Application of dog alveolar macrophage exosome in treatment of canine fossa cough

Abstract

The invention discloses application of a dog alveolar macrophage exosome in treatment of canine fossa cough, and belongs to the technical field of veterinary biological products. The canine alveolar macrophage exosome is a cell exosome obtained by using canine bordetella bronchiseptica exotoxin to stimulate canine alveolar macrophages. Compared with the conventionally-cultured canine alveolar macrophage exosome, the secretion amount of the canine alveolar macrophage exosome can be remarkably increased by using the canine bordetella bronchiseptica exotoxin to stimulate the canine alveolar macrophage exosome. Both the conventional exosome and the canine bordetella bronchiseptica exotoxin-induced exosome with the same dosage can weaken the inhibition effect of the canine bordetella bronchiseptica exotoxin on the activity of canine lung fibroblasts, and the toxin-induced exosome has a better canine lung fibroblast damage repair effect than the conventional exosome. The toxin-induced exosome has a remarkable treatment effect on the canine nest cough, the death rate of the nest cough can be reduced, and the clinical attack time is shortened.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and particularly relates to the application of canine alveolar macrophage exosomes in the treatment of kennel cough in dogs. Background Art

[0002] Kennel cough, also known as canine infectious tracheobronchitis, is a respiratory disease in dogs caused by multiple pathogens, commonly seen in puppies aged 2 - 5 months. Due to the low immunity of puppies, kennel cough has a high incidence and high mortality rate. Kennel cough is caused by the synergistic pathogenicity of multiple pathogens including Bordetella bronchiseptica, canine adenovirus type 2, canine distemper virus, canine herpesvirus, canine parainfluenza virus, etc. Puppies, elderly dogs, and dogs with potential health problems and low immunity are particularly prone to kennel cough. Due to the low immunity of diseased dogs, conventional antiviral and antibiotic treatments often have little effect, the kennel cough lingers for a long time, the treatment cost is high, and treatment failures often occur.

[0003] Alveolar macrophages play an important role in clearing pathogens in the lung respiratory tract and resisting pathogen infections. On the one hand, alveolar macrophages can non - specifically phagocytose invading bacteria, viruses and other pathogens and kill them. On the other hand, the exosomes secreted by alveolar macrophages under the stimulation of pathogenic microorganisms have a protective and damage - repairing effect on lung and tracheal mucosal cells, and can establish a strong anti - pathogenic microorganism defense line. Exosomes have a nanoscale bilayer membrane structure and contain lipids, microRNAs and proteins that play key roles in intercellular communication. Canine alveolar macrophage exosomes are expected to play an effective role in the treatment of kennel cough, but problems such as the low secretion amount of exosomes in conventional culture and the low therapeutic activity against kennel cough still need to be solved. Therefore, how to improve the secretion amount of canine alveolar macrophage exosomes and the therapeutic effect against kennel cough is the key to the application of canine alveolar macrophage exosomes in kennel cough treatment biological products. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of canine alveolar macrophage exosomes in the treatment of kennel cough, belonging to the technical field of veterinary biological products.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] First of all, the present invention provides the application of canine alveolar macrophage exosomes in the treatment of kennel cough, and the exosomes are exosomes produced by canine alveolar macrophages under the stimulation of Bordetella bronchiseptica exotoxin.

[0007] The preparation method of the exosomes includes the following steps:

[0008] (1) Isolate and culture canine alveolar macrophages;

[0009] (2) Prepare Bordetella bronchiseptica exotoxin for dogs;

[0010] (3) Passage canine alveolar macrophages into cell culture flasks, with the number of passages not exceeding 5 generations, the cell passage density being 5×10 6 / mL, culture in a carbon dioxide incubator for 24 h until the cell density reaches over 80%;

[0011] (4) Add Bordetella bronchiseptica exotoxin for dogs to the cell flask, culture for 4 h, discard the liquid in the cell flask, replace it with fresh cell maintenance medium, and continue to culture for 24 hours;

[0012] (5) Centrifuge to separate the cell culture supernatant, mix the exosome extraction reagent and the cell culture supernatant at a ratio of 1:1, let it stand for 16 hours, then centrifuge at 12000 rpm for 1 hour to collect the precipitate, and dissolve the exosomes with PBS at 1 / 10 volume of the original culture medium.

