Application of exosomes miR-200a-3p and miR-148a-3p in diagnosis and treatment of chicken chronic respiratory disease

Abstract

The invention discloses a method for treating chicken chronic respiratory diseases by using MG-sEV-miRNA-200, and belongs to the technical field of exosomes, and the method comprises the following steps: step 1, constructing an MG infected cell model, and carrying out fluorescence quantitative analysis on gga-miR-200a-3p in cells / exosomes in different time periods of infection; step 2, carrying out overexpression on the miR-200 in the ACE II cells, and extracting an exosome (MG-sEV-miRNA-200) rich in the miR-200; step 3, constructing a cell model for exosome treatment, and carrying out RNA-seq sequencing to identify an exosome treatment target and a pathway rich in gga-miR-200a-3p; step 4, constructing a chick embryo model infected by MG, and injecting MG-sEV-miRNA-200 to intervene in respiratory diseases caused by MG; 5, evaluating the curative effect; mG-sEV is taken as a medium, miRNA can be protected from being influenced by RNA enzyme activity, the gga-miR-200a-3p-rich MG-sEV has the advantages of low immunogenicity, low cytotoxicity, relatively high stability, relatively high biocompatibility and the like, and the MG-sEV rich in gga-miR-200a-3p can be used for remarkably relieving lung inflammation injury caused by MG infection and reducing the bacterium carrying amount of MG in lung tissues; the method has a wide application prospect in the treatment of chicken chronic respiratory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of exosome miR-200a-3p and miR-148a-3p in the diagnosis and treatment of chronic respiratory disease in chickens. Background Art

[0002] Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) is a common and serious infectious disease in the poultry industry, posing a huge threat to the healthy development of the poultry industry. MG infection spreads rapidly through the respiratory tract to other tissues and organs throughout the body, leading to multiple organ failure and death. It is easy to be secondary or concurrently infected with other pathogenic microorganisms, which further aggravates the harm. The infection rate is increasing year by year. So far, there is no safe and effective measure to control CRD. Therefore, early diagnosis and timely and effective control of MG infection and spread have important scientific significance and clinical value.

[0003] Exosomes (sEVs) are secretory organelles with a diameter of 30 to 200 nm. They have the same topological structure as cells and are rich in specific proteins, lipids, nucleic acids, and glycoconjugates. They deliver the contained bioactive substances to receptor cells, thereby regulating the biological functions of receptor cells. A large number of studies have shown that miRNAs play an important regulatory role in the occurrence and development of diseases, and exosomes carry stable miRNAs, which can be used as targets for disease diagnosis and treatment. Studies have confirmed that miRNAs in exosomes can change the biological activity of receptor cells (respiratory cells and immune cells), affect the lung microenvironment, and play an important role in the occurrence and development of respiratory diseases. The expression of miR-146b, which inhibits tumors, was significantly upregulated in the serum exosomes of chickens vaccinated with Marek's disease (MD) vaccine, while the expression of miR-21, which promotes tumors, was significantly downregulated. They can be used as biomarkers for MD vaccine protection and tumor diagnosis, respectively. After chickens were infected with avian influenza virus H5N1, 20 significantly different miRNAs were detected in serum exosomes, and the expression levels of these miRNAs in resistant chickens were significantly higher than those in sensitive chickens, indicating that exosome-derived miRNAs play an important role in anti-avian influenza. Exosomal gga-miR-20a-5p from chicken macrophage cell line HD11 maintains immune homeostasis and resists infection with highly pathogenic avian influenza virus H5N1 by regulating MAPK and apoptosis signaling pathways, regulating the expression of proinflammatory cytokines and cell apoptosis. At present, exosomal miRNA has great potential as a molecular marker for clinical diagnosis and treatment of CRD. Summary of the invention

