CN110522902B - Composition for establishing chronic kidney disease model and application thereof - Google Patents

The invention relates to a composition and a method for establishing a chronic kidney disease model, and a medicine for screening and treating chronic kidney disease by using the composition and the chronic kidney disease modelThe composition comprises ochratoxin A and cyclosporine A. The invention utilizes the nephrotoxicity of ochratoxin A and cyclosporine A to screen out the optimal concentration combination to establish a chronic kidney disease model, and the combination is given to a mouse at every other day by 0.25mg/kg.d ^‑1 Ochratoxin A administered at a dose of 20mg/kg per day ^‑1 Cyclosporine A (or ochratoxin A of 0.5 μ g/mL and cyclosporine A of 6 μ g/mL in vitro) successfully affects kidney function, and a chronic nephropathy model is established, which is administered separately at a high dose of 30mg/kg per day ^‑1 The change process of chronic nephropathy formed by the cyclosporine A is similar, and the use of the ochratoxin A reduces the dosage of the cyclosporine A, thereby obviously reducing the side effect on the heart and the liver, effectively relieving the side effect caused by high-concentration cyclosporine A, reducing the construction cost of a model, and providing a research basis for screening related medicines.

一种用于建立慢性肾病模型的组合物及其应用A kind of composition for establishing chronic kidney disease model and application thereof

技术领域technical field

本发明涉及疾病模型技术领域，具体涉及一种用于建立慢性肾病模型的组合物，还涉及一种慢性肾病模型的建立方法，以及上述组合物及慢性肾病模型在筛选治疗慢性肾病的药物中的应用，具体地，该组合物为由赭曲霉毒素A与环孢霉素A的联合使用。The invention relates to the technical field of disease models, in particular to a composition for establishing a chronic kidney disease model, a method for establishing a chronic kidney disease model, and the use of the composition and the chronic kidney disease model in screening drugs for the treatment of chronic kidney disease. The application, in particular, the composition is the combined use of ochratoxin A and cyclosporine A.

背景技术Background technique

由于内外界多种因素导致肾脏结构异常或功能异常，这就会造成慢性肾病的发生。成年人慢性肾病的发病率为8％-12％。慢性肾病发病特点有“三高”、“三低”：发病率高、伴发的心血管病患病率高、病死率高；全社会对慢性肾病的知晓率低、防治率低、伴发心血管病的知晓率低。慢性肾病起病隐匿，早期通常无明显临床表现，有临床症状时已错过最佳治疗或干预时机，再者由于我国现有医疗水平有限，继发性和药物诱导的慢性肾病不断增加，这就导致我国慢性肾病的防治工作形势面临严峻挑战。Due to a variety of internal and external factors leading to abnormal structure or function of the kidneys, this will lead to the occurrence of chronic kidney disease. The incidence of chronic kidney disease in adults is 8%-12%. The morbidity characteristics of CKD are "three highs" and "three lows": high morbidity, high prevalence of concomitant cardiovascular disease, and high mortality; low awareness of CKD in the whole society, low prevention and treatment rates, and concomitant Awareness of cardiovascular disease is low. The onset of chronic kidney disease is insidious, and there are usually no obvious clinical manifestations in the early stage. When there are clinical symptoms, the best time for treatment or intervention has been missed. Moreover, due to the limited medical level in our country, secondary and drug-induced chronic kidney disease is increasing. As a result, the prevention and treatment of chronic kidney disease in my country is facing severe challenges.

有研究表明引起慢性肾病的原因有原发性、继发性和药物等。其中药物有布洛芬、头孢霉素、环孢霉素等。环孢霉素A作为一种免疫抑制剂用于预防器官或组织移植所发生的排斥反应。不仅如此，环孢霉素A因为其存在肾毒性，经常被用于建立慢性环孢霉素肾病模型。因此，环孢霉素A诱导的慢性肾病动物模型被广泛用于筛选能有效治疗慢性肾病的药物。然而，环孢霉素A还对心脏、肝脏、神经系统和牙龈等具有毒副作用，导致动物模型安全性差、准确度低，从而限制了环孢霉素A的临床应用。目前文献报道仅有针对某种毒性作用研究，或是对慢性环孢霉素A肾病治疗药物的筛选，兼顾慢性环孢霉素A肾病模型的建立同时降低副作用的研究仍是空白。因此有必要探索新的、安全有效的方法来模拟人类慢性肾病模型，为后期筛选治疗慢性肾病药物的研究打下良好的动物模型基础。Studies have shown that the causes of chronic kidney disease include primary, secondary and drugs. Among them are ibuprofen, cephalosporin, cyclosporine and so on. Cyclosporine A is used as an immunosuppressant to prevent rejection of organ or tissue transplants. Not only that, cyclosporine A is often used to establish a chronic cyclosporine nephropathy model because of its nephrotoxicity. Therefore, cyclosporine A-induced chronic kidney disease animal models are widely used to screen drugs that can effectively treat chronic kidney disease. However, cyclosporine A also has toxic and side effects on the heart, liver, nervous system, and gums, resulting in poor safety and low accuracy of animal models, thus limiting the clinical application of cyclosporine A. At present, there are only studies on certain toxic effects reported in the literature, or the screening of drugs for the treatment of chronic cyclosporine A nephropathy, and the research on the establishment of a chronic cyclosporine A nephropathy model while reducing side effects is still blank. Therefore, it is necessary to explore new, safe and effective methods to simulate human chronic kidney disease models and lay a good animal model foundation for the later screening of drugs for the treatment of chronic kidney disease.

赭曲霉毒素是继黄曲霉毒素之后引起世界广泛关注的另一种真菌毒素。它是由七种曲霉和六种青霉菌产生的重要霉菌毒素群。其中毒性最大、分布最广、产毒量最高、对农产品的污染最重、与人类健康关系最密切的是赭曲霉毒素A。各种农产品、食物和饲料等广泛暴露于赭曲霉毒素A之下，动物误食含有赭曲霉毒素A的饲料后赭曲霉毒素A被机体吸收，在体内蓄积于肾脏、肝脏、血液以及肌肉中，人类再进食这些被赭曲霉毒素A污染的动物性食品等，这对人类健康和畜牧业发展是一个巨大的威胁。赭曲霉毒素A的主要靶器官是肾，哺乳动物肾脏作为一种排泄系统发挥着重要的作用，而赭曲霉毒素A可诱导急性或慢性肾损伤。绝大多数代谢产物和许多药理化合物都是通过尿排泄出来的，在肾脏复杂且不均匀的结构中，近端小管是赭曲霉毒素A等肾毒性化合物的主要靶点之一，这是因为大部分活性转运和生物转化过程都位于近端小管。巴尔干肾炎的主要原因就是人类饮食中的赭曲霉毒素A暴露。这种疾病的特点是近端小管上皮细胞变性和间质纤维化，导致多尿，血液和生化参数的各种变化。目前应用赭曲霉毒素A与其他药物联合诱导慢性肾病模型的研究国内外均未见报道。Ochratoxin is another mycotoxin that has attracted worldwide attention after aflatoxin. It is an important group of mycotoxins produced by seven species of Aspergillus and six species of Penicillium. Among them, ochratoxin A has the highest toxicity, the widest distribution, the highest toxin production, the heaviest pollution to agricultural products, and the closest relationship with human health. Various agricultural products, foods and feeds are widely exposed to ochratoxin A. After animals eat feed containing ochratoxin A by mistake, ochratoxin A is absorbed by the body and accumulates in the kidneys, liver, blood and muscles. Humans eat these animal foods contaminated with ochratoxin A, which is a huge threat to human health and the development of animal husbandry. The main target organ of ochratoxin A is the kidney, which plays an important role as an excretory system in mammals, and ochratoxin A can induce acute or chronic kidney damage. The vast majority of metabolites and many pharmacological compounds are excreted in the urine, and in the complex and heterogeneous structure of the kidney, the proximal tubule is one of the main targets of nephrotoxic compounds such as ochratoxin A because of the large Part of the active transport and biotransformation processes are located in the proximal tubule. The main cause of Balkan nephritis is ochratoxin A exposure in the human diet. The disease is characterized by degeneration of proximal tubule epithelial cells and interstitial fibrosis, resulting in polyuria and various changes in hematological and biochemical parameters. At present, there is no report on the use of ochratoxin A in combination with other drugs to induce chronic kidney disease models at home and abroad.

