KR102143183B1 - A composition for the diagnosis of asthma, comprising a reagent for measuring microbiome in the respiratory - Google Patents

Abstract

The present invention, by providing a composition for diagnosing asthma, provides a method for significantly predicting or grasping the degree of disease and early diagnosis of asthma through simple sample collection, and can be used to develop a diagnostic kit for rapid diagnosis.

Description

A composition for the diagnosis of asthma, comprising a reagent for measuring microbiome in the respiratory}

The present invention relates to a composition for diagnosing asthma, including an agent for measuring a specific microbial community, a diagnostic kit and a diagnostic method using the composition.

Fine dust is a fine particle suspended in the atmosphere with very complex components, and exposure to fine dust is known to increase mortality as well as incidence of respiratory and cardiovascular diseases. In general, the diameter of the fine particles is 0.1 to 2.5 μm, and the ultrafine particles mean a material having a diameter of less than 0.1 μm. Although the composition of fine dust is very diverse, it is mainly composed of carbon components (organic carbon, elemental carbon), ionic components (sulfate, nitrate, ammonium), and mineral components. Fine dust mainly causes inflammatory reactions in the bronchioles, and these actions are known to cause or worsen asthma and chronic obstructive pulmonary disease.

Microbiome refers to all microorganisms mainly present in human epithelial cells such as human intestine, respiratory tract, skin and genital organs, and all of their genetic information, and refers to a collection of genomes that means the entire genetic information of a single organism. do. These microbiomes are known to affect human health according to their composition. Most of the microorganisms living in the human body are symbiotic bacteria that do not harm the human body, and some of them are beneficial bacteria such as inhibiting the growth of pathogens or producing metabolites that human cells cannot make. However, although it exists as a general symbiotic bacteria in a specific body part, pathobiont that exhibits pathogenicity also exists according to environmental conditions such as condition and body parts.

With the recent development of gene sequencing technology, it has become possible to investigate the human microbiome in various parts of the human body.

Environmental microbes are associated with both protective and detrimental effects of chronic lung disease and can metabolize inhaled pollutants or modulate inflammatory responses to exposure. Thus, the biochemical and immunological profile of the microbial community can be a health indicator of the lung or bronchial environment. The effects of intestinal microbes on health have been relatively well studied, but the role of microbes in asthma induced by inflammation caused by inhalation of pollutants is generally unknown.

Republic of Korea Patent Publication No. 10-1831416

One object of the present invention is to provide a composition for diagnosis of asthma comprising an agent for detecting microorganisms in the genus Deinococcus .

Another object of the present invention is to provide a kit for diagnosing asthma comprising the composition.

Another object of the present invention includes the steps of (a) measuring the level of the genus Deinococcus from the suspected subject and (b) comparing the level of the microorganism measured in step (a) with a sample of a control It is to provide a method of providing information for the diagnosis of asthma.

The present inventors measured the level of microorganisms in the respiratory tract in an animal model inducing inflammation with titanium dioxide and compared it with the level of microorganisms in a control animal model, and in mice in which inflammation was induced, Brucella , Methylobacterium , and Acine It was confirmed that the relative abundance of microorganisms of the genus Acinetobacter , Deinococcus , and Enhydrobacter increased, while Pseudomonas , Mesorhizobium , and Micrococcus ( It was confirmed that the relative abundance ratio of microorganisms in the genus Micrococcus ) decreased. Accordingly, by analyzing changes in the microbial community in the lung, it was confirmed that it can be used for diagnosis of asthma, which is a pulmonary inflammatory disease, and the present invention was completed.

In order to achieve the above object, the present invention provides a composition for diagnosing asthma comprising an agent for detecting microorganisms in the genus Deinococcus in one aspect.

