CN110664836A

Movatterモバイル変換

Info

Publication number: CN110664836A
Application number: CN201910957941.7A
Authority: CN
Inventors: 和君; 傅江南; 赵月; 齐春丽; 方梅霞; 宋琳亮; 邢会杰
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-10-10
Filing date: 2019-10-10
Publication date: 2020-01-10

Abstract

Translated fromChinese

本发明提供了一种树鼩急性肺损伤模型的建立方法及其应用，麻醉树鼩后通过气管内给药180～200mg/kg剂量的脂多糖溶液，当树鼩肺部损伤面积达到一半以上时，即构建得到所述的树鼩急性肺损伤动物模型。通过本发明的建立方法，树鼩在70小时后均可肺部损伤达到一半以上；在96～120小时树鼩在临床上表现出消瘦、呼吸困难等症状，树鼩肺部的损伤面积在70％以上，血氧指标可达200mmHg以下。本发明的建模方法高效、成功率高，可有效节约成本；模型周期在70小时以上，显著地为急性肺损伤拓宽了研究周期空间；所建立的树鼩动物模型不可自我修复和可逆性恢复，临床症状与人类疾病更为相似，具有良好的产业化前景。

The invention provides a method for establishing an acute lung injury model of tree shrew and its application. After anesthetizing the tree shrew, a dose of 180-200 mg/kg of lipopolysaccharide solution is administered through the trachea. When the damage area of the tree shrew's lung reaches more than half , namely to construct the described tree shrew acute lung injury animal model. Through the establishment method of the present invention, the lung damage of tree shrew can reach more than half after 70 hours; in 96 to 120 hours, tree shrew clinically shows symptoms such as weight loss and dyspnea, and the damage area of tree shrew lungs is 70% % above, the blood oxygen index can reach below 200mmHg. The modeling method of the invention is efficient, has a high success rate, and can effectively save costs; the model period is more than 70 hours, which significantly widens the research period space for acute lung injury; the established tree shrew animal model cannot be self-repairing and reversible recovery , the clinical symptoms are more similar to human diseases, and it has a good industrialization prospect.

Description

Translated fromChinese

一种树鼩急性肺损伤模型的建立方法及其应用A method for establishing a tree shrew acute lung injury model and its application

技术领域technical field

本发明涉及动物模型的建模方法，特别涉及一种树鼩急性肺损伤模型的建立方法及其应用。The invention relates to a modeling method of an animal model, in particular to a method for establishing an acute lung injury model of tree shrew and its application.

背景技术Background technique

急性肺损伤(acute lung injury,ALI)是全身性炎症反应在肺部的表现，其临床表现为肺部炎症、毛细血管通透性增加进而导致肺水肿的产生，随着病情的加剧，可进一步发展为呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)，造成广泛肺泡损伤为病理特征，表现为蛋白性肺泡水肿和低氧血症。脓毒血症和多发性创伤是ARDS的最常见病因，炎性介质和抗炎介质的平衡失调是ALI/ARDS发生发展的关键环节，急性肺损伤一直是重症医学与呼吸病学亟需解决的关键问题。Acute lung injury (ALI) is a manifestation of systemic inflammatory response in the lungs, and its clinical manifestations are pulmonary inflammation, increased capillary permeability, which leads to the generation of pulmonary edema. The development of respiratory distress syndrome (ARDS), resulting in extensive alveolar damage as a pathological feature, manifested as proteinaceous alveolar edema and hypoxemia. Sepsis and multiple trauma are the most common causes of ARDS, and the imbalance of inflammatory mediators and anti-inflammatory mediators is a key link in the development of ALI/ARDS. Acute lung injury has always been an urgent problem in critical care medicine and respiratory medicine. The key issue.

现在常用的急性肺损伤动物模型有油酸诱导、机械通气诱导和脂多糖(Lipopolysaccharide,LPS)诱导的动物模型。其中脂多糖诱导方法因为操作简单、可行性高、动物存活率高，是目前国内外使用较多的制备急性肺损伤动物模型的方法。The commonly used animal models of acute lung injury include oleic acid-induced, mechanical ventilation-induced and lipopolysaccharide (LPS)-induced animal models. Among them, the lipopolysaccharide induction method is the most widely used method for preparing acute lung injury animal models at home and abroad because of its simple operation, high feasibility and high animal survival rate.

目前急性肺损伤研究最常用的模型动物是啮齿类和灵长类动物。由于啮齿类在进化上与人类相距甚远，以其为研究对象所得的实验数据对临床的指导意义不大；灵长类动物由于进化程度高，与人类亲缘关系最近，是目前实验研究中与人类最为相似的理想动物模型。但是它们价格过高，繁殖周期长，兼之动物伦理方面的原因，未能广泛应用。The most commonly used model animals for acute lung injury research are rodents and primates. Since rodents are far from humans in evolution, the experimental data obtained from rodents as research objects are of little significance for clinical guidance; primates, due to their high degree of evolution, have the closest relationship with humans, and are the most closely related to humans in current experimental research. The most similar ideal animal model for humans. However, they are too expensive, have a long breeding cycle, and have not been widely used for reasons of animal ethics.