发明内容SUMMARY OF THE INVENTION

为了克服现有技术中所存在的缺点与不足，本发明提供一种用于建立慢性肾病模型的组合物及其应用，其采用低剂量无毒性的赭曲霉毒素A与环孢霉素A 联合诱导慢性肾病模型的建立，并提供了组合物及慢性肾病模型在筛选治疗慢性肾病的动物模型中的应用。本发明通过使用低剂量无毒性的赭曲霉毒素A与低浓度的环孢霉素A联合诱导小鼠慢性肾病模型的建立，与正常高剂量环孢霉素A 诱导的慢性肾病模型相比，联合诱导的小鼠慢性肾病模型中，通过添加对机体或细胞无毒性的低剂量/浓度赭曲霉毒素A降低了环孢霉素A的用量，同时由此减少了因长期使用环孢霉素而导致的其他毒副作用。In order to overcome the shortcomings and deficiencies in the prior art, the present invention provides a composition for establishing a chronic kidney disease model and its application, which are induced by a combination of low-dose nontoxic ochratoxin A and cyclosporine A. The establishment of a chronic kidney disease model and the application of the composition and the chronic kidney disease model in screening animal models for treating chronic kidney disease are provided. The present invention induces the establishment of a mouse chronic kidney disease model by using low-dose non-toxic ochratoxin A combined with low-concentration cyclosporine A. Compared with the normal high-dose cyclosporine A-induced chronic kidney disease model, the combined In an induced mouse model of chronic kidney disease, the addition of low doses/concentrations of ochratoxin A that is not toxic to the body or cells reduces the dosage of cyclosporine A, and thus reduces the amount of cyclosporine caused by long-term use of cyclosporine. other toxic side effects.

为实现上述目的，本发明采用如下技术方案：To achieve the above object, the present invention adopts the following technical solutions:

本发明的第一个方面是提供一种用于建立慢性肾病模型的组合物，其包括赭曲霉毒素A与环孢霉素A。A first aspect of the present invention is to provide a composition for establishing a chronic kidney disease model, comprising ochratoxin A and cyclosporine A.

进一步地，所述组合物包括无毒性浓度的赭曲霉毒素A与低浓度的环孢霉素A。Further, the composition includes ochratoxin A at a nontoxic concentration and cyclosporine A at a low concentration.

进一步地，所述慢性肾病模型包括动物模型或体外细胞模型。Further, the chronic kidney disease model includes an animal model or an in vitro cell model.

进一步地，所述动物模型为小鼠慢性肾病模型，所述体外细胞模型为人肾小管上皮细胞肾病模型；更进一步地，所述小鼠采用C57 BL/6小鼠，所述人肾小管上皮细胞为人肾小管上皮细胞HK-2传代细胞系。可理解的是，上述动物及体外细胞可为本领域中任一合适的可用于建立慢性肾病模型的动物及体外细胞。Further, the animal model is a mouse chronic kidney disease model, and the in vitro cell model is a human renal tubular epithelial cell nephropathy model; further, the mouse is a C57 BL/6 mouse, and the human renal tubular epithelial cell For the human renal tubular epithelial cells HK-2 passage cell line. It is understood that the above-mentioned animal and in vitro cells can be any suitable animal and in vitro cells in the art that can be used to establish a chronic kidney disease model.

进一步地，当用于建立小鼠慢性肾病模型时，所述赭曲霉毒素A的施用剂量为0.15～0.5mg/kg.d^-1，例如可具体为0.15、0.2、0.25、0.3、0.35、0.4、0.45、 0.5mg/kg.d^-1等；所述环孢霉素A的施用剂量为5～25mg/kg.d^-1，例如可具体为5、 10、15、20、25mg/kg.d^-1等。Further, when used to establish a mouse chronic kidney disease model, the administered dose of ochratoxin A is 0.15-0.5 mg/kg.d ^-1 , for example, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 , 0.45, 0.5 mg/kg.d ^-1 , etc.; the dosage of cyclosporine A is 5 to 25 mg/kg.d ^-1 , for example, 5, 10, 15, 20, 25 mg/kg. d ^-1 etc.

更进一步地，当用于建立小鼠慢性肾病模型时，所述赭曲霉毒素A的施用剂量为0.25mg/kg.d^-1；所述环孢霉素A的施用剂量为10mg/kg.d^-1或20mg/kg.d^-1，更优选为20mg/kg.d^-1。Further, when used to establish a mouse chronic kidney disease model, the administered dose of the ochratoxin A is 0.25 mg/kg.d ⁻¹ ; the administered dose of the cyclosporine A is 10 mg/kg.d ^-1 or 20 mg/kg.d ^-1 , more preferably 20 mg/kg.d ^-1 .

进一步地，当用于建立人肾小管上皮细胞肾病模型时，所述赭曲霉毒素A 的施用剂量为0.01～25μg/mL，例如0.01、0.1、0.5、1、2、4、8、10、15、20、 25μg/mL等；所述环孢霉素A的施用剂量为0.1～50μg/mL，例如0.1、1、2、4、 6、8、10、20、30、40、50μg/mL等。Further, when used to establish a human renal tubular epithelial cell nephropathy model, the administration dose of ochratoxin A is 0.01-25 μg/mL, such as 0.01, 0.1, 0.5, 1, 2, 4, 8, 10, 15 , 20, 25 μg/mL, etc.; the dosage of cyclosporine A is 0.1-50 μg/mL, such as 0.1, 1, 2, 4, 6, 8, 10, 20, 30, 40, 50 μg/mL, etc. .

更进一步地，当用于建立人肾小管上皮细胞肾病模型时，所述赭曲霉毒素A 的施用剂量为0.5μg/mL；所述环孢霉素A的施用剂量为2、4或6μg/mL，更优选为6μg/mL。Further, when used to establish a human renal tubular epithelial cell nephropathy model, the administration dose of ochratoxin A is 0.5 μg/mL; the administration dose of cyclosporine A is 2, 4 or 6 μg/mL , more preferably 6 μg/mL.

本发明的第二个方面是提供一种采用任一上述的组合物建立动物慢性肾病模型的方法，其包括如下步骤：实验动物从试验开始的第一天起每间隔一天给予预定剂量的赭曲霉毒素A，从试验开始的第一天起每天同时给予预定剂量的环孢霉素A，给药方式为腹腔注射给药，给药周期为20～36天，获得动物慢性肾病模型。A second aspect of the present invention is to provide a method for establishing an animal chronic kidney disease model using any of the above compositions, comprising the steps of: the experimental animal is administered a predetermined dose of Aspergillus ochra every other day from the first day of the experiment For toxin A, a predetermined dose of cyclosporine A was simultaneously administered every day from the first day of the experiment, and the administration method was intraperitoneal injection, and the administration period was 20 to 36 days to obtain an animal model of chronic kidney disease.

进一步地，所述赭曲霉毒素A每间隔一天腹腔注射一次，共28天注射天数为14天，环孢霉素A每天腹腔注射一次，共28天注射天数为28天。Further, the ochratoxin A was intraperitoneally injected once every other day for a total of 28 days for 14 days, and the cyclosporine A was intraperitoneally injected once a day for a total of 28 days for 28 days.

进一步地，所述动物采用实验小鼠，小鼠先适应性喂养1周，室温，空气湿度45％～60％，每日光照12小时保持昼夜循环条件，适应性喂养期间各组均饲以蛋白质含量为20％的标准饲料；所有动物均饮用自来水(加热至沸腾消毒，冷却后使用)，垫料使用高压灭菌垫料，更换频率为1次/3天。Further, the animals were experimental mice. The mice were first adaptively fed for 1 week at room temperature, with an air humidity of 45% to 60%, and 12 hours of daily light to maintain a day-night cycle. During the adaptive feeding period, each group was fed with protein. Standard feed with a content of 20%; all animals drank tap water (heated to boiling for sterilization, and used after cooling), and the litter was autoclaved, and the replacement frequency was 1 time/3 days.

上述低剂量无毒性的赭曲霉毒素A与环孢霉素A的联合应用可以诱导小鼠慢性肾病的发生，与此同时，由于赭曲霉毒素A的添加，减少了环孢霉素A的剂量，也降低了因长期应用环孢霉素A导致的副作用；与阳性造模组中环孢霉素A按照30mg/kg.d^-1单独应用比较，优选按照赭曲霉毒素A剂量为0.25 mg/kg.d^-1与环孢霉素A剂量为20mg/kg.d^-1联合可以成功诱导慢性肾病动物模型。The combination of the above low-dose nontoxic ochratoxin A and cyclosporine A can induce chronic kidney disease in mice. At the same time, due to the addition of ochratoxin A, the dose of cyclosporine A is reduced, It also reduces the side effects caused by the long-term application of cyclosporine A; compared with the single application of cyclosporine A in the positive modeling group at 30 mg/kg.d ^-1 , the preferred dose of ochratoxin A is 0.25 mg/kg. The combination of d ^-1 and cyclosporine A at a dose of 20 mg/kg.d ^-1 can successfully induce the animal model of chronic kidney disease.