The term "asthma" used in the present invention is a generic name for various diseases characterized by inflammatory reactions of the airways leading to organs, bronchi, bronchioles, and alveoli, as well as damage and changes in airway tissues. Specifically, asthma is a condition in which the bronchi in the lungs becomes very sensitive, sometimes causing the bronchi to become narrowed, causing the bronchi to become short of breath and a crotch of breath, causing severe coughing. It is a disease. Typical symptoms of asthma are shortness of breath, cough, wheezing (wheezing, rough breathing), etc., symptom relievers (bronchodilators) that relieve narrowed bronchi in a short time, or disease regulators that prevent asthma attacks by suppressing allergic inflammation of the bronchi ( Anti-inflammatory drugs, leukotriene modulators), etc. are used as representative treatments. In the present invention, the asthma may be bronchial asthma, allergic asthma, atopic asthma, non-topic asthma, exercise-induced asthma, aspirin asthma, psychogenic asthma, or alveolar asthma, but is not limited thereto.

The term "diagnosis" as used in the present invention means identifying the presence or characteristics of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of asthma, and the onset of asthma can be confirmed early.

In an embodiment of the present invention, it was confirmed that airway hypersensitivity and inflammatory cells were increased in mice injected with titanium dioxide (nano dust) of 100 nm or less, and 16s rRNA was analyzed in the lung tissue of the mice that caused inflammation to The relative abundance of microorganisms detected in was compared and analyzed with the control group treated with physiological saline. 16s rRNA is V1 to by analyzed amplify the V3 domain, the analysis results, Brucella in lung tissue inflammation is induced mice (Brucella), tumefaciens (Methylobacterium) methyl, Acinetobacter (Acinetobacter), Day Noko Syracuse (Deinococcus ) And Enhydrobacter genera increased relative abundance of microorganisms, whereas Pseudomonas , Mesorhizobium , and Micrococcus were found to decrease in relative abundance of microorganisms. Confirmed. It was also confirmed that the number of inflammatory cells (total cells, macrophages, eosinophils) and the relative abundance of Deinococcus had a positive correlation.

Therefore, it is possible to diagnose asthma by measuring the level of microorganisms in the genus Deinococcus .

In addition, a Pseudomonas (Pseudomonas), meso hijo emptying (Mesorhizobium), micro nose kusu (Micrococcus), brucellosis (Brucella), tumefaciens (Methylobacterium) methyl, ahsine Sat bakteo (Acinetobacter) and yen dihydro bakteo (Enhydrobacter) By measuring the level of microorganisms in the genus selected from the group consisting of, it is possible to diagnose asthma.

Therefore, the composition of the present invention includes an agent that detects microorganisms in the genus Deinococcus , and includes Pseudomonas , Mesorhizobium , Micrococcus , Brucella , and methylo . tumefaciens (Methylobacterium), may include a ahsine Sat bakteo (Acinetobacter) and yen dihydro bakteo (Enhydrobacter) adding an agent for detecting the microorganism in the is selected from the group consisting of.

In the present invention, Deinococcus refers to a microorganism or a community thereof composed of species belonging to the genus Deinococcus taxonomically. In the present invention, deinococcus, as well as the previously reported strains, is preferably 70% or more, more preferably 80% or more, more preferably 90% when comparing the previously reported deinococcus and 16S rRNA sequence. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, Pseudomonas ( Pseudomonas ) refers to a microorganism composed of species (species) belonging to the genus Pseudomonas taxonomically or a community thereof. In the present invention, Pseudomonas, as well as the previously reported strains, is preferably 70% or more, more preferably 80% or more, and more preferably 90 when comparing the previously reported Pseudomonas and 16S rRNA sequences. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, Mesorhizobium refers to a microorganism or a community thereof composed of species belonging to the genus Mesorhizobium taxonomically. In the present invention, mesohizobium is preferably 70% or more, more preferably 80% or more, and even more preferably 90 when comparing the previously reported mesohizobium and 16S rRNA sequence as well as the previously reported strains. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, Micrococcus refers to a microorganism or a community thereof composed of species belonging to the genus Micrococcus taxonomically. Micrococcus in the present invention is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, when comparing the previously reported micrococcus and 16S rRNA sequences, as well as the previously reported strains, Most preferably, all microorganisms having a sequence homology of 95% or more are included in the scope of the present invention.

In the present invention, Brucella refers to a microorganism or a group thereof composed of species belonging to the genus Brucella taxonomically. In the present invention, Brucella, as well as conventionally reported strains, is preferably 70% or more, more preferably 80% or more, more preferably 90% or more, when comparing the conventionally reported Brucella and 16S rRNA sequence. Preferably, all microorganisms having a sequence homology of 95% or more are included in the scope of the present invention.