[0013] Further, the addition dose of the Bordetella bronchiseptica exotoxin for dogs in the canine alveolar macrophage medium is 20 μg / mL.

[0014] Further, the preparation method of the Bordetella bronchiseptica exotoxin for dogs includes the following steps:

[0015] (1) Take the cryopreserved Bordetella bronchiseptica, streak it on a 10% defibrinated sterile sheep blood Bordet-Gengou agar plate with an inoculation loop, and culture at 37°C for 36 h.

[0016] (2) Scrape the typical colonies and spread them evenly on a new 10% defibrinated sterile sheep blood Bordet-Gengou agar plate with a spreading rod, and culture at 37°C for 36 h.

[0017] (3) Wash the surface bacterial growth on each 120 mm plate with 10 ml of PBS and transfer it into a 50 ml shaking flask.

[0018] (4) Shake and culture the rinsed bacterial solution in a shaker at 37°C at 250 rpm / min for 2 hours.

[0019] (5) Centrifuge the bacterial solution at 12000 rpm / min for 10 minutes, collect the supernatant and filter it through a 0.22 μm filter to obtain the Bordetella bronchiseptica exotoxin for dogs, measure the protein concentration, and store it at -20°C for standby.

[0020] Secondly, the present invention provides a biological agent for stimulating the production of exosomes by canine alveolar macrophages, and the biological agent is Bordetella bronchiseptica exotoxin for dogs.

[0021] Further, the addition dose of the biological agent in the canine alveolar macrophage medium is 20 μg / mL.

[0022] Further, the preparation method of the biological agent comprises the following steps:

[0023] (1) Take the cryopreserved Bordetella bronchiseptica of dogs, streak it on a 10% defibrinated sterile sheep blood Bordet-Gengou agar plate with an inoculation loop, and culture it at 37°C for 36 h.

[0024] (2) Scrape the typical colonies and spread them evenly on a new 10% defibrinated sterile sheep blood Bordet-Gengou agar plate with a spreading rod, and culture it at 37°C for 36 h.

[0025] (3) Wash the surface bacterial flora on each 120-mm plate with 10 ml of PBS and transfer it into a 50-ml shaking culture tube.

[0026] (4) Shake and culture the rinsed bacterial liquid at 250 rpm / min on a shaker at 37°C for 2 h.

[0027] (5) Centrifuge the bacterial liquid at 12,000 rpm / min for 10 min, collect the supernatant, filter it with a 0.22-μm filter to obtain the exotoxin of Bordetella bronchiseptica of dogs, measure the protein concentration, and store it at -20°C for standby.

[0028] The beneficial effects of the present invention are as follows:

[0029] In the present invention, Bordetella bronchiseptica of dogs is cultured, the exotoxin of Bordetella bronchiseptica of dogs is extracted, and exosomes are prepared by stimulating canine alveolar macrophages with the exotoxin of Bordetella bronchiseptica of dogs. Compared with the conventional method for preparing exosomes of canine alveolar macrophages, using the exotoxin of Bordetella bronchiseptica of dogs for stimulation can significantly increase the secretion amount of exosomes of canine alveolar macrophages. When the exosomes are used for the repair of the injury of canine lung fibroblasts induced by the exotoxin of Bordetella bronchiseptica of dogs, the same dose of conventional exosomes and exosomes cultured with the exotoxin of Bordetella bronchiseptica of dogs can both weaken the inhibitory effect of the exotoxin of Bordetella bronchiseptica of dogs on the viability of canine lung fibroblasts, and the exotoxin-induced exosomes have a stronger effect on the repair of the injury of canine lung fibroblasts than the conventional exosomes. The exotoxin-induced exosomes have a significant therapeutic effect on canine kennel cough, can reduce the mortality rate of kennel cough and shorten the clinical onset time. Description of the Drawings

[0030] Figure 1 It is the detection result of the cytotoxicity of the exotoxin of Bordetella bronchiseptica of dogs to canine alveolar macrophages;

[0031] Figure 2 It is the gray scale detection result of the CD36 protein of exosomes of canine alveolar macrophages cultured with different concentrations of the exotoxin of Bordetella bronchiseptica of dogs;

[0032] Figure 3 It is the effect of exosomes of canine alveolar macrophages on the repair of the injury of canine lung fibroblasts. Detailed implementation manners

[0033] The following is a more detailed description of the present invention, which is elaborated through examples. It should be clear that these examples are only examples of the present invention, and their purpose is to illustrate the principles and functions of the present invention, rather than limiting the protection scope of the present invention.