目前脂多糖制备急性肺损伤模型中最常用的是小鼠模型。常用的诱导方法如下：将小鼠禁食12h，给予20mg/kg或10mg/kg LPS气管插管，24h采取样本；但目前的建模方法中，24h的时间主要检测的是细胞因子的变化情况。因此，剂量的多少影响小鼠的死亡时间、细胞因子的变化水平等。这种造模方法有诸多弊端：其一，实验小鼠与人类种属差异较大，所得实验数据指导意义不大；其二，实验小鼠的肺部自愈能力强，使得临床药物的筛选常常无效；其三，小鼠肺部血管内缺乏巨噬细胞(PIM)，肺部损伤反应相对较轻；其四，未有明显的呼吸困难症状，会突然死亡(die of shock)，因此，此模型只能检测ALI前期的炎症因子浸润期。At present, the mouse model is the most commonly used lipopolysaccharide for the preparation of acute lung injury model. The commonly used induction methods are as follows: the mice are fasted for 12 hours, given 20 mg/kg or 10 mg/kg LPS tracheal intubation, and samples are taken within 24 hours; however, in the current modeling method, the time of 24 hours is mainly to detect the changes of cytokines. . Therefore, the amount of the dose affects the death time of the mice, the change level of cytokines and so on. This modeling method has many drawbacks: first, the experimental mice are quite different from human species, and the experimental data obtained are of little significance; second, the lungs of the experimental mice have strong self-healing ability, which makes the screening of clinical drugs It is often ineffective; thirdly, the lack of macrophages (PIM) in the pulmonary blood vessels of mice has relatively mild lung injury; This model can only detect the inflammatory factor infiltration phase in the pre-ALI period.

临床上判定急性肺损伤的金标准如下：在排除心脏原因下由明确的诱因导致①CT改变或②血气分析(氧合指数(PaO₂/FiO₂)＜300mmHg为ALI，PaO₂/FiO₂＜200mmHg为ARDS)。但目前大多数的急性肺损伤动物模型以ALI的I期炎症因子的检测为主，周期均少于72h；此外啮齿类动物ALI模型通常会突然死亡，难以有明显的临床症状等。而在动脉血的检测研究方面，中大型动物模型如兔、犬、猪、羊模型能满足一定的取血量，但犬和兔体内缺乏肺血管内巨噬细胞(pulmonary intravascular macrophages,PIM)，致使其肺部损伤反应降低；猪、羊以反复肺泡灌洗，油酸模型居多。The gold standard for clinically judging acute lung injury is as follows: 1) CT changes or 2) blood gas analysis (oxygenation index (PaO₂ /FiO₂ ) <300mmHg is ALI, PaO₂ /FiO₂ <200mmHg, after excluding cardiac causes. for ARDS). However, most of the current acute lung injury animal models are mainly based on the detection of phase I inflammatory factors of ALI, and the cycle is less than 72 hours; in addition, rodent ALI models usually die suddenly, and it is difficult to have obvious clinical symptoms. In terms of arterial blood detection and research, medium and large animal models such as rabbit, dog, pig, and sheep models can meet a certain amount of blood collection, but dogs and rabbits lack pulmonary intravascular macrophages (PIM). Resulting in reduced lung injury response; pigs and sheep were subjected to repeated bronchoalveolar lavage and oleic acid models were mostly used.

树鼩作为低等灵长类动物，在模拟人类疾病方面更具有亲缘优势，可媲美灵长类动物，在生命医药领域中展示出了其他物种无法比拟的优势，已在病毒性感染、免疫相关疾病、癌症、肝炎感染模型、树鼩神经系统疾病等疾病研究中得到成功应用。As a lower primate, tree shrew has a genetic advantage in simulating human diseases, comparable to primates, and has demonstrated incomparable advantages in the field of life medicine. It has been used in viral infections, immune-related diseases It has been successfully applied in the research of diseases, cancer, hepatitis infection models, tree shrew nervous system diseases and other diseases.

但是树鼩的成本在1500～2000元/只，药物消耗约为600元/只，每只树鼩造模的平均成本在2100～2600元/只。虽然基于树鼩构建的动物病理模型在一些疾病研究已有应用，但尚未见树鼩急性肺损伤的模型的相关报道。不同疾病的关注焦点、检测指标大相径庭；而不同动物属种的差异给造模方法的稳定性、造模剂量适应性等带来了众多挑战，进一步增大了研究难度。However, the cost of tree shrew is 1500-2000 yuan per piece, the drug consumption is about 600 yuan per piece, and the average cost of modeling each tree shrew is 2100-2600 yuan per piece. Although animal pathological models based on tree shrew have been used in some disease studies, there are no reports on the model of tree shrew acute lung injury. The focus of attention and detection indicators of different diseases are quite different; and the differences of different animal species have brought many challenges to the stability of modeling methods and the adaptability of modeling doses, which further increased the difficulty of research.

因此，急性肺损伤疾病的研究和临床前药物评价的迫切需要与人类疾病症状更为相似的动物模型；如何高效地通过树鼩进行造模具有重要研究价值，对未来的临床应用将具有重要的指导意义。Therefore, there is an urgent need for animal models that are more similar to human disease symptoms in the research of acute lung injury diseases and preclinical drug evaluation; how to efficiently make models by tree shrew has important research value, which will be important for future clinical applications. Guiding significance.

发明内容SUMMARY OF THE INVENTION

本发明的首要目的在于克服现有技术的缺点与不足，提供一种树鼩急性肺损伤动物模型的构建方法，使用该方法建立的树鼩模型症状明显，并且过程容易控制，稳定性好，重复性强。The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a method for constructing an animal model of tree shrew acute lung injury. Strong sex.

本发明的另一目的在于提供所述的树鼩急性肺损伤动物模型的应用。Another object of the present invention is to provide the application of the described tree shrew acute lung injury animal model.