In the present invention, methylobacterium refers to a microorganism or a community thereof composed of species belonging to the genus methylobacterium taxonomically. In the present invention, methylobacterium is preferably 70% or more, more preferably 80% or more, and more preferably when comparing the previously reported methylobacterium and 16S rRNA sequence as well as the previously reported strains. All microorganisms having a sequence homology of 90% or more, most preferably 95% or more are included in the scope of the present invention.

Acinetobacter in the present invention (Acinetobacter) refers to a microorganism or a cluster made up of species (Species) belonging to the genus Acinetobacter taxonomically. In the present invention, Acinetobacter is preferably 70% or more, more preferably 80% or more, more preferably 90% when comparing the previously reported Acinetobacter and 16S rRNA sequence as well as the previously reported strains. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, Enhydrobacter refers to a microorganism or a community thereof composed of species belonging to the genus Enhydrobacter taxonomically. In the present invention, Enhydrobacter is preferably 70% or more, more preferably 80% or more, even more preferably 90% when comparing the previously reported Enhydrobacter and 16S rRNA sequences as well as the previously reported strains. All microorganisms having a sequence homology of at least 95%, most preferably at least 95% are included in the scope of the present invention.

In the present invention, the term "agent to detect" refers to asthma diagnostic markers in a sample, Deinococcus , Pseudomonas , Mesorhizobium , Micrococcus , Brucella ), means tumefaciens (Methylobacterium), materials which can be used to ahsine Sat bakteo (to detect the presence of the genus of microorganism Acinetobacter) or yen dihydro bakteo (Enhydrobacter) methyl. For example, Deinococcus ( Deinococcus ), Pseudomonas ( Pseudomonas ), Mesorhizobium ( Mesorhizobium ), Micrococcus ( Micrococcus ), Brucella ( Brucella ), Methylobacterium ( Methylobacterium ), Acinetobacter ( Acinetobacter) ) Or an organic biomolecule such as a protein, nucleic acid, lipid, glycolipid, glycoprotein or sugar (monosaccharide, disaccharide, oligosaccharide, etc.) that are specifically present in microorganisms in the genus Enhydrobacter can be specifically detected. Primers, probes, antisense oligonucleotides, aptamers, or antibodies.

In the present invention, a marker is a substance capable of distinguishing and diagnosing a normal group individual and an individual with asthma, and specifically, a microbial community showing an increase or decrease in the individual with asthma of the present invention. Means substance.

The term "primer" as used in the present invention includes all combinations of primer pairs consisting of forward and reverse primers that recognize a target gene sequence, but is preferably a primer pair that provides an analysis result having specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence inconsistent with the non-target sequence present in the sample, a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification can impart high specificity. .

The term "probe" used in the present invention refers to a substance capable of specifically binding to a target substance to be detected in a sample, and refers to a substance capable of specifically confirming the presence of a target substance in a sample through the binding. do. The type of probe molecule is not limited as a material commonly used in the art, but preferably may be a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a peptide, a polypeptide, a protein, RNA or DNA. Specifically, the probe is a biomaterial that includes an organism-derived or similar thing, or a thing produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and RNA. DNA includes cDNA, genomic DNA, and oligonucleotide, RNA includes genomic RNA, mRNA, and oligonucleotide, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

In the present invention, the term "antisense oligonucleotide" is a DNA or RNA containing a nucleic acid sequence complementary to a specific mRNA sequence, or a derivative thereof, which binds to a complementary sequence in the mRNA and inhibits translation of the mRNA into a protein. It works. The antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the mRNA of the genes and can bind to the mRNA. This can inhibit the translation of the gene mRNA, translocation into the cytoplasm, maturation or any other essential activity for overall biological function. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases. The antisense oligonucleotide may be synthesized in vitro by a conventional method and administered in vivo, or an antisense oligonucleotide may be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is the use of RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow antisense RNA to be transcribed using a vector whose origin is in the opposite direction of the multicloning site (MCS). It is preferable that the antisense RNA has a translation stop codon in the sequence so that it is not translated into a peptide sequence.