本发明的第三个方面是提供任一上述的组合物建立体外细胞慢性肾病模型的方法，其包括如下步骤：细胞贴壁18～30小时后，同时给予预定浓度的赭曲霉毒素A和预定浓度的环孢霉素A，共同培养32～54h，获得体外细胞慢性肾病模型。The third aspect of the present invention is to provide a method for establishing an in vitro cellular chronic kidney disease model with any of the above compositions, comprising the steps of: 18-30 hours after the cells adhere to the cell wall, simultaneously administering a predetermined concentration of ochratoxin A and a predetermined concentration cyclosporine A was co-cultured for 32-54 h to obtain an in vitro cellular chronic kidney disease model.

进一步地，细胞贴壁24小时后，同时给予预定浓度的赭曲霉毒素A和预定浓度的环孢霉素A，共同培养48h，获得体外细胞慢性肾病模型。Further, 24 hours after the cells adhered to the wall, a predetermined concentration of ochratoxin A and a predetermined concentration of cyclosporine A were simultaneously administered, and co-cultured for 48 hours to obtain an in vitro cellular chronic kidney disease model.

在上述体外细胞肾病模型的试验中，所述体外细胞采用人肾小管上皮细胞，用于联合应用的赭曲霉毒素A的浓度为0.5μg/mL，环孢霉素A的浓度2、4、6 μg/mL，联合组与单独环孢霉素A浓度为10μg/mL的毒性组进一步比较分析，优选按照以0.5μg/mL的赭曲霉毒素A与6μg/mL的环孢霉素A联合可以显著增加肾小管上皮细胞纤维化相关蛋白表达，与单独环孢霉素A毒性组无显著差异，以此证明了赭曲霉毒素A的加入确实可以减少环孢霉素A的用量。In the test of the above in vitro cellular nephropathy model, the in vitro cells were human renal tubular epithelial cells, the concentration of ochratoxin A for combined application was 0.5 μg/mL, and the concentration of cyclosporine A was 2, 4, 6 μg/mL, the combination group and the toxicity group with a single cyclosporine A concentration of 10 μg/mL were further compared and analyzed. The increase in the expression of fibrosis-related proteins in renal tubular epithelial cells was not significantly different from that in the cyclosporine A toxicity group, which proved that the addition of ochratoxin A could indeed reduce the dosage of cyclosporine A.

本发明的第四个方面是提供一种任一上述的组合物、任一上述的动物慢性肾病模型或任一上述的体外细胞慢性肾病模型在用于筛选治疗慢性肾病的药物中的应用。The fourth aspect of the present invention is to provide use of any of the above compositions, any of the above-mentioned animal chronic kidney disease models or any of the above-mentioned in vitro cellular chronic kidney disease models for screening drugs for the treatment of chronic kidney disease.

进一步地，所述筛选治疗慢性肾病的药物的步骤包括：Further, the step of screening a drug for the treatment of chronic kidney disease includes:

步骤(1)采用包括赭曲霉毒素A与环孢霉素A的组合物建立慢性肾病模型，所述慢性肾病模型为动物慢性肾病模型或体外细胞慢性肾病模型；Step (1) using a composition comprising ochratoxin A and cyclosporine A to establish a chronic kidney disease model, the chronic kidney disease model is an animal chronic kidney disease model or an in vitro cellular chronic kidney disease model;

步骤(2)将候选药物施用于步骤(1)所建立的慢性肾病模型；Step (2) applying the candidate drug to the chronic kidney disease model established in step (1);

步骤(3)用所述慢性肾病模型肾脏或细胞肾功能指标的损伤改善程度评价及筛选治疗慢性肾病的所述候选药物。Step (3) Evaluate and screen the candidate drug for the treatment of chronic kidney disease by the degree of improvement of the kidney or cellular renal function index of the chronic kidney disease model.

可理解的是，采用其他动物或体外细胞构建慢性肾病模型时，赭曲霉毒素A 与环孢霉素A的用量需根据采用的动物种属及细胞特性进行适应性调整。It is understandable that when other animals or in vitro cells are used to construct a chronic kidney disease model, the dosages of ochratoxin A and cyclosporine A need to be adapted according to the animal species and cell characteristics used.

本发明采用上述技术方案，具有如下技术效果：The present invention adopts the above-mentioned technical scheme, and has the following technical effects:

本发明采用赭曲霉毒素A与环孢霉素A联合可以有效建立慢性肾病模型，通过添加无毒性浓度的赭曲霉毒素A可减少环孢霉素A(低浓度)的用量，降低了长期使用高剂量环孢霉素A导致的副作用，而且制备方法安全，可以后续用于相关药物的筛选与作用评价，具有良好的前景。In the present invention, the combination of ochratoxin A and cyclosporine A can effectively establish a chronic kidney disease model, and the dosage of cyclosporine A (low concentration) can be reduced by adding ochratoxin A in a non-toxic concentration, and the high long-term use can be reduced. The side effects caused by the dose of cyclosporine A, and the preparation method is safe, can be used for subsequent screening and effect evaluation of related drugs, and has a good prospect.

本发明利用赭曲霉毒素A与环孢霉素A的肾毒性，筛选出最佳浓度组合建立小鼠慢性肾病模型，通过加入赭曲霉毒素A降低环孢霉素A用量的同时减少了因长期使用环孢霉素A而导致的对其他组织器官的毒副作用。与单独应用高浓度的环孢霉素A相比，低剂量无毒性的赭曲霉毒素A与低剂量的环孢霉素A 联合能显著提高模型小鼠平均日增重，使肾脏纤维化程度显著加剧，肾脏纤维化相关指标表达增加，除此之外，降低了心脏和肝脏的损伤，也无神经症状出现，一定程度上减弱了环孢霉素A的副作用。The present invention utilizes the nephrotoxicity of ochratoxin A and cyclosporine A, and selects the optimal concentration combination to establish a mouse chronic kidney disease model. Toxic side effects on other tissues and organs caused by cyclosporine A. Compared with the application of high concentration of cyclosporine A alone, the combination of low-dose nontoxic ochratoxin A and low-dose cyclosporine A can significantly increase the average daily weight gain of model mice and significantly increase the degree of renal fibrosis. Exacerbated, the expression of renal fibrosis-related indicators increased. In addition, the damage to the heart and liver was reduced, and no neurological symptoms appeared, which weakened the side effects of cyclosporine A to a certain extent.

在制备小鼠慢性肾病模型时，本发明采取腹腔注射的给药方式，相较口服灌胃减少小鼠的机械损伤等应激带来的死亡。不仅如此，现有技术中使用单一的长时间的高剂量环孢霉素A会导致心、肝、牙龈、高血压和神经副作用等，而本发明降低了环孢霉素A的用量，其所致的副作用也相应改善。因此，在达到传统小鼠模型效果的基础上，改善了原模型存在的副作用，并一定程度上降低了模型构建成本，为后续相关药物的筛选与作用评价提供研究基础。When preparing the mouse chronic kidney disease model, the present invention adopts the intraperitoneal injection mode of administration, which reduces the death of mice caused by stress such as mechanical damage compared with oral gavage. Not only that, the use of a single long-term high-dose cyclosporine A in the prior art can cause heart, liver, gingival, hypertension and neurological side effects, etc., and the present invention reduces the consumption of cyclosporine A. The resulting side effects are also improved accordingly. Therefore, on the basis of achieving the effect of the traditional mouse model, the side effects of the original model are improved, and the cost of model construction is reduced to a certain extent, providing a research basis for subsequent screening and evaluation of related drugs.

附图说明Description of drawings

图1为本发明一实施例中体内试验建模28天后赭曲霉毒素A与低浓度环孢霉素A的单独或联合应用对脏器指数(A、C)，对肾脏组织进行HE、免疫组化和Masson染色(B、D)，柱形图代表平均数±标准误；“*”和“#”表示差异显著(P<0.05)，“**”和“##”表示差异极显著(P<0.01)。Fig. 1 shows the organ index (A, C) of the single or combined application of ochratoxin A and low-concentration cyclosporine A after 28 days of in vivo test modeling in an embodiment of the present invention. Chloride and Masson staining (B, D), the bar graph represents the mean ± standard error; "*" and "#" indicate significant differences (P<0.05), "**" and "##" indicate extremely significant differences (P<0.05). P<0.01).

图2为本发明一实施例中体内试验建模28天后无毒性浓度赭曲霉毒素A与低浓度环孢霉素A的联合应用对尿比重、尿蛋白(A)、血清尿素氮、血清肌酐 Scr(B)和肾脏纤维化相关因子表达(C、D)的影响示意图,柱形图代表平均数±标准误；“*”和“#”表示差异显著(P<0.05)，“**”和“##”表示差异极显著(P<0.01)。Fig. 2 shows the effects of combined application of nontoxic concentration ochratoxin A and low concentration cyclosporine A on urine specific gravity, urine protein (A), serum urea nitrogen, serum creatinine Scr after 28 days of in vivo test modeling in an embodiment of the present invention (B) Schematic diagram of the effect of renal fibrosis-related factor expression (C, D), the bar graph represents the mean ± standard error; "*" and "#" indicate significant differences (P<0.05), "**" and "##" indicates a very significant difference (P<0.01).

