CN113180007B

CN113180007B - Exudative animal pneumonia model construction method

Info

Publication number: CN113180007B
Application number: CN202110480757.5A
Authority: CN
Inventors: 许小琴; 王银龙; 严静; 张玥; 王广泽; 汪培嘉
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-08-19
Anticipated expiration: 2041-04-30
Also published as: CN113180007A

Abstract

The invention discloses a construction method of an exudative animal pneumonia model, which comprises the following steps: injecting lipopolysaccharide into abdominal cavity of animal; the next day, several days in succession, the same amount of klebsiella pneumoniae was nasally inoculated every day to establish an animal model of pneumonia. The exudative pneumonia model is established by injecting low-dose lipopolysaccharide into abdominal cavity before attacking poison, so that the immunity of a rat is reduced, and the colonization of Klebsiella pneumoniae in the lung of the rat is facilitated, so that the exudative pneumonia model can be obtained within ten days, the molding period is greatly shortened, and a model basis is provided for later-stage prevention and treatment research.

Description

Exudative animal pneumonia model construction method

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method of an exudative animal pneumonia model.

Background

The exudative pneumonia is a kind of pneumonia, and the pathological changes mainly include a large amount of inflammatory exudation in the alveolar space, and are often accompanied with congestion and bleeding of the alveolar space, pulmonary interstitium and bronchia, which are the pathological bases for the occurrence of various pulmonary diseases. With the increasing severity of air pollution, the lung diseases of human beings and animals are growing, exudative pneumonia is taken as the basis of the attack of most lung diseases, and a great deal of attention is paid to scientifically, simply and efficiently manufacturing an animal model of exudative pneumonia, so that the understanding of people on the pneumonia mechanism can be accelerated, a theoretical basis is provided for the development of prevention and treatment, and the exudative pneumonia modeling method becomes the research direction of numerous scholars.

The existing establishment method of the klebsiella pneumoniae rat infection model mainly comprises repeated nasal drip infection, operation trachea drip and dispersion at long periods and intervals, the test period is long, and the operation molding has high requirements on conditions such as environment, equipment, personnel and the like, so that the large-batch molding of animals is difficult.

Disclosure of Invention

Aiming at the problem that the building period of the pneumonia animal model in the prior art is long, the invention provides a method capable of quickly building an exudative animal pneumonia model.

The technical purpose is achieved, the technical effect is achieved, and the invention is realized through the following technical scheme:

a construction method of an exudative animal pneumonia model comprises the following steps:

injecting lipopolysaccharide into abdominal cavity of animal;

the next day, several days continuously, the same amount of Klebsiella pneumoniae is inoculated to the nasal cavity every day, and an animal pneumonia model is established.

The method for constructing the exudative animal pneumonia model according to claim 1, wherein: the animals are SPF Wistar rats which are 6 weeks old and 200 +/-10 g in weight.

As a further improvement of the invention, the concentration of the lipopolysaccharide is 3.8-4.2mg/ml, and the injection amount is 45-55ul/100 g.

As a further improvement of the invention, the lipopolysaccharide is a polysaccharide derived from serotype O55: b5, which is extracted from Escherichia coli by benzene method, and has endotoxin level not less than 500000 EU/mg.

As a further improvement of the invention, the concentration of the Klebsiella pneumoniae in the bacterial liquid is 0.8-1.2 × 107 CFU/mL.

As a further improvement of the invention, the inoculation amount of the bacterial liquid is 45-55 ul/seed.

As a further improvement of the invention, the nasal cavity inoculation method is as follows: a hand-held pipettor is adopted to suck quantitative bacteria liquid, the bacteria liquid is dripped around the nasal cavity of the animal drop by drop, and the bacteria liquid is sucked into the lung through natural inspiration of the animal.

As a further improvement of the invention, the days of inoculation of the Klebsiella pneumoniae are 4.5-5.5 days.

The invention has the beneficial effects that: according to the establishment of the exudative pneumonia model, before toxicity attacking, the low-dose lipopolysaccharide is injected into the abdominal cavity, so that the immunity of a rat is reduced, and the colonization of Klebsiella pneumoniae in the lung of the rat is facilitated, so that the pneumonia model can be obtained within ten days, the molding period is greatly shortened, and the model basis is provided for later-stage prevention and treatment research.

Drawings

FIG. 1 is the statistics of clinical manifestations of rats after challenge, wherein a is the variation of body temperature of rats after challenge; b is the change condition of the breath frequency per minute of the rats after the challenge; c is the change of weight gain of the rat after toxin attack for seven days;

FIG. 2 is data statistics of the determination of inflammatory index in rats after challenge, wherein d is the change of blood term in rats; e is the change of IL-6 expression in rat serum; f is the change of the expression of TNF-alpha, IL-6 and IL-1 beta in rat alveolar lavage fluid;

FIG. 3 is a cross-sectional view of lung tissue from rats after challenge by autopsy.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified.

