A method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus

Abstract

The invention belongs to the field of biomedical animal models, and specifically discloses a method for establishing an adenovirus-induced acute exacerbation of pulmonary fibrosis animal model, which comprises at least the following steps: on the basis of establishing a mouse pulmonary fibrosis model with bleomycin, inducing acute exacerbation symptoms AE by tracheal administration of an adenovirus vector Ad without replication activity, so as to form an adenovirus-induced acute exacerbation of pulmonary fibrosis animal model AE-IPF; the invention adopts intratracheal injection administration under direct vision of a mouse laryngoscope, which can not only ensure the reliability and accuracy of administration, but also reduce damage to mice, improve survival rate and humanitarian protection; the administration time is preferably on the 14th day (mouse pulmonary fibrosis stage), and it is a single administration, which highly simulates clinical practice and relieves the pain of mice; creatively selecting inactivated adenovirus can not only simulate the possible induction of AE in IPF patients after viral infection, but also reduce environmental pollution and toxic side effects, and reduce the risk of infection for experimental personnel.

Description

Technical Field

[0001] The invention belongs to the field of biomedical animal models, and specifically discloses a method for establishing an adenovirus-induced acute exacerbation of pulmonary fibrosis animal model. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with unknown etiology, and the average survival period after diagnosis is only 2-3 years. Some patients may experience acute exacerbations (AE) during the course of the disease, which are manifested by acute exacerbation of dyspnea in a short period of time accompanied by deterioration of lung function, a significant increase in oxygen consumption, and diffuse lung damage (DAD), which leads to rapid deterioration of the patient's disease course or even death. At present, the treatment of stable IPF is still mainly anti-fibrosis and slowing down the decline of lung function, and there is no effective treatment when AE occurs. More importantly, the etiology and pathogenesis of AE-IPF are still unclear. In pathological histology, AE-IPF is manifested by DAD, massive infiltration of inflammatory cells, acute non-specific inflammatory changes, and matrix collagen production. Studies have found that multiple factors such as infection, drug toxicity, surgery or manipulation, and aspiration can induce AE, and immune-related lung injury and apoptosis of alveolar epithelial cells may be one of the important mechanisms of AE-IPF. Therefore, establishing a reasonable AE-IPF animal model is of great significance for studying its mechanism.

[0003] In recent years, a variety of AE-IPF animal models have been reported. The most common ones are: first, bleomycin (BLM) or fluorescein isothiocyanate (FITC) or transgenic methods are used to induce the establishment of a mouse pulmonary fibrosis model; on this basis, viruses, bacteria or other physical and chemical substances are used for stimulation to induce AE. However, there are many controversies in the modeling methods reported in the literature, mainly focusing on the method of administration, time and frequency of administration, and the choice of reagents to induce AE. Different AE-IPF animal models vary greatly.

[0004] In terms of administration methods, the literature currently reports nasal drops, oropharyngeal inhalation, intratracheal injection, organ tube incision administration, subcutaneous injection, etc.; nasal drops and oropharyngeal inhalation may result in problems such as drug failure to completely enter the lungs, drug loss, and inconsistent modeling effects due to large individual differences; intratracheal injection is related to the experience of the experimenter, and lack of experience may lead to accidental injection into the esophagus; organ tube incision causes great damage, and the infection and mortality rates of mice are high; subcutaneous injection may have absorption problems.

[0005] In terms of dosing time and frequency, the main problem currently is that the dosing time is too early. For example, if bleomycin is administered again within 14 days after administration to induce AE, the mouse model is still in the acute inflammatory stage, not the stage of obvious fibrosis, and the re-administration cannot accurately mimic the actual clinical situation. Some schemes use multiple dosing methods, which are cumbersome and complicated to operate, and are prone to increase the mortality rate of mice, and cannot reflect a certain factor "stimulating" AE, which is different from the "sudden" and rapid progression of AE in IPF patients in clinical practice.

[0006] Due to the variety of causes of AE, the literature reports that there are many types of stimulants that induce AE in animal models, such as bacteria, viruses with replication activity, physical and chemical reagents, etc. Bacteria and viruses with replication activity have risks such as high laboratory requirements and easy infection; physical and chemical reagents are quite different from the actual clinical situation. Summary of the invention

[0007] In order to solve the above problems, the present invention discloses a method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus, which has the advantages of accuracy, high efficiency, safety and ease.