图3为本发明一实施例中体内试验建模28天后无毒性浓度的赭曲霉毒素A 与低浓度环孢霉素A联合应用对体重指数(A)、心脏指数(B)、肝脏指数(C)、 HE染色(D)的影响示意图；其中，柱形图代表平均数±标准误；“*”和“#”表示差异显著(P<0.05)，“**”和“##”表示差异极显著(P<0.01)。Figure 3 is an example of the in vivo test modeling in an embodiment of the present invention after 28 days of non-toxic concentration of ochratoxin A combined with low-concentration cyclosporine A on body mass index (A), cardiac index (B), liver index (C) ), HE staining (D); the bar graph represents the mean ± standard error; "*" and "#" indicate significant differences (P<0.05), "**" and "##" indicate differences Very significant (P<0.01).

图4为本发明一实施例中体内试验建模28天后无毒性浓度的赭曲霉毒素A 与低浓度环孢霉素A联合应用对肝脏和心脏特异性酶(A、B)和纤维化相关因子(C-F)表达的影响示意图；其中，柱形图代表平均数±标准误；“*”和“#”表示差异显著(P<0.05)，“**”和“##”表示差异极显著(P<0.01)。Figure 4 is an example of an in vivo test modeled after 28 days of non-toxic concentrations of ochratoxin A combined with low concentrations of cyclosporine A on liver and heart specific enzymes (A, B) and fibrosis-related factors (C-F) Schematic diagram of the effect of expression; in which, the bar graph represents the mean ± standard error; "*" and "#" indicate significant differences (P<0.05), "**" and "##" indicate extremely significant differences ( P<0.01).

图5为本发明一实施例中体外细胞试验中赭曲霉毒素A与环孢霉素A单独或联合应用对HK-2细胞活性(A、B、E)和乳酸脱氢酶活性(C、D、F)的影响示意图；其中，柱形图代表平均数±标准误；“*”和“#”表示差异显著 (P<0.05)，“**”和“##”表示差异极显著(P<0.01)。Figure 5 shows the effects of ochratoxin A and cyclosporine A alone or in combination on HK-2 cell activity (A, B, E) and lactate dehydrogenase activity (C, D) in an in vitro cell test in an embodiment of the present invention , F) the influence diagram; wherein, the bar graph represents the mean ± standard error; "*" and "#" indicate significant difference (P<0.05), "**" and "##" indicate extremely significant difference (P<0.05) <0.01).

图6为本发明一实施例中体外细胞试验中赭曲霉毒素A与环孢霉素A单独或联合应用对HK-2细胞纤维化相关因子表达的影响示意图；其中，柱形图代表平均数±标准误；“*”和“#”表示差异显著(P<0.05)，“**”和“##”表示差异极显著(P<0.01)。6 is a schematic diagram showing the effect of ochratoxin A and cyclosporine A alone or in combination on the expression of fibrosis-related factors in HK-2 cells in an in vitro cell test in an embodiment of the present invention; wherein the bar graph represents the mean ± Standard error; "*" and "#" indicate significant difference (P<0.05), "**" and "##" indicate extremely significant difference (P<0.01).

具体实施方式Detailed ways

本发明涉及一种用于建立慢性肾病模型的组合物，其包括赭曲霉毒素A与环孢霉素A；本发明还涉及上述组合物的应用以及慢性肾病模型的建立方法。The present invention relates to a composition for establishing a chronic kidney disease model, which comprises ochratoxin A and cyclosporine A; the present invention also relates to the application of the above-mentioned composition and a method for establishing a chronic kidney disease model.

下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and embodiments. The following examples are only used to illustrate the technical solutions of the present invention more clearly, and cannot be used to limit the protection scope of the present invention.

下述实施例以小鼠为例构建动物慢性肾病模型，以人肾小管上皮细胞为例构建体外细胞模型。The following examples take mice as an example to construct an animal chronic kidney disease model, and take human renal tubular epithelial cells as an example to construct an in vitro cell model.

实施例1Example 1

本实施例为一较佳的实验动物慢性肾病模型的制备，其实验动物采用小鼠。This example is for the preparation of a preferred experimental animal model of chronic kidney disease, and the experimental animal is a mouse.

1.材料和方法1. Materials and methods

1.1 主要材料和试剂1.1 Main materials and reagents

6-8周龄的SPF级雌性C57BL/6小鼠70只，购自扬州大学。初始体重为16.25 ±0.25g，小鼠在无菌笼(7组，10只/每组)中进行适应性饲养一周后，环境温度保持22±2℃，相对湿度为45％～60％，12h光照昼夜交替。试验全程自由采食、饮水，每3天更换一次垫料，每天记录小鼠体重，对小鼠异常症状进行监测。所有试验管理均按照南京农业大学动物实验动物管理条例进行。70 SPF grade female C57BL/6 mice aged 6-8 weeks were purchased from Yangzhou University. The initial body weight was 16.25 ± 0.25 g. After the mice were adaptively reared in sterile cages (7 groups, 10 mice/group) for one week, the ambient temperature was kept at 22 ± 2 °C, and the relative humidity was 45% to 60% for 12 hours. Light alternates day and night. Food and water were freely consumed throughout the experiment, the bedding was changed every 3 days, the body weight of the mice was recorded every day, and the abnormal symptoms of the mice were monitored. All experimental management was carried out in accordance with the regulations of Nanjing Agricultural University for the management of experimental animals.

赭曲霉毒素A(OTA)购自Sigma公司(美国)，环孢霉素A(CsA)购自 Selleck公司(中国)，纯度均>99％。BCA蛋白浓度测定试剂盒、RIPA和PMSF 配制蛋白裂解液、蛋白预染marker均购自碧云天生物技术有限公司。Tween20、 4％甲醛、4％多聚甲醛、2.5％异氟烷、甲醇、NaCl、KCl、KH₂PO₄、NaHCO₃、 Na₂HPO₄·12H₂O、95％酒精、氯仿、异丙醇(分析纯)均购自南京寿德生物技术有限公司。β-actin一抗和抗兔二抗均购自美国Cell SignalingTechnology公司。 TGF-β1、Vimentin和α-SMA购自北京博奥森生物技术公司。Ochratoxin A (OTA) was purchased from Sigma Company (USA), cyclosporine A (CsA) was purchased from Selleck Company (China), and the purity was >99%. BCA protein concentration assay kit, RIPA and PMSF prepared protein lysate, and protein prestained marker were purchased from Biyuntian Biotechnology Co., Ltd. Tween20, 4% formaldehyde, 4% paraformaldehyde, 2.5% isoflurane, methanol, NaCl, KCl, KH ₂ PO ₄ , NaHCO ₃ , Na ₂ HPO ₄ 12H ₂ O, 95% alcohol, chloroform, isopropanol (analytical grade) were purchased from Nanjing Shoude Biotechnology Co., Ltd. β-actin primary antibody and anti-rabbit secondary antibody were purchased from Cell Signaling Technology, USA. TGF-β1, Vimentin and α-SMA were purchased from Beijing Boaosen Biotechnology Company.

1.2 模型建立和分组1.2 Model building and grouping

小鼠共70只(体重为16.25±0.25g)，分为7组，每组10只，分别为：A total of 70 mice (body weight 16.25±0.25g) were divided into 7 groups of 10 mice, respectively:

溶剂对照组(Control)Solvent Control (Control)

单独低剂量OTA组(0.25mg/kg.d^-1OTA)Low-dose OTA group alone (0.25mg/kg.d ^-1 OTA)

单独高剂量OTA组(0.5mg/kg.d^-1OTA)High-dose OTA group alone (0.5mg/kg.d ^-1 OTA)

OTA与CsA联合1组(0.25mg/kg.d^-1OTA+10mg/kg.d^-1CsA)OTA and CsA combined group 1 (0.25mg/kg.d ^-1 OTA+10mg/kg.d ^-1 CsA)

单独CsA阴性对照组(20mg/kg.d^-1CsA)CsA negative control group alone (20mg/kg.d ^-1 CsA)

OTA与CsA联合2组(0.25mg/kg.d^-1OTA+20mg/kg.d^-1CsA)OTA and CsA combined 2 groups (0.25mg/kg.d ^-1 OTA+20mg/kg.d ^-1 CsA)

阳性造模组(30mg/kg.d^-1CsA)。Positive modeling group (30 mg/kg.d ^-1 CsA).