In the present invention, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that binds to a target molecule with high affinity and specificity while having a stable tertiary structure by itself. For the purposes of the present invention, the aptamer is Deinococcus , Pseudomonas , Mesorhizobium , Micrococcus , Brucella , Methylobacterium , Acinetobacter ( Acinetobacter ) or Enhydrobacter ( Enhydrobacter ) means an aptamer that binds to a protein specifically present in microorganisms in the genus. The production of the aptamer can be easily prepared using techniques well known in the art.

In the present invention, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, antibodies are Deinococcus , Pseudomonas , Mesorhizobium , Micrococcus , Brucella , Methylobacterium , Acine It refers to an antibody that specifically binds to a protein present in a microorganism of the genus Acinetobacter or Enhydrobacter , and includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Producing antibodies can be readily prepared using techniques well known in the art.

In addition, the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. A functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

Specifically, the agent to be detected in the present invention is Deinococcus , Pseudomonas , Mesorhizobium , Micrococcus , Brucella , Methylobacterium , Acinetobacter ( Acinetobacter ) or Enhydrobacter ( Enhydrobacter ) It is preferable to specifically detect the genomic sequence of a microorganism in the genus and do not specifically bind to the genome sequence of other microorganisms. More specifically, Deinococcus ( Deinococcus ), Pseudomonas ( Pseudomonas ), Mesorhizobium ( Mesorhizobium ), Micrococcus ( Micrococcus ), Brucella ( Brucella ), Methylobacterium ( Methylobacterium ), Acinetobacter ( Acinetobacter) ) And Enhydrobacter may be a primer capable of amplifying 16S rRNA of one or more microorganisms selected from the group consisting of microorganisms of the genus.

In the present invention, 16S rRNA is an rRNA constituting the 30S subunit of a prokaryotic ribosome, and there is a conserved region common to all species and a hypervariable region capable of classifying a specific species. Microorganisms can be identified through sequencing. In particular, since there is little diversity between homogeneous species, diversity appears between other species, and thus prokaryotes can be usefully identified by comparing the sequence of 16S rRNA. In addition, 16S rDNA is a gene encoding 16S rRNA, so 16S rDNA can be used to identify microorganisms.

It can be used to amplify a specific 16S rRNA (or 16S rDNA) sequence, and through sequence amplification results in the production of a desired product, Deinococcus , Pseudomonas , Mesorhizobium , micro nose kusu (Micrococcus), brucellosis (Brucella), can detect the presence of the genus of microorganisms tumefaciens (Methylobacterium), sat ahsine bakteo (Acinetobacter) or yen dihydro bakteo (Enhydrobacter) methyl.

Specifically, in the present invention, the primers are Deinococcus , Pseudomonas , Mesorhizobium , Micrococcus , Brucella , Methylobacterium , The sequence amplification method using a primer specific for microorganisms of the genus Acinetobacter or Enhydrobacter may use various methods known in the art. For example, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, touchdown PCR, hot start PCR, nested PCR, Booster PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction , Vectorette PCR, TAIL-PCR (thermal asymmetric interlaced PCR), ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-maintaining sequence replication, or selective amplification of the target sequence may be used. , The scope of the present invention is not limited thereto

In addition, the agent for detecting a microorganism in the present invention may be an antibody, and the microorganism may be detected using an immunological method based on an antigen-antibody reaction. Analysis methods for this include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion, and rocket immunoelectricity. Electrophoresis, tissue immunostaining, immunoprecipitation assay, Complement Fixation Assay, FACS (Fluorescence activated cell sorter), or protein chip, etc., but are not limited thereto.

In addition, molecular and immunological methods widely used in the art can be used to detect the microorganism of the present invention.

In order to achieve the above object, the present invention provides a kit for diagnosing asthma, including the composition in another aspect.

The diagnostic composition of the present invention may be implemented and provided in the form of a diagnostic kit. The kit of the present invention not only includes detection agents such as primers, probes, antisense oligonucleotides, aptamers, or antibodies for detecting the microorganism, but also one or more other constituent compositions, solutions, or devices suitable for analysis methods. May be included.

