KR101801092B1 - Composition for Idiopathic pulmonary fibrosis prognosis and method of providing the information for the same - Google Patents
Composition for Idiopathic pulmonary fibrosis prognosis and method of providing the information for the same Download PDFInfo
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Abstract
The present invention relates to a kit and an information providing method for diagnosis of idiopathic pulmonary fibrosis through measurement of the protein or mRNA expression level of CCL8. According to the present invention, the level of protein expression of CCL8 can be measured and used as data for diagnosis and progress of idiopathic pulmonary fibrosis.
Description
The present invention relates to a kit and an information providing method for diagnosing idiopathic pulmonary fibrosis by measuring levels of genes (mRNA and the like) such as PF4V1 or measuring the level of CCL8 protein expression.
Idiopathic pulmonary fibrosis (IPF) is a chronic inflammation and fibrosis syndrome. It is characterized by extracellular matrix deposition, excessive alveolar epithelial cell proliferation, and proliferation of myofibroblasts. It is characterized by its progressive nature and does not show reactivity to immunosuppressants and corticosteroids [1]. The disease is characterized by chronic inflammation and fibrosis-related interactions [2, 3].
The search for whole gene expression in lung tissue helps to uncover new genes to understand the complex mechanisms of IPF. Thus, studies of transcripts using human IPF and mouse fibrosis lungs have revealed that IPF patients differ in gene expression. 470 genes have been reported to be expressed in mice with different sensitivities to pulmonary fibrosis [4]. In human studies, 164 genes have been reported to be differentially expressed in the lung tissue of IPF patients [5].
Recent studies have shown that fibrosing lungs differ in the expression of genes that regulate ECM formation and regression. When comparing IPF-fast and slow-onset patients, 437 genes with different expression characteristics are known to be involved in morphogenesis [6]. In addition, expression levels of 134 genes can be used to distinguish between progressive (fast-progressive) and stable (slow-progressive) patients [7].
IPF, along with normal lung tissue, shows a mixed pathology of alveolar inflammation, interstitial fibrosis, and honeycomb-like changes [8]. Furthermore, since the size of fibrosis and inflammation significantly changes in the course of the disease, ideally, it is necessary to selectively isolate homogeneous cells derived from diseased lungs, but this is a real problem. However, among the various cell types present in lung tissue, fibroblasts are comparatively easy to obtain and manage, and the biological characteristics of IPF fibroblasts are different from those of normal lung fibroblasts [9, 10].
It is an object of the present invention to provide a marker composition or the like for providing information for diagnosis of idiopathic pulmonary fibrosis by measuring the mRNA level of PF4V1 and the like and to provide a marker composition or the like which expresses CCL8 protein expression level or CCL8 mRNA level of the patient in a normal control sample And to provide a method of providing information for diagnosis and prediction of prognosis of idiopathic pulmonary fibrosis.
In order to accomplish the above object, the present invention provides a marker composition for the diagnosis of idiopathic pulmonary fibrosis according to the present invention, comprising at least one of PF4V1, MYOC, CCL8, ROR2, HBG2, D4S234E, KCNJ2, RGS18, PITX1, EPB41L3, LOC100132994, FLJ25037, IGFBP2, Or a combination of the above-mentioned genes.
When the PF4V1 gene is mRNA, it may have the sequence shown in SEQ ID NO: 1.
When the MYOC gene is mRNA, it may have the sequence shown in SEQ ID NO: 2.
When the CCL8 gene is mRNA, it may have the sequence shown in SEQ ID NO: 3.
When the ROR2 gene is mRNA, it may have the sequence represented by SEQ ID NO: 4.
When the HBG2 gene is mRNA, it may have the sequence represented by SEQ ID NO: 5.
When the D4S234E gene is mRNA, it may have the sequence shown in SEQ ID NO: 6.
When the KCNJ2 gene is mRNA, it may have the sequence represented by SEQ ID NO: 7.
When the RGS18 gene is mRNA, it may have the sequence shown in SEQ ID NO: 8.
When the PITX1 gene is mRNA, it may have the sequence shown in SEQ ID NO: 9.
When the EPB41L3 gene is mRNA, it may have the sequence shown in SEQ ID NO: 10.
When the LOC100132994 gene is mRNA, it may have the sequence shown in SEQ ID NO: 11.
When the FLJ25037 gene is mRNA, it may have the sequence represented by SEQ ID NO: 12.
When the IGFBP2 gene is mRNA, it may have the sequence represented by SEQ ID NO: 13.
The agent for detecting the gene may be a primer or a probe that specifically binds to the gene.
The probes specifically binding to the PF4V1, MYOC, CCL8, ROR2, HBG2, D4S234E, KCNJ2, RGS18, PITX1, EPB41L3, LOC100132994, FLJ25037 and IGFBP2 genes may have the sequence shown in SEQ ID NOs: 14 to 26, respectively .
In the case of the CCL8 gene, it may be combined with the CCL8 primer of the sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29.
The expression of these genes is shown to be up-regulated when the test sample is likely to be idiopathic pulmonary fibrosis, and FLJ25037, and IGFBP2, The gene may appear to be down-regulated if the sample is likely to be idiopathic pulmonary fibrosis.
At this time, biological specimens such as saliva, blood, and tissue can be applied to the specimen, and bronchial or pulmonary saliva or cells to be inspected can be applied to the specimen, and bronchial alveolar lavage fluid, pulmonary fibroblast, Can be applied.
The marker composition for diagnosis or progress prediction of idiopathic pulmonary fibrosis according to another embodiment of the present invention includes a preparation for detecting CCL8 protein or a preparation for detecting CCL8 gene.
The agent for detecting the CCL8 protein or the agent for detecting the CCL8 gene can be applied to a sample containing a bronchoalveolar lavage fluid or a lung-derived fibroblast.
The agent for detecting the CCL8 protein may be applied to a sample containing bronchoalveolar lavage fluid to provide information for diagnosing or predicting idiopathic pulmonary fibrosis based on the detected level of CCL8 protein expression.
Specifically, the diagnosis of idiopathic pulmonary fibrosis is likely to be diagnosed as idiopathic pulmonary fibrosis with an accuracy of 70% or more and a sensitivity of 80% or more when the CCL8 protein concentration in the bronchoalveolar lavage fluid is 1.31 pg / μg or more. The above values are based on the cut off value of ROC analysis results.
In addition, the progression of idiopathic pulmonary fibrosis can be predicted to be worse than when the concentration of CCL8 protein in the bronchoalveolar lavage fluid is 0.9025 pg / μg or more. The poor prognosis means that the survival rate is low for 1 to 8 years. That is, as a result of continuously observing the CCL8 protein concentration in the bronchoalveolar lavage fluid of the patients for about 8 years, the patients diagnosed with onset of idiopathic pulmonary fibrosis were observed for several times This means that the CCL8 protein concentration in bronchoalveolar lavage fluid is maintained at 0.9025 pg / μg or more.
The agent for detecting the CCL8 gene may be applied to a sample containing bronchoalveolar lavage fluid or pulmonary fibroblasts to confirm the expression level of CCL8 mRNA to provide information necessary for diagnosis of idiopathic pulmonary fibrosis.
The CCL8 protein detection method is one of methods for confirming the amount of the protein in the biological sample. The amount of the CCL8 protein expressed can be confirmed by including an antibody that specifically binds to the CCL8 protein. That is, the agent for detecting the CCL8 protein may be, for example, an antibody that specifically binds to CCL8 protein.
The antibody refers to a specific protein molecule directed to an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes a polyclonal antibody, a monoclonal antibody, and a recombinant antibody . As described above, the overexpression of CCL8 protein itself and its idiopathic pulmonary fibrosis has been identified, and the production of the antibody using the CCL8 protein can be easily performed using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injection of the protein antigens into the animal and collection from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.
Analysis methods for detecting the CCL8 protein include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion Immunohistochemistry Assay, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Immunoassay, Fluorescence Activated Immunoassay, Immunoprecipitation Assay, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Immunoassay Cell Sorter, FACS, and protein chips.
The agent for detecting the CCL8 gene may be a reagent capable of quantitatively detecting DNA or mRNA involved in overexpression of CCL8 protein, for example, a primer or a probe for CCL8 mRNA.
RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) , Northern blotting, and DNA chips may be used.
The kit for diagnosis or progress prediction of idiopathic pulmonary fibrosis according to another embodiment of the present invention includes the marker composition for predicting or predicting idiopathic pulmonary fibrosis described above. A detailed description of a preparation for detecting CCL8 protein or a preparation for detecting CCL8 gene contained in the marker composition will be omitted because it is redundant with the above description.
The screening method of the agent for improving idiopathic pulmonary fibrosis according to another embodiment of the present invention is a method for screening a substance for alleviating, treating or preventing symptoms of idiopathic pulmonary fibrosis by confirming whether it is a substance that controls the expression level of CCL8 protein or CCL8 gene It can be effectively screened.
Hereinafter, the present invention will be described in more detail.
To study the molecular mechanisms of IPF lungs, we performed an overall transcript analysis using IPF patients and fibroblasts from lung tissue of normal individuals with local lung lesions, and performed additional Using the BAL samples (bronchoalveolar lavage fluid, bronchoalveolar lavage fluid) and fibroblasts derived from lung tissue samples, the level of expression of CCL8 protein overexpressed in IPF patients was compared with that of normal controls. And can provide information useful for diagnosing an onset or predicting the progress of a disease.
In order to accomplish the above object, the present invention provides a method for detecting a cancer cell, comprising the steps of: (a) measuring the level of CCL8 protein represented by SEQ ID NO: 1 from a biological sample separated from a patient; (b) comparing the level of CCL8 protein to that of a normal control sample; And (c) determining that the level of the CCL8 protein is overexpressed in a normal control sample, thereby determining that the compound is idiopathic pulmonary fibrosis.
(A) measuring the mRNA level of the CCL8 gene represented by SEQ ID NO: 2 from the biological sample separated from the patient; (b) comparing the mRNA level of the CCL8 gene with the level of a normal control sample; And (c) determining that the level of the mRNA of the CCL8 gene is overexpressed in a normal control sample, thereby determining that it is idiopathic pulmonary fibrosis.
In the present invention, the sample may be a bronchoalveolar lavage fluid or a lung-derived fibroblast.
The present invention also provides an idiopathic pulmonary fibrosis diagnostic kit comprising a probe or a primer set having a sequence complementary to the CCL8 gene and an idiopathic pulmonary fibrosis diagnostic kit comprising an antibody or an aptamer that specifically binds to CCL8 protein .
(A) contacting a sample containing the CCL8 protein represented by SEQ ID NO: 1 with a sample to be analyzed; (b) measuring the amount or activity of the CCL8 protein; And (c) determining that the sample is a substance that improves idiopathic pulmonary fibrosis when the amount or activity of the CCL8 protein is measured to be regulated to be decreased.
(A) contacting a sample containing the gene encoding the CCL8 protein represented by SEQ ID NO: 1 with a sample to be analyzed; (b) measuring the expression level of the gene encoding the CCL8 protein; And (c) determining that the sample is a substance that improves idiopathic pulmonary fibrosis when the expression level of the gene encoding the CCL8 protein is measured to be regulated to be decreased, the screening method for idiopathic pulmonary fibrosis improvement agent do.
In the present invention, "diagnosis" refers to confirming the presence or characteristic of a pathological condition, and includes determining the susceptibility of an object to a specific disease or disease, determining whether an object currently has a specific disease or disease , And determining the prognosis of an object that has suffered a particular disease or disorder.
In the present invention, the term "biological sample" or "sample" refers to a lung or bronchial tissue, lung or bronchial tissue-derived cell, saliva, bronchoalveolar lavage fluid, or bronchoalveolar lavage fluid in which the level of gene or protein expression is differentiated by the onset of idiopathic pulmonary fibrosis, And the same sample.