OTA与CsA采取腹腔注射的方式。溶剂对照组给予等量的溶剂进行腹腔注射，单独赭曲霉毒素A的给药方式为从试验开始的第一天起每间隔一天给予赭曲霉毒素A，其剂量为0.25和0.5mg/kg.d^-1，共28天给药天数为14天；OTA与 CsA联合1组和2组除每隔一天给予0.25mg/kg.d^-1的赭曲霉毒素A之外，从试验开始的第一天起每天同时给予环孢霉素A，其剂量分别为10mg/kg.d^-1、20 mg/kg.d^-1，共28天给药天数为28天；阳性造模组从试验开始的第一天起每天单独给予环孢霉素A，其剂量为30mg/kg.d^-1，共28天给药天数为28天。所有实验动物于试验第29天眼球采血，采取肾脏、肝脏、心脏器官组织，一部分-80℃冻存，用于提取组织蛋白进行Western blotting检测，其余部分用10％福尔马林固定用于组织染色。检测脏器指数、血清尿素氮和组织纤维化等相关指标的变化。OTA and CsA were administered by intraperitoneal injection. The solvent control group was given the same amount of solvent for intraperitoneal injection, and ochratoxin A alone was administered with ochratoxin A every other day from the first day of the experiment at doses of 0.25 and 0.5 mg/kg.d ^-1 , a total of 28 days of administration for 14 days; OTA combined with CsA in groups 1 and 2, except that 0.25 mg/kg.d ^-1 of Ochratoxin A was administered every other day, from the first day of the experiment Cyclosporine A was given at the same time every day, the doses were 10 mg/kg.d ^-1 and 20 mg/kg.d ^-1 respectively, for a total of 28 days, the number of days of administration was 28 days; Cyclosporine A was administered alone every day from the first day at a dose of 30 mg/kg.d ^-1 for a total of 28 days of administration for 28 days. Blood was collected from the eyes of all experimental animals on the 29th day of the experiment, and kidney, liver, and heart organ tissues were collected, a part of which was frozen at -80°C for extraction of tissue protein for Western blotting detection, and the rest was fixed with 10% formalin for tissue use dyeing. Changes of related indexes such as organ index, serum urea nitrogen and tissue fibrosis were detected.

1.3 小鼠组织染色1.3 Mouse tissue staining

取小鼠肾脏、肝脏、心脏器官用4％多聚甲醛进行固定，常规石蜡包埋切片，厚度为5μm，进行肾组织、肝脏和心脏组织HE染色，肾脏组织还进行Masson、免疫组化染色。Kidneys, livers, and hearts of mice were fixed with 4% paraformaldehyde, and routinely embedded in paraffin into sections with a thickness of 5 μm. The kidneys, livers and hearts were stained with HE, and the kidneys were also stained with Masson and immunohistochemistry.

1.3.1 HE染色1.3.1 HE staining

石蜡切片脱蜡，依次将切片放入二甲苯I 20min-二甲苯II 20min-无水乙醇I5min-无水乙醇II 5min-75％酒精5min，自来水洗。之后切片入苏木素染液染3-5 min，自来水洗，分化液分化，自来水洗，返蓝液返蓝，流水冲洗。再将切片依次入85％、95％的梯度酒精脱水各5min，入伊红染液中染色5min。再依次放入无水乙醇I 5min-无水乙醇II 5min-无水乙醇III 5min-二甲苯I 5min-二甲苯II 5min透明，中性树胶封片。最后显微镜镜检，图像采集分析。The paraffin sections were dewaxed, and the sections were sequentially placed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min, and washed with tap water. After that, the sections were stained with hematoxylin staining solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, returned to blue with blue solution, and rinsed with running water. The sections were then dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin staining solution for 5 min. Then put in anhydrous ethanol I 5min-absolute ethanol II 5min-absolute ethanol III 5min-xylene I 5min-xylene II 5min in sequence, transparent, neutral resin seal. The final microscope examination, image acquisition and analysis.

1.3.2Masson染色1.3.2 Masson staining

石蜡切片脱蜡，依次将切片放入二甲苯I 20min-二甲苯II 20min-无水乙醇I5min-无水乙醇II 5min-75％酒精5min，自来水洗。之后切片重铬酸钾浸泡过夜，自来水洗。再将切片用铁苏木素A液与B液等比混合成铁苏木素染液，切片入铁苏木素3min，自来水洗，分化液分化，自来水洗，返蓝液返蓝，流水冲洗。切片入丽春红酸性品红浸染5-10min，自来水漂洗。磷钼酸水溶液浸染1-3min。磷钼酸之后不用水洗，直接入苯胺蓝染液染3-6min。之后切片用1％冰醋酸分化，两缸无水乙醇脱水。然后切片放入第三缸无水乙醇5min，二甲苯5min透明，中性树胶封片。最后显微镜镜检，图像采集分析。The paraffin sections were dewaxed, and the sections were sequentially placed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min, and washed with tap water. Afterwards, the slices were soaked in potassium dichromate overnight and washed with tap water. Then, the slices were mixed with iron hematoxylin solution A and solution B in equal ratio to form iron hematoxylin staining solution, sliced into iron hematoxylin for 3 min, washed with tap water, differentiated with differentiation solution, washed with tap water, returned to blue with blue solution, and rinsed with running water. Dip the slices into Ponceau red acid fuchsin for 5-10 min, and rinse with tap water. Phosphomolybdic acid aqueous solution for 1-3min. After phosphomolybdic acid, do not wash with water, directly into aniline blue dye solution for 3-6min. The sections were then differentiated with 1% glacial acetic acid and dehydrated in two cylinders of absolute ethanol. Then the sections were placed in the third cylinder of absolute ethanol for 5 minutes, xylene was transparent for 5 minutes, and the sections were sealed with neutral gum. The final microscope examination, image acquisition and analysis.

1.3.3 免疫组化1.3.3 Immunohistochemistry

石蜡切片脱蜡，依次将切片放入二甲苯I15min-二甲苯II 15min-二甲苯III15min-无水乙醇I 5min-无水乙醇II 5min-85％酒精5min-75％酒精5min-蒸馏水洗。组织切片置于盛满柠檬酸抗原修复缓冲液(pH 6.0)的修复盒中于微波炉内进行抗原修复，中火8min至沸，停火8min保温再转中低火7min，此过程中应防止缓冲液过度蒸发，切勿干片。自然冷却后将玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次，每次5min。之后切片放入3％双氧水溶液，室温避光孵育25min，将玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次，每次 5min，用于阻断内源性过氧化物酶。在组化圈内滴加3％BSA均匀覆盖组织，室温封闭30min(一抗是山羊来源的用兔血清封闭，其他来源的用BSA封闭)。轻轻甩掉封闭液，在切片上滴加PBS按一定比例配好的一抗，切片平放于湿盒内4℃孵育过夜。玻片置于PBS(pH7.4)中在脱色摇床上晃动洗涤3次，每次 5min。切片稍甩干后在圈内滴加与一抗相应种属的二抗(HRP标记)覆盖组织，室温孵育50min。结束后将玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3 次，每次5min。切片稍甩干后在圈内滴加新鲜配制的DAB显色液，显微镜下控制显色时间，阳性为棕黄色，自来水冲洗切片终止显色。苏木素复染3min左右，自来水洗，苏木素分化液分化数秒，自来水冲洗，苏木素返蓝液返蓝，流水冲洗。将切片依次放入75％酒精5min-85％酒精5min-无水乙醇I 5min-无水乙醇II 5min-二甲苯I 5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。最后显微镜镜检，图像采集分析。The paraffin sections were dewaxed, and the sections were washed in sequence with xylene I 15min-xylene II 15min-xylene III 15min-absolute ethanol I 5min-absolute ethanol II 5min-85% alcohol 5min-75% alcohol 5min-distilled water. The tissue sections were placed in a repair box filled with citric acid antigen retrieval buffer (pH 6.0) for antigen retrieval in a microwave oven, medium heat for 8 minutes to boiling, cessation of fire for 8 minutes, and then medium-low heat for 7 minutes. During this process, buffer solution should be avoided. Excessive evaporation, do not dry flakes. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After that, the slices were placed in 3% hydrogen peroxide solution, incubated at room temperature for 25 min in the dark, and the slides were placed in PBS (pH 7.4) and washed 3 times on a decolorizing shaker for 5 min each time to block endogenous peroxidase. . In the histochemical circle, 3% BSA was added dropwise to cover the tissue evenly, and the cells were blocked at room temperature for 30 minutes (primary antibodies from goat were blocked with rabbit serum, and those from other sources were blocked with BSA). Gently shake off the blocking solution, drop the primary antibody prepared in a certain proportion of PBS on the slices, and incubate the slices in a humid box at 4°C overnight. The slides were washed three times in PBS (pH 7.4) with shaking on a destaining shaker, 5 min each time. After the sections were slightly dried, a secondary antibody (HRP-labeled) corresponding to the primary antibody was added dropwise in the circle to cover the tissue, and incubated at room temperature for 50 min. After the end, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After the slices were slightly dried, the freshly prepared DAB color developing solution was added dropwise in the circle, and the color developing time was controlled under the microscope. Hematoxylin was counterstained for about 3 min, washed with tap water, differentiated with hematoxylin differentiation solution for a few seconds, rinsed with tap water, returned to blue with hematoxylin-returning solution, and rinsed with running water. Put the slices into 75% alcohol 5min-85% alcohol 5min-absolute ethanol I 5min-absolute ethanol II 5min-xylene 15min in turn to dehydrate and transparent, take out the slices from the xylene and air dry, and seal with neutral gum. piece. The final microscope examination, image acquisition and analysis.