[0008] The technical solution of the present invention is as follows:

[0009] A method for establishing an animal model of acute exacerbation of adenovirus-induced pulmonary fibrosis, comprising at least the following steps: on the basis of establishing a mouse pulmonary fibrosis model with bleomycin, inducing acute exacerbation symptoms AE by tracheal administration of adenovirus vector Ad without replication activity, forming an animal model of acute exacerbation of adenovirus-induced pulmonary fibrosis AE-IPF. At present, replication-deficient adenovirus (Ad) vectors, i.e., adenovirus empty vectors, refer to viral vectors that do not carry any exogenous DNA or RNA, and are widely used in gene transfection and gene therapy research. Although Ad empty vectors may cause some acute inflammatory reactions and lung damage, in normal mice, empty virus bodies do not cause chronic diseases such as pulmonary fibrosis, but can only cause mild inflammatory reactions. The applicant found that applying a certain dose of replication-deficient adenovirus (Ad) vectors on the basis of establishing a mouse pulmonary fibrosis model with bleomycin can induce acute lung damage in mice with pulmonary fibrosis, and acute exacerbation occurs. In this way, compared with the use of other active viral inducers, the infection risk and experimental difficulty are reduced.

[0010] Furthermore, in the method for establishing an animal model of acute exacerbation of adenovirus-induced pulmonary fibrosis, the tracheal administration is to insert a mouse intrapulmonary atomization drug delivery device into the mouse trachea to administer the Ad vector solution without replication activity. The use of tracheal injection not only ensures the accuracy of drug delivery, but also reduces unnecessary harm to mice and external environmental pollution.

[0011] Furthermore, in the above-mentioned method for establishing an animal model of acute exacerbation of adenovirus-induced pulmonary fibrosis, the Ad vector solution with no replication activity is prepared by dissolving a certain amount of Ad with no replication activity in phosphate buffer.

[0012] Furthermore, in the method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus, the Ad vector solution without replication activity is 0.5-2×10 8 pfu of replication-inactive Ad vector dissolved in 25-100 μL phosphate buffer. 8 pfu of replication-inactive Ad vector were dissolved in 50 μL of phosphate buffer.

[0013] Furthermore, in the above-mentioned method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus, in the bleomycin model for establishing mouse pulmonary fibrosis, the dosage of bleomycin is 3-5 mg / kg, preferably 5 mg / kg.

[0014] Furthermore, in the above-mentioned method for establishing an animal model of acute exacerbation of adenovirus-induced pulmonary fibrosis, the mice are 6-8 week old C57BL / 6J male mice.

[0015] Furthermore, the method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus, and establishing a mouse pulmonary fibrosis model with bleomycin comprises the following steps:

[0016] Induction of pulmonary fibrosis in mice by intratracheal injection of bleomycin:

[0017] ① On day 0, weigh the mice and mark them with ear tags; fully anesthetize the mice by nebulizing isoflurane and keep them warm;

[0018] ② After the mouse is anesthetized successfully, it is placed in a supine position on a mouse board, with the upper incisors fixed with wire and the limbs fixed with tape;

[0019] ③ Adjust the mouse board to an appropriate angle, with the mouse head facing upward, and use tweezers to gently pull the mouse tongue to one side;

[0020] ④ Hold the mouse laryngoscope in your left hand and insert it into the mouse's throat, press the tongue root and gently lift it up to fully expose the glottis, and you can see the regular opening and closing of the glottis; gently insert the mouse intrapulmonary atomization drug delivery device into the glottis and inject the corresponding volume of bleomycin solution;

[0021] ⑤ After modeling, it can be observed that the mouse's respiratory rate increases, the mouse's chest rises and falls significantly, and moist rales can be heard when the mouse is moved to the ear;

[0022] ⑥ The assistant touches and fixes the mouse, placing it in a prone position back into the cage; observe the mouse's state after it wakes up;

[0023] ⑦ Observe the mouse's activity, respiratory rate, eating, coat color changes, and survival status daily, and measure the mouse's weight every other day and keep records.

[0024] Furthermore, in the method for establishing the above-mentioned adenovirus-induced acute exacerbation of pulmonary fibrosis animal model, the time of tracheal administration of the adenovirus vector Ad with no replication activity is the pulmonary fibrosis stage of mice after administration of bleomycin.