The term "sample" used in reference to the screening method of the present invention means an unknown candidate used in screening to check whether the amount of gene expression is affected or affects the amount or activity of the protein . Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, and natural extracts.
As used herein, the term " primer " refers to a primer that, under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) in a suitable buffer at a suitable temperature, - means strand oligonucleotide. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.
The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the above-described nucleotide sequence, which is a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the gene sequence and acting as a primer. The design of such a primer can be easily carried out by those skilled in the art with reference to the above-described nucleotide sequence, and can be designed using, for example, a program for primer design (e.g.,
As used herein, the term " probe " refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.
The measurement of the expression level of the gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization (Sambrook et al. , Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Press (2001)).
In the case of carrying out according to the RT-PCR protocol, first, total RNA is isolated from the sample-treated cells, and single-stranded cDNA is prepared using oligo dT primer and reverse transcriptase. The PCR reaction can then be performed using single-stranded cDNA as a template and using a gene-specific primer set. Then, a method of electrophoresing the PCR amplification product and analyzing the formed band to measure changes in the expression level of the gene can be applied.
Changes in the amount of protein can be achieved through a variety of immunoassay methods known in the art. But are not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), capture-ELISA, inhibition or hard tissue analysis, and sandwich assays. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e. G., C 14, I 125, P 32 and S 35) The protein labeled with - a specific antibody can be used. When the method of the present invention is carried out by an ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating an extract from the treated cells on the surface of a solid substrate, (ii) (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody, and (iv) measuring the activity of the enzyme. Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates. The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. Examples of the enzyme include alkaline phosphatase, beta-galactosidase, Radish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as an enzyme that binds to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1 chromophore and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N (10-acetyl-3,7-dihydroxyphenoxaphone), TMB (3,3,5,5-tetramethylbenzidine), ABTS < (R) >(2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o-phenylenediamine (OPD). In the ELISA method, the measurement of the final enzyme activity or the measurement of the signal can be performed according to various methods known in the art. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.
According to the present invention, it is possible to diagnose and prognose idiopathic pulmonary fibrosis by measuring the level of protein expression of CCL8 or the level of CCL8 mRNA, and it can be used variously in the field of development of therapeutic agent for idiopathic pulmonary fibrosis.
1 is a graph showing the degree of expression of each gene in cultured fibroblasts of Control and Experimental Groups (IPF) as described in 2. Results and analysis of experimental results of the present invention and 2) Results from Lotting.
FIG. 2 is a diagram showing a result of a hierarchical cluster analysis described in 2. Results and analysis of experimental results of the present invention and 2) Results of gene expression profiling (in FIG. 2, A represents the difference of 178 genes between two groups (The p value by t- test is less than 0.05, the absolute fold change rate by TNoM test is more than 2), and B is the hierarchical clustering of the top 15 genes most related to the IPF. C is the top eight perturbed Gene Ontology nodes of the IPF patient compared to the control. The right side shows the statistical significance of the perturbation determined by the gene set test and the left side shows the ratio of enrichment. Black bars mean upregulation and white bars mean downregulation. D shows biological pathway analysis results of differentially expressed gene sets associated with IPF].
FIG. 3 is a graph showing the result of analyzing the results of the second experiment and the analysis of 4) the levels of CCL8 mRNA in fibroblasts.
FIG. 4 is a graph showing the results of analysis of CCL8 protein levels in bronchial alveolar lavage fluid (A) of the experimental results and analysis of the present invention. [A: CCL8 protein was detected in 23 samples of 35 normal controls and 86 samples of IPF patients Detected in 80 samples. Black circles and white circles represent CCL8 protein levels above and below 1.5 pg / mL, respectively. B: ROC curve for CCL8 protein concentration between two groups. The cutoff value of 1.31 pg / μg showed 73.6% accuracy, 45.7% specificity and 84.9% sensitivity to the IPF patient differentiation compared to the control group. C: Association of neutrophil count and CCL8 level in BAL solution (r = 0.297, p = 0.014). D: Kaplan-Meier plot of 69 individuals that were observed for 1 to 8 years. (Red line) showed a significant decrease (hazard ratio = 4.06, CI: 0.94-17.63, p = 0.0001) compared to the group with a survival rate of less than 0.9025 pg / 0.043).
FIG. 5 shows the result of analysis of CCL8 protein level in the serum of the 2. Experimental results and analysis of the examples of the present invention.
FIG. 6 is a photograph showing the result of immunofluorescence staining of 6) CCL8 of 2. Experimental results and analysis among examples of the present invention.
FIG. 7 is a view showing the sequence of CCL8 protein of SEQ ID NO: 27 and the CCL8 mRNA sequence of SEQ ID NO: 3 of the present invention. FIG.
Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
1. Preparation of sample and test method
Preparation of sample
Plasma, bronchoalveolar lavage fluid (BAL fluid) and lung tissue samples were obtained at Soonchunhyang University Hospital. The study protocol for the present invention was approved by the Hospital Ethics Committee (SCHBC-IRB-2006-05 and SCHBC-biobank-2014-005-2). Each patient gave written consent to participate in the study. The diagnostic criteria for IPF were based on the International Thoracic Society and the European Respiratory Society.
All subjects underwent interviews on their medical history and performed blood tests to exclude chest X-ray, pulmonary function test, high-resolution chest computed tomography (HRCT) and collagen vascular disease. None of the IPF patients had collagen vascular disease when tested for clinical signs or laboratory tests. IPF was diagnosed by HRCT in patients without pathologic samples (surgical IPF) with no UIP pattern or surgical lung biopsy (clinical IPF). To analyze the pathologically samples, each pathologist independently examined each slide after receiving information about the patient's age, sex, and HCRT results. IPF patients were evaluated using a series of FVC and DLCO measurements. The annual rate of FVC reduction [dFVC (% / year)] was calculated as: [dFVC (% / year)] = (last FVC - basis FVC) / reference FVC / year. The control group consisted of patients' spouses and hospital staff. In the control group, there were no respiratory symptoms, the expected FEV1 and FVC values were more than 80%, and normal chest radiogram results.
Fibroblast culture
Lung fibroblasts were cultured from 10 normal lung tissues that underwent surgery to remove surgical specimens from 14 IPF patients and
The lung samples were finely ground and placed in a 150 cm 2 cell culture flask with tissue culture media (TCM). The tissue culture medium was supplemented with DMEM (Lonza Walkersville, Inc., Walkersville, MD, USA), 10% fetal bovine serum (Thermo Fisher Scientific Inc., Rockford, IL, USA), 2 mmol / L glutamine and 1% penicillin- Amphotericin (Lonza Walkersville, Inc.). Cells were maintained in a 37 ° C, 5% CO 2 incubator and subcultured continuously until
Genomic DNA was purified using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) and total RNA was extracted using TRI reagent (Ambion, Carlsbad, CA, USA). Cell pellets were prepared in RIPA buffer for immunoblot analysis and protein concentrations were determined using BCA kit (Thermo Fisher Scientific Inc.).
Transcript Micro Away , Gene Ontology and Path Analysis
Total RNA was extracted from fibroblasts and amplified and purified using the Illumina® Total Prep ™ RNA Amplification Kit (Ambion, Carlsbad, Calif., USA) to convert into cDNA. Transcript analysis was performed using HumanHT-12 (BeadChip Illumina, San Diego, Calif., USA) with a sequence representing 47,315 probes. Gene expression data were deposited with the NCBI gene expression omnibus (series accession number GSE71351; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71351) . Gene Ontology and Pathway Prediction Was performed using a gene ontology database ( http://bioinfo.vanderbilt.edu/gotm/ ) and path-expression software (Onto-Tools; http://vortex.cs.wayne.edu/Projects.html ).
The quality and quantity of extracted RNA was tested with RNA quantitation reagent (Ribogreen ® , Invitrogen, Carlsbad, CA, USA). Fluorescence was measured using a fluorescence photometer (
A heatmap of the differentially expressed genes was constructed using GenomeStudiosoftware. Gene ontology WebGestalt (http:. //Bioinfo.vanderbilt edu / gotm /) was performed by using Gene Ontology database (GO), WebGestalt initial and bunpyo showing excessive represented Gene Ontology categories (p <0.05) . The P-value was calculated using the BINOMDIST function based on the overrepresentation of the gene ontology category as compared to all genes on the chip. Pathway-Express (Onto-Tools, Wayne State University, Detroit, MI, USA, http://vortex.cs.wayne.edu/Projects.) Was used to explore biologically relevant pathways affected by the input gene list . html ) was used.
CCL8 mRNA RT- PCR And real-time PCR
Total RNA purified using TRI reagent was treated with the Turbo DNA-Free ™ Kit (Ambion). Total RNA suspended in diethyl pyrocarbonated water was heated at 65 ° C for 5 min with 0.5 μg oligodeoxytimidine and 10 mM dNTPs, and then cooled on ice.
Amplification in 72 o C was carried out for 30 times for 7 minutes (5 minutes at 94 o C, 30 sec at 94 o C, 30 sec at 60 o C, 30 sec at 72 o C). The primer sequences used are as follows:
CCL8: sense 5'-TGGAGAGCTACACAAGAATCACC-3 ',
Antisense 5'-TGGTCCAGATGCTTCATGGAA-3 ';
β-actin: sense 5'-GGACTTCGAGCAAGAGATGG-3 ',
Antisense 5'-AGCACTGTGTTGGCGTACAG-3 '.
The PCR products were separated on a 1.0% agarose gel containing ethidium bromide in Tris-borate EDTA buffer at 100 V for 40 minutes and visualized in ultraviolet light. The CCL8 band intensity was normalized to that of beta-actin. Real-time PCR was performed using theStepOne ™ Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR mixture (20 μL) contained 1 μg cDNA, 1 μL 10 pmol forward, reverse primer and 10
CCL8 Measurement of protein level
CCL8 protein concentration was measured in bronchoalveolar lavage fluid (BAL) and plasma using an ELISA kit (Abnova, Taipei, Taiwan) and normalized to protein concentration.
BAL was performed on the right middle lobe of the identified or standard control population on HRCT without any immunosuppressive treatment. Total cell counts were calculated using a hemocytometer. The leukocyte percentages of 500 cells were performed on slides of BAL cells prepared by cell centrifugation and stained with Diff-Quik. The cell pellet was separated from the supernatant by centrifugation (500 G, 5 min) and the supernatant was stored at -80 ° C. CCL8 protein concentrations in BAL and serum were measured in normal control and IPF patients using an ELISA kit (Abnova, Taipei, Taiwan) according to the manufacturer's instructions. The lower limit of measurement was 1.5 pg / mL, and a value below this value was regarded as 0 pg / mL. The coefficient of variation for the inter- and intra-assay was less than 15%. Protein concentrations in BAL samples were measured using Micro BCA protein assay kit (Pierce, Rockford, IL, USA).
CCL8 And alpha-smooth muscle Actin (alpha-SMA) double Immunofluorescence dyeing
The paraffin block of IPF and the control lung tissue were divided into 4 μm thick sections, deparaffinized, rehydrated and hematocylin-eosin stained.
Fc-receptor blocker the fragments were incubated for 30 minutes (FC blocker, InnovexBiosciences, Richmond, CA, USA) and incubated in TBS with 5% BSA for 1 hour to prevent the non-specific binding, and then, 4 o 5 overnight at C Human CCL8 antibody (1: 100, Origene, Rockville, MD, USA) or multi-clone anti-human alpha-SMA antibody (1: 200, Abcam, Cambridge, Mass., USA). After washing with 1 * TBS, the sections were incubated with secondary antibodies: FITC-conjugated goat anti-rabbit antibody (1: 2000, Abcam) and PE- conjugated donkey anti- mouse antibody (1: 2000, Abcam ). The nuclei were contrasted with DAPI (Invitrogen, Carlsbad, CA, USA). Confocal laser scanning was performed using a microscope (LSM 510 META at 100 X magnification) connected to a CoolsnapPhotometrics HQ camera (Photometrics, Tucson, AZ, USA) and images were generated using a Zeiss LSM image browser.
