阅读说明：本技术 一种树鼩急性肺损伤模型的建立方法及其应用 (Method for establishing tree shrew acute lung injury model and application thereof ) 是由 和君 傅江南 赵月 齐春丽 方梅霞 宋琳亮 邢会杰 于 2019-10-10 设计创作，主要内容包括：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 a tree shrew acute lung injury model and application thereof, wherein a 180-200 mg/kg dose of lipopolysaccharide solution is administrated in a trachea after a tree shrew is anesthetized, and when the lung injury area of the tree shrew reaches more than half, the tree shrew acute lung injury animal model is established. By the establishing method, the lung injury of the tree shrews can reach more than half after 70 hours; the tree shrew shows symptoms such as emaciation, dyspnea and the like clinically within 96-120 hours, the lung injury area of the tree shrew is more than 70%, and the blood oxygen index can reach below 200 mmHg. The modeling method is efficient and high in success rate, and cost can be effectively saved; the model period is more than 70 hours, and the research period space is remarkably widened for acute lung injury; the established tree shrew animal model can not be self-repaired and can be reversibly restored, the clinical symptoms are more similar to human diseases, and the method has good industrialization prospect.)

1. A method for constructing an animal model of acute lung injury of tree shrews is characterized by comprising the following steps:

(1) after the healthy tree shrews are anesthetized, carrying out intratracheal administration on a lipopolysaccharide solution according to the dose of 180-200 mg/kg;

(2) and (4) observing after administration, and constructing the tree shrew acute lung injury animal model when the tree shrew lung injury area reaches more than half.

2. The method for constructing an animal model of tree shrew acute lung injury according to claim 1, wherein the specific operation of intratracheal administration in the step (1) is as follows:

fixing the tree shrew on a working plate in a dorsoviform posture, standing the working plate, inserting a laryngoscope into a trachea opening in the deep throat, and applying a lipopolysaccharide solution with the concentration of 180-200 mg/kg.

3. The method for constructing an animal model of tree shrew acute lung injury according to claim 2, which is characterized in that:

the laryngoscope is a children laryngoscope;

injecting a small amount of air immediately after the application of the lipopolysaccharide solution is completed;

the anesthesia method in the step (1) adopts a respiratory anesthesia method or an injection anesthesia method.

4. The method for constructing an animal model of tree shrew acute lung injury according to claim 3, which is characterized in that:

the laryngoscope is a children laryngoscope which wraps the front end of the laryngoscope lens by adhesive tape and keeps the laryngoscope lamp working normally;

the small amount of air is 0.2 mL;

the times of injecting a small amount of air are 3 times;

the anesthetic used in the anesthesia in the step (1) is one or two of sodium pentobarbital and isoflurane.

5. The method for constructing an animal model of tree shrew acute lung injury according to claim 4, which is characterized in that:

when the anesthetic is sodium pentobarbital, the specific mode of anesthesia is as follows: 3% sodium pentobarbital is injected into the abdominal cavity for anesthesia according to the dosage of the weight multiplied by 0.1% and the volume multiplied by 3 of the tree shrew;

when the anesthetic is isoflurane, the specific mode of anesthesia is as follows: anesthesia was performed with a dose of 3.5 and free oxygen of 0.8.

6. The method for constructing an animal model of tree shrew acute lung injury according to claim 1, which is characterized in that:

the intratracheal administration in the step (1) is non-exposed intratracheal administration;

the administration volume of the lipopolysaccharide solution in the step (1) is 400-500 mu L;

the healthy tree shrews in the step (1) are male adult tree shrews.

7. The method for constructing an animal model of tree shrew acute lung injury according to claim 1, which is characterized in that:

the intratracheal administration in the step (1) is completed administration at one time;

and (2) before anesthesia, the tree shrews are fasted for 10-12 hours, and water is not forbidden.

8. The method for constructing an animal model of tree shrew acute lung injury according to claim 1, which is characterized in that:

the intratracheal administration in the step (1) is intratracheal administration;

the healthy tree shrews in the step (1) are tree shrews with the weight of 120-160 g;

the anesthesia in the step (1) is in a deep anesthesia state.

9. The use of the method for constructing an animal model of tree shrew acute lung injury according to any one of claims 1 to 8 in the scientific study of acute lung injury or respiratory distress syndrome.

10. The application of the construction method of the tree shrew acute lung injury animal model in scientific research on acute lung injury or respiratory distress syndrome according to claim 9, wherein the application comprises performing at least one of the following researches when the tree shrew acute lung injury animal model clinically presents dyspnea and emaciation symptoms and lung lesions are more than 70 percent:

(1) performing histological evaluation on the lung and research on the mechanism by using a dissected material;

(2) collecting arterial blood for detection; the detection comprises blood gas analysis and blood routine test;

(3) collecting alveolar lavage fluid to detect the level of cell factor.

Technical Field

The invention relates to a modeling method of an animal model, in particular to a method for establishing a tree shrew acute lung injury model and application thereof.