本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:

一种树鼩急性肺损伤动物模型的构建方法，包括如下步骤：A method for constructing a tree shrew acute lung injury animal model, comprising the following steps:

(1)麻醉健康树鼩后，按180～200mg/kg的剂量进行气管内给药脂多糖溶液；(1) After anesthetizing healthy tree shrew, intratracheal administration of lipopolysaccharide solution was carried out at a dose of 180-200 mg/kg;

(2)给药后观察，当树鼩肺部损伤面积达到一半以上时，即构建得到所述的树鼩急性肺损伤动物模型。(2) Observation after administration, when the lung damage area of tree shrew reaches more than half, the animal model of acute lung injury of tree shrew is constructed and obtained.

步骤(1)中所述的健康树鼩优选为雄性成年树鼩，成年的雄性动物比雌性动物稳定，例如激素等方面；所述的健康树鼩优选为体重为120～160g的树鼩。The healthy tree shrew described in the step (1) is preferably a male adult tree shrew, and the adult male animal is more stable than the female animal, for example, in terms of hormones and the like; the healthy tree shrew is preferably a tree shrew with a body weight of 120-160 g.

所述的健康树鼩不含体内外寄生虫及体征正常。The healthy tree shrew did not contain internal and external parasites and had normal signs.

步骤(1)中所述的麻醉优选为深度麻醉状态。The anesthesia described in step (1) is preferably a deep anesthesia state.

步骤(1)中所述的麻醉的方法可采用呼吸麻醉法或注射麻醉法；所用的麻醉剂优选为戊巴比妥钠或异氟烷中的至少一种。The method of anesthesia described in the step (1) can adopt respiratory anesthesia method or injection anesthesia method; the anesthetic agent used is preferably at least one of pentobarbital sodium or isoflurane.

当所述的麻醉剂为戊巴比妥钠时，麻醉的具体方式优选为按树鼩体重×0.1％×3体积的剂量经腹腔注射3％戊巴比妥钠进行麻醉。When the anesthetic is pentobarbital sodium, the specific anesthesia method is preferably anesthesia by intraperitoneal injection of 3% pentobarbital sodium at a dose of tree shrew body weight × 0.1% × 3 volume.

当所述的麻醉剂为异氟烷时，麻醉的具体方式优选为：麻醉用量值为3.5，游离氧为0.8进行麻醉。When the anesthetic agent is isoflurane, the specific anesthesia method is preferably: the anesthesia dosage value is 3.5, and the free oxygen is 0.8 for anesthesia.

步骤(1)中所述的麻醉前，优选对树鼩禁食10～12小时，不禁水。Before the anesthesia described in step (1), the tree shrew is preferably fasted for 10 to 12 hours and cannot be restrained from water.

步骤(1)中所述的气管内给药优选为一次性完成给药。The intratracheal administration described in step (1) is preferably a one-time administration.

步骤(1)中所述的脂多糖溶液的给药体积优选为400～500μL，可使给药更为均匀。The administration volume of the lipopolysaccharide solution described in step (1) is preferably 400-500 μL, which can make administration more uniform.

步骤(1)中所述的气管内给药优选为气管导管内给药；优选为非暴露式气管内给药。The intratracheal administration described in step (1) is preferably intratracheal intubation administration; preferably non-exposed intratracheal administration.

步骤(1)中所述的气管内给药的具体操作优选为：将树鼩以背卧姿势固定于工作板，将工作板立起，将喉镜插入喉咙深部的气管开口处，施用浓度为180～200mg/kg的脂多糖溶液。The concrete operation of the intratracheal administration described in the step (1) is preferably: the tree shrew is fixed on the work board with the dorsal recumbent posture, the work board is erected, the laryngoscope is inserted into the trachea opening of the deep throat, and the administration concentration is 180～200mg/kg lipopolysaccharide solution.

所述的喉镜优选为儿童喉镜；进一步优选为将喉镜片的前端用胶布缠裹并保持喉镜灯可正常工作的儿童喉镜。The laryngoscope is preferably a children's laryngoscope; more preferably, it is a children's laryngoscope in which the front end of the laryngoscope blade is wrapped with adhesive tape and the laryngoscope lamp can work normally.

优选地，在完成施用脂多糖溶液后，立刻注入少量空气，以减少脂多糖溶液在给药容器上的残留。Preferably, a small amount of air is injected immediately after the administration of the lipopolysaccharide solution is completed to reduce the residue of the lipopolysaccharide solution on the administration container.

所述的少量空气优选为0.2mL；注入少量空气的次数优选为3次。The said small amount of air is preferably 0.2 mL; the number of times of injecting a small amount of air is preferably 3 times.

步骤(2)中所述的树鼩肺部损伤面积达到一半以上，即使不同的动物可能出现个体差异，通过本发明的构建方法树鼩一般在70小时后(多在70～120小时内)树鼩肺部损伤达到一半以上；部分树鼩在96～120小时在临床上表现出消瘦、呼吸困难等症状，树鼩肺部的损伤面积在70％以上，动脉血气分析的血氧指标氧合指数OI(PaO₂/FiO₂)可达200mmHg以下。The lung damage area of the tree shrew described in the step (2) reaches more than half, even if individual differences may occur in different animals, the tree shrew generally grows after 70 hours (mostly within 70 to 120 hours) by the construction method of the present invention. The lung damage of the shrew reached more than half; some tree shrew showed clinical symptoms such as weight loss and dyspnea within 96 to 120 hours. The damage area of the tree shrew's lung was more than 70%. The oxygenation index of the blood oxygen index of arterial blood gas analysis OI (PaO₂ /FiO₂ ) can reach below 200mmHg.

