and correctly reflect the disease process or evaluate the therapeutic effect of the drug.
  • the present invention provides a method of constructing an animal model more capable of reflecting human ulcerative colitis and an application thereof.
  • the present invention adopts the following technical solutions:
  • a method for constructing an animal model of ulcerative colitis comprising the steps of:
  • the Chinese garden dog is an adult dog.
  • the Chinese garden dog is 1 to 2 years old.
  • the Chinese garden dog has a body weight of 6.0 to 12.0 Kg.
  • the enema method is to inject the acetic acid solution into the colon of the Chinese rural dog, retaining for more than 10 s, and then rinsing the colon with physiological saline.
  • the acetic acid solution has a mass concentration of 5 to 10%, and more preferably the acetic acid solution is used in an amount of 1 mL to 2 mL per kg of body weight.
  • the gavage agent is a MgSO 4 solution
  • the concentration of the MgSO 4 solution is 5 to 10%, and the amount is 5 to 10 mL/kg.
  • the method for anesthesia of the Chinese rural dog is: intramuscular injection of Chinese rural dog by Shu Ningning.
  • the animal model of ulcerative colitis constructed by the above method of the present invention can be used for screening UC therapeutic drugs and evaluating the efficacy of UC therapeutic drugs.
  • the invention also provides a method for screening or evaluating a UC therapeutic drug, comprising the steps of:
  • test drug group a.
  • test drug group and the saline control group use the above-mentioned method to construct an animal model of ulcerative colitis
  • the normal control group is a healthy Chinese without modeling.
  • Pastoral dogs observe and record the physiological basic data and colon pathology of the test drug group, the saline control group and the normal control group;
  • the drug group to be tested is fed with the test drug, the saline control group and the normal control group are fed with physiological saline; the physiological basic data and colon of the test drug group, the saline control group and the normal control group are observed and recorded.
  • Pathological condition
  • step c Analyze the data recorded in step a and step b and the score of colon pathology, and evaluate the therapeutic effect of the drug to be tested; if the physiological basis of the drug group to be tested is closer to the corresponding measurement value of the normal control group than the physiological basic data of the saline control group Or, the drug group to be tested has a lower score of colon pathology than the saline control group, indicating that the drug to be tested has a therapeutic effect on UC.
  • the scoring standard of the colon pathological photograph is:
  • Grade 0 the mucous membrane is pale, the vascular network is clear, and it is branched; there is no redness and congestion under the mucosa, and the surface mucosa is normal;
  • Grade 1 The mucous membrane is still smooth, but the small area is congested, red and swollen, and the refraction is enhanced;
  • Grade 2 mucosal congestion, edema, granular, mucoid fragility, and easy bleeding;
  • Level 3 obvious mucosal congestion, edema, rough, a small amount of spontaneous bleeding or contact bleeding; more inflammatory secretions, multiple erosions and small area ulcer formation;
  • Grade 4 large area of mucous membrane congestion, edema, rough, obvious spontaneous bleeding and contact bleeding; multiple punctate erosion and large area ulcer formation;
  • the physiological basic data of the animal includes, but is not limited to, one or more of body temperature, body weight, stool shape, occult blood test, blood routine, blood biochemical index, and C-reactive protein.
  • the present invention has the following advantages:
  • the invention adopts Chinese garden dog (Canis lupus familiaris) as a model animal, and the animal has a wide source, and the dog is mainly a hybrid dog, which is less affected by external environmental factors, and the individual resistance is better, due to the dog's digestive system and human beings. More similar, the experimental results can be directly applied to the human body.
  • the acetic acid-induced canine ulcerative colitis model provided by the invention is easy to operate, has stable model, is easy to repeat, and has low cost, and provides more animal model selection for drug evaluation and therapeutic evaluation for the development of UC therapeutic drugs.
  • Figure 1 is a colonoscopy chart before and after modeling of 7% acetic acid 10mL;
  • Figure 2 is a colonoscopy chart before and after modeling 10% acetic acid 15mL;
  • Figure 3 is a colonoscopy chart before and after modeling of 5% acetic acid 20mL;
  • FIGs. 1 to 3 A, the intestine before the molding; B, the intestine on the first day after modeling; C, the intestine on the fourth day after modeling; D, the intestine on the seventh day after modeling.
  • Figure 4 is a corresponding picture of the colonoscopy score of acute ulcerative colitis in acetic acid-induced dogs.
  • the invention adopts Chinese garden dog as a model animal, and constructs an animal model of ulcerative colitis with acetic acid-induced canine ulcerative colitis. Specifically, the following steps are included:
  • the experimental Chinese dog used in the present invention is preferably an adult healthy Chinese garden dog.
  • a Chinese garden dog of 1 to 2 years old is preferably used, and the Chinese garden dog preferably has a body weight of 6.0 to 12.0 Kg.
  • the enema method is to inject the acetic acid solution into the colon of the Chinese rural dog, retaining for more than 10 s, and then rinsing the colon with physiological saline.
  • the specific enema operation is preferably: after anesthetizing the Chinese garden dog, the catheter is inserted into the colon 20cm from the anus of the Chinese garden dog, the acetic acid solution is slowly injected, the acetic acid enema is kept for more than 10s, and the Chinese garden dog adopts the position of the head low tail height. Wash the colon with saline.
  • a conventional animal anesthesia method in the art can be employed for the anesthesia method of the Chinese rural dog.
  • acetic acid is used to cause ulceration in the intestinal tract of an animal, which in turn causes inflammation.
  • the acetic acid solution used in the present invention has a mass concentration of 5 to 10%, more preferably 7 to 9%.
  • the amount of the acetic acid solution was appropriately adjusted according to the concentration of acetic acid.
  • the amount of the acetic acid solution used in the present invention is preferably 1 to 2 mL/kg of Chinese Garden Dog, and more preferably 1.3 to 1.7 mL/kg.
  • the time for enema of acetic acid in the present invention is 10 s or more, preferably 20 to 40 s. After enema, rinse with saline.
  • the amount of physiological saline to be used in the present invention is not particularly limited, but is preferably 20 to 100 mL, and more preferably 50 to 60 mL.
  • the model After the animal to be modeled is awakened, combined with the physical signs of the animal after modeling and colonoscopy, if symptoms such as blood in the stool, erosion of the intestinal mucosa, and congestion are present, the model is confirmed to be successful.
  • the above animal model of ulcerative colitis obtained by the present invention can be used for screening UC therapeutic drugs and evaluating the efficacy of UC therapeutic drugs.