[0025] Furthermore, the method for establishing the above-mentioned adenovirus-induced acute exacerbation of pulmonary fibrosis animal model comprises the following steps:

[0026] Intratracheal injection of replication-inactive Ad vector solution induced acute exacerbation of pulmonary fibrosis in bleomycin mice:

[0027] 1) On the 14th day after bleomycin administration, the mice were fully anesthetized by inhalation of isoflurane again to observe the anesthetic effect and any adverse reactions;

[0028] 2) After the mice were anesthetized, a replication-inactive Ad vector solution was injected intratracheally;

[0029] 3) After modeling, observe whether the respiratory rate of mice increases, the survival status and mortality of mice;

[0030] 4) The assistant touches and fixes the mouse, places it in the prone position back into the cage, and observes the state of the mouse after it wakes up;

[0031] 5) Observe the activity, respiratory rate, food intake, coat color changes, and survival of mice daily, and measure the weight of mice every other day and keep records;

[0032] 6) On the 20th day, the mice underwent chest CT examination. On the 21st day, the mice were killed by cervical cord segmentation, and blood, lung tissue, and bronchoalveolar lavage fluid samples were collected to evaluate the modeling effect.

[0033] Furthermore, the method for establishing the above-mentioned adenovirus-induced acute exacerbation of pulmonary fibrosis animal model is used in the development of drugs for treating / preventing idiopathic pulmonary fibrosis.

[0034] The present invention has the following beneficial effects:

[0035] This scheme uses the currently more recognized bleomycin to establish a mouse pulmonary fibrosis model. Regarding the method of administration, the present invention uses intratracheal injection under direct vision of a mouse laryngoscope to not only ensure the reliability and accuracy of the administration, but also reduce damage to mice, improve survival rate and humanitarian protection. The administration time is selected on the 14th day (mouse pulmonary fibrosis stage), and it is a single administration, which highly simulates clinical practice and alleviates the pain of mice. The creative selection of inactivated adenovirus can not only simulate the AE that may be induced in IPF patients after viral infection, but also reduce environmental pollution and toxic side effects, and reduce the risk of infection for experimental personnel. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Flow chart for modeling acute exacerbation of pulmonary fibrosis in mice;

[0037] Figure 2 This is the chest CT of mice after modeling (20 days after BLM, 7 days after Ad;

[0038] Figure 3 Pathological changes of mouse lung tissue after modeling (20 days after BLM, 7 days after Ad);

[0039] Figure 4 Analysis of hydroxyproline content in lung tissue of mice after modeling (20 days after BLM, 7 days after Ad);

[0040] Figure 5 This is the analysis of cell apoptosis in the lung tissue of mice after modeling (20 days after BLM and 7 days after Ad);

[0041] Figure 6 This is the analysis of markers of lung fibrosis in mice after modeling (20 days after BLM and 7 days after Ad). DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0044] Equipment, reagents, and mouse preparation:

[0045] Mouse:

[0046] 6-8 week old C57BL / 6J male mice were selected.

[0047] instrument:

[0048] Ear tags, adjustable mouse board, fixator (rubber band, tape), blunt forceps, mouse laryngoscope, mouse intrapulmonary aerosol drug delivery device.

[0049] Reagents:

[0050] Isoflurane, bleomycin powder, replication-inactive Ad vector (Heyuan Biotechnology (Shanghai) Co., Ltd., catalog number: H201), and phosphate buffer.

[0051] Preparation of bleomycin solution and Ad vector solution without replication activity:

[0052] Preparation of Bleomycin Solution:

[0053] Drug specification: 15mg / bottle.

[0054] On a sterile operating table, dissolve 15 mg of bleomycin in 7.5 ml of phosphate buffer (0.01 M) and shake thoroughly (the concentration of bleomycin solution is 2 μg / μl)

[0055] The concentration of bleomycin is 5 mg / kg. According to the weight of the mouse, a corresponding volume of solution is drawn.

[0056] Preparation of Ad vector solution without replication activity:

[0057] The size of the Ad vector without replication activity is 1.58×10 11 pfu / ml, 50μl / vial.

[0058] The proposed dosage for each mouse is 1×10 8 pfu, dissolved in 50 μl phosphate buffer, injected intratracheally

[0059] Therefore, the Ad vector stock solution without replication activity was diluted 1580 times (i.e., diluted to 7.9 ml) to obtain 1×10 8 pfu / 50 μl target solution.

[0060] The entire process of establishing the mouse model of acute exacerbation of pulmonary fibrosis is as shown in the attached Figure 1 shown.