所述的树鼩急性肺损伤动物模型的构建方法在急性肺损伤或呼吸窘迫综合征的科学研究中的应用。The application of the method for constructing the tree shrew acute lung injury animal model in the scientific research of acute lung injury or respiratory distress syndrome.

所述的科学研究包括急性肺损伤的疾病发生发展机制研究和评价新型急性肺损伤治疗药物等等。The scientific research includes the research on the development mechanism of acute lung injury and the evaluation of new acute lung injury therapeutic drugs.

所述的应用包括当树鼩急性肺损伤动物模型临床上出现呼吸困难及消瘦症状，肺部病变在70％以上时，进行如下至少一项研究：The application includes at least one of the following studies when the animal model of acute lung injury in tree shrew has symptoms of dyspnea and weight loss clinically, and the lung lesions are more than 70%:

(1)剖检取材进行肺部病理评价和机制的研究；(1) Pulmonary pathological evaluation and mechanism research on autopsy materials;

(2)采集动脉血进行检测；所述的检测包括血气分析、血常规检验等；(2) collecting arterial blood for detection; the detection includes blood gas analysis, blood routine test, etc.;

(3)采集肺泡换洗液检测细胞因子水平。(3) Collecting alveolar change washing fluid to detect cytokine levels.

本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:

(1)本发明首次成功构建了低等灵长类动物树鼩的急性肺损伤模型，模型种属较现有技术中的实验动物具有明显的优势。利用本发明的树鼩急性肺损伤模型研究该病的发生发展机制和评价新型急性肺损伤治疗药物，对未来的临床应用具有重要的指导意义。(1) The present invention successfully constructs the acute lung injury model of the lower primate tree shrew for the first time, and the model species has obvious advantages over the experimental animals in the prior art. Using the tree shrew acute lung injury model of the present invention to study the occurrence and development mechanism of the disease and evaluate new acute lung injury therapeutic drugs has important guiding significance for future clinical applications.

(2)本发明的造模剂量非常重要，但由于模型种属的不同，本发明的造模剂量无法简单地从常规的物种换算系数计算得到。本发明既要保证树鼩肺部损伤面积达到急性肺损伤检测指标的要求(肺部损伤面积应至少一半以上)，又期望树鼩能表现出临床症状(呼吸困难、消瘦等)，本发明的研究团队围绕理想的药物剂量进行了大量、长时间的创新临床研究。(2) The modeling dosage of the present invention is very important, but due to the difference of the model species, the modeling dosage of the present invention cannot be simply calculated from the conventional species conversion factor. The present invention not only needs to ensure that the lung damage area of the tree shrew meets the requirements of the acute lung injury detection index (the area of lung damage should be at least half), but also expects the tree shrew to show clinical symptoms (difficulty in breathing, weight loss, etc.). The research team has conducted a large number of long-term innovative clinical studies around the ideal drug dosage.

(3)给药方式的选择及辅助器械的改造：在本发明的研究过程中，曾选择腹腔注射的方式，考虑到损伤反应及药物吸收率的问题，最终改用气管插管的方式(非暴露式气管插管)。同时，因本发明所用的药物浓度大，管壁上的药物残留不可忽略，注入药物时，立刻注入200μL空气以降低药物损耗而引起的误差。更优选的，由于正常儿童所用器械如果在插入气管部位直接用于树鼩，很容易导致器械损伤，为了便于气管插管的成功进行，如图1所示，本发明将儿童喉镜片的前端用胶布缠裹(保持喉镜灯可正常工作)，将改造后儿童喉镜进行树鼩的气管插管方式，不会因器械的问题而造成损伤，成功率接近100％。(3) Selection of the mode of administration and modification of auxiliary equipment: In the research process of the present invention, the method of intraperitoneal injection was selected. Considering the problems of injury reaction and drug absorption rate, the method of tracheal intubation was finally used (non- exposed endotracheal tube). At the same time, because the drug concentration used in the present invention is large, the drug residue on the tube wall cannot be ignored. When injecting the drug, 200 μL of air is injected immediately to reduce the error caused by the loss of the drug. More preferably, since the instruments used by normal children are directly used on tree shrew at the site of insertion into the trachea, it is easy to cause instrument damage. In order to facilitate the successful intubation of the trachea, as shown in FIG. Wrapped with tape (to keep the laryngoscope lamp working properly), the modified children's laryngoscope can be used for tree shrew tracheal intubation, which will not cause damage due to equipment problems, and the success rate is close to 100%.

(4)本发明的建模方法高效、成功率高，可有效节约成本；模型周期在70h以上，为急性肺损伤显著地拓宽了研究周期空间；临床症状与人类疾病更为相似，具有良好的产业化前景。(4) The modeling method of the present invention is efficient, has a high success rate, and can effectively save costs; the model period is more than 70 hours, which significantly widens the research period space for acute lung injury; the clinical symptoms are more similar to human diseases, and have good industrialization prospects.

(5)本发明的模型可持续70h以上，与常规的大小鼠模型相比，本发明构建得到的树鼩模型未出现自我修复和可逆性恢复等过程。(5) The model of the present invention can last for more than 70 hours. Compared with the conventional rat and mouse models, the tree shrew model constructed by the present invention does not have processes such as self-healing and reversible recovery.