Statistical analysis
The data were analyzed using SPSS v. 20.0. The difference in gene expression between the normal control and IPF groups was judged to be statistically significant when the complete multiple difference was ≥2 and the p-value <0.05 at the median using t- tests and non-parametric TNoM assays. Comparisons of CCL8 concentrations between the two groups were performed using the Mann-Whitney U test. ROC (receiver operating characteristic) curves, AUC, and cutoff values were calculated using MedCalc. Correlations between CCL8 levels and other indicators were analyzed by Spearman correlation coefficient. Data are presented as intermediate values with 25% and 75% quartiles for skewed variables, or as medians ± SEM for those with normal distributions. The optimal cut level of CCL8 was calculated using the Cutoff Finder, and the survival rate was assessed by the Kaplan-Meier's method and compared using the log-rank test. A value of p < 0.05 was considered statistically significant.
2. Experimental Results and Analysis
One) IPF And control group
The experiment was carried out in two steps: transcript analysis and confirmation of CCL8 in BAL solution (see Table 1).
Eight IPF fibroblasts and four control fibroblasts were used for transcript analysis, and functional vital capacity (FVC) and measurement of diffusing capacity (DLCO) were significantly lower in IPF patients than in controls (p <0.05).
BAL samples were obtained from 86 IPF patients. IPF patients had significantly greater neutrophil and eosinophil percentages and lower FVC and DLCO values in total cell numbers and BAL compared to the 35 normal controls (p <0.05). The IPF group consisted of 34 patients with surgical IPF and 52 patients with clinical IPF. There were no significant clinical or physiological differences between these two groups (Table 2).
[4]
[8]
[54 (48-59)]
[64 (50-72)]
(35-72)
(59-75) *
(Male / female)
[1/3]
[4/4]
(CS / ES / NS)
[0/0/4]
[0/4/4]
[6/2]
(years)
(1.6-5.5)
(% pred.)
[98 (87-106)]
[84 (56-99)] *
(87-119)
(52-79) *
(% pred.)
(88.2-117)
(71-93) *
(% pred.)
[90 (76-120)]
[60 (39-87)] *
(57-108)
(46-71) *
(15.8-2.8)
(6.0-21.0)
(13.8-153.2) †
(88.4-93.2)
(39.4-77.5) †
(0-1.2)
(8.6-36.1) †
(0-0.6)
(1.6-3.4) †
(0-1.2)
(1.2-7.5)
1. Fibroblast cultures were treated with lung fibroblasts (n = 10) obtained from normal lung tissue from which cancer cells had been removed as control, and IPF fibroblasts (n = 14) obtained from biopsy specimens were used as an experimental group. The numbers superimposed on the control and the IPF indicate the number of transcript analysis performed.
2. CS (current smoker): current smoker, ES (ex-smoker): old smoker, NS (never-smoker)
3. dFVC (%) is the annual reduction rate of FVC calculated by FVC / year (last FVC minus base line FVC) / baseline.
4. ND: not determined
5. All data were expressed as mean and interquartile ranges, with * as P <0.05 and † as P <0.001 based on the control group.
2) IPF And the entire gene expression profiling results of the control group
Cultured fibroblasts expressed alpha-SMA (myofibroblast markers) but not E-cadherin (epithelial cell markers) by Western blotting (FIG. 1). Referring to FIG. 1, the intensity of alpha-SMA normalized on beta-actin was much higher in IPF lung-derived fibroblasts (p = 0.011), whereas the intensity of vimentin was similar to that of control.
The significant difference in gene expression was calculated as the "perfect match intensity-mismatch intensity" of the probe pair. The genes with a P value of less than 0.01 were treated as not expressed to reduce the number of false positives, and the remaining 15,020 genes were analyzed.
Comparing gene expression levels between the two groups, 178 genes showed different expression levels at the expression level according to t- test and TNoM (p-values <0.05 and multiple differences> 2 between the two groups, Table 3 to Table 7 Reference).
(burglar)
(burglar)
(p-value)
(p-value)
(burglar)
(burglar)
(p-value)
(p-value)
(burglar)
(burglar)
(p-value)
(p-value)
(burglar)
(burglar)
(p-value)
(p-value)
(burglar)
(burglar)
(p-value)
(p-value)
2 (A) showing hierarchical cluster analysis results showed different expression patterns (see FIG. 2A). Of 109 genes, expression was increased in the IPF group compared to the control group, whereas expression of 69 genes was decreased. The top 15 genes that differ by more than 10-fold in gene expression between the two groups are shown in Table 8 and B.
(mean ± SE)
(mean ± SE)
( p-value )
( p-value )
In the IPF patients, 13 genes ( PF4V1 , MYOC , CCL8 , ROR2 , HBG2 , D4S234E , KCNJ2 , RGS18 , PITX1 , EPB41L3 , FGF7 , LOC100132994 and POSTN ) Two genes ( FLJ25037 And IGFBP2 ) decreased. PF4V1 expression was most upregulated (59.2-fold) and IGFBP2 expression was most down-regulated (-11.38-fold). Expression of CCL8 was 22.8 times higher in the IPF group than in the control group.
3) Confirm ontology and pathway analysis of expressed gene
Gene ontology analysis was performed to predict the function of 178 genes with different expression levels between the two groups.
The difference between the data set and the normal path was measured by a ratio of enrichment and the gene set test determined the statistical significance of the small change. Compared to expectations, the observed number of genes was larger in the total 16 ontology categories (corrected p-values <0.05; Table 9).
ring
Ontology
category
Number of genes
gene
Number
gene
Number
Importance (Pcorr <0.05)
(raw P-value)
control
The
process
function
Configuration
Element
control
function
(P = 4.6 * 10 -5 , ratio = 167.6), cell adhesion molecule binding (p = 0.06) 0.0005, ratio = 19.2) (see Fig. 2C).
The differentially expressed genes in IPF patients have been shown to play an important role in eight pathways involving adhesion binding, melanoma, cytokine-cytokine receptor interactions (see Table 10 and Figure 2 D).
Input gene
Path gene
p-value
p-value
Receptor interaction
From the above results, it can be seen that CCL8 binds to the ontology category of the extracellular domain, receptor binding, heparin binding, G-protein associated receptor binding, chemokine activity, carbohydrate derivative binding and glycosaminoglycan binding and cytokine-cytokine receptor binding interaction pathway Respectively.
4) In fibroblasts CCL8 mRNA Check level
14 IPF pulmonary fibroblasts and 10 control pulmonary fibroblasts showed increased expression of CCL8 gene. The level of CCL8 mRNA normalized to the level of β-actin mRNA was confirmed by RT-PCR (p = 0.0001; A and B in FIG. 3) and real-time PCR (p = 0.022; ), And the expression level of IPF fibroblasts was about three times higher than that of control fibroblasts. CCL8 mRNA levels were determined by transcript analysis (r = 0.615, p = 0.033; D in Fig. 3), which strongly correlated with the results of real-time PCR in 12 individuals.
5) Of bronchodilator CCL8 Identify protein levels
CCL8 protein levels were measured using bronchoalveolar lavage fluid (BAL fluid) from 86 IPF patients and 35 normal controls. CCL8 protein was observed in 23 of 35 control subjects and 80 of 86 IPF patients (Fig. 4A).
The concentration of CCL8 protein normalized to the total protein concentration was significantly higher in IPF patients than in the control group [5.70 (0.57-10.65 pg / μg) vs. 0.44 (0.00-0.88 pg / μg), p <0.0001]. The ROC curve shows a clear difference between the IPF patient and the normal control (AUC = 0.808; Fig. 4B). The cleavage value of 1.31 pg / μg shows 73.6% accuracy, 45.7% specificity, and 84.9% sensitivity. CCL8 levels were strongly associated with the number of neutrophils (r = 0.297, p = 0.014) in BAL (Fig. 4C).
CCL8 concentrations were analyzed in 69 individuals who were followed up for 1-8 years with respect to survival. When individuals were divided into two groups with a cutoff value of 0.9025 pg / μg calculated by the Cutoff Finder, the survival rate was much lower in the CCL8 group> 0.9025 pg / μg compared to the CCL8 group <0.9025 pg / μg (Hazard ratio = 4.06; CI: 0.94-17.63; p = 0.043; FIG. 4D). CCL8 protein levels were not associated with other clinical and physiological parameters.
Serum CCL8 levels were measured in 35 normal controls and 66 IPF patients. There was no difference between the two groups (p = 0.167; Fig. 5A), and there was no correlation between the serum of 60 IPF patients and CCL8 levels in BAL (p = 0.567;
6) Of CCL8 Immunofluorescent staining Check the result
CCL8 and alpha-SMA dual immunofluorescent staining was performed in three IPF lung tissues and three control lung tissues to confirm CCL8 expression by myofibroblasts in IPF lungs. In the control lungs, peripheral blood vessels and bronchial stains were stained. Alpha-SMA was stained strongly by fibroblasts between the organs, but most of CCL8 was stained but not entirely stained (see FIG. 6).
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.