[0061] Example 1

[0062] Experiment 1: Inducing pulmonary fibrosis in mice by intratracheal injection of bleomycin:

[0063] ①. On day 0, weigh the mice and mark them with ear tags; fully anesthetize the mice by nebulizing isoflurane and keep them warm;

[0064] ② After the mouse is anesthetized successfully, it is placed in a supine position on a mouse board, with the upper incisors fixed with wire and the limbs fixed with tape;

[0065] ③. Adjust the mouse board to an appropriate angle, with the mouse head facing upward, and use tweezers to gently pull the mouse tongue to one side;

[0066] ④. Hold the mouse laryngoscope in your left hand and insert it into the mouse's throat, press the tongue root and gently lift it up to fully expose the glottis, which can be seen opening and closing regularly; gently insert the mouse intrapulmonary atomization drug delivery device into the glottis, and inject the corresponding volume of bleomycin solution;

[0067] ⑤. After modeling, it can be observed that the mouse's respiratory rate increases, the mouse's chest rises and falls significantly, and moist rales can be heard when the mouse is moved to the ear;

[0068] ⑥ The assistant touches and fixes the mouse, placing it back into the cage in a prone position; observe the mouse's state after it wakes up.

[0069] ⑦. Observe the mouse's activity, breathing rate, eating, coat color changes, and survival status every day, and measure the mouse's weight every other day and keep records.

[0070] Example 2

[0071] Experiment 2: Intratracheal injection of replication-inactive Ad vector solution induced acute exacerbation of pulmonary fibrosis in bleomycin mice:

[0072] ①. On the 14th day, the mice were fully anesthetized by inhalation of isoflurane again, and the anesthetic effect and adverse reactions were observed;

[0073] ②. After the mouse is anesthetized successfully, 50 μl of Ad vector solution without replication activity is injected into the trachea in the same way as in process 3.1.

[0074] ③. After modeling, observe whether the respiratory rate of mice increases, the survival status and mortality of mice;

[0075] ④. The assistant touches and fixes the mouse, places it back into the cage in a prone position, and observes the mouse's state after it wakes up.

[0076] ⑤. Observe the mouse's activity, breathing rate, eating, coat color changes, and survival status every day, and measure the mouse's weight every other day and keep records.

[0077] ⑥. On the 20th day, the mice underwent chest CT examination. On the 21st day, the mice were killed by cervical cord disconnection, and lung tissue samples were collected to evaluate the modeling effect.

[0078] Experimental Results

[0079] As attached Figure 2As shown, chest CT of mice after modeling (20 days after BLM, 7 days after Ad) showed that mice in the BLM+Ad co-intervention group had diffuse ground-glass exudates in both lungs, which were significantly more severe than those in the NS group, BLM group, Ad group, and BLM+BLM group.

[0080] As attached Figure 3 As shown in the figure, pathological changes of lung tissue in mice after modeling (20 days after BLM, 7 days after Ad): HE staining results of AE showed that the lung tissue of mice in the BLM+Ad co-intervention group had severe inflammatory response, which was significantly aggravated compared with mice in other groups; Masson staining results of FJ showed that the lung tissue of mice in the BLM+Ad co-intervention group had more severe collagen deposition, which was more obvious than that in other groups.

[0081] As attached Figure 4 As shown in the figure, analysis of hydroxyproline content in lung tissue of mice after modeling (20 days after BLM, 7 days after Ad): the hydroxyproline content in lung tissue of mice in the BLM+Ad co-intervention group was significantly higher than that in BLM and NS, and the difference was significant (all P values ​​were < 0.05). However, compared with the BLM+BLM group, although it was increased, the difference was not significant (P value > 0.05).

[0082] As attached Figure 5 As shown, analysis of cell apoptosis in the lung tissue of mice after modeling (20 days after BLM, 7 days after Ad): TUNEL immunofluorescence staining showed that cell apoptosis in the lung tissue of mice in the BLM+Ad co-intervention group was significantly increased compared with the single BLM, double BLM and NS groups (×200 times).

[0083] As attached Figure 6 As shown, the markers of fibrosis in the lung tissue of mice after modeling (20 days after BLM, 7 days after Ad): WB results showed that the protein expression levels of Collagen-1, α-SMA and TGF-β1 in the lung tissue of mice in the BLM+Ad co-intervention group were significantly increased compared with BLM and NS, and the expression was also increased compared with the two BLM model groups.