附图说明Description of drawings

图1是本发明改造后的儿童喉镜示意图。Fig. 1 is the schematic diagram of the modified children's laryngoscope of the present invention.

图2是本发明改造后的留置针示意图。Fig. 2 is the schematic diagram of the indwelling needle after the modification of the present invention.

图3是树鼩LPS诱导后micro-CT检测结果图。Figure 3 is a graph showing the results of micro-CT detection in tree shrew after LPS induction.

图4是树鼩肺脏组织切片HE染色结果图。Figure 4 is a graph showing the results of HE staining of tree shrew lung tissue sections.

具体实施方式Detailed ways

下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

实施例中所用试剂如无特殊说明均可从市场常规购得。The reagents used in the examples can be routinely purchased from the market unless otherwise specified.

实施例1Example 1

1.材料与方法：1. Materials and methods:

1.1实验材料1.1 Experimental materials

体重120～160g的雄性树鼩5只，无体内外寄生虫，表征正常的树鼩；购自中国科学院昆明动物研究所。Five male tree shrew weighing 120-160 g, without internal and external parasites, were normal tree shrew; purchased from Kunming Institute of Zoology, Chinese Academy of Sciences.

1.2主要试剂1.2 Main reagents

脂多糖(LPS)购自圣克鲁斯生物技术公司(Santa Cruz Biotechnology)；异氟烷购自深圳市瑞沃德生命科技有限公司。Lipopolysaccharide (LPS) was purchased from Santa Cruz Biotechnology; isoflurane was purchased from Shenzhen Reward Life Technology Co., Ltd.

1.3主要溶液的配制1.3 Preparation of the main solution

200mg/kg LPS溶液配制：按树鼩体重130g，称取LPS晶体26mg，溶于400μL PBS(pH＝7.35～7.45)中。200 mg/kg LPS solution preparation: According to the tree shrew body weight of 130 g, 26 mg of LPS crystals were weighed and dissolved in 400 μL of PBS (pH=7.35-7.45).

1.4主要仪器1.4 Main instruments

喉镜：Simeite 2012-58Laryngoscope: Simeite 2012-58

留置针：BD 20GIndwelling needle: BD 20G

血气针：BD血气针(2mL)Blood gas injection: BD blood gas injection (2mL)

麻醉机：瑞沃德生命科技有限公司510IPAnesthesia machine: Reward Life Technology Co., Ltd. 510IP

小动物活体成像微型CT：Super Nova CT(SNC-100)Small animal in vivo imaging micro-CT: Super Nova CT (SNC-100)

血气分析仪：西门子RXPIDPoint 500Blood gas analyzer: Siemens RXPIDPoint 500

1.5实验方法1.5 Experimental method

(1)选取5只雄性树鼩，10～12h禁食，不禁水；(1) Select 5 male tree shrews, fast for 10-12 hours, and cannot help water;

(2)腹腔注射前，对树鼩进行麻醉，麻醉方法可采用呼吸麻醉法或注射麻醉法。(2) Before the intraperitoneal injection, the tree shrew was anesthetized, and the anesthesia method could be respiratory anesthesia or injection anesthesia.

研究中对树鼩进行呼吸麻醉，异氟烷用量值为3.5，游离氧为0.8；或根据动物体重，腹腔注射3％戊巴比妥钠按树鼩体重×0.1％×3的体积进行深度麻醉，均可使树鼩进入深度麻醉状态，实施后续造模操作。In the study, the tree shrew was subjected to respiratory anesthesia, the dose of isoflurane was 3.5, and the free oxygen was 0.8; or according to the animal body weight, intraperitoneal injection of 3% sodium pentobarbital was performed according to the volume of tree shrew body weight × 0.1% × 3 for deep anesthesia , can make the tree shrew enter a state of deep anesthesia, and implement subsequent modeling operations.

(3)将树鼩以背卧姿势固定在啮齿动物工作板上，将橡皮筋置于树鼩上门牙上，树鼩的背部应完全平放在工作板上，立起工作板，固定，胸部不应偏向两侧，并用橡皮筋将其完全固定；用直镊将树鼩的舌头向左放一边，轻轻地把改造过的儿童喉镜(如图1所示，将喉镜片的前端用胶布缠裹)插入喉咙深部的气管开口处，直到可以看到喉部；打完麻药后，动物会产生较多的痰液，在喉镜照射下，立刻用改造过的BD 20G的留置针(如图2所示，出针处用胶带固定)进行吸痰；(3) Fix the tree shrew on the rodent work board in a dorsal recumbent position, place the rubber band on the upper incisor of the tree shrew, the back of the tree shrew should be completely flat on the work board, erect the work board, fix it, chest It should not be deviated to the sides, and it should be completely fixed with a rubber band; use straight forceps to set the tongue of the tree shrew to the left, and gently place the modified children's laryngoscope (as shown in Figure 1, with the front end of the laryngeal blade The trachea opening in the deep throat is inserted into the trachea opening until the larynx can be seen; after the anesthetic is administered, the animal will produce a lot of sputum. Under the irradiation of the laryngoscope, the modified BD 20G indwelling needle ( As shown in Figure 2, the needle outlet is fixed with tape) for sputum suction;