<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> Composition for Idiopathic pulmonary fibrosis prognosis and method of providing the information for the same <130> DP20160001 <160> 29 <170> Kopatentin 2.0 <210> 1 <211> 744 <212> RNA <213> Homo sapiens <400> 1 actgcctgca gaaccccagc ccgactttcc ctgcgcactg ggatcctgct ggaacctcag 60 ctgcaacatg agctccgcag ccaggtcccg cctcacccgc gccacccgcc aggagatgct 120 gttcttggcg ttgctgctcc tgccagttgt ggtcgccttc gccagagctg aagctgaaga 180 agatggggac ctgcagtgcc tgtgtgtgaa gaccacctcc caggtccgtc ccaggcacat 240 caccagcctg gaggtgatca aggccggacc ccactgcccc actgcccaac tcatagccac 300 gctgaagaat gggaggaaaa tttgcttgga tctgcaagcc ctgctgtaca agaaaatcat 360 taaggaacat ttggagagtt agctactagc tgcctaagtg tgcactttca atctaactgt 420 gaaagaatct tctgatgttt gtattatcct tcttatatta tattaacgaa ataaatcaag 480 ttgtggtata gtcaatctat ttcttaataa tactgcaaaa ataatgctga cacatcacaa 540 tttcatattt taaaatttcc agaattttaa gcaaaaagca ttatgaagga aggcttggtt 600 taataaagac tgattttgtt cagtgttata tgttagctga tacatatttg ttcatttatg 660 tgattgcagt actttatagc tacatattta ccttgaatgt tacaattagc ttgccaataa 720 atattagtag ctcttaagca ttac 744 <210> 2 <211> 2061 <212> RNA <213> Homo sapiens <400> 2 cctttgggcc tgagatgcca gctgtccagc tgctgcttct ggcctgcctg gtgtgggatg tgggggccag 120 gacagctcag ctcaggaagg ccaatgacca gagtggccga tgccagtata ccttcagtgt 180 ggccagtccc aatgaatcca gctgcccaga gcagagccag gccatgtcag tcatccataa 240 cttacagaga gacagcagca cccaacgctt agacctggag gccaccaaag ctcgactcag 300 ctccctggag agcctcctcc accaattgac cttggaccag gctgccaggc cccaggagac 360 ccaggagggg ctgcagaggg agctgggcac cctgaggcgg gagcgggacc agctggaaac 420 ccaaaccaga gagttggaga ctgcctacag caacctcctc cgagacaagt cagttctgga 480 ggaagagaag aagcgactaa ggcaagaaaa tgagaatctg gccaggaggt tggaaagcag 540 cagccaggag gtagcaaggc tgagaagggg ccagtgtccc cagacccgag acactgctcg 600 ggctgtgcca ccaggctcca gagaagtttc tacgtggaat ttggacactt tggccttcca 660 ggaactgaag tccgagctaa ctgaagttcc tgcttcccga attttgaagg agagcccatc 720 tggctatctc aggagtggag agggagacac cggatgtgga gaactagttt gggtaggaga 780 gcctctcacg ctgagaacag cagaaacaat tactggcaag tatggtgtgt ggatgcgaga 840 ccccaagccc acctacccct acacccagga gaccacgtgg agaatcgaca cagttggcac 900 ggatgtccgc caggtttttg agtatgacct catcagccag tttatgcagg gctacccttc 960 taaggttcac atactgccta ggccactgga aagcacgggt gctgtggtgt actcggggag 1020 cctctatttc cagggcgctg agtccagaac tgtcataaga tatgagctga ataccgagac 1080 agtgaaggct gagaaggaaa tccctggagc tggctaccac ggacagttcc cgtattcttg 1140 gggtggctac acggacattg acttggctgt ggatgaagca ggcctctggg tcatttacag 1200 caccgatgag gccaaaggtg ccattgtcct ctccaaactg aacccagaga atctggaact 1260 cgaacaaacc tgggagacaa acatccgtaa gcagtcagtc gccaatgcct tcatcatctg 1320 tggcaccttg tacaccgtca gcagctacac ctcagcagat gctaccgtca actttgctta 1380 tgacacaggc acaggtatca gcaagaccct gaccatccca ttcaagaacc gctataagta 1440 cagcagcatg attgactaca accccctgga gaagaagctc tttgcctggg acaacttgaa 1500 catggtcact tatgacatca agctctccaa gatgtgaaaa gcctccaagc tgtacaggca 1560 atggcagaag gagatgctca gggctcctgg ggggagcagg ctgaagggag agccagccag 1620 ccagggccca ggcagctttg actgctttcc aagttttcat taatccagaa ggatgaacat 1680 ggtcaccatc taactattca ggaattgtag tctgagggcg tagacaattt catataataa 1740 atatccttta tcttctgtca gcatttatgg gatgtttaat gacatagttc aagttttctt 1800 gtgatttggg gcaaaagctg taaggcataa tagtttcttc ctgaaaacca ttgctcttgc 1860 atgttacatg gttaccacaa gccacaataa aaagcataac ttctaaagga agcagaatag 1920 ctcctctggc cagcatcgaa tataagtaag atgcatttac tacagttggc ttctaatgct 1980 tcagatagaa tacagttggg tctcacataa ccctttacat tgtgaaataa aattttctta 2040 cccaaaaaaa aaaaaaaaaa a 2061 <210> 3 <211> 1351 <212> RNA <213> Homo sapiens <400> 3 gtgatggaga gcaccagcaa agccttaggg cccatccctg gcctcctgtt acccacagag 60 gggtaggccc ttggctctct tccactatga cgtcagcttc cattcttcct ttcttataga 120 caattttcca tttcaaggaa atcagagccc ttaatagttc agtgaggtca ctttgctgag 180 cacaatccca tacccttcag cctctgctcc acagagccta agcaaaagat agaaactcac 240 aacttccttg ttttgttatc tggaaattat cccaggatct ggtgcttact cagcatattc 300 aaggaaggtc ttacttcatt cttccttgat tgtgaccatg cccaggctct ctgctcccta 360 taaaaggcag gcagagccac cgaggagcag agaggttgag aacaacccag aaaccttcac 420 ctctcatgct gaagctcaca cccttgccct ccaagatgaa ggtttctgca gcgcttctgt 480 gcctgctgct catggcagcc actttcagcc ctcagggact tgctcagcca gattcagttt 540 ccattccaat cacctgctgc tttaacgtga tcaataggaa aattcctatc cagaggctgg 600 agagctacac aagaatcacc aacatccaat gtcccaagga agctgtgatc ttcaagacca 660 aacggggcaa ggaggtctgt gctgacccca aggagagatg ggtcagggat tccatgaagc 720 atctggacca aatatttcaa aatctgaagc catgagcctt catacatgga ctgagagtca 780 gagcttgaag aaaagcttat ttattttccc caacctcccc caggtgcagt gtgacattat 840 tttattataa catccacaaa gagattattt ttaaataatt taaagcataa tatttcttaa 900 aaagtattta attatattta agttgttgat gttttaactc tatctgtcat acatcctagt 960 gaatgtaaaa tgcaaaatcc tggtgatgtg ttttttgttt ttgttttcct gtgagctcaa 1020 ctaagttcac ggcaaaatgt cattgttctc cctcctacct gtctgtagtg ttgtggggtc 1080 ctcccatgga tcatcaaggt gaaacacttt ggtattcttt ggcaatcagt gctcctgtaa 1140 gtcaaatgtg tgctttgtac tgctgttgtt gaaattgatg ttactgtata taactatgga 1200 attttgaaaa aaaatttcaa aaagaaaaaa atatatataa tttaaaacta aaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1351 <210> 4 <211> 4099 <212> RNA <213> Homo sapiens <400> 4 agccagccct tgccgtggcc ggagccgagc ggcgcatccg ggccggagaa gaggacgacg 60 acgaggtcct cgaagtggac ccgtttgcga agcgccaggg agaaggagga gcggacgcat 120 cgtagaaagg ggtggtggcg cccgaccccg cgccccggcc cgaagctctg agggcttccc 180 ggcccccact gcctgcggca tggcccgggg ctcggcgctc ccgcggcggc cgctgctgtg 240 catcccggcc gtctgggcgg ccgccgcgct tctgctctca gtgtcccgga cttcaggtga 300 agtggaggtt ctggatccga acgacccttt aggacccctt gatgggcagg acggcccgat 360 tccaactctg aaaggttact ttctgaattt tctggagcca gtaaacaata tcaccattgt 420 ccaaggccag acggcaattc tgcactgcaa ggtggcagga aacccacccc ctaacgtgcg 480 gtggctaaag aatgatgccc cggtggtgca ggagccgcgg cggatcatca tccggaagac 540 agaatatggt tcacgactgc gaatccagga cctggacacg acagacactg gctactacca 600 gtgcgtggcc accaacggga tgaagaccat taccgccact ggcgtcctgt ttgtgcggct 660 gggtccaacg cacagcccaa atcataactt tcaggatgat taccacgagg atgggttctg 720 ccagccttac cggggaattg cctgtgcacg cttcattggc aaccggacca tttatgtgga 780 ctcgcttcag atgcaggggg agattgaaaa ccgaatcaca gcggccttca ccatgatcgg 840 cacgtctacg cacctgtcgg accagtgctc acagttcgcc atcccatcct tctgccactt 900 cgtgtttcct ctgtgcgacg cgcgctcccg gacacccaag ccgcgtgagc tgtgccgcga 960 cgagtgcgag gtgctggaga gcgacctgtg ccgccaggag tacaccatcg cccgctccaa 1020 cccgctcatc ctcatgcggc ttcagctgcc caagtgtgag gcgctgccca tgcctgagag 1080 ccccgacgct gccaactgca tgcgcattgg catcccagcc gagaggctgg gccgctacca 1140 tcagtgctat aacggctcag gcatggatta cagaggaacg gcaagcacca ccaagtcagg 1200 ccaccagtgc cagccgtggg ccctgcagca cccccacagc caccacctgt ccagcacaga 1260 cttccctgag cttggagggg ggcacgccta ctgccggaac cccggaggcc agatggaggg 1320 cccctggtgc tttacgcaga ataaaaacgt acgcatggaa ctgtgtgacg taccctcgtg 1380 tagtccccga gacagcagca agatggggat tctgtacatc ttggtcccca gcatcgcaat 1440 tccactggtc atcgcttgcc ttttcttctt ggtttgcatg tgccggaata agcagaaggc 1500 atctgcgtcc acaccgcagc ggcgacagct gatggcctcg cccagccaag acatggaaat 1560 gcccctcatt aaccagcaca aacaggccaa actcaaagag atcagcctgt ctgcggtgag 1620 gttcatggag gagctgggag aggaccggtt tgggaaagtc tacaaaggtc acctgttcgg 1680 ccctgccccg ggggagcaga cccaggctgt ggccatcaaa acgctgaagg acaaagcgga 1740 ggggcccctg cgggaggagt tccggcatga ggctatgctg cgagcacggc tgcaacaccc 1800 caacgtcgtc tgcctgctgg gcgtggtgac caaggaccag cccctgagca tgatcttcag 1860 ctactgttcg cacggcgacc tccacgaatt cctggtcatg cgctcgccgc actcggacgt 1920 gggcagcacc gatgatgacc gcacggtgaa gtccgccctg gagccccccg acttcgtgca 1980 ccttgtggca cagatcgcgg cggggatgga gtacctatcc agccaccacg tggttcacaa 2040 ggacctggcc acccgcaatg tgctagtgta cgacaagctg aacgtgaaga tctcagactt 2100 gggcctcttc cgagaggtgt atgccgccga ttactacaag ctgctgggga actcgctgct 2160 gcctatccgc tggatggccc cagaggccat catgtacggc aagttctcca tcgactcaga 2220 catctggtcc tacggtgtgg tcctgtggga ggtcttcagc tacggcctgc agccctactg 2280 cgggtactcc aaccaggatg tggtggagat gatccggaac cggcaggtgc tgccttgccc 2340 cgatgactgt cccgcctggg tgtatgccct catgatcgag tgctggaacg agttccccag 2400 ccggcggccc cgcttcaagg acatccacag ccggctccga gcctggggca acctttccaa 2460 ctacaacagc tcggcgcaga cctcgggggc cagcaacacc acgcagacca gctccctgag 2520 caccagccca gtgagcaatg tgagcaacgc ccgctacgtg gggcccaagc agaaggcccc 2580 gcccttccca cagccccagt tcatccccat gaagggccag atcagaccca tggtgccccc 2640 gccgcagctc tacgtccccg tcaacggcta