[0084] From the above experimental results, we can conclude that: we used BLM-induced pulmonary fibrosis mice, gave Ad vectors on the 14th day after BLM modeling, and then observed BLM-induced pulmonary fibrosis mice on the 21st day. Compared with the normal saline control group and BLM group mice, diffuse damage to the lung tissue, inflammation and fibrosis reactions were significantly aggravated, and cell apoptosis was significantly increased. The results suggest that a single direct intratracheal injection of a replication-inactive Ad vector can induce AE in BLM mice, and an animal model of acute exacerbation of pulmonary fibrosis can be successfully established.

[0085] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus, characterized in that: At least the following steps are included: Based on the establishment of a mouse pulmonary fibrosis model with bleomycin, the adenovirus vector Ad with no replication activity was administered through the trachea to induce acute exacerbation symptoms AE, forming an animal model of adenovirus-induced acute exacerbation of pulmonary fibrosis AE-IPF; The tracheal administration is to use a mouse intrapulmonary atomization drug delivery device to insert into the mouse trachea to administer an Ad vector solution without replication activity; The Ad vector solution without replication activity is prepared by dissolving a certain amount of Ad without replication activity in phosphate buffer; The Ad vector solution without replication activity is 0.5-2×10 8 pfu of replication-inactive Ad vector dissolved in 25-100 µL phosphate buffer; In the bleomycin-induced mouse pulmonary fibrosis model, the dosage of bleomycin is 3-5 mg / kg; The mice are 6-8 weeks old C57BL / 6J male mice; The establishment of mouse pulmonary fibrosis model with bleomycin includes the following steps: Induction of pulmonary fibrosis in mice by intratracheal injection of bleomycin: ① On day 0, weigh the mice and mark them with ear tags; fully anesthetize the mice by nebulizing isoflurane and keep them warm; ② After the mouse is anesthetized successfully, it is placed in a supine position on a mouse board, with the upper incisors fixed with wire and the limbs fixed with tape; ③ Adjust the mouse board to an appropriate angle, with the mouse head facing upward, and use tweezers to gently pull the mouse tongue to one side; ④ Hold the mouse laryngoscope in your left hand and insert it into the mouse's throat, press the tongue root and gently lift it up to fully expose the glottis, and you can see the regular opening and closing of the glottis; gently insert the mouse intrapulmonary atomization drug delivery device into the glottis and inject the corresponding volume of bleomycin solution; ⑤ After modeling, it can be observed that the mouse's respiratory rate increases, the mouse's chest rises and falls significantly, and moist rales can be heard when the mouse is moved to the ear; ⑥ The assistant touches and fixes the mouse, placing it in a prone position back into the cage; observe the mouse's state after it wakes up; ⑦ Observe the mouse activities, respiratory rate, food intake, coat color changes, and survival status daily, and measure the mouse weight every other day and keep records.

2. The method for establishing an adenovirus-induced acute exacerbation of pulmonary fibrosis animal model according to claim 1, characterized in that: The time of intratracheal administration of the adenoviral vector Ad without replication activity is the pulmonary fibrosis stage of mice after administration of bleomycin.

3. The method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus according to claim 2, characterized in that: The following steps are involved: Intratracheal injection of replication-inactive Ad vector solution induced acute exacerbation of pulmonary fibrosis in bleomycin mice: 1) On the 14th day after bleomycin administration, the mice were again fully anesthetized by nebulized isoflurane, and the anesthetic effect and adverse reactions were observed; 2) After the mice were anesthetized, a replication-inactive Ad vector solution was injected intratracheally; 3) After modeling, observe whether the respiratory rate of mice increases, the survival status and mortality of mice; 4) The assistant touches and fixes the mouse, placing it in a prone position back into the cage; observe the mouse's state after it wakes up; 5) Observe the mouse's activity, respiratory rate, food intake, coat color changes, and survival status daily, and measure the mouse's weight every other day and keep records; 6) On the 20th day, the mice underwent chest CT examination. On the 21st day, the mice were killed by cervical cord segmentation, and blood, lung tissue, and bronchoalveolar lavage fluid samples were collected to evaluate the modeling effect.

4. Use of the method for establishing an animal model of acute exacerbation of pulmonary fibrosis induced by adenovirus according to any one of claims 1 to 3 in the development of drugs for treating / preventing acute exacerbation of idiopathic pulmonary fibrosis.

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