  • the invention also provides a method for screening and evaluating UC therapeutic drugs, comprising the following steps:
  • test drug group a saline control group, and a normal control group
  • test drug group and the saline control group use the animal model of ulcerative colitis constructed by the method of the present invention, and the normal control group does not perform modeling.
  • Healthy Chinese tourism dogs observe and record the physiological basic data and colon pathology of the test drug group, the saline control group and the normal control group;
  • the drug group to be tested is fed with the test drug, the saline control group and the normal control group are fed with physiological saline, and the number of treatment days is determined according to the clinical drug use period of the drug to be tested, and the drug group to be tested and the physiological saline control are observed and recorded.
  • step c analyzing the data recorded in step a and step b and the pathological photograph of the colon, and evaluating the therapeutic effect of the drug, if the physiological basis data of the test group is closer to the corresponding measurement value of the normal control group, or The score of the colonic pathological photograph of the drug group to be tested was lower than that of the saline control group, indicating that the drug to be tested has the effect of treating UC.
  • the colon pathology photograph formed by the animal model of ulcerative colitis used in the present invention is preferably according to the following scoring criteria:
  • Grade 0 the mucous membrane is pale, the vascular network is clear, and it is branched; there is no redness and congestion under the mucosa, and the surface mucosa is normal;
  • Grade 1 The mucous membrane is still smooth, but the small area is congested, red and swollen, and the refraction is enhanced;
  • Grade 2 mucosal congestion, edema, granular, mucoid fragility, and easy bleeding;
  • Level 3 obvious mucosal congestion, edema, rough, a small amount of spontaneous bleeding or contact bleeding; more inflammatory secretions, multiple erosions and small area ulcer formation;
  • Grade 4 large area of mucosa congestion, edema, rough, obvious spontaneous bleeding and contact bleeding; multiple punctate erosion and large area ulcer formation.
  • the physiological basic data of the animal includes, but is not limited to, body temperature, body weight, stool shape, occult blood test, blood routine, blood biochemical index, and C-reactive protein.
  • test equipment and test reagents are used in the specific examples of the present invention, but they are not to be construed as limiting the scope of the present invention.
  • Ultra-pure water machine (Aike Instrument; DZG-303A type);
  • Macro camera Nikon DIGITAI CAMERA D7100, macro lens: AF-SMicro 60/2.8GED;
  • Occult blood kit 20mL, 5mL, 1mL syringe, 2mm diameter polyethylene catheter, vacuum blood collection device, beaker, filter paper, weighing paper, animal dissection equipment, electronic platform scale, ziplock bag, garbage bag, conventional surgical instruments, HH.W21. 600 stainless steel electronic constant temperature water tank.
  • Glacial acetic acid (Tianjin Fuchen Chemical Reagent Co., Ltd., batch number: 20151020);
  • Formaldehyde (Tianjin Fuchen Chemical Reagent Factory, batch number: 20150408);
  • Disodium hydrogen phosphate (Tianjin Sailboat Chemical Reagent Technology Co., Ltd., batch number: 20131008);
  • Starch colon-soluble capsules Starch colon-soluble capsules, sulfasalazine colon-soluble capsules.
  • the experimental animals were randomly divided into 3 groups, 10 mL 7% acetic acid group, 15 mL 10% acetic acid group and 20 mL 5% acetic acid group; 10 mL MgSO 4 solution 5 mL/kg one day before model establishment, the intestinal tract was cleaned, and the water was fasted for 24 hours.
  • the mucosal surface of the colon in the three groups of dogs was smooth, intact and intact, and the color was normal and pink, showing no inflammatory reaction; the first day after modeling Visible rectal mucosal erosion, multiple superficial ulcers, with hyperemia and edema, diffuse distribution; microscopic examination on the 7th day after modeling showed that the colonic mucosa of all experimental animals was still congested and eroded to form a pseudomembrane; It was shown that all the experimental animals had acute ulcerative inflammation in the colon and the modeling was successful.
  • Acetic acid-induced acute UC model colonoscopy score Acetic acid-induced acute UC model colonoscopy score
  • the colonic lesions of the UC active period are diffuse congestive edema, and the vascular texture is blurred or disordered; as the disease progresses, the mucosa becomes rough, and the fine particles scattered inside are brittle, with natural bleeding or contact bleeding; when the lesion is heavier, There are multiple superficial ulcers, severe ulcers become large and deep, and mucus and blood seepage can occur in the intestinal lumen.
  • Canine parvovirus infection is also called canine viral enteritis or hemorrhagic enteritis. It is classified into enteric type and myocardial type. The clinical manifestations are characterized by acute hemorrhagic enteritis and non-suppurative myocarditis. Enteritis occurs in adult dogs and puppies older than 3 months. The virus invades the intestinal membrane, causing the dog's loss of appetite, vomiting, bloody stools and body temperature. Myocarditis usually occurs in puppies, and the virus invades the heart's muscles and usually kills within 72 hours.
  • Canine distemper virus infection manifests a variety of symptoms, which are related to viral virulence, environmental conditions, host age and immune status, and can be divided into five characteristics: hyperacute, acute, digestive tract, neurological and skin symptoms. Sexual type, once characteristic symptoms appear, it is extremely poor.
  • the rats were administered by fasting according to the group, 2 capsules/kg body weight, once in the morning and evening, and fed for 2 hours after drug administration for 7 days.
  • group A (model group) was fed with starch colonic capsules.
  • Group B was fed with Weikangling Capsule,
  • Group C was given with Osalazine capsule, and
  • Group D was given with sulfasalazine colonic capsule.
  • the endoscope was used for 1 day before and after the modeling and the third and seventh days after the administration, and the colon mucosa was examined for ulceration, ulceration, and hemorrhage.
  • the disease activity index (DAI, Disease Activity Index) was scored for each group. And record the dog's body temperature, weight and stool shape. The DAI scoring method is shown in Table 1.
  • normal feces formable feces
  • paste paste-like feces that do not stick to the anus
  • diarrhea liquefied feces sticking to the anus.
  • Weight loss (%) (weight at a certain point in time - weight before modeling) / weight before modeling ⁇ 100%
  • DAI (score of weight loss + score of stool trait + score of occult blood) / 3.
  • Fecal occult blood also known as occult blood refers to a small amount of bleeding in the digestive tract, red blood cells are destroyed by digestion, no abnormal changes in the appearance of the stool, and bleeding that cannot be confirmed by the naked eye or under the microscope.
  • the occult blood test kit was used to detect a small amount of large white porcelain plate with a cotton swab, o-toluidine solution was added dropwise, and hydrogen peroxide solution was added dropwise, and the occult blood level was scored according to the color development time and the degree of color development.