ccagccggtg ccggcctatg gggcctacct 2700 gcccaacttc tacccggtgc agatcccaat gcagatggcc ccgcagcagg tgcctcctca 2760 gatggtcccc aagcccagct cacaccacag tggcagtggc tccaccagca caggctacgt 2820 caccacggcc ccctccaaca catccatggc agacagggca gccctgctct cagagggcgc 2880 tgatgacaca cagaacgccc cagaagatgg ggcccagagc accgtgcagg aagcagagga 2940 ggaggaggaa ggctctgtcc cagagactga gctgctgggg gactgtgaca ctctgcaggt 3000 ggacgaggcc caagtccagc tggaagcttg agtggcacca gggcccgggg ttcggggata 3060 gaagccccgc cgagacccca cagggacctc agtcaccttt gagaagacac catactcagc 3120 aatcacaaga gcccgccggc cagtgggctt gtttgcagac tgggtgaggt ggagccctgc 3180 tcctctctgt cctctgacac agagagctgc cctgcctagg agcacccaag ccaggcaggg 3240 ggtctggcag cacggcgtcc tggggagcag gacacatggt catccccagg gctgtataca 3300 ttgattctgg tggtagactg gtagtgagca gcaaatgcct ttcaagaaaa taggtggcag 3360 cttcactcca tgtcatatat ggagtgaata tttcaaaacg ttgggaataa gggcctgcaa 3420 aaggcagcga ggaggcacct cgggtcttga ggttcctgac aaccgatctg gtctgttggt 3480 ttgaggatga aggggctcca tttctgctgc ctccctgctg agaatattct ccctttagca 3540 gccaaagatt cgctggaacg gaggctgccc tctgctgcct gttggggtcg gaagacaagg 3600 ggcttctgaa atgggagttc ctgagataca acaaaatgtg tgccttcaaa gaaactgaca 3660 gctttgtatt tggtgaaatg gttttaatta tactccatgt gtattttgcc cacttttttt 3720 gggaattcaa gggaaagtgt ttcttgggtt tggaatgttc agaggaagca gtattgtaca 3780 gaacacggta ttgttatttt tgttaagaat catgtacaga gcttaaatgt aatttatatg 3840 tttttaatat gccattttca ttgaagtatt ttggtcttaa gatgacttta gtaatttaac 3900 tgtttatgtt acccacgttg ggatccagtt ggtcttggtt tgcttctctc tgtaccacgt 3960 gcacatgagg tccattcatt ttacagcccc tgttacacac agacccacag gcagccgtct 4020 gtgccccgca cacattgttg gtcctatttg taaatcccac acccggtgta tccaataaag 4080 tgaaacaaag catgtgaaa 4099 <210> 5 <211> 583 <212> RNA <213> Homo sapiens <400> 5 acactcgctt ctggaacgtc tgaggttatc aataagctcc tagtccagac gccatgggtc 60 atttcacaga ggaggacaag gctactatca caagcctgtg gggcaaggtg aatgtggaag 120 atgctggagg agaaaccctg ggaaggctcc tggttgtcta cccatggacc cagaggttct 180 ttgacagctt tggcaacctg tcctctgcct ctgccatcat gggcaacccc aaagtcaagg 240 cacatggcaa gaaggtgctg acttccttgg gagatgccat aaagcacctg gatgatctca 300 agggcacctt tgcccagctg agtgaactgc actgtgacaa gctgcatgtg gatcctgaga 360 acttcaagct cctgggaaat gtgctggtga ccgttttggc aatccatttc ggcaaagaat 420 tcacccctga ggtgcaggct tcctggcaga agatggtgac tggagtggcc agtgccctgt 480 cctccagata ccactgagct cactgcccat gatgcagagc tttcaaggat aggctttatt 540 ctgcaagcaa tcaaataata aatctattct gctaagagat cac 583 <210> 6 <211> 2418 <212> RNA <213> Homo sapiens <400> 6 ggccacacgc gcgcacccac ccgcgcgcac ccagcccccg ggagaggcag gaagggaggc 60 ggcggcgcga ggaggaggga gcggccgtgg agcccaatcg ttcgctcccc ttcccgggtc 120 cgcgcgcggc gccgcctccg ccattgctgc gagcaggagc aggagacgcg gagctcggag 180 cgctgagctg acctgccgga gccgggcgtg ggctgcagcc tcggagctcc cggaacgatg 240 gtgaagttgg ggaacaattt cgcagagaag ggcaccaagc agccgctgct ggaggatggc 300 ttcgacacca ttcccctgat gacgcccctc gatgtcaatc agctgcagtt cccgcccccg 360 gataaggtgg tcgtgaaaac taagaccgag tatgaacctg accgcaagaa agggaaagca 420 cgtcctcccc aaattgctga gttcaccgtc agcatcacgg agggtgtcac cgagaggttt 480 aaggtctccg tgttggtcct cttcgccctg gccttcctca cctgcgtcgt cttcctggtt 540 gtctacaagg tgtacaagta tgaccgcgcc tgccccgatg ggttcgtcct caagaacacc 600 cagtgcatcc cagaaggctt ggagagctac tacgcggagc aagactccag tgcccgggag 660 aaattttaca cagtcataaa ccactacaac ctggccaagc agagcatcac gcgctccgta 720 tcgccctgga tgtcagttct gtcagaagag aagctgtccg agcaggagac tgaagcggct 780 gagaagtcag cttagcggga tgggcaagtt ccttacaatg tgtcacttgc aaataacaaa 840 gggactttga gggacatttc attaaatata attactgata ctttagaggt tactcattta 900 cggtgcaatt gcttctgttt gctaatgctg ctttgcaaat aaaacttgct gccgaccacc 960 cacgggcata aaatcaagtg catttcagca ttgcctaaag agctctgaca ccacttttca 1020 tgttaagatc ttcatttagc tcctttactg ggatttattg gatgctgtaa aaaaataaat 1080 ttacactgga tatgcgaagg gtttggatct cagataaatg cattttgtgg aattgatttt 1140 ctgaaccgac cctgctgtct gcaaaccttc ctccatagcc atatctagag tgatctctcg 1200 ctgtgctaag agcaagccta cttcgcattt cttcctcggc catcagcggg taacagtgct 1260 gactgctgcc aaggtgcact gtagtaagta agtggcatag agaacgagga aaaagacccc 1320 cccacccctc cctgtgctga ctcctgagtc ggggtgggac ggccctgcaa tgtggcaggc 1380 tcggtgtgga ttgactactc cctcccctct gactctggca tcttggtaag gagggagggg 1440 caccacccat ctgggtggag acagctgggg tttgctccag tgtgtgtaga ctggcagagc 1500 atgtgaggga gagggctggg aagagacggc cctgggcccg tgaggtgcag aactctccct 1560 gcaggtagtc tgtctgctct gtgatgtgct gataggaaag agcccccact ggccctctgg 1620 ttcacttcta ttatgaataa atttgcacca atagccccat tagtttttaa agaaatgagc 1680 tgggtgggaa aagtggaaat gtaatttctg gtaagcttga gctactttaa tttttattct 1740 atgaaactga tatcccttga tgtgggaaac aggcaggaaa ggctgtgggg tgggggtggg 1800 ggactggtgt ggcaccgtgc aaacaaaatg acaaatttca actcacaatc tttgtaagaa 1860 aattgttcca gtttgaatct tgatattaaa gtttatgttt acaaaactgc cagttagatg 1920 aactaagtgt gtaaaacaaa tagaaaagac attcgcagac atttcttcta tgttattgaa 1980 tgtgttgttt ctggtgtacg tgtcaaaagt ttcagttttt tagatcagaa ataaacgaca 2040 aatagtgtga gatgtgttgt gaacaggcat ggtgaccgtg gtcagcgcca ctcttgtttc 2100 ctgaaatgtg cttctaagac agaaaatgta ttttctcatc aagggtgtct ggagacacag 2160 accgtgacct tggcgcagcg gtgtgcatca gaggcgtgtg ctgagaaggg tggtgttaag 2220 ctgtggaaaa tgactcaaga gcaataaatc agtgccaaag cttccctgcg catctgaata 2280 gacacaagtg aagcccgtgt gcgcacagtg cagaggccga gcctgtattt ccaggtagac 2340 gtggacttta ttgactgtga attcatttac atgtaacttc tgacatttca ctctgtgcaa 2400 ataaagaaca tgaggata 2418 <210> 7 <211> 5397 <212> RNA <213> Homo sapiens <400> 7 gcgcactgga gccctggcca gcgcgcagcc ttcccggcgc cggcgggctg ggtcttggga 60 attctggttt gctttggctc actcgctttt tacaaaccac tggatcttac atgcctctgt 120 accccccact tccactccat gtccccatgc tcctgcgcca gcaacaggac atgttctctg 180 gatgtcagct gagtcattaa agtaactctg catgtcagta gacagacctt ggtagaacca 240 caaggctccc agagacaccc atctctcctc atttttttgg tgtgtgtgtc ttcaccgaac 300 attcaaaact gtttctccaa agcgttttgc aaaaactcag actgttttcc aaagcagaag 360 cactggagtc cccagcagaa gcgatgggca gtgtgcgaac caaccgctac agcatcgtct 420 cttcagaaga agacggtatg aagttggcca ccatggcagt tgcaaatggc tttgggaacg 480 ggaagagtaa agtccacacc cgacaacagt gcaggagccg ctttgtgaag aaagatggcc 540 actgtaatgt tcagttcatc aatgtgggtg agaaggggca acggtacctc gcagacatct 600 tcaccacgtg tgtggacatt cgctggcggt ggatgctggt tatcttctgc ctggctttcg 660 tcctgtcatg gctgtttttt ggctgtgtgt tttggttgat agctctgctc catggggacc 720 tggatgcatc caaagagggc aaagcttgtg tgtccgaggt caacagcttc acggctgcct 780 tcctcttctc cattgagacc cagacaacca taggctatgg tttcagatgt gtcacggatg 840 aatgcccaat tgctgttttc atggtggtgt tccagtcaat cgtgggctgc atcatcgatg 900 ctttcatcat tggcgcagtc atggccaaga tggcaaagcc aaagaagaga aacgagactc 960 ttgtcttcag tcacaatgcc gtgattgcca tgagagacgg caagctgtgt ttgatgtggc 1020 gagtgggcaa tcttcggaaa agccacttgg tggaagctca tgttcgagca cagctcctca 1080 aatccagaat tacttctgaa ggggagtata tccctctgga tcaaatagac atcaatgttg 1140 ggtttgacag tggaatcgat cgtatatttc tggtgtcccc aatcactata gtccatgaaa 1200 tagatgaaga cagtccttta tatgatttga gtaaacagga cattgacaac gcagactttg 1260 aaatcgtggt catactggaa ggcatggtgg aagccactgc catgacgaca cagtgccgta 1320 gctcttatct agcaaatgaa atcctgtggg gccaccgcta tgagcctgtg ctctttgaag 1380 agaagcacta ctacaaagtg gactattcca ggttccacaa aacttacgaa gtccccaaca 1440 ctcccctttg tagtgccaga gacttagcag aaaagaaata tatcctctca aatgcaaatt 1500 cattttgcta tgaaaatgaa gttgccctca caagcaaaga ggaagacgac agtgaaaatg 1560 gagttccaga aagcactagt acggacacgc cccctgacat agaccttcac aaccaggcaa 1620 gtgtacctct agagcccagg cccttacggc gagagtcgga gatatgactg actgattcct 1680 tctctggaat agttacttta caacacggtc tgttggtcag aggcccaaaa cagttataca 1740 gatgacggta ctggtcaaga tgggtcaagc aagcggccac aagggactga ggcaagcaca 1800 atggtttcaa agaaagactg taagctccat gattagcata aagcactaac catgtctcca 1860 tgtgacccga tggcacatag atgttgtaga ataagttatg ggtttttatg ttttgttttg 1920 tgtttttcca aaacttgaac ttgcaggcaa gccttggttg ggtatttgat ttatccagaa 1980 tgcttctctt tagggaacaa ggatgttttt aatggcataa caaaggcaag actctgcctt 2040 aatttttgaa aagctgctaa ctacatgaac acaaatgtgt atttttgttg cagtgtagtt 2100 ttccttttgt gtaattttaa agtcagtgtt gaattttatt gaaagctcat gatgcgcttc 2160 aaagtggcaa gtatttggct attaactgcc aaaacaagag cctgattttt tgaggccagt 2220 aattcgtttg ctagaattga ttttttttct ctctctcttt gttacataag ggcattatgt 2280 aacactagcc gaatggtagc ctctgggttg ttgttttttt cttttcctcc atgatgttaa 2340 tgggttatct caaattttaa gttaaactac ctaaaataaa taccaaagat aatgcatatt 2400 tttgcacagt ggagcttaca cttaaaagaa aacaaagccc catgggctgc cttgaaatca 2460 agagacaata actttgaacc tcagcaagac cttgaaccgc