[0034] Example 1 Preparation of Bordetella bronchiseptica exotoxin for dogs

[0035] (1) Take the cryopreserved Bordetella bronchiseptica, and streak it on a 10% defibrinated sterile sheep blood Bordet-Gengou agar plate with an inoculation loop, and culture it at 37°C for 36 h.

[0036] (2) Scrape the typical colonies and spread them evenly on a new 10% defibrinated sterile sheep blood Bordet-Gengou agar plate with a spreading rod, and culture it at 37°C for 36 h.

[0037] (3) Wash the surface bacterial lawn of each 120 mm plate with 10 ml of PBS and transfer it into a 50 ml shaking culture tube.

[0038] (4) Shake and culture the rinsed bacterial liquid at 250 rpm / min on a shaker at 37°C for 2 hours.

[0039] (5) Centrifuge the bacterial liquid at 12,000 rpm / min for 10 minutes, collect the supernatant and filter it with a 0.22 μm filter to obtain the Bordetella bronchiseptica exotoxin for dogs. Measure the concentration of the toxin protein with a BCA protein content assay kit and store it at -20°C for later use.

[0040] Example 2 Isolation and culture of canine alveolar macrophages

[0041] (1) After sacrificing the beagle dog, dissect it, lift the lungs, ligate the trachea, take out the fresh lungs and quickly transfer them to the laboratory biosafety cabinet.

[0042] (2) Open the trachea, wash the inside of the lungs with 50 mL of sterile PBS, and gently press the lung lobes to release the lung macrophages, and repeat 4-5 times.

[0043] (3) Collect the above alveolar lavage fluid, filter the impurities with sterile gauze, and isolate the pulmonary mononuclear macrophages from the lavage fluid according to the instructions of the peripheral blood mononuclear cell separation liquid kit. Then resuspend them at a cell density of 1×10 6 cells / mL in DMEM complete medium containing 10% FBS. Place the cell bottle in an incubator at 37°C and 5% CO2, statically culture it for 4 h, then replace the DMEM complete medium to remove the non-adherent cells, and check the cell growth status every 24 h and replace the culture medium.

[0044] (4) When the cells grow to 80%-90% of the bottom of the flask, start subculturing. Freeze the cells when they reach the 2nd passage. The canine alveolar macrophages used in this study are from the 3rd to 5th passages.

[0045] Example 3 Detection of the cytotoxicity of Bordetella bronchiseptica exotoxin on canine alveolar macrophages

[0046] (1) Prepare a single-cell suspension of canine alveolar macrophages and inoculate the cells into a 96-well plate. Culture the cells in a carbon dioxide incubator with a CO2 concentration of 5% until the cell density reaches approximately 80%.

[0047] (2) Prepare cell culture media containing different concentrations of Bordetella bronchiseptica exotoxin: 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL.

[0048] (3) Remove the culture media from the 96-well cell culture plate and replace it with the different concentrations of Bordetella bronchiseptica exotoxin media prepared in step (2). The 0 μg / mL group is the control group.

[0049] (4) After culturing in the cell culture incubator for 24 h, use CCK-8 to measure the OD450 value and calculate the cell survival rate. The cell survival rate = (OD450 of the experimental group - OD450 of the blank control group) / (OD450nm of the control group - OD450 of the blank control group).

[0050] It can be seen from Figure 1 that within the range of 0-20 μg / mL, as the concentration of Bordetella bronchiseptica exotoxin increases, the cell survival rate of canine alveolar macrophages remains basically stable, and there is no significant difference among the groups. However, in the groups of 40 μg / mL, 80 μg / mL, and 100 μg / mL Bordetella bronchiseptica exotoxin, the cell survival rates of canine alveolar macrophages decreased to 85.61%, 62.76%, and 36.14% respectively, indicating that when the concentration of Bordetella bronchiseptica exotoxin in the culture medium reaches 40 μg / mL and above, it will cause toxicity to canine alveolar macrophages.