  • Blood cell analyzer analyzes the total number of white blood cells (WBC), total lymphocytes (LYM), number of monocytes (MONO), total neutrophils (GRAN), percentage of lymphocytes (LYM%), monocytes in the blood of experimental animals. Percentage (MON%), neutrophil percentage (GRA%), hemoglobin (HGB), hematocrit (HCT), total red blood cell count (RBC), mean red blood cell volume (MCV), mean red blood cell hemoglobin concentration (MCH), mean Hemoglobin concentration (MCHC), red blood cell distribution width (RDW%), total platelet count (PLT), mean platelet volume (MPV).
  • USHIOINCPoint-readerV was used to check the serum C-reactive protein content before modeling, 1, 4, and 7 days after modeling.
  • the sections were observed to observe the colonic mucosal epithelium: normal morphology of epithelial cells; infiltration and infiltration of goblet cells, crypt cells, and inflammatory cells.
  • the rectum is given acetic acid.
  • the dog is irritated and disturbed for about 10 minutes. He frequently looks back at the abdomen and beats the abdomen with his forelimbs. A small part of the dog's cage is bitten with a mouth, and the pain is called. 80% of the dogs start blood in the stool in 10-20 minutes. He was anxious and heavy, bent over his back, frequently blamed, and frequently walked in the dog cage, and he had a lot of blood, mostly blood.
  • group A model group
  • group A colonic mucosa congestion, edema, mucosal surface roughening, diffuse distribution, uniform size of fine particles, tissue becomes brittle, natural bleeding or contact bleeding, there are cavities Mucinous secretions; 7d after modeling, the colonic mucosa of the model group was still congested and eroded to form a pseudomembrane.
  • group B (weikangling capsule group) had colonic mucosal erosion and edema, and there were still ulcers
  • group C (Osalaqin capsule group) had less colonic mucosal edema, and the ulcer was smaller and less
  • group D (sulmonazopyridine enteric-coated capsule group) colonic mucosa visible many scattered red spots, needle-like or patchy, irregular shape, irregular arrangement.
  • occult blood test 1 day before model establishment, the occult blood test was negative (-), and the 1st and 4th occult blood tests were strongly positive (+++), and the stool was visible with blood, 7d after administration. Fecal occult blood test is still positive (++), but the bleeding is reduced.
  • the reference range for white blood cells is 6 ⁇ 10 9 /L - 17 ⁇ 10 9 /L.
  • the biochemical test index only changed for BUN. During the test period, BUN was within the normal range or slightly below the normal range. The remaining biochemical test indicators did not differ at each test point.
  • the CRP reference range is 1-10mg/L.