[0004] The purpose of the present invention is to provide a method for treating chronic respiratory diseases in chickens using MG-sEV-miRNAs, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical scheme: a method for treating chronic respiratory diseases in chickens using MG-sEV-miRNAs, comprising the following steps: step one, screening miR-200 and miR-148 enriched in exosomes (MG-sEV) derived from MG-infected chicken alveolar type II epithelial cells (ACEII); step two, overexpressing miR-200 and miR-148 in ACEII cells to extract exosomes (MG-sEV-miRNAs) rich in miR-200 and miR-148; step three, constructing an in vitro cell model infected with MG to confirm the therapeutic targets of MG-sEV-miRNAs; step four, constructing a chicken embryo model infected with MG, and injecting MG-sEV-miRNAs to intervene in respiratory diseases caused by MG; step five, evaluating the efficacy; BRIEF DESCRIPTION OF THE DRAWINGS

[0006] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0007] Figure 1 Temporal expression profiles of gga-miR-200a-3p and gga-miR-148a-3p;

[0008] Figure 2 The cellular localization map of gga-miR-200a-3p and gga-miR-148a-3p after MG infection;

[0009] Figure 3 For the therapeutic target analysis of exosomal gga-miR-200a-3p and gga-miR-148a-3p;

[0010] Figure 4 The chicken embryo treatment model for exosomal gga-miR-200a-3p and gga-miR-148a-3p was constructed;

[0011] Figure 5 To evaluate the therapeutic effect of exosomal gga-miR-200a-3p and gga-miR-148a-3p in chick embryo lung;

[0012] Figure 6To evaluate the effect of exosomal gga-miR-200a-3p and gga-miR-148a-3p in reducing MG content in lung tissue. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0014] Step 1:

[0015] A cell model of MG infection was constructed, and gga-miR-200a-3p and gga-miR-148a-3p in cells / exosomes at different infection time periods were analyzed by fluorescence quantitative analysis. Primers were designed using Oligo 7.0 software, and the relative expression of target genes and reference genes was detected using SYBR GEEN reagents in a Roche 480 quantitative PCR instrument;

[0016] The experimental results are as follows Figure 1 As shown, qPCR results showed that the levels of gga-miR-200a-3p and gga-miR-148a-3p were significantly upregulated at 3 h after MG infection, but were upregulated in exosomes derived from CP-II cells 6 h after MG infection, and gga-miR-200a-3p and gga-miR-148a-3p were secreted into exosomes after MG infection.

[0017] Step 2:

[0018] Overexpress gga-miR-200a-3p and gga-miR-148a-3p in ACEII cells to extract exosomes rich in gga-miR-200a-3p and gga-miR-148a-3p (MG-sEV-miRNAs). When the cell density reaches 50%-60% and the cells are in good condition, transfection is performed. Taking a 6-well plate as an example, the transfection steps are as follows:

[0019] ① 1 hour before transfection, aspirate the cell culture medium, wash the cells twice with PBS without double antibody, aspirate the PBS, and add 2 mL Reduced Serum Medium;

[0020] ②Pipette 1.5μL LipofectamineTM 2000 and mix it with 50μL Reduced Serum Medium;

[0021] ③Pipette 2.5 μL of miR-200 and miR-148 mimics and mix them into 50 μL Reduced Serum Medium, let stand at room temperature for 5 minutes;

[0022] ④ Mix the mixture of (2) and (3) and let it stand at room temperature for 20 minutes to form a complex;

[0023] ⑤ Pipette 100 μL of the mixture into the prepared cells, gently shake the cell culture plate to mix evenly, and replace the complete culture medium (containing 1% double antibody) 4-6 hours after transfection;

[0024] ⑥ Culture in a cell culture incubator at 37°C and 5% CO2 for 24-72 hours;