[0051] Example 4 Preparation of exosomes from canine alveolar macrophages

[0052] (1) The canine alveolar macrophages are seeded at a cell density of 1×10 6Passage at a density of cells / mL, culture in a carbon dioxide incubator with a CO2 concentration of 5% for 24 h until the cell density reaches over 80%. Add 0 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL of Bordetella bronchiseptica exotoxin to groups 1 - 4 respectively, with 3 replicates in each group. Continue to culture for 24 h, centrifuge at 12000 rpm / min for 10 minutes to collect the cell culture supernatant. According to the instructions of the exosome extraction kit, mix the exosome extraction reagent and the cell culture supernatant at a ratio of 1:1, let it stand for 16 hours, then centrifuge at 12000 rpm / min for 1 hour to collect the precipitate, and dissolve the exosomes with PBS at 1 / 10 volume of the original culture medium.

[0053] (2) Mix RIPA lysis buffer with the exosomes in equal volume, let it stand on ice for 1 hour, and shake it for 1 - 2 minutes every 10 minutes during this period. Centrifuge at 12000 rpm / min for 15 minutes at 4°C to collect the supernatant. Mix the sample and 2×Loading buffer at a volume ratio of 1:1 and boil in a water bath for 10 minutes. Perform SDS - PAGE electrophoresis on the prepared sample, transfer it to a PVDF membrane, block it with a 5% skim milk solution for 1 hour, incubate with CD63 and GAPDH antibodies purchased from Abcam at 37°C for 1 hour respectively, and rinse 3 times with PBS; dilute the goat anti - rabbit secondary antibody at a ratio of 1:2000, incubate at 37°C for 1 hour, and rinse 3 times with PBS; finally, develop and expose with ECL luminescent reagent. Analyze the protein gray value using Quantity One v462 software, compare it with the internal reference protein band, and obtain the relative gray ratio. The gray value of the target protein = OD value of the target protein / OD value of the corresponding internal reference.

[0054] It can be seen from Figure 2 that the amount of exosome - marker protein CD63 produced by canine alveolar macrophages cultured in the medium supplemented with 0 μg / mL, 10 μg / mL, and 20 μg / mL of Bordetella bronchiseptica exotoxin shows an obvious increasing trend with the increase in the concentration of Bordetella bronchiseptica exotoxin, while the amount of exosome - marker protein CD63 produced by canine alveolar macrophages cultured in the 40 μg / mL Bordetella bronchiseptica exotoxin group is lower than that in the 20 μg / mL group. Thus, it can be seen that stimulating with 20 μg / mL of Bordetella bronchiseptica exotoxin added to the cell culture medium can make canine alveolar macrophages secrete the largest amount of exosomes.

[0055] Example 5 Effect of Canine Alveolar Macrophage Exosomes on the Injury Repair of Canine Lung Fibroblasts

[0056] (1) After sacrificing the beagle dogs by dissection, excise the lung tissue and quickly transfer it to the laboratory biosafety cabinet. Wash away the attached blood with PBS and place it in a petri dish. Cut it into pieces with scissors and transfer it to the lung tissue digestion solution (DMEM medium containing 1% Penicillin-Streptomycin solution and 100 mg type I collagenase with a volume of 10 mL), and digest the lung tissue in a constant temperature shaker at 37 °C for about 30 minutes.

[0057] (2) Filter the digested tissue through a 100 μm cell sieve into a 15 mL centrifuge tube and centrifuge at 1500 rpm / min for 10 minutes.

[0058] (3) After centrifugation, discard the supernatant completely, add 2 - 3 mL of red blood cell lysate, shake vigorously to completely resuspend the cell pellet, let it stand at room temperature for 5 minutes and then centrifuge again at 1500 rpm / min for 10 minutes.

[0059] (4) Remove the supernatant, resuspend the cell pellet with an appropriate amount of DMEM medium containing 1% Penicillin-Streptomycin solution and 10% FBS, evenly spread the cells in a cell culture dish, and place it in a 37 °C carbon dioxide incubator for culture.