  • a UC model of a dog is prepared by perfusion of 10-20 mL of a 5-10% acetic acid solution, and the endoscopic results show that the experimental animal is successfully induced acute UC.
  • the clinical first-line drug Weikangling, Aishaqin, and sulfasalazine validation models were used to evaluate the efficacy of the drug.
  • Inflammatory cell infiltration etc., and after 7 days, the ulcer lesions have been reduced or even shed, inflammatory cell infiltration is also significantly reduced, a large number of fibroblasts proliferate, indicating that the experimental animals are self-healing; while the experimental dogs given UC clinical drugs in the same period After 3 days of administration, the ulcer was much reduced compared with the model group, and the infiltration of inflammatory cells was also greatly reduced, indicating that the inflammation of the colon was improved and the colonic mucosa was gradually repaired.

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Abstract

涉及一种实验动物模型，公开了一种溃疡性结肠炎动物模型的构建方法和应用。该溃疡性结肠炎动物模型以中华田园犬为模型动物，采用醋酸致犬溃疡性结肠炎，实验结果基本能直接推广应用于人体。造模方式易操作，模型稳定，易重复，成本低，为UC治疗药物的研发提供了更多成药性评价、疗效评价的动物模型选择。

Description

一种溃疡性结肠炎动物模型的构建方法和应用
本申请要求于2017年11月27日提交中国专利局、申请号为201711205144.0、发明名称为“一种溃疡性结肠炎动物模型的构建方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及实验动物模型领域，具体涉及一种溃疡性结肠炎动物模型的构建方法及其应用。
背景技术
溃疡性结肠炎(Ulcerative Colitis，UC)是一种以溃疡形成和慢性炎症为主要病理特点的慢性非特异性溃疡性结肠炎，其是炎症性肠病(Inflammatory Bowel Disease，IBD)的一种。UC病因不明，以腹泻、腹痛及黏液脓血便等消化道症状为主要临床表现；病变多累及直肠和乙状结肠，也可遍及整个结肠，主要侵犯大肠黏膜与黏膜下层，呈阶段性和弥漫性分布。UC发作期与缓解期反复交替、病程冗长，且治愈难度大，治愈后复发率高，并与结肠癌发病存在一定相关性，愈后较差，因此被世界卫生组织列为现代难治疾病之一，受到医学界的普遍重视。建立与临床症状相似、重复性好、操作简单的适用于UC研究的动物模型，不仅为研究疾病规律提供依据，还为开发研制治疗UC的新药提供合适的条件。
常用的UC疾病研究/药效评价实验动物模型为对健康的实验动物采用化学刺激法、免疫法、复合法等诱导产生肠道溃疡灶和炎症反应。其中采用醋酸化学刺激的方法使实验动物的肠道黏膜屏障结构破坏，继而启动炎症，引起炎症介质，其发病灶的病理变化及炎症病症与人类花生四烯酸代谢异常引起的肠道炎性性质相似，且制备简单、成本低、成功率高、重复性好，短期内即可出现明显症状，UC诱导方法相对简便。
目前UC动物模型中普遍采用的实验动物为大鼠，家兔、豚鼠和小鼠也偶有用之；然而，包括兔形目和啮齿目的啮齿类动物因食性不同，其消 化系统的生理、毒理和对疾病的反应与人类迥然不同，因此不能直接推广于人体。
目前以犬建立的UC动物模型其采用的实验犬为成年的德国牧羊犬或比格犬，此类犬在饲养期间易受外界环境因素影响，其成年犬的体型较大导致人工诱导UC的操作程序难以标准化、从而导致与UC相关的混杂因素难以得到有效控制。且其个体抵抗力较差、易在造模过程中诱发其它疾病甚或死亡。再者，为避免德国牧羊犬或比格犬在造模过程中产生其它疾病或死亡，文献方法中公开的诱导UC发生的化学试剂其剂量较低，因此引发的炎症程度与人类临床表现差异较大，无法客观、正确反应疾病过程或评价药物的治疗效果。
发明内容
为了解决上述技术问题，本发明提供了一种构建更能反映人类溃疡性结肠炎动物模型的方法及其应用。
为实现本发明的目的，本发明采用如下技术方案：
一种溃疡性结肠炎动物模型的构建方法，包括以下步骤：
中华田园犬灌胃清洗肠道，禁食不禁水24h；
麻醉中华田园犬，用醋酸溶液对麻醉后的中华田园犬进行灌肠，待中华田园犬自然苏醒，即得溃疡性结肠炎动物模型。
优选的，所述中华田园犬为成年犬。
进一步优选的，所述中华田园犬为1～2岁。
更进一步的，所述中华田园犬的体重为6.0～12.0Kg。
优选的，所述灌肠方式为将醋酸溶液灌入中华田园犬结肠内，保留10s以上，再用生理盐水冲洗结肠。
进一步优选的，所述醋酸溶液的质量浓度为5～10％，更优选醋酸溶液的用量为每公斤体重1mL～2mL。
优选的，所述灌胃用药剂为MgSO 4溶液；
更优选所述MgSO 4溶液的浓度为5～10％，用量为5～10mL/kg。
优选的，所述中华田园犬麻醉的方法为：用舒眠宁对中华田园犬进行肌肉注射。
本发明上述方法构建得到的溃疡性结肠炎动物模型能够用于筛选UC治疗药物，评价UC治疗药物的药效。
本发明还提供了一种筛选或评价UC治疗药物的方法，包括以下步骤：
a、设置待测药物组、生理盐水对照组和正常对照组，其中待测药物组和生理盐水对照组使用上述方法构建的溃疡性结肠炎动物模型，正常对照组为不进行造模的健康中华田园犬；观察并记录待测药物组、生理盐水对照组和正常对照组动物的生理基础数据和结肠病理情况；