(4)用喉镜找到会厌软骨位，插入导管针，取出导管针在导管口放置一小撮棉花验证是否插入气管内；给药剂量如表1所示，用移液枪注射200mg/kg/次LPS溶液，1次注射400～500μL，立刻注入0.2mL的空气，重复注入空气2次(每次0.2mL)以减少LPS在导管内的残留；以相同的方式通过气管插管用移液枪注射相同体积400μL的PBS溶液(pH＝7.35～7.45)作为对照组(亦曾注射500μL pH＝7.35～7.45的PBS溶液作为对照组，效果无明显差异)。(4) Find the epiglottis cartilage position with a laryngoscope, insert a catheter needle, take out the catheter needle and place a small amount of cotton at the catheter port to verify whether it is inserted into the trachea; the dosage is shown in Table 1, inject 200mg/kg/ 1 injection of 400-500 μL of LPS solution, immediately inject 0.2 mL of air, and repeat the injection of air 2 times (0.2 mL each time) to reduce the residue of LPS in the catheter; in the same way, use a pipette for tracheal intubation The same volume of 400 μL of PBS solution (pH=7.35-7.45) was injected as a control group (500 μL of PBS solution of pH=7.35-7.45 was also injected as a control group, with no significant difference in effect).

(5)移除导管，将树鼩轻轻从工作台拿下，并将头部和胸部用折叠的纸巾小幅度垫高，以保证其顺畅的呼吸；对树鼩状态进行监测直到其苏醒，约4～6h。(5) Remove the catheter, gently take the tree shrew off the workbench, and slightly raise the head and chest with a folded tissue to ensure smooth breathing; monitor the tree shrew until it wakes up, About 4 ~ 6h.

(6)苏醒后将动物放回饲养笼中，观察动物状况直至72h；(6) Put the animal back into the rearing cage after waking up, and observe the condition of the animal until 72h;

(7)72h后，树鼩进行micro-CT检测肺部病变情况，肺部病变达到1/2且还未出现临床症状的，每天进行检测肺部病变情况的检测；(7) After 72 hours, the tree shrew performed micro-CT to detect the lung lesions. If the lung lesions reached 1/2 and no clinical symptoms appeared, the detection of the lung lesions was carried out every day;

(8)临床上出现呼吸困难，消瘦等症状的，肺部病变在70％以上的，即刻剖检取材检测；(8) Patients with clinical symptoms such as dyspnea and weight loss, and the lung lesions are more than 70%, immediately autopsy and test materials;

(9)将树鼩固定于手术台上，碘伏消毒腹部；用手术剪剖开腹部，用2mL血气针插入腹主动脉抽血4mL(分别做血气分析、血常规并保留血清)；(9) Fix the tree shrew on the operating table, and disinfect the abdomen with iodophor; open the abdomen with surgical scissors, insert 2 mL of blood gas needle into the abdominal aorta to draw 4 mL of blood (respectively do blood gas analysis, blood routine and reserve serum);

(10)碘伏消毒胸部及喉部，手术间剪开胸腔及喉部，取出肺部连带气管；(10) Disinfect the chest and larynx with iodophor, cut the chest cavity and larynx in the operating room, and take out the lungs and the trachea;

(11)用灭菌的4mL的PBS灌注气管至肺部，吹打3次，吸出肺泡换洗液待测细胞因子等；(11) Perfuse the trachea into the lungs with sterilized 4 mL of PBS, pipette 3 times, and aspirate the alveoli to change the washing fluid to be tested for cytokines, etc.;

(12)肺组织部分固定部分冻存。(12) Partially fixed part of lung tissue and part cryopreserved.

(13)以下为动物给药方案：(13) The following is the animal dosing schedule:

表1树鼩的给药剂量Table 1 Dosage of tree shrew

(14)5只树鼩按本发明方法造模，所有树鼩在第70h后肺部损伤达均到一半以上，5只动物均造模成功。(14) Five tree shrews were modeled according to the method of the present invention, and the lung damage of all tree shrews reached more than half after the 70th hour, and all five animals were successfully modeled.

(15)树鼩micro-CT测定肺部病变：使用平生Super Nova CT(SNC-100)以肺窗和纵膈窗扫描，分析动物肺部病变情况。(15) Determination of lung lesions by micro-CT in tree shrew: The lung and mediastinum windows were scanned with the Super Nova CT (SNC-100) of Lifetime to analyze the lung lesions of animals.

(16)树鼩肺部病理变化：将树鼩左下肺叶固定24h，纵向切割肺叶，制作病理切片。(16) Pathological changes of tree shrew lung: The left lower lung lobe of tree shrew was fixed for 24 hours, and the lung lobe was cut longitudinally to make pathological sections.

(17)树鼩肺湿/干重(W/D)情况：将树鼩右上叶取出称重，放入80℃烘箱72h，称重，记录。(17) Wet/dry weight (W/D) of tree shrew lungs: The right upper lobe of tree shrew was taken out and weighed, placed in an oven at 80°C for 72 hours, weighed, and recorded.

2.实验结果2. Experimental results

树鼩气管插管200mg/kg的LPS溶液，树鼩肺部损伤面积至少在70％以上(见图3)；病理切片显示肺间质轻度至中度弥漫性增宽，轻度至中度水肿，肺间质和肺泡腔内可见明显大量泡沫细胞分布，肺脏可见嗜中性粒细胞浸润，巨噬细胞增多，支气管腔内可见脱落上皮细胞、泡沫细胞及嗜酸性分泌物，肺泡结构消失，肺不张(见图4)；树鼩肺水肿情况见表2。The tree shrew was intubated with 200 mg/kg LPS solution, and the lung damage area of the tree shrew was at least 70% (see Figure 3); pathological sections showed mild to moderate diffuse widening of the lung interstitium, mild to moderate Edema, a large number of foam cells can be seen in the pulmonary interstitium and alveolar cavity, neutrophil infiltration in the lungs, increased macrophages, exfoliated epithelial cells, foam cells and eosinophilic secretions can be seen in the bronchial cavity, and the alveolar structure disappears. Atelectasis (see Figure 4); tree shrew pulmonary edema is shown in Table 2.