[0060] (5) When the canine lung fibroblasts grow to confluence, digest the cells with 1% trypsin, adjust the cell density to 1×10 6 / well, inoculate into a 96-well plate, place it in a 37 °C, 5% carbon dioxide incubator for 24 h, discard the normal cell wells, and add 80 μg / mL of Bordetella bronchiseptica exotoxin to the remaining wells.

[0061] (6) The first group is the model group of Bordetella bronchiseptica exotoxin-induced injury of canine lung fibroblasts. After adding 80 μg / mL of Bordetella bronchiseptica exotoxin, add 50 μL of cell culture medium; the second group is the conventional exosome group, add 50 μL of exosomes produced by canine alveolar macrophages without induction by Bordetella bronchiseptica exotoxin in Example 4 to each well; the third group is the exosome group induced by Bordetella bronchiseptica exotoxin culture. Dilute the exosomes produced by canine alveolar macrophages cultured with 20 μg / mL of Bordetella bronchiseptica exotoxin in Example 4 with the culture medium, and dilute according to the gray value of the exosome marker protein CD63 to be the same as the CD63 gray value of the exosomes in the third group.

[0062] (7) After adding Bordetella bronchiseptica exotoxin and exosomes to canine lung fibroblasts, the four groups of cells were cultured in an incubator at 37 °C and 5% carbon dioxide for 6 hours. The OD450 value was measured using CCK-8, and the cell survival rate was calculated. The cell survival rate = (OD450 of the experimental group - OD450 of the blank control group) / (OD450nm of the control group - OD450 of the blank control group). The data were expressed as the mean of three independent experiments.

[0063] It can be seen from Figure 3 that after adding 80 μg / mL Bordetella bronchiseptica exotoxin to canine lung fibroblasts, the cell survival rate decreased to 62.76%; the cell survival rate of the conventional exosome group was 79.05%, and the cell survival rate of the exosomes cultured with Bordetella bronchiseptica exotoxin was 97.85%. It can be seen that both conventional exosomes and exosomes cultured with Bordetella bronchiseptica exotoxin at the same dose can weaken the inhibitory effect of Bordetella bronchiseptica exotoxin on the viability of canine lung fibroblasts, and the exosomes cultured with Bordetella bronchiseptica exotoxin have a stronger effect on repairing the damage of canine lung fibroblasts than conventional exosomes.

[0064] Example 6 Therapeutic effect of canine alveolar macrophage exosomes on dogs with kennel cough

[0065] Puppies aged 2 - 5 months with typical respiratory symptoms who visited a pet hospital were diagnosed with kennel cough after being tested for pathogens including Bordetella bronchiseptica, canine adenovirus type 2, canine distemper virus, canine herpesvirus, and canine parainfluenza virus, and at least two pathogens were mixedly infected. After communicating with the dog owners, the dog owners were asked to choose conventional treatment, exosome treatment, or combined treatment for the diseased dogs. The conventional treatment group was treated with conventional antibiotics and antiviral drugs according to the drug sensitivity test and the severity of the disease in the diseased dogs; the exosome treatment group was injected with exosomes produced by canine alveolar macrophages cultured with Bordetella bronchiseptica exotoxin in Example 4, and 0.2 ml / kg of exosomes was intramuscularly injected according to the body weight of the puppies, once a day; the combined treatment group was treated with conventional antibiotics and antiviral drugs, and at the same time, 0.2 ml / kg of exosomes was intramuscularly injected, once a day. 50 test kennel cough dogs were included in each of the three groups, and the treatment conditions of each test dog were recorded, and the cure rate, average cure time, and mortality rate were statistically analyzed.

[0066] As can be seen from the results in Table 1, the cure rate of the conventional treatment group is low, only 68%, the mortality rate is as high as 32%, and the average cure time is as long as 9 days; the effect of the exosome treatment group is better than that of the conventional treatment group, the cure rate is 86%, and the average cure time is 5 days; the combined treatment group has the best effect, the cure rate is as high as 94%, and the average cure time is shortened to 3 days. Therefore, the canine alveolar macrophage exosomes of the present invention have a good therapeutic effect on canine infectious tracheobronchitis, solving the problems of poor clinical treatment effect, low cure rate and long treatment time of canine infectious tracheobronchitis. The combined treatment of canine alveolar macrophage exosomes with conventional antibiotics and antiviral drugs has a better effect.