b、待测药物组灌喂待测药物，生理盐水对照组和正常对照组灌喂生理盐水；观察并记录处理后待测药物组、生理盐水对照组和正常对照组动物的生理基础数据和结肠病理情况；
c、分析步骤a和步骤b记录的数据及结肠病理照片评分，对待测药物的治疗效果进行评价；若待测药物组较生理盐水对照组的生理基础数据更接近于正常对照组的相应测量值，或待测药物组较生理盐水对照组的结肠病理照片评分低，则说明待测药物具有治疗UC的作用。
本发明中，优选所述结肠病理照片的评分标准为：
0级：黏膜苍白，血管网清晰，呈分支状；黏膜下无红肿、充血，其表面黏膜正常；
1级：黏膜尚光滑，但小面积充血、红肿，折光增强；
2级：黏膜充血、水肿，呈颗粒状，黏膜脆性增加，接触易出血；
3级：黏膜明显充血、水肿、粗糙，少量自发性出血点或接触性出血；有较多炎性分泌物，多发性糜烂与小面积溃疡形成；
4级：黏膜大面积充血、水肿、粗糙，明显自发性出血和接触性出血；多发性点状糜烂与大面积溃疡形成；
若待测药物组结肠病理照片的评分低，则说明待测药物治疗UC的效果好。
本发明中，所述动物的生理基础数据包括但不限于体温、体重、大便形状、隐血检查、血常规、血液生化指标和C-反应蛋白中的一项或几项。
与现有技术相比，本发明具有以下优点：
本发明采用中华田园犬(Canis lupus familiaris)作为模型动物，动物来源广，且该犬主要是以杂交犬居多，受外界环境因素影响较小，个体抵 抗力较好，由于犬的消化系统与人类较为近似，实验结果基本能直接推广应用于人体。本发明提供的醋酸致犬溃疡性结肠炎模型的造模方式易操作，模型稳定，易重复，成本低，为UC治疗药物的研发提供了更多成药性评价、疗效评价的动物模型选择。
附图说明
图1为7％醋酸10mL造模前后肠镜检查图；
图2为10％醋酸15mL造模前后肠镜检查图；
图3为5％醋酸20mL造模前后肠镜检查图；
图1～3中：A、未造模前肠道；B、造模后第一天肠道；C、造模后第四天肠道；D、造模后第七天肠道。
图4为本发明醋酸致家犬急性溃疡性结肠炎肠镜检查评分等级对应图片。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明采用中华田园犬作为模型动物，用醋酸致犬溃疡性结肠炎构建溃疡性结肠炎动物模型。具体包括以下步骤：
中华田园犬灌胃清洗肠道，禁食不禁水24h；
麻醉中华田园犬，用醋酸溶液对麻醉后的中华田园犬进行灌肠，待中华田园犬自然苏醒，即得溃疡性结肠炎动物模型。
本发明所用实验中华田园犬优选为成年健康中华田园犬，在本发明具体实施例中优选采用年龄1～2岁的中华田园犬，中华田园犬体重优选为6.0～12.0Kg。
本发明对灌胃及灌肠的具体操作没有特殊限定，采用本领域技术人员的常规技术操作即可。优选所述灌肠方式为将醋酸溶液灌入中华田园犬结肠内，保留10s以上，再用生理盐水冲洗结肠。具体的灌肠操作优选为，将中华田园犬麻醉后，用导管自中华田园犬肛门处插入至结肠内20cm，缓慢注入醋酸溶液，保留醋酸灌肠10s以上，将中华田园犬采用头低尾高的体位用生理盐水冲洗结肠。
本发明中，对中华田园犬的麻醉方式可以采用本领域中的常规动物麻醉方法。在本发明具体实施例中，优选采用舒眠宁对中华田园犬进行肌肉注射。按照说明书记载的麻醉剂量进行操作。
本发明中采用醋酸使动物的肠道产生溃疡，继而引发炎症。本发明所用醋酸溶液的质量浓度为5～10％，更优选为7～9％。根据醋酸的浓度适度调整醋酸溶液的用量。本发明中醋酸溶液的用量优选1～2mL/kg中华田园犬，更优选为1.3～1.7mL/kg。
本发明用醋酸灌肠的时间为10s以上，优选为20～40s。灌肠后用生理盐水进行冲洗。本发明对生理盐水的用量没有特殊限定，优选为20～100mL，更优选为50～60mL。
待造模后的动物苏醒后，结合造模后动物的体征情况及肠镜检查，若出现便血、肠粘膜糜烂、充血等症状，则确认造模成功。
本发明得到的上述溃疡性结肠炎动物模型能够用于筛选UC治疗药物，评价UC治疗药物的药效。
本发明还提供了一种筛选与评价UC治疗药物的方法，包括以下步骤：
a、设置待测药物组、生理盐水对照组和正常对照组，其中待测药物组和生理盐水对照组使用本发明所述方法构建的溃疡性结肠炎动物模型，正常对照组为不进行造模的健康中华田园犬；观察并记录待测药物组、生理盐水对照组和正常对照组动物的生理基础数据和结肠病理情况；
b、待测药物组灌喂待测药物，生理盐水对照组和正常对照组灌喂生理盐水，根据待测药物的临床用药周期确定处理天数，观察并记录处理后待测药物组、生理盐水对照组和正常对照组动物的生理基础数据和结肠病理情况；
c、分析步骤a和步骤b记录的数据及结肠病理照片，对药物的治疗效果进行评价，若待测药物组较生理盐水对照组的生理基础数据更接近于正常对照组的相应测量值，或待测药物组较生理盐水对照组的结肠病理照片评分低，则说明待测药物具有治疗UC的作用。
本发明中所使用的溃疡性结肠炎动物模型所形成的结肠病理照片，优选按照以下评分标准：
0级：黏膜苍白，血管网清晰，呈分支状；黏膜下无红肿、充血，其表面黏膜正常；
1级：黏膜尚光滑，但小面积充血、红肿，折光增强；
2级：黏膜充血、水肿，呈颗粒状，黏膜脆性增加，接触易出血；
3级：黏膜明显充血、水肿、粗糙，少量自发性出血点或接触性出血；有较多炎性分泌物，多发性糜烂与小面积溃疡形成；
4级：黏膜大面积充血、水肿、粗糙，明显自发性出血和接触性出血；多发性点状糜烂与大面积溃疡形成。
若待测药物组结肠病理照片的评分低，则说明待测药物治疗UC的效果好。
本发明中，所述动物的生理基础数据包括但不限于体温、体重、大便形状、隐血检查、血常规、血液生化指标和C-反应蛋白。
根据生理基础数据及结肠评分的变化，评价UC药物的药效及治疗效果。
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例对本发明进行详细的说明。
如无特殊说明，本发明具体实施例中采用以下试验器材及试验试剂，但是不能把它们理解为对本发明保护范围的限定。
1.试验器材：
医学图像分析系统(重庆澳浦光电技术有限公司；BL-2000)；
高速冷冻离心机(湖南湘立科学仪器有限公司；TLC16M)；
超纯水机(艾柯仪器；DZG-303A型)；
分析天平(赛多利斯科学仪器有限公司；BSA-124S型)；
电子内窥镜(上海澳华光电内窥镜有限公司；VET-8015)；
全自动动物血液细胞分析仪(南京普朗医疗设备有限公司；XFA6030)；
微距相机：尼康DIGITAI CAMERA D7100型，微距镜头：AF-SMicro 60/2.8GED；
6号导尿管(杭州博菲医疗器械有限公司；批号：A16048)；