cggttcattt tgcaccttat 2520 tcagaaaata gagcatcata ctcaccgagt ctagtcagtg tagtgctttt aaaaattttg 2580 tcctttcatg taactttttt attttaagag gaagaagaag aaaggggcac acacacacaa 2640 taccgacgtc tatcctttcc tgctaggcag tgctggccag gctcatgtgt agtgtgcgag 2700 atggtgatgt actcttatat ttttctgggc ttttcctttt gcacattcca aaattcattt 2760 cataagacaa gatcttcata ggacctcctt ggcatcctgg cattctcaaa actgagccat 2820 ccagcatgaa agataaatgg gtttaaaccc ttgctgctga atttattgcc tggactgtca 2880 ggacatcacc agcccacctt caccttaggg aagatgccac acctggcctc cacacttgct 2940 cttctgatca gtctgtctgg attgagtcct acagtgtcag atagggcggc aaatgccaaa 3000 gcagggaaac agggaggtgt ggacaagcca gtttgatgca gcacttcaga tcaagtgctt 3060 aggaaggaga ggaaacttgc cttttttatg gcagaggata gtaatgaaaa tgtctcagta 3120 ttttagggtc aatgagagcc ataaaaatat aacataatca caagtaaagg agataatggt 3180 ctaaaacagc tatttccctt ttctgtgtgc atacttatga ctgaatgtga gctaagcatt 3240 ttctcctgtg gagccctaga gcaggttact aaggaaggac acattgtttt ccagaagcct 3300 cccctgcctg gctgactgcc ttgctagaaa cataattttt tttttctcac tgaagctcaa 3360 taatggaact cttttttttt ttttttttaa tttaaagttc cctatttgtg aattctggga 3420 ttactgactt ttctttttaa ttggagtctc aaaatcaact ctcttatggt attatatctc 3480 tgtatgccat taaaaaacag cttgttctag aatcatgtat tttgtaaact gatgtttgtg 3540 atggtctctg gttcttgaac agccatatct gaatgccgtg cctgcaaaac tatgacaatt 3600 tttgctgttt tcagccttca gatttgatgg cttgggaaac tgaggtgtta ttttcaatga 3660 aacaaagaaa gagatgttaa gcaagtggtt gttttagatc caaatgtaaa ggcaggtttg 3720 ggaaggtgtt taaagagttg gaggaattgg ggattgagtt gtaaagaaaa cttacagaag 3780 aggcaacaat ttggttcttg acagtgagag gatattgagg gcttcagctg ctgctattat 3840 gatgttttgc aaaggaaaat aatcaaacca aagagtattc agtgatatgt aaattaaatg 3900 aagatacagt ggagaatggg ggtgaccaca aaagaggctc cccctaaaca cacagtgctg 3960 ccacttaaaa agacttgaga aatttgaaag ggggtgggta tggggggggc aagaaagagg 4020 gagggaaatc tttcaactta tttctgaaaa agagaaaaaa atataaaatt tctggtgcac 4080 aggtttgttt tttcaagaaa attttgcaga agctatgttt ttaaagtgta cattttataa 4140 agtttatcag atattttcat atttaaagcc aaatgtaaat agaggtctgt aaagaaaaat 4200 aattgccata gaaagtataa tttcagtgca gtaatttctg agagctagta cctatatgct 4260 accggttagc atggttttag caaatatata ccagccttat aaggttcgta ttgctatgtt 4320 cttctgttat ttatttcagc atggactgtt catttgaaac ctttttctag ttattagcgt 4380 tttaacagtt acaagcttta aatggcaatt tttttttttt tttttttttt tttttttttt 4440 ttgtcaagag ccaagacaca ggtaatgcac gacattgatt gctgcatttt accttcaaaa 4500 tatttgtcct tattgactgg gtctccttaa ttaatgtaca catgtcatta gaatgcagac 4560 ggaggggact caccatgaat atctggggtt gattcccaga tgtgtgttgc ttctctattg 4620 caagcagatt ccctgttgga tttacttcgg atttattccc ttttaaagaa tttttgccca 4680 tatctggaag ggcactatat ttttgggagg agccatagat tcctggttat cctattttta 4740 aacaaaatgt agacaaagtg aactctattt tgattattga gaaaggagta gttttctatc 4800 cctctaagag tatacttgaa tcagacattt taaggatgtc actatggcac tgttgtcatt 4860 tccaaattcc tagaaaagtt tgttttactt tgtttttatt ctgttaatgc attctttctt 4920 ctctttactt cctttcttac cagtacactc ctatctcaac tctgtttatt tgatgagttc 4980 tgtcccgtaa atcatatttc ccttacaatt aataaatgtc acttcatatt ttataataaa 5040 ccactcagta aaagcaaaag cttgtcctga gaagtagagt gagttctttt tcactctgtg 5100 tctaataatg ttaaggtggg aaaaaaaaaa gtgtggcata gctacctgcc catccccaac 5160 cctcagcaaa gtagaatctc ttttctggta attttgggtt tccgctctgg gctctggcaa 5220 gttgaacaat cctagccatt gacaatcgtg atagttatta ttttcccatt tgctgtcttt 5280 ttgtatctaa agtcttccta ttgtactgca caaaccatgg attgtacata tttttatata 5340 ttatgtctta ttttattatt tctaaataaa aaaattaaaa attgaaaaaa aaaaaaa 5397 <210> 8 <211> 2158 <212> RNA <213> Homo sapiens <400> 8 agttctgcat ttctgcagag acagaaagaa acgcagctct tgacttcttt tttgtaaaca 60 ttactgtaag agttgtgata actttttatt ctactatgta tatgtatgga atagtattaa 120 taaatgaact agggaaggat gtaataaatt agacatctct tcattttaga gagaagatgg 180 aaacaacatt gcttttcttt tctcaaataa atatgtgtga atcaaaagaa aaaacttttt 240 tcaagttaat acatggttca ggaaaagaag aaacaagcaa agaagccaaa atcagagcta 300 aggaaaaaag aaatagacta agtcttcttg tgcagaaacc tgagtttcat gaagacaccc 360 gctccagtag atctgggcac ttggccaaag aaacaagagt ctcccctgaa gaggcagtga 420 aatggggtga atcatttgac aaactgcttt cccatagaga tggactagag gcttttacca 480 gatttcttaa aactgaattc agtgaagaaa atattgaatt ttggatagcc tgtgaagatt 540 tcaagaaaag caagggacct caacaaattc accttaaagc aaaagcaata tatgagaaat 600 ttatacagac tgatgcccca aaagaggtta accttgattt tcacacaaaa gaagtcatta 660 caaacagcat cactcaacct accctccaca gttttgatgc tgcacaaagc agagtgtatc 720 agctcatgga acaagacagt tatacacgtt ttctgaaatc tgacatctat ttagacttga 780 tggaaggaag acctcagaga ccaacaaatc ttaggagacg atcacgctca tttacctgca 840 atgaattcca agatgtacaa tcagatgttg ccatttggtt ataaagaaaa ttgattttgc 900 tcatttttat gacaaactta tacatctgct tctaacatat cgcatgttta tgttaagatt 960 tggtcccatc ctttaaactg aaatatgtca tgtgaaatta ttttaaaaat gtaaaaacaa 1020 aactttctgc taacaaaata catacagtat ctgccagtat attctgtaaa accttctatt 1080 tgatgtcatt ccatttataa tcagaaaaaa aacttatttc ttaatcaaaa ggcagtacaa 1140 aaaaagtaat aatgttttat aagattgtag agttaagtaa aagttaagct tttgcaaagt 1200 tgtcaaaagt tcaaacaaaa gtctagttgg gattttttac caaagcagca taatatgtgt 1260 tatataaaca taataatact cagatatcca aatgttcaga tagcattttt cataatgaat 1320 gttctctttt ttttggtaat agtgtagaag tgatctggtt cttacaatgg gagatgaaga 1380 acatttatta ttgggttact actaaccctg tcccaagaat agtaatatca cctctagtta 1440 taagccagca acaggaactt ttgtgaagac acattcatct ctacagaact tcagattaaa 1500 tataatctag attaatgact gagaataaga tccacatttg aactcattcc taagtgaaca 1560 tggacgtacc cagttataca aagtacttct gttggtcaca gaaacatgac cagattttgc 1620 atatctccag gtagggaact aagtagacta ccttatcacc ggctaagaaa acttgctact 1680 aaactattag gccatcaatg gcttgaataa aaaccagaga aggtttttcc caggacgtct 1740 catgtttggc cctttagaat tggggtagaa atcagaaatg agatgagggg aagaagcaag 1800 gagtctaagg ccctagcgat ttgggcatct gccacattgg ttcatattca gaaagtgtta 1860 tctcattgat tatattcttg ttaagcaaat ctccttaagt aattattatt caaataagat 1920 tatactcata catctatatg tcactgtttt aaagagatat ttaattttta atgtgtgtta 1980 catggtctgt aaatatttgt atttaaaaat gccatgcatt aggctttgga aatttaatgt 2040 tagttgaaat gtaaaatgtg aaaactttag atcatttgta gtaataaata tttttaactt 2100 cattcataca gttaagttta tctgacaata aaagctctga ctgaaaaaaa aaaaaaaa 2158 <210> 9 <211> 2383 <212> RNA <213> Homo sapiens <400> 9 cctggcgcag ggactgctgg aacctggctg tgcgcgctgt cgctttaaga cagactctgc 60 cggcgccgtc cggagcctta gaaaccggcc ccggatcgcg agccggagcc ggagccggag 120 ccggggccgg ccgggctgct gaggcccgag cggcaggagc gcagcgcgga gcgctgagcc 180 aggcgcccag tcgcgagaag ctgccgccgc ctctgcccgc ccggcgccgc agccccgggc 240 ggtccatggg gcgggcacgg cgtcgctgca ggcgccggca gccctggagg gcagccgctt 300 aggcgctgcg ctcttgtccc cgcaggtcgc agccagggcg gcggggcgcg cccagccccg 360 gcccctggag cgcccgccgc ggtccccacc tccatggacg ccttcaaggg gggcatgagc 420 ctggagcggc tgccggaggg gctccggccg ccgccgccgc caccccatga catggggccc 480 gccttccacc tggcccggcc cgccgacccc cgcgagccgc tcgagaactc cgccagcgag 540 tcgtctgaca cggagctgcc agagaaggag cgcggcgggg aacccaaggg gcccgaggac 600 agtggtgcgg gaggcacggg ctgcggcggc gcagacgacc cagccaagaa gaagaagcag 660 cggcggcaac gtacgcactt cacaagccag cagttgcaag agctagaggc cacgttccag 720 aggaaccgct accccgacat gagcatgagg gaggagatcg ccgtgtggac caacctcacc 780 gagccgcgcg tgcgggtctg gttcaagaac cggcgagcca agtggcgtaa gcgcgagcgt 840 aaccagcagc tggacctgtg caagggtggc tacgtgccgc agttcagcgg cctagtgcag 900 ccctacgagg acgtgtacgc cgccggctac tcctacaaca actgggccgc caagagcctg 960 gcgccagcgc cgctctccac caagagcttc accttcttca actccatgag cccgctgtcg 1020 tcgcagtcca tgttctcagc acccagctcc atctcctcca tgaccatgcc gtccagcatg 1080 ggcccaggcg ccgtgcctgg catgcccaac tcgggcctca acaacatcaa caacctcacc 1140 ggctcctcgc tcaactcggc catgtcgccg ggcgcttgcc cgtacggcac tcccgcctcg 1200 ccctacagcg tctaccggga cacgtgcaac tcgagcctag ccagcctgcg gctcaagtcc 1260 aaacagcact cgtcgtttgg ctacggcggc ctgcagggcc cggcctcggg cctcaacgcg 1320 tgccagtaca acagctgacc gccccgccgc accacgcggg ccggcggccg gagcggggaa 1380 gggcgcgggc gcgggcgcgg aggacgcacg cggggccccg gctcgcaagc cccagctcac 1440 cgcgccgcgg acctcacacc tgcgcagccc cctcctccca cttcccactc cgggttggtt 1500 ttgtgtttgc ttttccggac cccactctgc cctccaaaaa gacaaaaaaa aaaaaaaaaa 1560 aaaaaaagca aaaagacgtc ggagaaaagt gccgcgaaaa aatggatgag ttgcaatttc 1620 tctcgggatg gcgcgggtgg tgtgtgtgtt cccacgggcc ccggaggccc actccgcgga 1680 gggcacgcgg cgcggtaggc gagcgccgag gcccagcggc cgggggagga cgacctcgta 1740 tcccgcgtcc ccgccgcgct ggatccggac tgagtggccg ggcctgcgga ctggatgtgc 1800 ggggcctgga cttgcctagg atttcccgac cccgtacaaa ccaagttgcc ctctccgagc 1860 taggcccggc cgagagcgcc ttagctcgag tcggatccgt gttggggcgg gcgttgggtt 1920 