JP2010145294A - Method of screening curative medicine of non-idiopathic interstitial pneumonia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of detecting non-idiopathic interstitial pneumonia and a method of screening a curative medicine of non-idiopathic interstitial pneumonia. <P>SOLUTION: The method of detecting non-idiopathic interstitial pneumonia includes the step of measuring the expression level of periostin gene or periostin protein in a testpiece derived from a drug administered patient, a radiation irradiated patient, a collagen disease patient or an infected patient, and performing a cell or a tissue with capability producing periostin microbial infection treatment, drug-treatment, and/or radiation treatment, and the method of preventing non-idiopathic interstitial pneumonia or screening a curative medicine includes the step of measuring the expression level of the periostin gene or periostin protein when a candidate is brought into contact with them. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting non-idiopathic interstitial pneumonia and a screening method for a therapeutic agent for non-idiopathic interstitial pneumonia.

  Interstitial pneumonia is a lung disease characterized by fibrosis. Histologically, it is characterized by the deposition of matrix proteins such as collagen and the accompanying destruction of alveolar structures. Representative examples are drug-induced drug-induced lung injury and idiopathic interstitial pneumonia of unknown cause. In the treatment of drug-induced lung injury, removal of the causative agent is the primary principle, and pirfenidone is being put into practical use as a therapeutic agent for idiopathic interstitial pneumonia. However, since there is no other therapeutic agent, it is difficult to treat interstitial pneumonia. Interstitial pneumonia, as it progresses, impairs gas exchange in lung tissue and often results in death. For this reason, if a therapeutic drug can be developed by targeting a molecule related to fibrosis in interstitial pneumonia, it will be useful for patients.

On the other hand, the present inventors have found that periostin (osteoblast-specific factor 2) is a novel component in fibrosis of bronchial asthma, and measurement of the expression level of periostin gene is useful as a test method for allergic diseases And a method for measuring the expression level of the periostin gene was developed (Patent No. 4155561, G. Takayama et al., J Allergy Clin Immunol, vol.118, 98-104, 2006) .
No.4155561 patent G. Takayama et al., J Allergy Clin Immunol, vol.118, 98-104, 2006

  An object of this invention is to provide the screening method of the therapeutic agent of non-idiopathic interstitial pneumonia.

  As a result of intensive studies to solve the above problems, the present inventor has found that a substance that suppresses the expression of periostin (osteoblast-specific factor 2) is useful as a therapeutic agent for non-idiopathic interstitial pneumonia. The headline and the present invention were completed.

That is, the present invention is as follows.
(1) Non-idiopathic stroma including a step of measuring the expression level of periostin gene or periostin protein in a test sample derived from a patient who has received a drug, a patient who has received radiation, a collagen disease patient or an infectious disease patient To detect pneumonia.
(2) (i) Expression level of periostin gene or periostin protein in a test sample derived from a patient who received a drug, a patient who received radiation, a collagen disease patient or an infectious disease patient, and (ii) periostin in a normal sample The method according to (1), comprising a step of comparing the expression level of the gene or periostin protein.
(3) (i) The expression level of periostin gene or periostin protein in a test sample derived from a patient who has received a drug, a patient who has received radiation, a collagen disease patient or an infectious disease patient is (ii) periostin in a normal sample The method according to (3), comprising a step of determining that the patient has non-idiopathic interstitial pneumonia or suspected of non-idiopathic interstitial pneumonia when the gene or periostin protein expression level is higher. Method.
(4) Any of (1) to (3), wherein the non-idiopathic interstitial pneumonia is at least one selected from the group consisting of drug-induced interstitial pneumonia and interstitial pneumonia based on radiation irradiation 2. The method according to item 1.
(5) The method according to any one of (1) to (4), wherein the test sample is lung tissue and / or blood.
(6) The method according to any one of (1) to (5), wherein the measurement is performed by an immunoassay.
(7) including a step of measuring the expression level of the periostin gene or periostin protein when a cell or tissue having the ability to produce periostin is subjected to microbial infection treatment, drug treatment and / or radiation treatment and contact with a candidate substance, A screening method for a preventive or therapeutic agent for non-idiopathic interstitial pneumonia.
(8) (i) the expression level of the periostin gene or periostin protein when a cell or tissue having the ability to produce periostin is treated with a microbial infection, a drug and / or a radiation and contacted with a candidate substance; ) Comparing the expression level of the periostin gene or periostin protein in the case where cells or tissues having the ability to produce periostin are treated with a microbial infection, drug and / or radiation but not contacted with a candidate substance, (7) The method described in 1.
(9) (i) The expression level of the periostin gene or periostin protein when a cell or tissue having the ability to produce periostin is treated with a microbial infection, a drug and / or radiation and brought into contact with a candidate substance is (ii) When a cell or tissue capable of producing periostin is treated with a microbial infection, drug and / or radiation, but not in contact with the candidate substance, the candidate substance is non-idiopathic when it is lower than the expression level of the periostin gene or periostin protein The method according to (8), comprising a step of selecting as a prophylactic or therapeutic agent for interstitial pneumonia.
(10) Non-idiopathic interstitial pneumonia is selected from the group consisting of drug-induced interstitial pneumonia, interstitial pneumonia based on radiation, interstitial pneumonia based on collagen disease, and interstitial pneumonia based on infection 10. The method according to any one of (7) to (9), wherein the method is at least one.
(11) The method according to any one of (7) to (10), wherein the biological sample is lung tissue and / or blood.
(12) The method according to any one of (7) to (11), wherein the measurement is based on an immunoassay.
(13) In any one of (1) to (6), comprising an antibody against periostin, or a periostin gene detection probe or amplification primer set designed from the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5 The kit for the screening method according to any one of the above-mentioned detection or (7) to (12).