表2 5只树鼩的W/DTable 2 W/D of 5 tree shrews

实施例2Example 2

180mg/kg LPS溶液配制：按树鼩体重130g，称取LPS晶体23.4mg，溶于400μL PBS中。180mg/kg LPS solution preparation: According to the tree shrew body weight of 130g, 23.4mg of LPS crystals were weighed and dissolved in 400μL of PBS.

取2只雄性树鼩，无体内外寄生虫，表征正常的树鼩，体重在150g左右；购自中国科学院昆明动物研究所。Two male tree shrew, without internal and external parasites, were characterized as normal tree shrew, weighing about 150 g; purchased from Kunming Institute of Zoology, Chinese Academy of Sciences.

除了气管插管给药的LPS溶液的剂量为180mg/kg外，本实施例的实验方法与操作同实施例1。The experimental method and operation of this example are the same as those of Example 1, except that the dose of LPS solution administered by tracheal intubation is 180 mg/kg.

(1)动物禁食12h，不禁水；气管插管给药180mg/kg的LPS溶液，给药量见表3。(1) Animals were fasted for 12 hours and could not help water; 180 mg/kg of LPS solution was administered through tracheal intubation, and the dosage is shown in Table 3.

表3给药剂量Table 3 Dosage

(2)结果：LPS诱导的后，树鼩在3～4天左右出现明显的呼吸症状，食欲废绝；血气分析发现，氧合指数OI(PaO₂/FiO₂)分别为166mmHg、188mmHg，均在200mmHg以下，达到ARDS的要求；CT发现树鼩的病变面积均已达到80％以上。(2) Results: After LPS induction, tree shrew had obvious respiratory symptoms and lost appetite in about 3 to 4 days; blood gas analysis showed that the oxygenation index OI (PaO₂ /FiO₂ ) were 166 mmHg and 188 mmHg, respectively. Below 200mmHg, the requirements of ARDS were met; CT findings showed that the lesion area of tree shrew had reached more than 80%.

表4树鼩的W/DTable 4 W/D of tree shrew

对比例1Comparative Example 1

发明人团队在研究过程中，曾参照现有技术中的树鼩与其他实验动物等效剂量换算系数尝试获得本发明的造模剂量。During the research process, the inventor team tried to obtain the modeling dose of the present invention with reference to the equivalent dose conversion factor of tree shrew and other experimental animals in the prior art.

根据文献吴婷婷，屈会化，胡丽娜，孙晔，戴进，孙慧，李翼飞，赵琰*，王庆国*，基于树鼩体表面积的树鼩与人类及其他实验动物等效剂量换算系数的测算，中华中医药杂志，2015年1月第30卷第1期.记载，得知树鼩在Meeh-Rubner氏公式中的K值为10.83±1.73；单位体质量上体表面积相对比值为0.65±0.10；按体表面积比值折算等效剂量的系数中，小鼠剂量(20g)至树鼩剂量(130g)的换算系数为4.134。According to the literature Wu Tingting, Qu Huihua, Hu Lina, Sun Ye, Dai Jin, Sun Hui, Li Yifei, Zhao Yan*, Wang Qingguo*, the calculation of the equivalent dose conversion factor of tree shrew and human and other experimental animals based on the body surface area of tree shrew, Chinese Journal of Traditional Chinese Medicine, January 2015, Vol. 30, No. 1. It was recorded that the K value of tree shrew in Meeh-Rubner's formula was 10.83±1.73; the relative ratio of body surface area per unit body mass was 0.65±0.10; In the coefficient of converting the equivalent dose according to the ratio of body surface area, the conversion coefficient from the mouse dose (20 g) to the tree shrew dose (130 g) is 4.134.

LPS造模一般以2种途径：腹腔注射和气管插管，前者是以肺内毛细血管内皮损伤为主，后者以肺泡上皮损伤为主。常用的小鼠模型中，也是用这两种造模方式。LPS modeling is generally done in two ways: intraperitoneal injection and tracheal intubation. The former is mainly based on endothelial injury of capillaries in the lung, while the latter is mainly based on alveolar epithelial damage. These two modeling methods are also used in commonly used mouse models.

在腹腔注射模型中，小鼠多以1.5～5mg/kg LPS的剂量能检测到明显的肺部毛细血管内皮通透性的改变(参考文献1～2)，以体表系数计算，体重125.3g和128g的树鼩腹腔分别注射1mg(类比于小鼠为2mg/kg的剂量)和2.65mg(类比于小鼠为5mg/kg的剂量)的LPS。In the intraperitoneal injection model, mice with a dose of 1.5 to 5 mg/kg LPS can detect significant changes in pulmonary capillary endothelial permeability (References 1 to 2). Calculated by the body surface coefficient, the body weight is 125.3 g. and 128 g of tree shrew were injected intraperitoneally with 1 mg (comparable to the dose of 2 mg/kg in mice) and 2.65 mg (comparable to the dose of 5 mg/kg in mice) of LPS, respectively.