Background

Acute Lung Injury (ALI) is a manifestation of systemic inflammatory reaction in the lung, and its clinical manifestations include pulmonary inflammation, increased capillary permeability and further pulmonary edema, and with the aggravation of the disease, it can further progress into respiratory distress syndrome (ARDS), causing extensive alveolar injury as a pathological feature, manifested as proteinic alveolar edema and hypoxemia. Sepsis and multiple trauma are the most common causes of ARDS, imbalance between inflammatory mediators and anti-inflammatory mediators is a key link in the occurrence and development of ALI/ARDS, and acute lung injury is always a key problem to be solved urgently in severe medicine and respiratory science.

Among the acute lung injury animal models that are commonly used today are oleic acid-induced, mechanical ventilation-induced, and Lipopolysaccharide (LPS) induced animal models. The lipopolysaccharide induction method is a method for preparing the acute lung injury animal model which is frequently used at home and abroad at present because of simple operation, high feasibility and high animal survival rate.

The most commonly used model animals for acute lung injury studies are rodents and primates at present. Since rodents are evolutionarily far from humans, experimental data obtained by using them as research subjects have little clinical guidance; because of high degree of evolution, primates are most closely related to humans and are the most similar ideal animal model to humans in experimental studies at present. However, they are too expensive, have long reproduction periods, and are not widely used due to animal ethics.

Currently, the mouse model is the most commonly used lipopolysaccharide in preparing acute lung injury models. The commonly used induction method is as follows: the mice are fasted for 12h, and are given LPS tracheal intubation of 20mg/kg or 10mg/kg, and samples are taken for 24 h; however, in the current modeling method, the change of the cell factors is mainly detected in 24 h. Therefore, the magnitude of the dose affects the death time of the mouse, the level of change in cytokines, and the like. The molding method has the following defects: firstly, the species difference between the experimental mouse and the human is large, and the guiding significance of the obtained experimental data is not large; secondly, the self-healing capacity of the lung of the experimental mouse is strong, so that the clinical drug screening is often ineffective; thirdly, macrophages (PIMs) are lacked in pulmonary vessels of the mice, and the lung injury response is relatively light; fourth, there are no obvious symptoms of dyspnea and sudden death (die of shock), so this model can only detect inflammatory factor infiltration in the early stage of ALI.

The gold criteria for clinical determination of acute lung injury were either ① CT changes due to a clear predisposition to exclude cardiac causes or ② blood gas analysis (oxygenation index (PaO)₂/FiO₂) Less than 300mmHg is ALI, PaO₂/FiO₂ARDS < 200 mmHg). However, most of the current acute lung injury animal models mainly detect phase I inflammatory factors of ALI, and the period is less than 72 hours; in addition, the rodent ALI model usually dies suddenly and is difficult to have obvious clinical symptoms, and the like. In the aspect of arterial blood detection research, medium and large animal models such as rabbit, dog, pig and sheep models can meet a certain blood sampling amount, but the dogs and rabbits lack lung intravascular macrophages (PIMs) in vivo, so that the lung injury reaction is reduced; pigs and sheep were treated with repeated alveolar lavage, and the oleic acid model was abundant.

The tree shrew as a low-grade primate has more genetic advantages in the aspect of simulating human diseases, can be comparable with the primate, shows incomparable advantages of other species in the field of life medicine, and is successfully applied to the research of diseases such as viral infection, immune-related diseases, cancers, hepatitis infection models, tree shrew nervous system diseases and the like.

However, the cost of the tree shrews is 1500-2000 yuan/piece, the medicine consumption is about 600 yuan/piece, and the average cost of modeling each tree shrew is 2100-2600 yuan/piece. Although the animal pathology model constructed based on the tree shrew has been applied to some disease researches, no relevant report of the tree shrew acute lung injury model is found. The focus of attention and detection indexes of different diseases are greatly different; the difference of different animal species brings a plurality of challenges to the stability of the molding method, the adaptability of molding dosage and the like, and further increases the research difficulty.

Therefore, there is an urgent need for animal models that more closely resemble the symptoms of human disease in the study of acute lung injury disease and in the evaluation of preclinical drugs; how to efficiently model the tree shrews has important research value and important guiding significance for future clinical application.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the method for constructing the tree shrew acute lung injury animal model.

The invention also aims to provide application of the tree shrew acute lung injury animal model.

The purpose of the invention is realized by the following technical scheme:

a method for constructing an animal model of acute lung injury of tree shrews comprises the following steps:

(1) after the healthy tree shrews are anesthetized, carrying out intratracheal administration on a lipopolysaccharide solution according to the dose of 180-200 mg/kg;

(2) and (4) observing after administration, and constructing the tree shrew acute lung injury animal model when the tree shrew lung injury area reaches more than half.

The healthy tree shrews in the step (1) are preferably male adult tree shrews, wherein the adult male animals are more stable than the female animals, such as hormones; the healthy tree shrews are preferably tree shrews with the weight of 120-160 g.

The healthy tree shrews do not contain parasites in vivo and in vitro and have normal physical signs.

The anesthesia in step (1) is preferably a deep anesthesia state.

The anesthesia method in the step (1) can adopt a respiratory anesthesia method or an injection anesthesia method; the anesthetic used is preferably at least one of sodium pentobarbital or isoflurane.