1.4 血清生化及肾功能损伤检测1.4 Detection of serum biochemical and renal function damage

将采集到的血清稀释作血清生化和肾功能检测，全自动生化分析仪测定血清中谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)、尿素氮(BUN) 和肌酐水平，测定出后根据稀释比例计算出原始水平。对采集到的尿液使用手持式折射仪进行尿比重(UG)和尿蛋白(UP)的测定。采用Western blotting方法检测肾损伤相关因子蛋白的表达。The collected serum was diluted for serum biochemical and renal function tests, and an automatic biochemical analyzer was used to determine serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), urea nitrogen (BUN) and The creatinine level is determined and the original level is calculated according to the dilution ratio. Urine specific gravity (UG) and urinary protein (UP) were measured using a hand-held refractometer on the collected urine. The expression of kidney injury-related factor protein was detected by Western blotting method.

1.5 Western Blot分析1.5 Western Blot analysis

30％丙烯酰胺(Acrylamide)100mL配制方法为29g Acrylamide、1g BIS，定容后用0.45μm的滤器除去杂质，用棕色瓶子存储于4℃。1M Tris-HCl (pH＝6-8)2.25mL、Glycerol5mL、SDS 0.5g、溴酚蓝5mg配制5×SDS-PAGE loading buffer(使用前添加5％的β-巯基乙醇)。Tris Base 60.6g、Glycine 144.1g、 SDS 5g用1L的容量瓶定容，配置成1L的5×SDS-PAGE电泳缓冲液，室温保存。如下表1配制12％的SDS-PAGE分离胶和5％浓缩胶。The preparation method of 100 mL of 30% acrylamide is 29 g of Acrylamide and 1 g of BIS. After constant volume, use a 0.45 μm filter to remove impurities, and store at 4° C. in a brown bottle. 1M Tris-HCl (pH=6-8) 2.25mL, Glycerol 5mL, SDS 0.5g, bromophenol blue 5mg to prepare 5×SDS-PAGE loading buffer (add 5% β-mercaptoethanol before use). Tris Base 60.6g, Glycine 144.1g, and SDS 5g were made up with a 1L volumetric flask, prepared into 1L of 5×SDS-PAGE running buffer, and stored at room temperature. Prepare 12% SDS-PAGE separating gel and 5% stacking gel as shown in Table 1 below.

表1 SDS-PAGE分离胶和浓缩胶配方Table 1 SDS-PAGE separating gel and stacking gel formulations

20mg组织加入400-500μL细胞裂解液裂解细胞，收集细胞置于1.5mL EP 管中，用超声波细胞破碎仪破碎细胞，4℃12000rpm离心15min，取上清，采用BCA法测蛋白浓度。蛋白与loading buffer按4：1混合，于95℃变性5min，于-80℃可长期保存待用。Add 400-500 μL of cell lysate to 20 mg of tissue to lyse the cells, collect the cells and place them in a 1.5 mL EP tube, disrupt the cells with an ultrasonic cell disrupter, centrifuge at 12,000 rpm at 4°C for 15 min, take the supernatant, and measure the protein concentration by BCA method. The protein and loading buffer were mixed at a ratio of 4:1, denatured at 95°C for 5 minutes, and stored at -80°C for long-term use.

1.7 数据分析1.7 Data Analysis

采用SPSS 18.0软件对试验数据进行差异显著性检验用单因素方差 (One-wayANOVA)统计分析。应用最小显著差法(Least-significant difference) 分别对各均值进行多重比较，当P<0.05时为差异有统计学意义，使用GraphPad Prism 5软件进行作图。SPSS 18.0 software was used to perform statistical analysis on the test data with one-way ANOVA. The Least-significant difference method was used to perform multiple comparisons on each mean, and when P<0.05, the difference was considered statistically significant, and GraphPad Prism 5 software was used for graphing.

2.结果2. Results

2.1 小鼠的一般情况和脏器指数2.1 General condition and organ index of mice

试验过程中，各组小鼠表现有明显不同，试验第一周小鼠外观体征均正常，粪便呈颗粒状，垫料干燥无异味。在处理两周后，置换垫料时发现与对照组和空白组相比，联合组和阳性对照组小鼠垫料更加潮湿，酸臭气味更加明显，但小鼠毛色未见明显异常。不仅如此，单独给予高剂量环孢霉素A(30mg/kg.d^-1)的阳性造模组小鼠出现神经症状，表现为转圈运动，日增重率在试验初期逐渐上升，第13天后日增重率开始下降，且体重日增重率低于联合组，直至试验终点。而 OTA与CsA联合组小鼠没有出现任何神经症状，体重随处理时间逐渐上升，未见下降(见图3的A部分)。During the test, the performance of mice in each group was significantly different. In the first week of the test, the appearance and signs of the mice were normal, the feces were granular, and the litter was dry and had no peculiar smell. After two weeks of treatment, when the litter was replaced, it was found that compared with the control group and the blank group, the litter of the mice in the combined group and the positive control group was more humid, and the sour odor was more obvious, but the coat color of the mice was not significantly abnormal. Not only that, the mice in the positive model group given high dose of cyclosporine A (30 mg/kg.d ^-1 ) alone developed neurological symptoms, manifested as circling movements, and the daily weight gain rate gradually increased at the beginning of the test, and after the 13th day. The daily weight gain rate began to decrease, and the daily weight gain rate was lower than that of the combination group until the end of the trial. However, the mice in the combined OTA and CsA group did not have any neurological symptoms, and the body weight gradually increased with the treatment time, but did not decrease (see Part A of Figure 3).

2.2 小鼠肾功能变化2.2 Changes in renal function in mice

根据肾脏指数结果显示，0.5mg/kg.d^-1OTA处理组出现显著性下降(见图1 的A部分)，肾脏HE染色结果显示，在经过赭曲霉毒素A单独处理后，0.25 mg/kg.d^-1OTA处理组未出现显著组织病变，而0.5mg/kg.d^-1OTA处理组肾脏组织中发现炎性细胞浸润、肾小球分叶以及肾小管损伤(见图1的B部分)。于是选择对小鼠无显著性影响的OTA剂量与环孢霉素A联合处理，肾脏脏器指数结果显示，在赭曲霉毒素A(0.25mg/kg.d^-1)与环孢霉素A(20mg/kg.d^-1)联合的情况下小鼠肾脏指数显著降低且与单独给予高剂量环孢霉素A(30mg/kg.d^-1) 的阳性造模组相比无显著性差异，效果优于低剂量环孢霉素A(10mg/kg.d^-1)与赭曲霉毒素的联合(见图1的C部分)。HE染色结果显示，在赭曲霉毒素A(0.25 mg/kg.d^-1)与环孢霉素A(20mg/kg.d^-1)联合的情况下，肾脏组织中肾小管细胞碎裂、炎性细胞浸润以及肾小球轻度萎缩。Masson染色结果显示，在赭曲霉毒素A(0.25mg/kg.d^-1)与环孢霉素A(20mg/kg.d^-1)联合的情况下，胶原蛋白蓝染颜色较深，范围更广，与阳性造模组相比，蓝染颜色更深(见图1的D部分)。通过对尿液、血清生化及肾损伤相关蛋白的检测发现，与对照组相比，赭曲霉毒素A(0.25mg/kg.d^-1)与环孢霉素A(20mg/kg.d^-1)联合时尿液的尿比重和尿蛋白显著升高(见图2的A部分)，血清中尿素氮和肌酐含量显著升高(见图2 的B部分)，效果与阳性造模组所形成的肾损伤指标相比无显著性差异；肾损伤相关蛋白TGF-β1和α-SMA的表达显著增加，Vimentin的表达显著下降，且效果优于低剂量环孢霉素A(10mg/kg.d^-1)与赭曲霉毒素A的联合(见图2的C 和D部分)。在单独环孢霉素A(20mg/kg.d^-1)阴性对照组中，并未见到上述所有变化，由此可见赭曲霉毒素A在改变小鼠肾脏功能方面起到了关键作用。According to the results of kidney index, the 0.5mg/kg.d ^-1 OTA treatment group showed a significant decrease (see part A of Figure 1). The results of kidney HE staining showed that after ochratoxin A alone treatment, 0.25 mg/kg .d ^-1 OTA treatment group did not show significant tissue lesions, while inflammatory cell infiltration, glomerular lobulation and renal tubular damage were found in the kidney tissue of 0.5 mg/kg .d ^-1 OTA treatment group (see Figure 1, part B ). Therefore, the dose of OTA that had no significant effect on mice was selected to be combined with cyclosporine A. The results of the kidney organ index showed that in the presence of ochratoxin A (0.25 mg/kg.d ^-1 ) and cyclosporine A ( 20mg/kg.d ^-1 ) combined with the mouse kidney index was significantly reduced and there was no significant difference compared with the positive model group given high-dose cyclosporine A (30mg/kg.d ^-1 ) alone, The effect was better than the combination of low-dose cyclosporine A (10 mg/kg.d ^-1 ) and ochratoxin (see Part C of Figure 1 ). The results of HE staining showed that in the case of the combination of ochratoxin A (0.25 mg/kg.d ^-1 ) and cyclosporine A (20 mg/kg.d ^-1 ), the renal tubular cells were fragmented, inflammatory Sex cell infiltration and mild glomerular atrophy. Masson staining results showed that in the case of ochratoxin A (0.25mg/kg.d ^-1 ) combined with cyclosporine A (20mg/kg.d ^-1 ), collagen blue staining was darker and the range was wider Broad, blue staining was darker compared to the positive model group (see Figure 1, part D). By the detection of urine, serum biochemical and kidney injury-related proteins, compared with the control group, ochratoxin A (0.25mg/kg.d ^-1 ) and cyclosporine A (20mg/kg.d ^{-1 )} ) combined with urine specific gravity and urine protein significantly increased (see Figure 2 part A), serum urea nitrogen and creatinine content significantly increased (see Figure 2 part B), the effect and positive modeling group formed There was no significant difference in the renal injury indexes between the two groups; the expressions of renal injury-related proteins TGF-β1 and α-SMA were significantly increased, and the expression of Vimentin was significantly decreased, and the effect was better than that of low-dose cyclosporine A (10 mg/kg.d ^-1 ) Combination with Ochratoxin A (see Figure 2, Parts C and D). In the negative control group of cyclosporine A (20 mg/kg.d ^-1 ) alone, all the above changes were not seen, which shows that ochratoxin A plays a key role in changing the renal function of mice.