[0067] Table 1 Clinical treatment trial of canine infectious tracheobronchitis

[0068] Grouping Cure rate Long average cure time Mortality rate Conventional treatment group 68％ 9 32％ Exosome treatment group 86％ 5 14％ Combined treatment group 94％ 3 6％

Claims

1. A use of canine alveolar macrophage exosomes in treating kennel cough, characterized in that: The exosomes are exosomes produced by canine alveolar macrophages under the stimulation of canine bronchiseptica Bordetella exotoxin.

2. The use according to claim 1, characterized in that: The method for preparing exosomes comprises the following steps: (1) Isolation and culture of canine alveolar macrophages; (2) preparing canine bronchiseptica Bordetella exotoxin; (3) Canine alveolar macrophages were subcultured into cell culture flasks. The number of subcultures did not exceed 5, and the cell subculture density was 5×10 6 / mL, culture in a carbon dioxide incubator for 24 h until the cell density reaches more than 80%; (4) Add canine bronchiseptica exotoxin to the cell bottle, culture for 4 hours, discard the liquid in the cell bottle, replace with fresh cell maintenance medium, and continue to culture for 24 hours; (5) The cell culture supernatant was separated by centrifugation, and the exosome extraction reagent and the cell culture supernatant were mixed in a ratio of 1:

1. After standing for 16 hours, the precipitate was collected by centrifugation at 12000 rpm for 1 hour, and the exosomes were dissolved in PBS with 1 / 10 volume of the original culture medium.

3. The canine bronchiseptica Bordetella exotoxin according to claim 2, characterized in that: The dosage of the canine bronchiseptica Bordetella exotoxin added to the canine alveolar macrophage culture medium is 20 μg / mL.

4. The canine bronchiseptica Bordetella exotoxin according to claim 3, characterized in that The method for preparing the canine bronchiseptica Bordetella exotoxin comprises the following steps: (1) Take frozen canine bronchiseptica Bordetella, streak it on a 10% defibrinated sterile sheep blood Abalone-Jiang plate with an inoculation loop, and culture it at 37°C for 36 h; (2) Scrape typical colonies onto new 10% defibrinated sterile sheep blood abalone-Jiang plates and spread them evenly with a spreading rod, and incubate at 37°C for 36 h; (3) Rinse the surface bacterial moss of each 120 mm plate with 10 ml PBS and place into a 50 ml shaking tube; (4) The washed bacterial solution was cultured in a shaker at 37°C and 250 rpm / min for 2 h; (5) The bacterial solution was centrifuged at 12000 rpm / min for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to obtain the canine bronchiseptica Bordetella exotoxin. The protein concentration was determined and the solution was stored at -20°C for future use.

5. A biological preparation for stimulating the production of exosomes in canine alveolar macrophages, characterized in that: The biological agent is canine bronchiseptica Bordetella exotoxin.

6. The biological agent according to claim 5, characterized in that The dosage of the biological preparation added to the canine alveolar macrophage culture medium is 20 μg / mL.

7. The biological agent according to claim 6, characterized in that The preparation method of the biological agent comprises the following steps: (1) Take frozen canine bronchiseptica Bordetella, streak it on a 10% defibrinated sterile sheep blood Abalone-Jiang plate with an inoculation loop, and culture it at 37°C for 36 h; (2) Scrape typical colonies onto new 10% defibrinated sterile sheep blood abalone-Jiang plates and spread them evenly with a spreading rod, and incubate at 37°C for 36 h; (3) Rinse the surface bacterial moss of each 120 mm plate with 10 ml PBS and place into a 50 ml shaking tube; (4) The washed bacterial solution was cultured in a shaker at 37°C and 250 rpm / min for 2 h; (5) The bacterial solution was centrifuged at 12000 rpm / min for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to obtain the canine bronchiseptica Bordetella exotoxin. The protein concentration was determined and the solution was stored at -20°C for future use.

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