The rearing referred to in the following examples is a conventional rearing method in the art and rearing is carried out in a general laboratory environment of ordinary indoor decontamination grade, at a conventional temperature. Feed: conventional antibiotic-free feed. Drinking water: common domestic water.

The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.

First, material preparation

Experimental animals: SPF Wistar rats, which are 6 weeks old and have a body weight of 200 + -10 g.

The Klebsiella pneumoniae is purchased from China institute of veterinary medicine, is preserved in the veterinary microorganism strain preservation management center, and has the preservation number of CVCC 519.

Firstly, the Klebsiella Pneumoniae (KPC) 10 preserved in a refrigerator at the temperature of-80 ℃ in the laboratory is takenResuscitating 0 mu L in 10mL LB liquid culture medium, after shaking culture at 37 ℃ for 24h, drawing a three-zone line on a sterile Macconk agar plate, culturing at 37 ℃ for 24h, selecting a single colorless transparent colony to inoculate in 10mL LB enrichment medium, after shaking culture at 37 ℃ for 24h, taking 100 mu L of bacterial liquid in 10mL LB liquid culture medium, shaking culture at 37 ℃ for 8h, diluting through gradient, coating on the Macconk agar plate for counting, and diluting the bacterial liquid concentration to 1x10 by using sterile PBS (phosphate buffer solution) ⁷ CFU/mL or 1X10 ⁹ CFU/mL。

Lipopolysaccharide: from serotype O55: b5, which is extracted from Escherichia coli by benzene method, and has endotoxin level not less than 500000 EU/mg. The solution was prepared at 4mg/ml with PBS.

Second, the experimental procedure

50 rats were randomly divided into 10 rats in group A (high dose H-Kpc), 10 rats in group B (low dose L-Kpc), 10 rats in group C (LPS), 10 rats in group D (LPS + L-Kpc), and 10 rats in group E (blank). Adaptive feeding for three days. Group D: the first day of the experiment was intraperitoneally injected with lipopolysaccharide, and four consecutive days thereafter, were inoculated by nasal drip with 1 × 10 ⁷ CFU/ml Kpc50 ul; group A: nasal drip inoculation 1x10 starting on the next day of the experiment ⁹ CFU/ml Kpc50ul for four consecutive days; group B: nasal drip inoculation 1x10 starting on the next day of the experiment ⁷ CFU/ml Kpc50ul for four consecutive days; group C: intraperitoneal injection of lipopolysaccharide was performed on the first day of the test; A. b, C, D four groups were sacrificed three days (72 h) after the end of molding. Group E: the blank control group, beginning on the second day of the experiment, was treated by nasal instillation of an equal amount of PBS, and A, B, C groups were treated three days after the challenge, and the rats were euthanized with 3% sodium pentobarbital.

Wherein, the nasal drip inoculation process comprises the following steps: the rat is fixed, 50ul of bacteria liquid is sucked by a hand-held pipette, slowly and dropwise added to the periphery of the nasal cavity of the rat, and when the rat inhales naturally, if the rat has excessive stress, 0.1 ml/mouse of 3% sodium pentobarbital solution can be injected into the abdominal cavity for auxiliary inoculation.

Third, experimental results

A. Clinical symptoms are as follows:

after inoculation, rats were stunted, had a decreased appetite, increased body temperature, increased number of breaths and were accompanied by an increase in lightObvious clinical symptoms such as cough and the like. As can be seen from the physiological index tests shown in FIG. 1, the indexes of the rats in the group D are obviously different from those in the group E, and the specific expression is as follows: after the molding is finished, the body temperature of the rats in the D group is extremely higher than that of the rats in the E group (P<0.01), and significantly higher than group B, C (c) ((c)P<0.05) see FIG. 1 (a) in detail; the D group observes that the rats have cough from 3 days pm3:00 of the first toxin attack, 8 rats have obvious cough symptoms after the model building is finished, the total number of the rats is 8/10, and the rats are 3/10, 2/10 in the B group, 0/10 in the C group and 0/10 in the E group which are higher than those in the A group, and are shown in a detailed picture 1 (B); the number of breaths per minute of rats in group D is significantly higher than that in group E: (P<0.01) and very significantly higher than group B (P<0.01）。

B. Inflammatory factor index:

after the detoxification is finished, after the rats are anesthetized, the abdominal cavity is cut open, and the abdominal venous blood is taken; the skin of the neck is cut open, and the congestion and bleeding of the lung can be seen in the process of the autopsy; and preparing alveolar lavage fluid by using PBS, centrifuging serum and alveolar lavage fluid for 10min at 3000pr/min in a centrifuge, and measuring the expression levels of TNF-alpha, IL-6 and IL-1 beta by an Elisa method. The results obtained are shown in FIG. 2.