Specifically day Noko kusu in (Deinococcus), Pseudomonas (Pseudomonas), meso hijo emptying (Mesorhizobium), micro nose kusu (Micrococcus), brucellosis (Brucella), tumefaciens (Methylobacterium) methyl, Acinetobacter (Acinetobacter) Alternatively, it may be a diagnostic kit comprising a primer specific for bacteria of the genus Enhydrobacter . A diagnostic kit including a primer may include essential elements for performing an amplification reaction such as PCR. For example, the PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitor, DEPC-water ), or sterilized water.

In addition, it may be a diagnostic kit characterized in that it includes essential elements necessary to perform the DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, it may be a diagnostic kit characterized in that it contains the essential elements necessary to perform the ELISA. ELISA kits contain antibodies specific for the protein. Antibodies are antibodies with high specificity and affinity for each marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the ELISA kit may contain an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or antibodies capable of binding. Other materials may be included.

In addition, preferably, it may be a rapid kit, characterized in that it contains the essential elements necessary to perform a rapid test that can know the analysis result within 5 minutes. The rapid kit contains antibodies specific for the protein. Antibodies are antibodies with high specificity and affinity for each marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. In addition, the rapid kit may include an antibody specific for a control protein. Other rapid kits include reagents that can detect bound antibodies, for example, nitrocellulose membranes in which specific antibodies and secondary antibodies are immobilized, membranes bound to beads bound to antibodies, absorption pads and sample pads, etc. It may contain substances and the like.

In order to achieve the above object, the present invention is another aspect of (a) measuring the level of the genus Deinococcus from a suspected individual, and (b) measuring the level of microorganisms measured in the step (a) as a control It provides a method of providing information for diagnosing asthma comprising the step of comparing with a sample of.

In the step (a), the “suspect subject” may be collected from the lungs of a patient suspected of asthma, and specifically, may be a bronchial alveolar lavage fluid (BALF) collected from a suspected asthma patient, or a lung tissue sample.

The present invention Day Noko Syracuse (Deinococcus) level measuring in the are data Noko Syracuse (Deinococcus) specific to the present protein, nucleic acid, lipid, glycolipid, protein or (monosaccharide sugar per to the genus microorganisms present in a sample of the suspected object in, It means measuring the level of organic molecules such as disaccharides, oligosaccharides).

For measuring the level of the organic molecule, a primer, a probe, an antisense oligonucleotide, an aptamer, or an antibody capable of specifically detecting the organic molecule may be used, and specifically, a primer that detects 16s rRNA may be used.

In the step (b), the control sample is taken from the lungs of a normal individual, and preferably may be a bronchial alveolar lavage fluid (BALF) or lung tissue collected from a normal individual.

Deinococcus ( Deinococcus ), Pseudomonas ( Pseudomonas ), Mesorhizobium ( Mesorhizobium ), Micrococcus ( Micrococcus ), Brucella ( Brucella ), Methylobacterium ( Methylobacterium) ), Acinetobacter ( Acinetobacter ) and Enhydrobacter ( Enhydrobacter ) levels of microorganisms in the genus were confirmed to change compared to the normal control bar, Deinococcus ( Deinococcus ) level measured in the suspected subject compared to the normal control and a diagnosis of asthma, wherein the Pseudomonas (Pseudomonas), meso hijo emptying (Mesorhizobium), micro nose kusu (Micrococcus), brucellosis (Brucella), tumefaciens (Methylobacterium) methyl, Acinetobacter (Acinetobacter) and yen It may further comprise the step of measuring the level of microorganisms in the genus selected from the group consisting of Hydrobacter ( Enhydrobacter ).

In addition, Brucella in the lung inflammation is induced mice (Brucella), bar hayeotneun confirmed that tumefaciens (Methylobacterium), ahsine Sat bakteo (Acinetobacter), Day Noko Syracuse (Deinococcus) and yen dihydro bakteo (Enhydrobacter) is increased to methyl , brucellosis (Brucella), tumefaciens (Methylobacterium), ahsine Sat bakteo (Acinetobacter), Day Noko Syracuse (Deinococcus) and yen dihydro bakteo (Enhydrobacter) the level of microbial control microbial levels of the genus selected from the group consisting of methyl than If it increases, the step of diagnosing asthma may be further included.