2.3 小鼠其他脏器毒副作用的变化2.3 Changes in the side effects of other organs in mice

对小鼠肝脏和心脏的损伤相关指标进行检测，结果显示，与阳性造模组相比较，心脏和肝脏的脏器指数无显著性变化(见图3的B和C部分)，但在HE 染色观察组织病理变化时发现，赭曲霉毒素A(0.25mg/kg.d^-1)与环孢霉素A(20 mg/kg.d^-1)联合缓解了阳性造模组中对心脏和肝脏的组织损伤(见图3的D部分)。另外，代表肝脏和心脏损伤的特异性酶也发生了变化，赭曲霉毒素A(0.25 mg/kg.d^-1)与环孢霉素A(20mg/kg.d^-1)联合组与阳性造模组相比，ALT和CK 都被显著降低，ALP和LDH都有降低的趋势(见图4的A和B部分)。除心脏中TGF-β1蛋白的表达与阳性造模组无显著性差异，赭曲霉毒素A(0.25 mg/kg.d^-1)与环孢霉素A(20mg/kg.d^-1)联合能显著降低心脏中纤维化相关因子α-SMA和肝脏中纤维化相关因子TGF-β1和α-SMA的蛋白表达水平(见图4的 C-F部分)。The injury-related indicators of the liver and heart of mice were detected, and the results showed that compared with the positive model group, there was no significant change in the organ index of the heart and liver (see parts B and C of Figure 3), but in HE staining When observing histopathological changes, it was found that the combination of ochratoxin A (0.25 mg/kg.d ^-1 ) and cyclosporine A (20 mg/kg.d ^-1 ) alleviated the effects on the heart and liver in the positive model group. Tissue damage (see Figure 3, part D). In addition, specific enzymes representing liver and heart injury also changed, and the combination group of ochratoxin A (0.25 mg/kg.d ^-1 ) and cyclosporine A (20 mg/kg.d ^-1 ) was associated with positive Compared with the modules, both ALT and CK were significantly decreased, and both ALP and LDH tended to decrease (see Parts A and B of Figure 4). Except that the expression of TGF-β1 protein in the heart was not significantly different from the positive model group, the combination of ochratoxin A (0.25 mg/kg.d ^-1 ) and cyclosporine A (20 mg/kg.d ^-1 ) could The protein expression levels of the fibrosis-related factors α-SMA in the heart and the fibrosis-related factors TGF-β1 and α-SMA in the liver were significantly decreased (see Figure 4, section CF).

2.4 成本经济效益2.4 Cost-effectiveness

本实施例中环孢霉素A购自Selleck(中国，上海蓝木化工有限公司)，价格为786元人民币50mg；赭曲霉毒素A购自Sigma-Aldrich(美国圣路易斯)，价格为4700元人民币5mg，按照上述制备方法进行操作，小鼠平均体重为20g，以下述动物模型建立方法为例：采取0.25mg/kg赭曲霉毒素A每隔一天进行腹腔注射，20mg/kg环孢霉素A每天进行腹腔注射，试验周期为28天，每只老鼠建模成本为241.86元人民币；而现有技术中所报道的小鼠慢性环孢霉素肾病模型使用的环孢霉素A浓度为30mg/kg，试验周期28天，此方法所需成本为264.10 元人民币。在成本水平上，上述模型较现有技术降低了22.24元人民币/每只老鼠。因此，本实施例在改善了原模型存在的副作用的同时，并极大降低了成本。In this example, cyclosporine A was purchased from Selleck (China, Shanghai Bluewood Chemical Co., Ltd.), and the price was 786 yuan for 50 mg; Ochratoxin A was purchased from Sigma-Aldrich (St. Louis, USA), and the price was 4,700 yuan for 5 mg, According to the above preparation method, the average body weight of mice is 20g, and the following animal model establishment method is used as an example: 0.25mg/kg ochratoxin A is used for intraperitoneal injection every other day, and 20mg/kg cyclosporine A is administered intraperitoneally every day. Injection, the test period is 28 days, and the modeling cost per mouse is 241.86 yuan; while the concentration of cyclosporine A used in the mouse chronic cyclosporine nephropathy model reported in the prior art is 30 mg/kg. With a cycle of 28 days, the cost of this method is RMB 264.10. In terms of cost, the above model is 22.24 yuan/mouse lower than the existing technology. Therefore, the present embodiment greatly reduces the cost while improving the side effects of the original model.

实施例2Example 2

本实施例为一较佳的体外细胞慢性肾病模型的制备，其采用的细胞为人肾小管上皮细胞。This example is for the preparation of a preferred in vitro cellular chronic kidney disease model, and the cells used are human renal tubular epithelial cells.

1.材料和方法1. Materials and methods

1.1 主要材料和试剂1.1 Main materials and reagents

无污染的人肾皮质近曲小管上皮细胞传代细胞系HK-2购自上海歌凡生物科技有限公司，细胞在含有10％灭活胎牛血清，1％青链霉素的1640(Invitrogen, USA)培养基中培养，培养箱中温度为37℃，二氧化碳浓度为5％，pH值为7.2-7.4。 RPMI-1640培养基和胎牛血清均购自Gibco(Invitrogen)上海贸易有限公司。青链霉素混合液购自索莱宝生物科技有限公司。预染蛋白Marker购自德国Thermo Fisher公司。β-actin一抗和抗兔二抗均购自美国Cell Signaling Technology公司。 TGF-β1、Vimentin和α-SMA购自北京博奥森生物技术公司。The contamination-free human renal cortex proximal convoluted tubule epithelial cell line HK-2 was purchased from Shanghai Gefan Biotechnology Co., Ltd., and the cells were cultured in 1640 (Invitrogen, USA) containing 10% inactivated fetal bovine serum and 1% penicillin ) medium, the temperature in the incubator is 37°C, the carbon dioxide concentration is 5%, and the pH value is 7.2-7.4. Both RPMI-1640 medium and fetal bovine serum were purchased from Gibco (Invitrogen) Shanghai Trading Co., Ltd. The penicillin-streptomycin mixture was purchased from Soleibo Biotechnology Co., Ltd. Prestained protein markers were purchased from Thermo Fisher, Germany. β-actin primary antibody and anti-rabbit secondary antibody were purchased from Cell Signaling Technology Company of the United States. TGF-β1, Vimentin and α-SMA were purchased from Beijing Boaosen Biotechnology Company.

1.2 细胞培养1.2 Cell Culture

传代细胞系HK-2培养在含10％胎牛血清，100U/mL青霉素和100U/mL链霉素的RPMI-1640培养基中，培养温度为37℃，二氧化碳浓度为5％。OTA和 CsA用DMSO溶解成分别为2mg/mL和50mg/mL的溶液，分装至西林瓶中保存，-20℃储藏备用。最终毒素工作浓度用细胞培养基根据需要现用现配。The passaged cell line HK-2 was cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin at a temperature of 37°C and a carbon dioxide concentration of 5%. OTA and CsA were dissolved in DMSO to obtain solutions of 2 mg/mL and 50 mg/mL, respectively, which were packaged in vials and stored at -20°C for later use. Final toxin working concentration was prepared as needed in cell culture medium.