[0025] Subsequently, the exosomes were extracted, the cell culture supernatant was collected, and the exosomes were extracted by ultracentrifugation. The specific steps were as follows: the cell supernatant was centrifuged at 1500×g at 4°C for 15 min to remove dead cells; 4°C, 10000×g, centrifuged for 30 min to remove cell debris; 4°C, 14000×g, centrifuged for 30 min, and the supernatant was taken; ultracentrifuged at 4°C, 110000×g, centrifuged for 2 h, the precipitate was collected, the precipitate was resuspended with PBS, and ultracentrifuged again at 4°C, 110000×g, and centrifuged. Centrifuge for 1 hour, discard the supernatant, collect the precipitate, which is the exosome precipitate, resuspend the exosomes with 100μl sterile PBS, and store at -80℃ for later use; verify the extracted exosomes by transmission electron microscopy and particle size analysis. The results of electron microscopy and particle size analysis show that the obtained exosomes are cup-shaped, double-membrane vesicles with a size of about 40-160nm; determine the exosome concentration by BCA protein concentration: 100mg / ml-200mg / ml exosomes can be extracted from every 50ml supernatant;

[0026] Step 3:

[0027] To identify therapeutic targets and pathways of exosomes enriched with gga-miR-200a-3p and gga-miR-148a-3p, RNA-seq sequencing was performed on MG-infected cells after incubation with exosomes. The results showed that 512 downregulated genes and 877 upregulated genes were identified in cells treated with exosomes. Enrichment analysis of all the above differentially expressed genes was performed, and the KEGG results showed that the differentially expressed genes were mainly concentrated in pathways related to actin cytoskeleton regulation, endocytosis, and autophagy. GO enrichment results showed that they were mainly related to actin filament movement, endocytic vesicle action, and lung development. This indicates that the activity of HD11 in taking up lung-derived exosomes may involve cytoskeleton movement and endocytosis processes, and is closely related to autophagy induced after exosome treatment.

[0028] To further clarify the therapeutic effect of exosomes, the differentially expressed genes were classified into two subsets: up-regulated and down-regulated genes, and KEGG pathway enrichment analysis was performed on them. The results showed that the up-regulated genes after exosome treatment were mainly enriched in autophagy, MAPK, FOXO, Toll-like receptors and other immune-related pathways, indicating that exosome treatment can activate the autophagy pathway of recipient cells. The down-regulated genes after exosome treatment were enriched in the metabolism and oxidative stress pathways, indicating that exosomes have a potential inhibitory effect on oxidative stress induced by MG infection.

[0029] Step 4:

[0030] The SPF eggs were placed in a sterile biochemical incubator with a relative humidity of 55%-60% and a temperature of 37.8±0.1℃ for incubation. During the period, sterile eggs and dead embryos were detected by egg candling. At 8 days of incubation, the position of the allantoic cavity of the chicken embryo was marked with a pencil by egg candling. At 13 days of incubation, the embryo eggs were disinfected with alcohol and iodine in a sterile clean bench, and a 1mm hole was drilled at the marked position with a sterile egg drill; 0.8mL MG-HS (1×10 9 CCU / mL) was injected into the allantoic cavity of chicken embryos, and the non-infected group was injected with the same volume of saline as a control; 24 hours later, the embryos were candled to detect nonspecific dead embryos. 72 hours after MG infection, embryo eggs were randomly selected from each group, DNA was extracted to detect MG marker genes, and the infection status was determined. Subsequently, exosomes (about 3-5×1010 particles / mL per egg, 200ul) and agomiR (5nmol agomiR diluted in 200μL saline) were injected using a syringe.

[0031] The results of the small animal imaging system showed that fluorescence signals could be detected in the heart, liver and lungs 6 hours after the exosomes were ingested, and in all organs of the body (including the heart, liver, spleen, lungs, kidneys, trachea and intestines) 12 hours later. Among them, the fluorescence signals in the heart, liver and lungs were the strongest, and a certain amount of fluorescence signals could also be detected in the trachea.