隐血试剂盒、20mL、5mL、1mL注射器、直径2mm聚乙烯导管、 真空采血器、烧杯、滤纸、称量纸、动物解剖器械、电子台秤、自封袋、垃圾袋、常规手术器械、HH.W21.600型不锈钢电子恒温水箱。
2.试验试剂：
冰醋酸(天津福晨化学试剂有限公司，批号：20151020)；
生理盐水(贵州天地药业责任有限公司，批号：A16021507)；
甲醛(天津福晨化学试剂厂，批号：20150408)；
磷酸氢二钠(天津市风船化学试剂科技有限公司，批号：20131008)；
磷酸二氢钠(天津市风船化学试剂科技有限公司，批号：20140318)；10％MgSO 4溶液；
胃康灵胶囊(四川好医生攀西药业责任有限公司，批号：20170113)；
奥沙拉秦胶囊(天津力生制药股份有限公司，批号：1608005)；
舒眠宁II注射液(南京大学小动物疾病研究室，批号：c20170225)；
淀粉结肠溶胶囊、柳氮磺吡啶结肠溶胶囊。
实施例1
一、试验动物
成年中华田园犬(Canis lupus familiaris)，年龄1～2岁，体重6.0～12.0Kg，由贵州大学动物医院提供，实验前饲喂2周，注射英特威四联苗2次(时间间隔2周)，临床检查健康者用于本试验。
二、建立实验模型
1.试验犬的分组及生理基础数据测定
健康的成年中华田园犬12只，随机分为3组，每组4只，雌雄各半。试验期间所有试验动物，单笼饲养，每日提供同等量的食物和饮用水，造模前一天，测定所有家犬的相关数据。
1.1犬常规体征的观察：每天给予犬适量的食物，并记录体重、体温、呼吸频率(次/分)、以及12h内(9:00至21:00)的大便次数、大便性状、精神状态。
1.2血常规检测及正常血浆样品收集：动物适应性饲养后，清晨进食前对其进行静脉采集抗凝血4mL、2mL各一管，测定造模动物的血液生化指标、C反应蛋白，共取2次，全血管经3500rmp、4℃离心10min后取出血清-80℃保存备用。
2.UC模型的建立
实验动物随机分为3组，分别为10mL7％醋酸组、15mL10％醋酸组和20mL5％醋酸组；造模前一天10％MgSO 4溶液5mL/kg灌胃，清洁肠道，禁食不禁水24h后，舒眠宁II注射液0.1mL/kg麻醉家犬，用聚乙烯6号导尿管自肛门处插入至结肠内20cm，缓慢注入相应体积、相应浓度的醋酸溶液，保留灌肠15s，头低尾高，用50mL生理盐水冲洗。造模完成后平躺放回饲养笼中待其自然苏醒，观察并记录造模后的状态变化。
3.模型评价
3.1一般情况观察：造模后每天记录犬体温、呼吸频率(次/分)、大便次数、大便性状、精神状态并记录家犬的便血情况。
3.2肠镜检测：分别于造模后的第1、4、7、10、14天舒眠宁Ⅱ注射液0.1mL/kg麻醉，用生理盐水冲洗犬结肠降结肠段和乙状结肠段(约从肛门至结肠内部20cm处)，使结肠内容物尽可能的冲洗出，在将VET-8015型电子内窥镜插入家犬结肠中，并于插入20cm处依次向肛门端移动拍照保存并以肠镜对模型进行评分。
3.3结肠病理切片：于造模后第10、14天舒眠宁Ⅱ注射液0.1mL/kg麻醉，然后空气栓塞处死，取出结肠，剖开，洗净，平铺拍照；然后沿结肠纵切分为三份，将中间段结肠置于10％福尔马林液中浸泡固定，石蜡包埋，HE染色做病理切片，剩余结肠-80℃冰箱中保存。
4.UC犬模型造模结果
4.1大体观察结果：直肠给予醋酸10分钟左右犬烦躁不安，频频回头观腹，用前肢抓腹敲打腹部，少部分用嘴啃咬狗笼，发出疼痛叫声，80％的犬在10-20分钟左右开始便血，里急后重，弯腰弓背，频频努责，在狗笼里频繁走动，便血量多，大多都为鲜血。
4.2肠镜检查结果：
根据图1～图3的内视镜显示，3组实验犬在造模前结肠部位粘膜表面光滑、完整没有损伤、色泽正常呈粉红色、未显出炎症反应；而造模后的第1天，可见直肠黏膜糜烂、多发性浅表溃疡，伴充血、水肿，呈弥漫性分布；造模后第7天的镜检结果提示，所有实验动物的结肠粘膜仍充血、 糜烂，形成伪膜；结果显示所有实验动物的结肠都出现了急性溃疡性炎症，造模成功。
实施例2
醋酸致犬急性UC模型肠镜检查评分标准
UC活动期的结肠病变粘膜成弥散性充血水肿，血管纹理模糊或紊乱；随着病情进展，粘膜变得粗糙，里面弥散分布的细颗粒质脆，有自然出血或接触出血；病变较重时，有多发性浅溃疡，重度溃疡变得大而深，肠腔可有粘液及血液渗出。
目前国内外评判UC的内镜评分方法有多种，不同的评分方法对于粘膜愈合和疾病活动程度各有侧重。其中Baron内镜评分方法主要关注粘膜出血程度，Mayo肠镜评分方法侧重于粘膜的整体特征，具体评分等级如下：
表1 Baron内镜分级
分级 内镜表现
0 血管纹理清晰可见，无自发性或接触性出血
1 粘膜有损害但无出血，介于0～2之间
2 粘膜中度出血；接触性出血，但无自发性出血
3 严重出血；自发性出血。接触性出血
表2 Mayo肠镜UC评分
Figure PCTCN2018075705-appb-000001
国内对动物肠镜检查并无标准，并且动物自发性溃疡性结肠炎相对于醋酸诱导的急性溃疡性结肠炎炎症相对较轻，无法直观评价实验动物模型的严重程度及其疾病进程。故结合临床患者溃疡性结肠炎的疾病严重程度 及临床分期，着重从粘膜损伤及肠道出血红肿情况，将醋酸致家犬急性溃疡性结肠炎的肠镜评分标准制定如下：
表3 醋酸致家犬急性溃疡性结肠炎肠镜检查评分标准
Figure PCTCN2018075705-appb-000002
实施例3
1.试验犬的选择
选取健康的纯种金毛犬(英文名：Golden Retriever)10只、纯种比格犬(英文名：Beagle)10只、中华田园犬(Canis lupus familiaris)10只，雌雄各半，犬龄1～2岁；试验期间三种试验犬分别饲养于三个独立且相同的试验环境中，单笼饲养。
2.醋酸诱导试验犬溃疡性结肠炎的模型建立
造模前一天10％MgSO 4溶液5mL/kg灌胃，清洁肠道，禁食不禁水24h后，按照0.15mL/kg肌肉注射舒眠宁麻醉，用聚乙烯导管自肛门处插入至结肠内20cm，缓慢注入7％醋酸溶液20mL，保留灌肠1min，头低尾高，用50mL生理盐水冲洗。
3.试验犬并发症及存活率观测
3.1并发症检测：分别于造模后的第2、4、6、8、10天取试验犬的粪便检测犬瘟热病毒、犬细小病毒的感染情况；并记录实验犬的体重及一般健康情况。
3.2存活记录：分别于造模后每天观察试验犬的存活情况。
4.结果
4.1并发症检测：纯种金毛犬于第4天检测出犬瘟热病毒阳性2只，犬细小病毒阳性2只；第8天测出犬瘟热病毒阳性3只，犬细小病毒阳性6只；第10天测出犬瘟热病毒阳性5只，犬细小病毒阳性9只。纯种比格犬于第6天检测出犬瘟热病毒阳性2只，犬细小病毒阳性3只；第10天测出犬瘟热病毒阳性3只，犬细小病毒阳性5只。中华田园犬于造模后的第2至10天均未测出犬瘟热病毒阳性或犬细小病毒阳性。