tgggggggacg gtgcccccag cccaggatcg ggcactcagt ggagccgcac acggccccgg 1980 cgcgcctggt agagcctcgc tggccccgcg ccccggagcc ctatattaag gccacggagc 2040 gacagcgggc agtgcgggcc tggcgggagg tgggggaggt ccatctcaga acaccccagc 2100 cttgagctta gctgcaggcc caggccctct gctctgctcc cgggctagga ggtggccctc 2160 tgtctgggcg aacagccccc tcctcaccgc ccgccgtgca agagtcgagc cggcagagca 2220 aggggcgcgg ccccagggcc ctgcgcccac tttgcacacc cgctctccgg cccgcgcccc 2280 tgtttacagc gtccctgtgt atgttggact gactgtaata aatctgtcta tatcgacttg 2340 tccatgtacg tctattaaaa ccatagtccg agcgtgctaa gca 2383 <210> 10 <211> 4111 <212> RNA <213> Homo sapiens <400> 10 agaggccagg ccatcatcta agtgggaata aagggctaaa gctattggca ttggcagctg 60 tctccaggcg tcgtccgcgg agcctgaccc tgcgccagga cggaggaggc tgtgcccatg 120 taactgtcct gaagtactgc ggtggccccc tcccccgcgg ctgggagggc aggcaggacg 180 aggttggctt tcctcgcacg gtctctgggc gtgggccccc gccgcagctc cgcggagcct 240 cggtgtctcc tgcaacaggg ggcgggggga acagcggcga gcagccctgg gctgcggctt 300 cctccccacg cccgagtctc ctgcgcaccg ccgccgagga cgcgcgcccg agcctagtcc 360 ccacgccgcg gcgcgcccgg gctccctgct gatcccagaa caatcaacca tgacgaccga 420 atctggatca gactcggaat ccaagccgga ccaggaggcc gagccccagg aggcggcggg 480 ggcgcagggg cgcgcggggg cgcccgtgcc ggagccgccc aaggaggagc agcagcaggc 540 cctggagcag ttcgccgccg ctgcagcgca cagcaccccg gtgcggaggg aggtcactga 600 caaggaacag gagtttgctg ccagggctgc aaaacagctc gaatatcagc aattagaaga 660 cgataaactt tctcagaaat catctagcag taaactctct cggtctccat taaagattgt 720 caaaaagcct aaaagcatgc agtgcaaagt gatacttctc gatggatcag aatatacctg 780 tgatgtagag aaacgctcca gaggacaagt gctgtttgat aaagtgtgtg aacacttgaa 840 cttgctagag aaagactact ttgggcttac gtatcgagat gctgaaaacc agaagaattg 900 gttggaccct gctaaggaaa taaaaaaaca ggttcgaagt ggtgcttggc acttttcatt 960 taatgtgaaa ttttatccac cagaccctgc ccaactatct gaagatatca ccaggtacta 1020 cctctgcttg cagttgcgag atgacatcgt gtccggaagg ctgccctgct cctttgttac 1080 cctggccttg ctgggctcct acactgtcca gtcagagctc ggagactatg acccagatga 1140 atgtgggagc gattacatta gtgagttccg ctttgcacca aaccacacta aagaactgga 1200 agacaaagtg atcgagctgc acaagagcca cagaggaatg acgccagcag aagcagagat 1260 gcatttcttg gaaaatgcca aaaaattatc aatgtatggg gtagatttac atcatgctaa 1320 ggactcagaa ggggtagaaa ttatgttagg agtttgtgca agtggtctgt tgatatatcg 1380 cgaccggctg cgaataaaca gatttgcctg gcccaaggtt ctaaagattt catacaaacg 1440 gaacaacttt tacattaaga tccggccggg agagtttgaa caatttgaaa gcaccattgg 1500 gtttaagctg ccaaaccatc gagctgccaa gcgtttatgg aaagtatgtg ttgagcatca 1560 tacatttttc agactactgt taccagaagc acctcccaag aaattcctaa ccttgggttc 1620 caagtttcgt tatagtggca ggacacaagc gcaaacgaga agagccagtg cgttgataga 1680 tcgcccagca ccttactttg aacgctcatc cagcaaacgt tataccatgt ctcgcagctt 1740 ggatggagca tcagtgaatg aaaaccatga aatatacatg aaggattcta tgtctgctgc 1800 agaggttggt actggccagt acgccacaac aaaaggcatc tctcagacca acttgatcac 1860 cactgtgact ccggagaaga aggctgagga ggagcgggac gaggaagagg acaaacggag 1920 gaagggggaa gaagtcacgc ccatctcggc catccggcac gagggaaaga ctgacagtga 1980 gcgcacggac accgcagccg acggggagac cactgccact gaggagctag aaaaaactca 2040 agatgacctg atgaaacatc aaaccaacat tagcgagctg aaaagaacct tcttagaaac 2100 ctcaacagac actgccgtaa cgaatgaatg ggagaagagg ctttccacct cccccgtgcg 2160 actggccgcc aggcaggagg atgcccccat gatcgaacca cttgtccctg aagagaaaat 2220 ggaaaccaag acggagtcca gtggaataga gacggaaccc accgtgcacc acctgccgct 2280 tagcactgag aaggtggtgc aggagaccgt gttggtggag gagcggcgtg tggtgcacgc 2340 gagtggggat gcttcttact cggcgggaga cagcggggat gctgcagcac agcccgcatt 2400 cacaggcatt aaagggaaag agggctctgc cttgacggag ggggctaaag aggaaggagg 2460 ggaggaggtc gctaaagctg tcctggaaca ggaagagaca gccgctgctt cccgtgagcg 2520 acaagaggag cagagtgcag ccatccacat ttcagaaact ttggaacaaa aacctcattt 2580 tgagtcctca acggtgaaga cggaaaccat cagttttggc agtgtttcac cgggaggagt 2640 aaagctagaa atttccacga aggaagtgcc agtagttcac accgaaacca aaaccatcac 2700 atatgaatca tcacaggtcg atccaggcac agatctggag ccaggcgtgc tgatgagtgc 2760 acagacgatc acatctgaaa ccaccagtac caccaccact acgcacatca ccaaaactgt 2820 gaaagggggc atttcagaga caagaattga gaagcgaata gtcatcacgg gggatgcaga 2880 cattgaccat gaccaggcgc tggctcaggc aattaaagag gccaaagagc agcaccctga 2940 catgtcagtg accaaagtag tggtccataa agagacagag atcacaccag aagatggaga 3000 ggattgacca gaggaataac ttagcttgca catgaatgca gtcatgcaaa ccgttaggaa 3060 aaccagagcc tatatggagt tccctcttct aacccaactg acttgtatct gtccgtggaa 3120 aatttcagtc cagaagaatt gaccttgacc attaataaag acactggcag agagatcttc 3180 ccataataaa gcaatctgat tcagcatcac taaaccgata atgcatgaag caacgataaa 3240 attacaaaag agcagcattt ttaattttca caaaatgtct cagttttcag ctatacctgc 3300 acgttcataa ccaacaatat aaaccgtggt ctcatgtaac acataaacaa ttcatgcctt 3360 tcatagttta ttattattaa agtctaaaca aaattgcaat ttcttaggta accttatatt 3420 tacaataaat gaagattacc ctcaaatgct agaagctgtc taggtccgtc cggtgtgtca 3480 gattttcctc agattagatg tgccaataac caagtttatt cagtaaacaa cttgtacttg 3540 tttcatctgg ttttattact ctcacccata aacagtaatg actctctgac cctctggaaa 3600 tatgtaatgc ttccaatctt gctttgtgta tctcatttaa tttgttataa ggtagtactg 3660 attttagcat attaatgcga tttcttcctt gttgtttgct ttggtctgtg ttcaatccag 3720 agagcttaaa ttgtcattat tttgggaaga aaacctgtat ttttgttagt ttacaatatt 3780 atgaaatttc acttcaggag aaactgctgg gcttcctgtg gctttgtttt cttagttact 3840 ttttccgtgc cgtgtatttt ttaattgatt tttcttcttt tacttgaaaa gaaagtgttt 3900 tttttcaaa tctggtccat atttacattc tagttcagag ccaagcctta aactgtacag 3960 aatttccact gtaattaaaa ctatttagtg ttagttataa atagccttca aaaagagaga 4020 ttctccatta cacgatcacc tgcatcacag cccatggtga atgtatgttt ctgcatagcg 4080 aaataaaaat ggcaaatgca ctgaaagctc t 4111 <210> 11 <211> 1851 <212> RNA <213> Homo sapiens <400> 11 gcggtggtgt cccgaaggcc tcactgtttc tgtcgcgtgg cccgctcagc tgggcttggc 60 tgggctcagc tctccgctcc cgccgttacc aggacgaccg tctccccgca gcccaggctg 120 cccggactgg cgccatgagc tcccccgaca aggtgtccgt ttgtggggcc ggtttcgacc 180 ggggcccca 240 gccacggcct cgatttgggg gtgccgggca gcggcgatgg caagagcgag agcgggttca 300 cagatccaga gggcttcagc ttcgagtctg agagcgaatt gatagagcaa ggaagggtgg 360 tgctctgggg ccgggaaggc cggccaggca ccccggtgga tgaccaaggg gacgttgtgg 420 actactcatt ctacctggct gacgaaccag ccgccatcgt gccgccgccc agcgtccagg 480 gacacccgtt cccagaaggt gccgctgccg aagggtcggc tgagaattgg gcagatgcgg 540 aggtcggtcc cagtgggaga gacgtgctgg gccacagccc tggaaaatgg cagcaggcct 600 ctgccggccg tctccacctc tgcggtcctg ggccagtgcg ggcctggaag aacccggaaa 660 ggggctcgaa gagcagatgg agcctccgcg tggatcccca gcagccctct gcgaaaggcc 720 ccaccaggct gcctacccac gactctgatt ccgcagatga gagcagcgac ttaccactga 780 tgaaggtagg catttgccgc aacgaaggaa gccaggccaa gcccggcagc cccaagaagc 840 gagcagacac atccagacag gcaagcttcc actgcaagga gagttacctg cctgtgccgg 900 gccgtttcct gacctctgct ccccgcggac tcactccagt cgcagagagg ccggctgtgg 960 gagagctgga ggactctccc cagaagaaaa tgcagagcag ggcctgggga aaggtggagg 1020 tcaggcccag ctgctcagga gctgctgctg caggggccct gccccagggc ctttcgagga 1080 ggaagatggc cggggggaag aagtccctag ggggtgcctc tcaactggcc ctggggagag 1140 gctttcctgc ctgcggagag agactctcag ccgctccccc ggagccggcc accttcccgc 1200 cattctctgg tgtgcggcca caggggatgt ccaagaaacc ccaaaagcct aagcacagca 1260 gccctgggaa gaaaccagca gggaggaaga ccagggagtc ccaggctgcg gccagagaag 1320 ataatgaccc aaatagagat gaggtcccaa gagcccaact tcccacccac aggccaggac 1380 tgcctcgcct gtctgtgcgt cgtggagaat tcagcagtag cgaccccaac atcagagctc 1440 cccaacttcc gggaacttca gagccctcgg cctacagccc gggaggcctc gtgcccagac 1500 gccatgcacc ctccggtaac cagcagccgc ctgtccatcc cccaagaccg gaaaggcagc 1560 agcagccccc gggagcccag ggctgtcctc ggtgcatctg gctgcagagg gaaattgagg 1620 accttacaca gcagctagcg gccatgcagt tcctcactga caagttccag gacctttgaa 1680 gttggagcca gcatcttcct acaagatgaa cagctgccac ctttggagct ccggagctgc 1740 agccaagcgg gttccctcca tatcctgttc agccagggct tcctctcttc cgctgcattt 1800 gcccccttcc caacgcagtt caaagcaatt tgaaataaag tcgttctcat a 1851 <210> 12 <211> 2561 <212> RNA <213> Homo sapiens <400> 12 gttgggcggt ggcgggggtg atgcagcggc cgctgagggt ggcgcagggg ccccggccag 60 cccggggctg cagcagtgcg gacagctcca gaagctcatc ggcatctcca ttggcagcct 120 gcgcgggctg ggcaccaagt gcgctgtgtc caacgacctt accgagcagg agatacggac 180 cctggaacat tgtcccaatt ccttcttcta atgaagaaat acgcttagtt gatgatgcgt 240 ttggaaaaat ttgtcacatg gtcggtgatg gctcttgggt ggttcatgtt caggcagcaa 300 aactgttggg ctctatggag caagtcagtt ctcatttctt ggagcagacc cttgacaaga 360 agctgatgtc agatctgagg aggaaacgta ctgcacatga gcgtgccaag gaactttaca 420 gttcagggga gttttccagt ggcagaaagt gggaagatga tgctcccaag gaagaagtag 480 ataccggggc tgtgaacttg attgagtcag gagcttgtgg agcttttgtt catgggttgg 540 aagatgagat