When the anesthetic is sodium pentobarbital, the specific mode of anesthesia is preferably to perform anesthesia by injecting 3% sodium pentobarbital into the abdominal cavity according to the dose of the weight of the tree shrew multiplied by 0.1% multiplied by 3 volume.

When the anesthetic is isoflurane, the specific mode of anesthesia is preferably as follows: anesthesia was performed with a dose of 3.5 and free oxygen of 0.8.

Before anesthesia in the step (1), preferably, the tree shrews are fasted for 10-12 hours without water prohibition.

The intratracheal administration in step (1) is preferably completed in one time.

The preferable administration volume of the lipopolysaccharide solution in the step (1) is 400-500 mu L, so that the administration is more uniform.

The intratracheal administration in the step (1) is preferably intratracheal administration; non-exposed intratracheal administration is preferred.

The specific operation of intratracheal administration in step (1) is preferably: fixing the tree shrew on a working plate in a dorsoviform posture, standing the working plate, inserting a laryngoscope into a trachea opening in the deep throat, and applying a lipopolysaccharide solution with the concentration of 180-200 mg/kg.

The laryngoscope is preferably a children laryngoscope; further preferably, the children laryngoscope is characterized in that the front end of the laryngoscope lens is wrapped by rubberized fabric and the laryngoscope lamp can be kept to work normally.

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.

The small amount of air is preferably 0.2 mL; the number of times of injecting the small amount of air is preferably 3 times.

The lung injury area of the tree shrew in the step (2) is more than half, even if different animals may have individual differences, the tree shrew lung injury area is more than half after 70 hours (more than 70-120 hours) by the construction method of the invention; part of tree shrews show symptoms such as emaciation, dyspnea and the like clinically in 96-120 hours, the damaged area of the lung of the tree shrew is more than 70%, and the oxygenation index OI (PaO) of the blood oxygen index of arterial blood gas analysis₂/FiO₂) Can reach below 200 mmHg.

The construction method of the tree shrew acute lung injury animal model is applied to scientific research of acute lung injury or respiratory distress syndrome.

The scientific research comprises the research of the disease occurrence and development mechanism of acute lung injury, the evaluation of novel acute lung injury treatment drugs and the like.

The application comprises that when the tree shrew acute lung injury animal model clinically has dyspnea and emaciation symptoms, and the lung lesion is more than 70%, at least one study is carried out as follows:

(1) performing histological evaluation on the lung and research on the mechanism by using a dissected material;

(2) collecting arterial blood for detection; the detection comprises blood gas analysis, blood routine test and the like;

(3) collecting alveolar lavage fluid to detect the level of cell factor.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention successfully constructs the acute lung injury model of the tree shrew of the lower primate for the first time, and the model has obvious advantages compared with the experimental animals in the prior art. The tree shrew acute lung injury model is used for researching the occurrence and development mechanism of the disease and evaluating a novel acute lung injury treatment drug, and has important guiding significance for future clinical application.

(2) The molding dose of the present invention is very important, but cannot be simply calculated from the conventional species conversion coefficient due to the difference of model species. The invention not only ensures that the lung injury area of the tree shrew meets the requirement of the acute lung injury detection index (the lung injury area is at least half of the lung injury area), but also expects the tree shrew to show clinical symptoms (dyspnea, emaciation and the like), and the research team of the invention carries out a large amount of long-time innovative clinical research around the ideal drug dosage.

(3) Selection of administration modes and modification of auxiliary devices: in the course of the present invention, the intraperitoneal injection method was selected, and the method of intubation (non-exposure intubation) was finally used in consideration of the problems of injury reaction and drug absorption rate. Meanwhile, because the concentration of the medicine used by the invention is high, the medicine residue on the tube wall is not negligible, and 200 mu L of air is injected immediately to reduce the error caused by the medicine loss when the medicine is injected. More preferably, if the instrument used by a normal child is directly used for the tree shrew at the position inserted into the trachea, the instrument is easy to be damaged, and in order to facilitate the successful implementation of the tracheal intubation, as shown in fig. 1, the front end of the laryngoscope lens of the child is wrapped by the adhesive tape (the laryngoscope lamp can be kept to work normally), and the modified child laryngoscope is used for the tracheal intubation of the tree shrew, so that the damage caused by the problem of the instrument is avoided, and the success rate is close to 100%.

(4) The modeling method is efficient and high in success rate, and cost can be effectively saved; the model period is more than 70h, so that the research period space is remarkably widened for acute lung injury; the clinical symptoms are more similar to human diseases, and the method has good industrialization prospect.

(5) The model of the invention can last for more than 70h, and compared with the conventional big and small mouse models, the tree shrew model constructed by the invention has no self-repairing and reversible recovery processes.

Drawings

Fig. 1 is a schematic view of a modified children's laryngoscope according to the invention.

FIG. 2 is a schematic view of the modified indwelling needle of the present invention.

FIG. 3 is a graph of the results of micro-CT detection after LPS induction of tree shrew.

Fig. 4 is a graph of the results of HE staining of the lung tissue sections of tree shrew.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.