The results shown in fig. 2 can be obtained by A, B, C, D that the inflammatory index of rats in the model group is changed to different degrees, and the inflammatory index of rats in the group D is more significant than that in the group E, and is specifically shown in the following steps: after the modeling is finished, the total number of white blood cells of the rats in the group D is remarkably higher than that of the rats in the group E (P<0.01), wherein the neutrophil count is significantly higher than in group E: (P<0.05) see FIG. 2 (d) for details; the expression level of IL-6 in the serum of the rats in the group D is obviously higher than that in the group E: (A), (B)P<0.05); the IL-6 expression level in the alveolar lavage fluid of the rats in the group D is obviously higher than that of the rats in the group E (P<0.05) and significantly higher than group C (P<0.05) see FIG. 2 (e) for details; the expression level of IL-1 beta is obviously higher than that of the group E: (P<0.05); the expression level of TNF-alpha is very much higher than that of group E: (P<0.01), and significantly higher than A, B, C groups (c)P<0.05) see FIG. 2 (f) for details.

C. Pathological section observation of rat lung

After the rat is killed by anesthesia, the middle leaf of the left lung is taken and placed in 4% formaldehyde solution for fixation for 36 hours; trimming the tissue block to 0.5x0.5x0.5 cm, and running water overnight; gradient dehydration of 70% ethanol (overnight) -80% ethanol (4 h) -90% ethanol (2 h) -95% ethanol (1 h) -100% (1) ethanol (1 h) -100% (2) ethanol (1 h); xylene (1) 5 min-xylene (2) 2min is transparent; wax (1) for 0.5 h-wax (2) for 1.5 h; embedding; the slice thickness is 0.3 mm; the spreading temperature is 42 ℃; the dried slices are kept in a thermostat at 37 ℃ overnight; dewaxing 100% ethanol (2 min) -95% ethanol (2 min) -90% ethanol (2 min) -80% ethanol (2 min) -70% ethanol (2 min); sappanin for 10 min; washing with water for 5 min; carrying out alcohol differentiation for 1s by hydrochloric acid; washing with water for 5 min; ammonia water anti-blue for 5 s; washing with water for 5 min; eosin 1 s; washing with water for 5 min; drying at room temperature; sealing neutral gum into a sheet; and (6) microscopic examination.

The microscopic examination result is shown in the figure: g represents the phenomena of rupture of bronchial mucosa of the rat lung, shedding of part of columnar epithelial cells, different sizes of alveolar structural disorder and thinning of alveolar walls after toxicity attack; h represents the phenomenon of massive inflammatory exudates in the alveolar cavities of rats after challenge; i represents the phenomenon of the shedding of the tracheal mucosa of the rat after the toxicity attack; it can be concluded that A, B, C, D groups of rats all showed different pathological changes in lung tissues, and the D group of rats were closer to the typical pathological changes in EP. The lung tissues of rats in group A can be seen as thickened bronchial tube walls, narrow tube cavities and a large amount of inflammatory exudation in the alveolar cavities; the lung tissues of rats in group D can be seen in bronchial mucosa rupture, part of columnar epithelial cells fall off, alveolar structural disorder is different in size, alveolar walls are thinned, and a large amount of inflammatory exudates can be seen in alveolar cavities; the E group of rats has complete lung tissue bronchial mucosa, tidy and rich cilia and normal alveolar structure; B. group C rats had thickened bronchial lumen walls.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. an exudative animal pneumonia model construction method, is characterized in that, comprises the following steps:

Intraperitoneal injection of lipopolysaccharide into animals;

The next day, for several consecutive days, the same amount of Klebsiella pneumoniae was inoculated into the nasal cavity every day to establish an animal pneumonia model;

The concentration of the lipopolysaccharide is 3.8-4.2mg/ml, and the injection volume is 45-55ul/100g.

2. a kind of exudative animal pneumonia model construction method according to claim 1, is characterized in that: described animal selects SPF Wistar rat, and described SPF Wistar rat is 6 weeks old, body weight 200 ± 10g.

3. a kind of exudative animal pneumonia model construction method according to claim 1, is characterized in that: described lipopolysaccharide is from the Escherichia coli of serotype O55:B5, with benzene method to extract and obtain, in which The toxin level is not less than 500,000 EU/mg.

4 . The method for constructing an exudative animal pneumonia model according to claim 1 , wherein the concentration of the Klebsiella pneumoniae in the bacterial solution is 0.8-1.2×10 ⁷ CFU/mL. 5 .

5. a kind of exudative animal pneumonia model construction method according to claim 4 is characterized in that: the inoculation amount of described bacterial liquid is 45-55ul/ only.

6. a kind of exudative animal pneumonia model construction method according to claim 1, is characterized in that: the method for described nasal cavity inoculation is: adopt hand-held pipette to inhale quantitative bacterial liquid, drip drop by drop around animal nasal cavity, Inhale into the lungs by the animal's natural inspiration.

7 . The method for constructing an exudative animal pneumonia model according to claim 1 , wherein the days of inoculation of the Klebsiella pneumoniae are 4.5-5.5 days. 8 .