gtatgaggtt cgtattgctg ctgtggaggc cctctgcatg ttggcccagt 600 cttcaccctc ttttgctgag aagtgccttg atttcctagt tgacatgttc aacgatgaaa 660 ttgaggaagt acgtctgcag tccatacata ccatgagaaa aatctctaac aacatcaccc 720 tccgagaaga tcagcttgac actgtcctgg ctgtgctaga ggattcatcc agaggtattc 780 gagaggctct tcatgaactc ttatgctgta ctaatgtttc aaccaaagaa gggattcatc 840 ttgcattggt ggagctgctg aaaaatttaa ccaagtaccc tactgatagg gactccatat 900 ggaagtgctt gaagtttctg ggaagtcggc atccaacgct ggtgcttccc ttggtgccag 960 agcttctgag cacccaccca ttttttgaca cagctgaacc agacatggat gatccagctt 1020 atattgcagt tttggtactt attttcaatg ctgctaaaac ctgtccaaca atgcgagcat 1080 tgttctcaga tcacaccgtc aggcactatg cctacctccg agacagtctt tctcatcttg 1140 ttcctgcctt gaggttgcca ggtagaaaac tggtgtcatc agctgtttct cccagcatca 1200 tacctcaaga ggatccttcg cagcagctcc tgcagcagag ccttgaagga gtgtgtagtc 1260 ttcagcactt ggaccctcag ggagcccagg agctgctgga attcaccatc aggccttgca 1320 gt; agcagcgaaa cagactttac cccttatttg ggatgtgtga aaaattttta caggaagtag 1440 acttttttca gaggacacat gaaccattaa caagaaaaat atttaaataa gaaactcacc 1500 aagccaggag aatatgaata cctggatgtg ccccctagga atctggatct cttctctttt 1560 ccccaccctg ccctcaactc cccttacata catacagcct ggatcacaag acccaagaaa 1620 accattccac cagctgctga gagaagagcc acaaaaggac ccagaacttc tccatggcta 1680 ccttagagca ccctaaaatg tgctaacagc cattgttagt attcaggagc tgtttattcc 1740 cattttcaca ctacactaaa gaattttaaa caatcacaag atagactggc tggaagttgg 1800 tgcttggaac atcccacgtc accgaaaagc acagtgctgc aatctcagaa accccaggct 1860 cagccagcag agagaggagg agcagctgga cattggagac tatgattgga cattagagag 1920 aaggagcttg acttcacaga gaaagcacag tgggctgagt gagtggagtt tgcccctgcc 1980 agcacccgtg cactccagtt cccacctgtg aaggtgtcag agagatattc tgcttaaaaa 2040 tcagagtgac tccatcttaa atagcagctg ggtgagataa ggctgagacc tactgggctg 2100 cattcccagg aggttaggca ttctaacctt ctatcaggat gagatagaag gccagcataa 2160 gatataggtc acgaagactc tgctgataaa acaggatgtg gtttaaaaaa atgccagcca 2220 gaacccacca aaaccaagat ggtgatgctc attctcactg ctcattatat gccagtataa 2280 tgcatttgca tgcaaaaaga ctctaccacc agtgccatga cagcttacag atgccatggc 2340 aagatctgga agttaccctg tagcctctaa aatggggaag aaccctcagt tctggaaaat 2400 ttctgcccct ttcctgaaaa actcatgaat aatctaccct ttgtttagca tataatcaag 2460 aaacaactat aagtatagtc aatcgagcag ccaatctcac cgctctgtct atggagtagt 2520 cattcttttc tttacatctc taataaaatt gctttcactt t 2561 <210> 13 <211> 1439 <212> RNA <213> Homo sapiens <400> 13 tgcggcggcg agggaggagg aagaagcgga ggaggcggct cccgcgctcg cagggccgtg 60 ccacctgccc gcccgcccgc tcgctcgctc gcccgccgcg ccgcgctgcc gaccgccagc 120 atgctgccga gagtgggctg ccccgcgctg ccgctgccgc cgccgccgct gctgccgctg 180 ctgccgctgc tgctgctgct actgggcgcg agtggcggcg gcggcggggc gcgcgcggag 240 gtgctgttcc gctgcccgcc ctgcacaccc gagcgcctgg ccgcctgcgg gcccccgccg 300 gttgcgccgc ccgccgcggt ggccgcagtg gccggaggcg cccgcatgcc atgcgcggag 360 ctcgtccggg agccgggctg cggctgctgc tcggtgtgcg cccggctgga gggcgaggcg 420 tgcggcgtct acaccccgcg ctgcggccag gggctgcgct gctatcccca cccgggctcc 480 gagctgcccc tgcaggcgct ggtcatgggc gagggcactt gtgagaagcg ccgggacgcc 540 gagtatggcg ccagcccgga gcaggttgca gacaatggcg atgaccactc agaaggaggc 600 ctggtggaga accacgtgga cagcaccatg aacatgttgg gcgggggagg cagtgctggc 660 cggaagcccc tcaagtcggg tatgaaggag ctggccgtgt tccgggagaa ggtcactgag 720 cagcaccggc agatgggcaa gggtggcaag catcaccttg gcctggagga gcccaagaag 780 ctgcgaccac cccctgccag gactccctgc caacaggaac tggaccaggt cctggagcgg 840 atctccacca tgcgccttcc ggatgagcgg ggccctctgg agcacctcta ctccctgcac 900 atccccaact gtgacaagca tggcctgtac aacctcaaac agtgcaagat gtctctgaac 960 gggcagcgtg gggagtgctg gtgtgtgaac cccaacaccg ggaagctgat ccagggagcc 1020 cccaccatcc ggggggaccc cgagtgtcat ctcttctaca atgagcagca ggaggctcgc 1080 ggggtgcaca cccagcggat gcagtagacc gcagccagcc ggtgcctggc gcccctgccc 1140 cccgcccctc tccaaacacc ggcagaaaac ggagagtgct tgggtggtgg gtgctggagg 1200 attttccagt tctgacacac gtatttatat ttggaaagag accagcaccg agctcggcac 1260 ctccccggcc tctctcttcc cagctgcaga tgccacacct gctccttctt gctttccccg 1320 ggggaggaag ggggttgtgg tcggggagct ggggtacagg tttggggagg gggaagagaa 1380 atttttattt ttgaacccct gtgtcccttt tgcataagat taaaggaagg aaaagtaaa 1439 <210> 14 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> PF4V1 Probe <400> 14 gccacccgcc aggagatgct gttcttggcg ttgctgctcc tgccagttgt 50 <210> 15 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> MYOC Probe <400> 15 ttcttggggt ggctacacgg acattgactt ggctgtggat gaagcaggcc 50 <210> 16 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> CCL8 Probe <400> 16 gtcattgttc tccctcctac ctgtctgtag tgttgtgggg tcctcccatg 50 <210> 17 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ROR2 Probe <400> 17 gaggtccatt cattttacag cccctgttac acacagaccc acaggcagcc 50 <210> 18 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> HBG2 Probe <400> 18 agaattcacc cctgaggtgc aggcttcctg gcagaagatg gtgactggag 50 <210> 19 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> D4S234E Probe <400> 19 ttggcgcagc ggtgtgcatc agaggcgtgt gctgagaagg gtggtgttaa 50 <210> 20 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> KCNJ2 Probe <400> 20 gtgtggcata gctacctgcc catccccaac cctcagcaaa gtagaatctc 50 <210> 21 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> RGS18 Probe <400> 21 gaaggttttt cccaggacgt ctcatgtttg gccctttaga attggggtag 50 <210> 22 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> PITX1 Probe <400> 22 ggcccgcgcc cctgtttaca gcgtccctgt gtatgttgga ctgactgtaa 50 <210> 23 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> EPB41L3 Probe <400> 23 gctgtctagg tccgtccggt gtgtcagatt ttcctcagat tagatgtgcc 50 <210> 24 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> LOC100132994 Probe <400> 24 gcagggcctg gggaaaggtg gaggtcaggc ccagctgctc aggagctgct 50 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> FLJ25037 Probe <400> 25 agtcaatcga gcagccaatc tcaccgctct gtctatggag tagtcattct 50 <210> 26 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> IGFBP2 Probe <400> 26 caaacaccgg cagaaaacgg agagtgcttg ggtggtgggt gctggaggat 50 <210> 27 <211> 99 <212> PRT <213> Homo sapiens <400> 27 Met Lys Val Ser Ala Leu Leu Cys Leu Leu Le Met Ala Ala Thr 1 5 10 15 Phe Ser Pro Gln Gly Leu Ala Gln Pro Asp Ser Val Ser Ile Pro Ile 20 25 30 Thr Cys Cys Phe Asn Val Ile Asn Arg Lys Ile Pro Ile Gln Arg Leu 35 40 45 Glu Ser Tyr Thr Arg Ile Thr Asn Ile Gln Cys Pro Lys Glu Ala Val 50 55 60 Ile Phe Lys Thr Lys Arg Gly Lys Glu Val Cys Ala Asp Pro Lys Glu 65 70 75 80 Arg Trp Val Arg Asp Ser Met Lys His Leu Asp Gln Ile Phe Gln Asn 85 90 95 Leu Lys Pro <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CCL8 primer sense <400> 28 tggagagcta cacaagaatc acc 23 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCL8 primer antisense <400> 29 tggtccagat gcttcatgga a 21
Claims (3)
(b) comparing the expression level of said gene with the expression level of a normal control sample.
Wherein the expression level of the gene of PF4V1, MYOC, CCL8, ROR2, HBG2, D4S234E, KCNJ2, RGS18, PITX1, EPB41L3 or LOC100132994 is higher than the expression level of the gene of the normal control sample,
Wherein the expression level of the FLJ25037 or IGFBP2 gene measured in the step (a) is lower than the expression level of the gene of the normal control sample, it is judged to be idiopathic pulmonary fibrosis.
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| KR20200046507A (en) | 2018-10-24 | 2020-05-07 | 서울여자대학교 산학협력단 | Method and devices for pulmonary fibrosis diagnosis |
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| JP2006526140A (en) | 2002-12-24 | 2006-11-16 | バイオサイト インコーポレイテッド | Marker for differential diagnosis and method of using the same |
| JP5786020B2 (en) | 2010-04-16 | 2015-09-30 | アボットジャパン株式会社 | Methods and reagents for diagnosing rheumatoid arthritis |
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| JP2006526140A (en) | 2002-12-24 | 2006-11-16 | バイオサイト インコーポレイテッド | Marker for differential diagnosis and method of using the same |
| JP5786020B2 (en) | 2010-04-16 | 2015-09-30 | アボットジャパン株式会社 | Methods and reagents for diagnosing rheumatoid arthritis |
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| J. Endocrinol. Invest, Vol 24, pp. 856-864(2001) |
| PLOS ONE, Volume 9, e107055(2014. 9)* |
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| KR20200046507A (en) | 2018-10-24 | 2020-05-07 | 서울여자대학교 산학협력단 | Method and devices for pulmonary fibrosis diagnosis |
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