[0032] Step 5:

[0033] Lungs treated with exosomes enriched with gga-miR-200a-3p and gga-miR-148a-3p were collected, and pathological sections and MG bacterial loads of lung tissues were detected to evaluate the effects of exosomal gga-miR-200a-3p and gga-miR-148a-3p in treating chicken respiratory diseases. Results As shown in Figure 5, MG infection led to lung pathological conditions such as alveolar septum reduction, lymphocyte infiltration, and diffuse pulmonary hemorrhage. Treatment with MG-sEV-miRNAs or gga-miR-200a-3p and gga-miR-148a-3p mimics significantly improved the above pathological damage. In addition, injection of MG-sEV-miRNAs or gga-miR-200a-3p and gga-miR-148a-3p mimics significantly inhibited the adhesion protein level and bacterial load of MG in lung tissues.

[0034] Based on the above, the advantages of the present invention are that the MG-sEV in the present invention is a natural enrichment of miRNA-200-148, which can protect miRNA from the influence of RNase activity, and has the advantages of low immunogenicity and low cytotoxicity, and high stability and biocompatibility; the miR-200 and miR-148 in the present invention are a type of anti-inflammatory gene, which can alleviate the inflammatory damage of lung tissue caused by MG infection, thereby inhibiting the adhesion protein level and bacterial load of MG in the lung tissue, and the lung tissue is the target organ of MG infection and the main organ of respiratory disease damage; therefore, the present invention uses MG-sEV as a therapeutic medium to treat chronic respiratory diseases in chickens induced by MG infection.

[0035] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for treating chronic respiratory diseases in chickens using MG-sEV-miRNA-200-148, comprising the following steps: Step 1, screening miR-200 enriched in exosomes (MG-sEV) derived from MG-infected chicken alveolar type II epithelial cells (ACEII); step 2, overexpressing miR-200 in ACEII cells to extract exosomes rich in miR-200-148 (MG-sEV-miRNA-200-148); step 3, constructing an in vitro cell model of MG infection to confirm the therapeutic target of MG-sEV-miRNA-200-148; step 4, constructing a chicken embryo model of MG infection, and injecting MG-sEV-miRNA-200-148 to intervene in respiratory diseases caused by MG; step 5, evaluating the efficacy; characterized in that: according to claim 1, the miRNA related to the treatment of chronic respiratory diseases in chickens is characterized in that the miRNAs are gga-miR-200a-3p and gga-miR-148a-3p.

2. The method for treating chronic respiratory diseases in chickens using MG-sEV-miRNA-200-148 according to claim 1, characterized in that: In the step 2, the culture system used for MG-sEV-miRNA-200-148 is a serum-free medium.

3. The method for treating chronic respiratory diseases in chickens using MG-sEV-miRNA-200-148 according to claim 1, characterized in that: In the step 2, the specific steps of extracting MG-sEV-miRNA-200-148 are as follows: taking the cell supernatant at 1500×g, centrifuging at 4°C for 15min to remove dead cells; centrifuging at 4°C, 10000×g, centrifuging for 30min to remove cell debris; centrifuging at 4°C, 14000×g, centrifuging for 30min, taking the supernatant; ultracentrifuging at 4°C, 110000×g, centrifuging for 2h, collecting the precipitate, resuspending the precipitate with DPBS, ultracentrifuging again at 4°C, 110000×g, centrifuging for 1h, discarding the supernatant, collecting the precipitate, which is the exosome precipitate, resuspending the exosomes with 100μl sterile PBS, and storing at -80°C for later use.

4. The method for treating chronic respiratory diseases in chickens using MG-sEV-miRNA-200-148 according to claim 1, characterized in that: In step 4, the chick embryo used in the chick embryo treatment model was 13 days old, and the MG dose injected into the allantoic cavity was 0.8 mL MG-HS (1×10 9 CCU / mL).

5. The method for treating chronic respiratory diseases in chickens using MG-sEV-miRNA-200-148 according to claim 1, characterized in that: In the step 4, the exosome dosage injected into the allantoic cavity of the chicken embryo used in the chicken embryo treatment model is about 3-5×1010 particles / mL per egg, 200ul; the amount of injected miRNA is 5nmol agomiR.

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