In addition, it was confirmed that the level of microorganisms in the genus Pseudomonas , Mesorhizobium , and Micrococcus in the lungs of the mice in which inflammation was induced was decreased compared to the level of microorganisms in the control. If the level of the microorganism selected from the group consisting of ( Pseudomonas ), Mesorhizobium and Micrococcus decreases than that of the control, the step of diagnosing asthma may be further included.

As described above, the present invention provides a method for significantly predicting or grasping the early diagnosis and disease degree of asthma through simple sample collection by providing a composition for diagnosing asthma, and provides a diagnostic kit for rapid diagnosis. It can be used for development.

Figure 1 relates to the analysis of a mouse model induced by titanium dioxide, specifically a shows the method of inducing titanium dioxide in the mouse model, b is the airway hypersensitivity measurement result, c is the total number of cells and macrophages, eosinophils, neutrophils And d is a microscopic image obtained by H&E staining of lung tissue.
2A to 2C are results of measuring Chao1, Shannon, and Simpson, which are microbial diversity indexes in the lungs of a mouse model. Specifically, a is the Chao1 abundance estimation, b is the Shannon uniformity, and c is the measurement result of Simpson's diversity.
2D is a main coordinate analysis plot for investigating differences in lung microbiome using a weighted Unifrac distance metric.
2E is a result of analyzing the frequency of 16s rRNA genes in the lungs of a mouse model.
2F is a heat map showing 11 major operating taxonomic units (OTUs) among the bacterial communities inferred from the mouse lung sequence, and each mouse is shown individually (n=6-8).
3 is a result of analyzing the bacterial community composition of a lung sample of a mouse model (a to h). The relative abundance of the lung microbiome in each group was shown by boxplot analysis, and the boxes represent the inner quartile value range of the median indicated by a black line.
FIG. 4 relates to the relationship between the number of inflammatory cells and the relative abundance of Deinococcus , specifically analyzing the correlation between the total number of cells, the number of macrophages, or the number of eosinophils and the relative abundance of Deinococcus .

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

<Materials and methods>

1. Mouse model study

This study was carried out in accordance with the Steering Committee of the Animal Laboratory of Soonchunhyang University Bucheon Hospital, and the experimental animals were raised in an environment free of specific pathogens. BALB/c female mice of 6 to 8 weeks of age (n = 8, Orient Bio, Seongnam, Korea) were used in the study. Mice were exposed to titanium dioxide (TiO ₂ , 100 nm or less) for 1 hour at 200 μg/m ³ for 5 days using an ultrasonic nebulizer, and control mice were exposed to physiological saline. On the 6th day, airway hyperreactivity (AHR) was measured, and on the 7th day, 1.05X phosphate buffered saline (PBS) was added to both lungs of the mouse and gently inhaled to collect and analyze bronchial airway lavage (BAL). . The total number of cells was measured using a hemocytometer, and the differential cell count was performed using a cytocentrifugation and Diff-Quik staining (Dade Diagnostics, Deerfield, IL). The lungs were fixed overnight in 4% buffered formalin and then embedded in paraffin. Lung micro-sections (4㎛) were stained with hematoxylin and eosin (H&E staining) and used for histological evaluation.

2. Measurement of airway response to methacholine

Airway responses were measured using conscious mice 1 day after the last exposure (day 6), as described above. The mice were placed in a barometric plethysmographic chamber, measured for 3 minutes, and an average value was calculated as a baseline measurement value. Thereafter, while increasing the concentration of aerosolized methacholine from 5 to 100 mg/ml, it was sprayed through the inlet of the chamber for 3 minutes, and the change in atmospheric pressure was measured for 3 minutes. The average of the measured values after spraying for each concentration was calculated to analyze the enhanced pause (Penh). Penh is calculated as (exhalation time/relaxation time-1) × (maximum expiratory flow/maximum inspiratory flow), a dimensionless value representing a function of the maximum expiration rate for the maximum intake box pressure signal, and a function of the expiration time. Penh was used as a measure of the airway response to methacholine. The results were expressed as a percent increase in Penh after administration at each concentration of methacholine, and the baseline Penh (saline exposure) was expressed as an actual value. Penh values were evaluated and averaged for 3 minutes after each nebulization.