4.2存活率：造模后的第6天，纯种金毛犬出现死亡，死亡前口吐白沫，肛部有大量的排泄物；造模后第8天纯种金毛犬和纯种比格犬均出现死亡，死亡前口吐白沫，肛部有大量的酱色排泄物。造模后第10天，纯种金毛犬存活4只，存活率40％，存活的金毛犬饮食量少、状态虚弱、无力、眼角有脓状眼屎、呼吸有轰鸣声，可见多次粘液便的排便现象；纯种比格犬存活8只，存活率80％，存活的比格犬饮食量正常，但多数无力、少动，部分眼角有脓状眼屎，呼吸正常，可见多次粘液便的排便现象；中华田园犬存活10只，存活率100％，饮食、活动正常，眼角正常，呼吸正常，可见多次稀便或粘液便的排便现象。
5.结论
从造模后第10天仍存活的试演犬的排便及其它相关症状可以判断，本实验采用的UC造模法能成功地对所述3种犬诱发溃疡性结肠炎。即，本实验验证了犬UC模型用于评价药物疗效的可行性。
犬细小病毒感染又叫犬病毒性肠炎或出血性肠炎，有肠炎型(enteric)和心肌炎型(myocardial)两类，临床表现以急性出血性肠炎和非化脓性心肌炎为特征。肠炎型发生在成年犬类和大于3个月的幼犬身上。病毒会入侵肠膜，导致患病犬食欲不振、呕吐、血便和体温上升。心肌炎型通常发生在幼犬身上，病毒会侵入心脏的肌肉，通常72小时内会导致死亡。犬瘟热病毒感染表现症状多种多样，与病毒毒力、环境条件、宿主的年龄及免疫状态有关，主要可分为超急性型、急性型、消化道症状、神经症状和皮肤症状5种特征性类型，一旦出现特征性症状，则愈后极差。
由于本试验中，实验动物采单笼饲养、饲养环境天天消毒、饲养人员受过专门培训，因此本试验中，金毛犬和比格犬感染犬细小病毒和犬瘟热 病毒主要是由于此2种犬比起中华田园犬更易感染该类病毒并发病。
由于成年犬感染犬细小病毒和犬瘟热病毒的主要症状为急性肠炎，因此，此2种病毒感染并发症会一定程度的干扰药效评价；加之，此2种并发症的高死亡率导致了动物实验成本的增高。
基于醋酸诱导的UC模型对金毛犬和比格犬而言，有较高比例的并发症及死亡，因此，本模型采用抗感染能力更强、体型较小因而更易造模的中华田园犬为实验动物。
实施例4
实验模型验证
1.试验犬的分组及生理基础数据测定
选取合格的成年中华田园犬16只，随机分为4组，每组4只，雌雄各半。试验期间所有试验动物，单笼饲养，每日提供同等量的食物和饮用水，造模前一天，测定所有家犬的相关数据。
2.醋酸诱导家犬溃疡性结肠炎的模型建立
造模前一天10％MgSO 4溶液5mL/kg灌胃，清洁肠道，禁食不禁水24h后，按照0.15mL/kg肌肉注射舒眠宁麻醉，用聚乙烯导管自肛门处插入至结肠内20cm，缓慢注入7％醋酸溶液20mL，保留灌肠1min，头低尾高，用50mL生理盐水冲洗。
3.给药方式
造模后第1d起，按分组空腹灌喂给药，2粒/kg体重，早晚各一次，喂药2h后进食，连续给药7d；其中：A组(模型组)灌喂淀粉结肠溶胶囊、B组灌喂胃康灵胶囊、C组灌喂奥沙拉秦胶囊、D组灌喂柳氮磺吡啶结肠溶胶囊。
4.疗效判定
4.1大体观察
用电子内窥镜分别于造模的前后1d和给药后第3d、7d进行观察，记录结肠粘膜是否出现溃疡、溃烂和出血等；对各组进行疾病活动指数(DAI,Disease Activity Index)评分；并记录实验犬体温、体重以及大便形状。DAI评分方法如表1。
表4 疾病活动指数(DAI)评分
分数 体重减轻(％) 粪便性状 隐血情况
0 <1 正常 隐血(－)
1 1～5 介于两者之间 隐血(+)
2 5.01～10 糊状便 隐血(++)
3 10.01～15 介于两者之间 隐血(+++)
4 >15 腹泻 肉眼可见出血
注：正常粪便＝可成形的粪便；糊状＝不与肛门相粘的糊状粪便；腹泻＝黏于肛门的液化粪。
体重减轻(％)＝(某时间点的体重一造模前体重)/造模前体重×100％
DAI＝(体重下降的评分+大便性状的评分+隐血情况的评分)/3。
4.2隐血检查
大便隐血(又称便潜血)是指消化道少量出血，红细胞被消化破坏，粪便外观无异常改变，肉眼和显微镜下均不能证实的出血。采用隐血试剂盒检测，用棉签挑起少许大便于白瓷板上，滴加邻联甲苯胺液，再滴加过氧化氢液，根据显色时间和显色程度进行隐血等级评分。
4.3血常规
血细胞分析仪分析实验动物血液中的白细胞总数(WBC)、淋巴细胞总数(LYM)、单核细胞数(MONO)、中性粒细胞总数(GRAN)、淋巴细胞百分比(LYM％)、单核细胞百分比(MON％)、中性粒细胞百分比(GRA％)、血红蛋白(HGB)、红细胞比容(HCT)、红细胞总数(RBC)、红细胞平均体积(MCV)、平均红细胞血红蛋白浓度(MCH)、平均血红蛋白浓度(MCHC)、红细胞分布宽度(RDW％)、血小板总数(PLT)、平均血小板体积(MPV)。
4.4血液生化指标
用IDEXX生化分析仪分析血清中的胆红素(CHOL)、总蛋白(TP)、白蛋白(ALB)、尿素氮(BUN)、肌酐(CREA)、葡萄糖(GLU)、总胆固醇(TBIL)、血钙(CA)、磷酸根(PHOS)、谷丙转氨酶(ALT)、碱性磷酸酶(ALKP)、淀粉酶(AMYL)、球蛋白(GLOB)含量。
4.5C-反应蛋白(CRP)
用USHIOINCPoint-readerV分别检查造模前、造模后1、4、7天血清中C-反应蛋白含量。
4.6光学显微镜病理切片观察(HE)
制作切片观察结肠粘膜上皮：上皮细胞正常形态；杯状细胞、隐窝细胞、炎细胞浸润浸润情况等。
5.以临床用于治疗UC的药物炎症犬UC模型结果
5.1大体观察结果
直肠给予醋酸，10分钟左右犬烦躁不安，频频回头观腹，用前肢抓腹敲打腹部，少部分用嘴啃咬狗笼，发出疼痛叫声，80％的犬在10-20分钟左右开始便血，里急后重，弯腰弓背，频频努责，在狗笼里频繁走动，便血量多，大多都为鲜血。
5.2内窥镜检查：造模前，结肠粘膜呈粉红色，粘膜表面光滑。造模后1d，可见模型组直肠黏膜糜烂、多发性浅表溃疡，伴充血、水肿，呈弥漫性分布。
模型组在造模后4d，A组(模型组)结肠黏膜充血、水肿，黏膜面变粗糙，出现弥漫分布、大小较一致的细颗粒，组织变脆，有自然出血或接触出血，腔内有黏液性分泌物；造模后7d，模型组结肠黏膜仍充血、糜烂，形成伪膜。
药物组内窥镜检查结果：用药后4d，B组(胃康灵胶囊组)结肠黏膜糜烂、水肿，仍有溃疡；C组(奥沙拉秦胶囊组)结肠粘膜水肿减轻，溃疡较小而少；D组(柳氮磺吡啶肠溶胶囊组)结肠粘膜可见许多散在分布的红色小斑，呈针头样或斑片状，形态不规则，排列无规律。