考虑到成本及急性肺损伤动物体重会下降的特性，这两只树鼩的体重每天以5～8g在增长，观察至第5天，此造模剂量失败。Considering the cost and the characteristics that the body weight of the animals with acute lung injury will decrease, the body weight of these two tree shrews increased by 5-8 g per day, and it was observed that the modeling dose failed on the 5th day.

对比例2Comparative Example 2

鉴于对比例1按现有技术报道的常规体表系数换算得到的剂量造模失败，同时考虑到成本及腹腔注射的吸收率的问题，本对比例选用气管插管这种更为直接的肺损伤模式进行探索。In view of the failure of the dose modeling obtained by the conversion of the conventional body surface coefficient reported in the prior art in Comparative Example 1, and taking into account the problem of cost and the absorption rate of intraperitoneal injection, this comparative example selects the more direct lung injury of tracheal intubation. mode to explore.

进一步将提高给药剂量至37.5mg/kg(类比于小鼠为9mg/kg的剂量)和108mg/kg(类比于小鼠为26mg/kg的剂量)按实施例1的方法进行气管插管给药，这两只树鼩3天体重增长趋势与正常树鼩一致，体重每天以5～8g在增长，此造模剂量失败。Further increase the administration dose to 37.5mg/kg (comparable to the dose of 9mg/kg in mice) and 108mg/kg (comparable to the dose of 26mg/kg in mice) and perform endotracheal intubation according to the method of Example 1. Medicine, these two tree shrew 3-day body weight growth trend is consistent with the normal tree shrew, the body weight is increasing by 5 ~ 8g per day, this modeling dose failed.

参考文献3报道小鼠气管插管30mg/kg、40mg/kg高剂量的LPS，会引发小鼠重症肺损伤；给予10mg/kg、20mg/kg的剂量，可引发中度的肺损伤。Reference 3 reported that high doses of 30 mg/kg and 40 mg/kg of LPS in tracheal intubation of mice can cause severe lung injury in mice; administration of 10 mg/kg and 20 mg/kg doses can cause moderate lung damage.

考虑到树鼩在麻醉过程中展示的对麻药的耐受性，本对比例先以20*4.134＝82.68mg/kg的剂量按实施例1的方法进行气管插管给药，如：体重121.3g的树鼩，10mg的LPS给药，观察称重5天，树鼩状态良好，体重增加12g，此剂量建模失败。Considering the tolerance of tree shrew to anesthetics during anesthesia, this comparative example firstly administered tracheal intubation at a dose of 20*4.134=82.68mg/kg according to the method of Example 1, such as: body weight 121.3g The tree shrew was given 10 mg of LPS and observed and weighed for 5 days. The tree shrew was in good condition and gained 12 g in weight. This dose model failed.

体重131g的树鼩，增加剂量至164mg/kg，类比于小鼠为40mg/kg，给药第3天，树鼩的肺损伤为40％，未超过一半，远高于常规剂量的造模剂量在树鼩上仍旧无法达到急性肺损伤的要求。Tree shrew weighing 131g, increased the dose to 164mg/kg, analogous to 40mg/kg in mice, on the 3rd day of administration, the lung damage of tree shrew was 40%, not more than half, much higher than the conventional dose for modeling The requirements for acute lung injury are still not met in tree shrew.

进一步增加药物剂量至325mg/kg，2只受试树鼩体重分别为132g和128g，均在给药25h左右死亡。When the drug dose was further increased to 325 mg/kg, the two tested tree shrews weighed 132 g and 128 g respectively, and both died within 25 hours of administration.

由以上实施例和对比例可知，不同实验动物之间的等效剂量换算系数是实验动物的重要基础数据，更是实验动物应用于现代生物医学研究中的先决条件。但在研究中发现，在构建急性肺损伤标准的树鼩动物模型中，本发明的造模剂量无法简单地从常规的物种之间等效剂量换算系数等现有技术或常规技术手段获得(如对比例1)；甚至采用高剂量给药，树鼩的建模依旧不如人意(如对比例2)。It can be seen from the above examples and comparative examples that the equivalent dose conversion factor between different experimental animals is an important basic data for experimental animals, and it is also a prerequisite for the application of experimental animals in modern biomedical research. However, in the research, it was found that in the establishment of the standard tree shrew animal model of acute lung injury, the modeling dose of the present invention cannot be simply obtained from the prior art or conventional technical means such as the equivalent dose conversion factor between conventional species (such as Comparative Example 1); even with high doses, tree shrew modeling was still not satisfactory (as in Comparative Example 2).

发明人团队经过长达1年的大量创新临床研究，出乎意料地发现180～200mg/kg的剂量可使树鼩约在第三天均可达到一半的肺部损伤，4～5天树鼩肺部的损伤面积在70％以上，且有明显的呼吸困难、消瘦的临床症状，血氧指标亦在200mmHg以下，这在大鼠、小鼠模型中是无法做到的。本发明的建模方法高效、成功率高，对树鼩动物模型的临床应用具有重要的指导意义。After a year of extensive innovative clinical research, the inventor team unexpectedly found that the dose of 180-200mg/kg can make tree shrews half of the lung damage in the third day, and tree shrew in 4-5 days. The damage area of the lungs is more than 70%, and there are obvious clinical symptoms of dyspnea and weight loss, and the blood oxygen index is also below 200mmHg, which cannot be achieved in rat and mouse models. The modeling method of the invention is efficient and has a high success rate, and has important guiding significance for the clinical application of the tree shrew animal model.

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上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.