3. Lung microbiota analysis

DNA was isolated from lung tissue from 200 μg TiO ₂ /m ³ (n = 6) and saline exposure (n = 6) groups using a QIAamp DNA mini kit (Qiagen), and DNA extracts of the two groups were transferred to a sequencing center. sent. Sequence analysis was performed using Illumina HiSeq 2500. The V1 to V3 regions of 16S rRNA were also amplified by PCR.

PCR was performed under conditions of initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and final stretching steps at 72°C for 5 minutes. Then, PCR products were visualized and confirmed using Gel Doc System (BioRad, Hercules, CA, USA) through 2% agarose gel electrophoresis.

The amplification product was purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). Purified products of equal concentration were mixed, and non-specific amplified products were removed using AMpure beads kit (Agencourt Bioscience, MA, USA). The size and quality of PCR products purified by Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) were analyzed using a DNA 7500 chip. The mixed amplification products were applied to emulsion PCR and then deposited on a picotite plate. Chunlab, Inc. using the GS Junior System (Roche, Branford, CT, USA) according to the manufacturer's instructions. (Seoul, Korea).

The composition and ratio of the spices shared by two samples or multiple sample sets were calculated, and the overall phylogenetic distance between communities was estimated using Fast UniFrac, and principal coordinate analysis (PCoA). ) To visualize. The shared OTU was identified using XOR analysis to compare the operational taxonomic unit (OTU) between samples (CL Community Program, Chunlab Inc.).

4. Statistical Analysis

Continuous variables were compared using Mann-Whitney test, and categorical variables were analyzed using Chi-square test or Fisher's exact test. Data are expressed as median [quartile] for continuous variables and number (%) for categorical variables. If the adjusted p value was less than 0.05, it was considered statistically significant. All statistical analyzes were performed using SPSS version 22 (SPSS, Chicago, IL, USA).

<Result>

1. Titanium dioxide (TiO ₂ ) Lung dysfunction analysis in exposed mouse model

As a result of analyzing BAL of mice 48 hours after exposure to titanium dioxide (TiO ₂ ) nanoparticles of 100 nm or less, compared to vehicle-treated control mice, airway hypersensitivity (Fig. 1B) and total BAL cell count (Fig. 1c) Showed a significant increase in. Titanium dioxide (TiO ₂ ) induced an increase in inflammatory cells in which macrophages were remarkably present in BAL (FIG. 1B). In addition, as a result of lung histology analysis, it was confirmed that titanium dioxide (TiO ₂ ) induces inflammatory cell infiltration (mainly neutrophils and monocytes) and epithelial remodeling in the lung interstitial and alveolar compartment (Fig. 1d). That is, it was confirmed that asthma was induced in the mouse model.

2. Microbial community profiling: Pyrosequencing results and bacterial community composition analysis

DNA was extracted from lung tissue and PCR was performed with a universal primer targeting a region of the 16S rRNA gene. The PCR amplification products were sequenced and collected into operational classification units, and taxonomically annotated to evaluate the composition and structure of the lung microbial community (FIG. 2E). In the lung tissue of TiO ₂ group of mice was confirmed that the bacteria are dominant genus of Pseudomonas (Pseudomonas), brucellosis (Brucella) and Acinetobacter (Acinetobacter), accounted for each 17.6%, 14.3% and 13.9%. Further, the method hijo emptying (Mesorhizobium), yen dihydro bakteo (Enhydrobacter), methyl tumefaciens (Methylobacterium), Day Noko Syracuse (Deinococcus), separation tank emptying (Rhizobium), Cri Seo tumefaciens (Chryseobacterium), spin and emptying (Sphingobium ), Dietzia , and Brevundimonas were found to have a relative abundance ratio of 2 to 8%, respectively.