用药后7d，B组结肠黏膜轻微水肿，溃疡愈合；C组渗出物吸收，肠黏膜色泽变得苍白，黏膜正常光泽但未完全恢复；D组结肠内可见浓汁，但结肠粘膜表面光滑，未见溃疡性病灶。
5.3DAI评分结果
表5 DAI评分结果
Figure PCTCN2018075705-appb-000003
注：表中各个数据取的是各组实验动物DAI的平均值。
DAI评分结果：从表5可看出，造模后的第1dDAI值最高，给药后的第4到第7天DAI逐渐下降。同期相比，模型组DAI值最高。
5.4大便隐血结果：
隐血实验结果：造模前1d的犬大便隐血检测为阴性(—)，给药后第1d和第4d隐血实验检测为强阳性(+++)，肉眼可见大便带鲜血，给药后第7d，大便隐血检测虽仍为阳性(++)，但出血情况减轻。
5.5血常规检测结果：
白细胞变化结果见表6。给药后第4d，A组(模型组)和C组白细胞总数高，B组和D组的白细胞总数则恢复到正常范围；给药后第7d，B组和D组白细胞总数维持在正常范围内，C组则持续下降至接近正常范围。
表6 WBC检测结果(均值±标准差,n＝4)
Figure PCTCN2018075705-appb-000004
注：白细胞的参考范围为6×10 9/L-17×10 9/L。
5.6生化检测结果
生化检测指标只有BUN有变化，在试验期间，BUN在正常范围内或略低于正常范围。其余的生化检测指标在各检测时点无差异。
5.7C-反应蛋白(CRP)检测结果
CRP变化结果见表7。给药后第4d，各用药组CRP下降，其中D组下降最明显；给药后第7d，D组的CRP为7.975±2.425，恢复到正常范围。
表7 CRP检测结果(mg/L)(均值±标准差,n＝4)
Figure PCTCN2018075705-appb-000005
注：CRP参考范围为1-10mg/L。
本发明中，采用直肠灌注10～20mL的5～10％醋酸溶液，制作犬的UC模型，内视镜结果显示实验动物被成功诱发急性UC。采用临床一线用药胃康灵、爱沙拉秦、柳氮磺吡啶验证模型用于药效评价的结果显示：用药后4d，胃康灵胶囊组结肠黏膜糜烂、水肿，仍有溃疡；爱沙拉秦胶囊组结肠粘膜水肿减轻，溃疡较小而少；柳氮磺吡啶结肠溶胶囊组结肠粘膜可见许多散在分布的红色小斑，呈针头样或斑片状，形态不规则，排列无规律；用药后7d，胃康灵胶囊组结肠黏膜轻微水肿，溃疡愈合；爱沙拉秦胶囊组渗出物吸收，肠黏膜色泽变得苍白，黏膜正常光泽但未完全恢复；柳氮磺吡啶结肠溶胶囊组结肠内可见浓汁，但结肠粘膜表面光滑，未见溃疡性病灶。模型组的4只犬的大便稀带血、结肠形态、组织学观察均呈现出明显的炎症症状，同时通过造模3d与7d后的组织学观察发现，3d后溃疡灶依然明显，并有明显的炎性细胞浸润等，而7d后可见溃疡灶已减少甚至脱落，炎性细胞浸润也明显减少，大量成纤维细胞增生，说明实验动物正在自愈过程中；而同期给予UC临床用药的试验犬在给药后的3d后溃疡较模型组减少许多，炎性细胞的浸润也大幅度减少，说明结肠的炎症得到改善，结肠粘膜逐渐修复。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的 精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (14)

  1. 一种溃疡性结肠炎动物模型的构建方法，包括以下步骤：
    中华田园犬灌胃清洗肠道，禁食不禁水24h；
    麻醉中华田园犬，用醋酸溶液对麻醉后的中华田园犬进行灌肠，待中华田园犬自然苏醒，即得溃疡性结肠炎动物模型。
  2. 根据权利要求1所述的构建方法，其特征在于，所述中华田园犬为成年犬。
  3. 根据权利要求2所述的构建方法，其特征在于，所述中华田园犬为1～2岁。
  4. 根据权利要求2或3所述的构建方法，其特征在于，所述中华田园犬的体重为6.0～12.0Kg。
  5. 根据权利要求1所述的构建方法，其特征在于，所述灌肠方式为将醋酸溶液灌入中华田园犬结肠内，保留10s以上，再用生理盐水冲洗结肠。
  6. 根据权利要求5所述的构建方法，其特征在于，所述醋酸溶液的质量浓度为5～10％。
  7. 根据权利要6所述的构建方法，其特征在于，所述醋酸溶液的用量为每公斤体重1mL～2mL。
  8. 根据权利要求1所述的构建方法，其特征在于，所述灌胃用药剂为MgSO 4溶液。
  9. 根据权利要求8所述的构建方法，其特征在于，所述MgSO 4溶液的浓度为5～10％，用量为5～10mL/kg。
  10. 根据权利要求1所述的构建方法，其特征在于，所述中华田园犬麻醉的方法为：用舒眠宁对中华田园犬进行肌肉注射。
  11. 权利要求1～10任意一项所述方法构建得到的溃疡性结肠炎动物模型在筛选UC治疗药物中的应用。
  12. 一种筛选或评价UC治疗药物的方法，其特征在于，包括以下步骤：
    a、设置待测药物组、生理盐水对照组和正常对照组，其中待测药物组和生理盐水对照组使用权利要求1～4任意一项所述方法构建的溃疡性结肠炎动物模型，正常对照组为不进行造模的健康中华田园犬；观察并记录待测药物组、生理盐水对照组和正常对照组动物的生理基础数据和结肠病理情况；
    b、待测药物组灌喂待测药物，生理盐水对照组和正常对照组均灌喂生理盐水；观察并记录处理后待测药物组、生理盐水对照组和正常对照组动物的生理基础数据和结肠病理情况；
    c、分析步骤a和步骤b记录的数据及结肠病理照片评分，对待测药物的治疗效果进行评价；若待测药物组较生理盐水对照组的生理基础数据更接近于正常对照组的相应测量值，或待测药物组较生理盐水对照组的结肠病理照片评分低，则说明待测药物具有治疗UC的作用。
  13. 根据权利要求12所述的方法，其特征在于，所述结肠病理照片的评分标准为：
    0级：黏膜苍白，血管网清晰，呈分支状；黏膜下无红肿、充血，其表面黏膜正常；
    1级：黏膜尚光滑，但小面积充血、红肿，折光增强；
    2级：黏膜充血、水肿，呈颗粒状，黏膜脆性增加，接触易出血；
    3级：黏膜明显充血、水肿、粗糙，少量自发性出血点或接触性出血；有较多炎性分泌物，多发性糜烂与小面积溃疡形成；
    4级：黏膜大面积充血、水肿、粗糙，明显自发性出血和接触性出血；多发性点状糜烂与大面积溃疡形成；
    若待测药物组结肠病理照片的评分低，则说明待测药物治疗UC的效果好。
  14. 根据权利要求12所述的方法，其特征在于，所述动物的生理基础数据包括体温、体重、大便形状、隐血检查、血常规、血液生化指标和C-反应蛋白中的一项或几项。
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