1.3 细胞毒性试验1.3 Cytotoxicity test

细胞以密度为1×10⁴个细胞/孔接种于96孔细胞板上培养24小时后，更换含4％血清的培养基，分别加入不同浓度的OTA(0、0.01、0.1、0.5、1、2、4、 8、10和20μg/mL)和不同浓度的CsA(0、1、2、4、6、8、10、20、30和40μg/mL)，共同培养48小时(对照组中含有0.25％终浓度的DMSO)。之后选择无毒性的 OTA浓度与无毒性低浓度CsA联合处理。在处理时间结束前3h，于每孔中加入 15μL 5mg/mL MTT溶液，继续培养3h后，弃去上清，每孔加入200μL DMSO，振荡4min后置于酶标仪上于主波长为490nm，次波长为655nm处测定OD值。每个处理组5个重复。The cells were seeded on 96-well cell plates at a density of 1×10 ⁴ cells/well for 24 hours, then the medium containing 4% serum was replaced, and different concentrations of OTA (0, 0.01, 0.1, 0.5, 1, 2, 4, 8, 10 and 20 μg/mL) and different concentrations of CsA (0, 1, 2, 4, 6, 8, 10, 20, 30 and 40 μg/mL), co-cultured for 48 hours (the control group contained 0.25% final concentration of DMSO). Then, the non-toxic OTA concentration and the non-toxic low-concentration CsA were selected for combined treatment. 3 hours before the end of the treatment time, 15 μL of 5 mg/mL MTT solution was added to each well, and after culturing for 3 hours, the supernatant was discarded, and 200 μL of DMSO was added to each well. The OD value was measured at the secondary wavelength of 655 nm. 5 replicates per treatment group.

1.4 乳酸脱氢酶活性试验1.4 Lactate dehydrogenase activity test

收集细胞培养基，4℃12000rpm离心15分钟，取上清于-20℃待测。使用乳酸脱氢酶(LDH)试剂盒检测，使用酶标仪于波长450nm处测定吸光度。一个单位的酶活性定义为相当于每分钟氧化1μmol还原型烟酰胺腺嘌呤二核苷酸的量。结果表示为对照组的百分比。每个处理组5个重复。The cell culture medium was collected, centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was collected at -20°C for testing. Lactate dehydrogenase (LDH) kit was used for detection, and absorbance was measured at a wavelength of 450 nm using a microplate reader. One unit of enzymatic activity was defined as the amount equivalent to the oxidation of 1 μmol of reduced nicotinamide adenine dinucleotide per minute. Results are expressed as a percentage of the control group. 5 replicates per treatment group.

1.5 Western Blot分析1.5 Western Blot analysis

同实施例1。Same as Example 1.

1.6 数据分析1.6 Data Analysis

采用SPSS 18.0软件对试验数据进行差异显著性检验用单因素方差 (One-wayANOVA)统计分析。应用最小显著差法(Least-significant difference) 分别对各均值进行多重比较，当P<0.05时为差异有统计学意义，使用GraphPad Prism 5软件进行作图。SPSS 18.0 software was used to perform statistical analysis on the test data with one-way ANOVA. The Least-significant difference method was used to perform multiple comparisons on each mean, and when P<0.05, the difference was considered statistically significant, and GraphPad Prism 5 software was used for graphing.

2.结果2. Results

2.1 赭曲霉毒素A和环孢霉素A浓度的筛选2.1 Screening of ochratoxin A and cyclosporine A concentrations

体外细胞试验中，通过单独赭曲霉毒素A与环孢霉素A处理结果显示，环孢霉素A在浓度为10μg/mL时存在显著细胞毒性，此时细胞活性显著降低，乳酸脱氢酶活性显著升高，赭曲霉毒素A在1μg/mL存在显著细胞毒性(见图5 的A-D部分)。因此选择无细胞毒性的环孢霉素A(2、4、6μg/mL)与0.5μg/mL 的赭曲霉毒素A进行联合。结果显示6μg/mL的环孢霉素A与0.5μg/mL的赭曲霉毒素A联合时显著降低了HK-2细胞的活性且显著升高了乳酸脱氢酶活性(见图5的E-F部分)。In the in vitro cell test, the results of single ochratoxin A and cyclosporine A treatment showed that cyclosporine A had significant cytotoxicity at a concentration of 10 μg/mL, at this time, the cell activity was significantly reduced, and the lactate dehydrogenase activity was significantly reduced. Significantly elevated, ochratoxin A was significantly cytotoxic at 1 μg/mL (see Figure 5, Parts A-D). Therefore, non-cytotoxic cyclosporine A (2, 4, 6 μg/mL) was selected in combination with 0.5 μg/mL ochratoxin A. The results showed that cyclosporine A at 6 μg/mL combined with ochratoxin A at 0.5 μg/mL significantly reduced the activity of HK-2 cells and significantly increased the activity of lactate dehydrogenase (see Figure 5, part E-F). .

2.2 无毒性浓度的赭曲霉毒素A和低浓度的环孢霉素A联合应用对HK-2细胞中肾损伤相关蛋白表达的影响2.2 The effect of combined use of nontoxic concentration of ochratoxin A and low concentration of cyclosporine A on the expression of kidney injury-related proteins in HK-2 cells

在体外细胞试验中，当低浓度的赭曲霉毒素A和环孢霉素A联合处理HK-2 细胞时，蛋白印迹结果显示，在低浓度的环孢霉素A(2、4、6μg/mL)与0.5μg/mL 的赭曲霉毒素A进行联合时，6μg/mL的环孢霉素A与0.5μg/mL的赭曲霉毒素 A与单独的环孢霉素A(10μg/mL)均可以显著降低肾损伤相关蛋白Vimentin 的表达，显著升高TGF-β1和α-SMA的蛋白表达水平，且二者无显著性差异，6 μg/mL的环孢霉素A与0.5μg/mL的赭曲霉毒素A的联合应用效果优于4μg/mL 的环孢霉素A与0.5μg/mL的赭曲霉毒素A的联合(见图6)。In in vitro cell experiments, when low concentrations of ochratoxin A and cyclosporine A were combined to treat HK-2 cells, the results of western blotting showed that at low concentrations of cyclosporine A (2, 4, 6 μg/mL) ) combined with 0.5 μg/mL ochratoxin A, 6 μg/mL cyclosporine A and 0.5 μg/mL ochratoxin A and cyclosporine A alone (10 μg/mL) can significantly Reduce the expression of kidney injury-related protein Vimentin, significantly increase the protein expression levels of TGF-β1 and α-SMA, and there is no significant difference between the two, 6 μg/mL cyclosporine A and 0.5 μg/mL Aspergillus ochra The combined application effect of toxin A was better than the combination of 4 μg/mL cyclosporine A and 0.5 μg/mL ochratoxin A (see Figure 6).

综合上述检测指标可以得出，本发明联合给予0.25mg/kg.d^-1赭曲霉毒素A 与20mg/kg.d^-1环孢霉素A，可以使得尿蛋白和尿比重升高，血清尿素氮和肌酐含量上升，肾小管等出现病理学变化，肾脏纤维化程度加深，与单独给予高剂量 30mg/kg.d^-1环孢霉素A形成的慢性环孢霉素肾病的变化过程相似，与此同时，减少环孢霉素A的用量显著降低了对心脏和肝脏的副作用，说明成功建立了慢性肾病模型，0.25mg/kg.d^-1赭曲霉毒素A与20mg/kg.d^-1环孢霉素A(或在体外 0.5μg/mL赭曲霉毒素A与6μg/mL环孢霉素A)联合后成功影响肾脏功能，并有效缓解高浓度环孢霉素A导致的副作用。Based on the above detection indicators, it can be concluded that the combined administration of 0.25 mg/kg.d ^-1 ochratoxin A and 20 mg/kg.d ^-1 cyclosporine A in the present invention can make urine protein and urine specific gravity increase, and serum urea Nitrogen and creatinine content increased, renal tubules and other pathological changes appeared, and the degree of renal fibrosis was ^deepened . At the same time, reducing the dosage of ^cyclosporine A significantly reduced the side effects on the heart and liver, indicating that a chronic ^kidney disease model was successfully established. Cyclosporine A (or 0.5 μg/mL ochratoxin A and 6 μg/mL cyclosporine A in vitro) successfully affected renal function and effectively alleviated the side effects caused by high concentrations of cyclosporine A.

通过上述实施例证明，本发明建立了的慢性肾病模型，可用于筛选治疗针对慢性肾病的药物及治疗方法。The above examples prove that the chronic kidney disease model established by the present invention can be used to screen drugs and treatment methods for chronic kidney disease.

以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。The specific embodiments of the present invention have been described above in detail, but they are only used as examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.