By analyzing the 16S RNA tag, the complexity of the community was measured by the diversity index of Chao1, Shannon and Simpson (1-), and as a result of the analysis, it was confirmed that the complexity of the community was not changed by TiO ₂ treatment. (Fig. 2a, b and c). Although the effect of TiO ₂ on community complexity was not statistically significant, it was confirmed that the bacterial growth observed in the analysis results contributed to the microbial ecosystem in the lungs.

Next, the composition of the microbial community in the lung was analyzed to confirm the change in the composition of the microbial community in the lung in which inflammation was induced. A matrix based on Yue and Chandlans measurement, which measures the difference in the structure of clusters, was used to distinguish samples by Principal Coordinates Analysis (PCoA) (FIG. 2D). As a result of the analysis, it was confirmed that 29.3% of the total variation in the data can be explained by the main coordinate PC1.

In addition, through the frequency analysis of bacterial microorganisms in the TiO ₂ exposed group and the control group mice, 11 major genus of bacteria having a large relative abundance difference were identified (FIG. 2F). The 11 in the bacterium is Pseudomonas (Pseudomonas), ahsine Sat bakteo (Acinetobacter), brucellosis (Brucella), meso hijo emptying (Mesorhizobium), yen dihydro bakteo (Enhydrobacter), tumefaciens (Methylobacterium) methyl, separation tank emptying ( Rhizobium ), Chryseobacterium , Brevundimonas , Deinococcus and Micrococcus were identified.

Next, the relative abundance of microorganisms were selected for the bacteria in the relative abundance ratio is greater eight genus (genus) difference in TiO ₂ exposed group and the control group mice on the basis of the analysis results, the presence of the control group mice, and TiO ₂ exposed group Compared using box plot analysis (Fig. 3a-h). As a result of the analysis, it was confirmed that the relative abundance of bacteria in the genus Pseudomonas , Mesorhizobium , and Micrococcus decreased in the TiO ₂ exposure group compared to the control group mice. On the other hand it was confirmed that brucellosis (Brucella), methyl tumefaciens (Methylobacterium), ahsine Sat bakteo (Acinetobacter), Day Noko Syracuse (Deinococcus) and yen dihydro bakteo (Enhydrobacter) relative presence of in the bacteria ratio is increased from TiO ₂ exposed group .

Specifically, it was confirmed that in the lungs of mice induced airway inflammation with TiO ₂ , Micrococcus genus decreased 11 times from 1.3 to 0.1%, and Deinococus genus increased 3.8 times from 0.8 to 3.38%. (P = 0.025).

Additionally, the relationship between airway inflammatory cells and microbial communities was confirmed. As a result of the analysis, it was confirmed that the number of total cells, macrophages, and eosinophils had a positive correlation with the relative abundance in Deinococcus (Fig. 4).

Claims (9)

delete delete delete delete delete (a) measuring microbial levels from bronchial alveolar lavage fluid or lung tissue samples collected from control and suspected individuals;
(b) comparing the relative abundance ratio of the microbial level of the sample of the suspected object measured in step (a) with the microbial level of the control sample; And
(c) measuring the number of airway inflammatory cells in the sample of the suspected individual and comparing the correlation between the number of inflammatory cells and the level of microorganisms of the suspected individual,
The microorganism day Noko Syracuse (Deinococcus), Pseudomonas (Pseudomonas), meso hijo emptying (Mesorhizobium), micro nose kusu (Micrococcus), brucellosis (Brucella), tumefaciens (Methylobacterium) methyl, Acinetobacter (Acinetobacter) And Enhydrobacte genus,
Tumefaciens (Methylobacterium) in Brucella (Brucella) is i) microbial level of suspicious objects samples compared to control samples in the step (b), methyl, ahsine Sat bakteo (Acinetobacter), Day Noko Syracuse (Deinococcus) and yen dihydro bakteo ( Enhydrobacter ) increases, ii) Pseudomonas ( Pseudomonas ), Mesorhizobium ( Mesorhizobium ) and Micrococcus ( Micrococcus ) when the genus decreases, diagnosed as asthma,
How to provide information for diagnosing asthma. delete delete The method of claim 6,
If the number of airway inflammatory cells in the sample of the suspected individual in step (c) has a positive correlation with the level of the microorganism of the suspected individual, it is determined as asthma, and the microorganism is of the genus Deinococcus . How to provide information for

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