  The present invention provides a screening method for a therapeutic agent for non-idiopathic interstitial pneumonia. According to the present invention, a compound that targets periostin, that is, a substance that suppresses the expression and function of periostin can be developed as a therapeutic agent for non-idiopathic interstitial pneumonia.

  Until now, no therapeutic drug targeting molecules important for the pathogenesis of interstitial pneumonia has been put to practical use other than Pirrespa (pirfenidone). For this reason, this invention becomes useful for development of the therapeutic agent which improves the symptom of the patient with interstitial pneumonia.

  Hereinafter, the present invention will be described in detail.

  The present invention measures the expression level of periostin in a sample obtained from a patient who has been administered a drug, a patient who has received radiation therapy, a collagen disease patient, or an infectious disease patient, and uses the obtained measurement result as an index. Detects whether or not is non-idiopathic interstitial pneumonia. In addition, the present invention measures the expression level of the periostin gene or protein in the tissue or cell sample by contacting the candidate substance with the tissue or cell sample that has been subjected to drug treatment and / or radiation irradiation treatment. Using the result as an index, a therapeutic drug for non-idiopathic interstitial pneumonia is screened.

1. Outline The present inventors have derived periostin (osteoblast-specific factor 2) as a component in fibrosis of bronchial asthma, and derived the relationship between the expression level and allergy by measuring the expression level of the periostin gene. It was. From the findings that allergies occur when expression increases, it was found that allergic diseases can be examined (G. Takayama et al., J Allergy Clin Immunol, vol.118, 98-104, 2006). . In addition, since the expression level of periostin is increased in lung tissue and peripheral blood of idiopathic interstitial pneumonia, the present inventors considered that periostin may be involved in the pathogenesis of interstitial pneumonia. The inventors have found that idiopathic interstitial pneumonia can be detected by measuring the expression level of periostin.

  The present inventors consider that a substance that suppresses the expression level of periostin is also useful for non-idiopathic interstitial pneumonia, and the expression level of periostin by tissue staining using lung tissue of a patient with drug-induced lung injury Was measured.

  As a result, strong expression of periostin was observed in all specimens derived from patients with drug-induced lung injury. In contrast, no periostin expression was observed in normal lung tissues. In addition, periostin expression was increased in interstitial pneumonia model mice by inhalation of bleomycin. When bleomycin was inhaled to wild-type mice and periostin-deficient mice, the survival rate of mice decreased with fibrosis in wild-type mice, whereas the survival rate in periostin-deficient mice hardly decreased. It has been clarified that the presence of is a major factor in determining the prognosis after the onset of non-idiopathic interstitial pneumonia.

  Based on the above findings, the present inventors have played an important role in the pathogenesis of non-idiopathic interstitial pneumonia, and periostin has been used in the development of therapeutic agents for non-idiopathic interstitial pneumonia. We have succeeded in clarifying that the search for targeted compounds is useful.

2. Periostin Periostin is a protein of about 90 kDa, also called osteoblast specific factor 2 (OSF2os; Horiuchi K, Amizuka N, Takeshita S, Takamatsu H, Katsuura M, Ozawa H, Toyama Y, Bonairald LF, Kudo A.; Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta.J Bone Miner Res.1999 Jul; 14 (7): 1239-49.). It is known that there are several variants of periostin that can be distinguished by the difference in C-terminal length due to alternative splicing.

  Proteins and genes to be detected in the detection method of the present invention are periostin proteins and genes encoding the same.

  An example of the amino acid sequence of the full-length protein of periostin is shown in SEQ ID NO: 2, and the gene encoding the amino acid sequence shown in SEQ ID NO: 2, that is, the base sequence of the transcription product by all exons of the periostin gene (Accession No. D13666) is arranged The number 1 is shown. Further, the amino acid sequences of other splicing variants of human periostin are shown in SEQ ID NOs: 4 and 6, and the base sequences of periostin genes encoding these amino acid sequences are shown in SEQ ID NOs: 3 and 5, respectively (Accession Nos. AY918092, AY140646).

  In the present invention, in addition to the amino acid sequence represented by SEQ ID NO: 2, a variant derived from the amino acid sequence represented by SEQ ID NO: 2 (for example, periostin comprising the amino acid sequence represented by SEQ ID NO: 4 or 6) is encoded. The expression level of the periostin gene or periostin protein containing these amino acid sequences is measured.

3. Method for detecting or diagnosing non-idiopathic interstitial pneumonia according to the present invention The amount of periostin gene or periostin protein is a non-idiopathic interstitial pneumonia, a patient who has received a drug, a patient who has undergone radiation therapy, a collagen disease patient, or an infectious disease patient. In tissue samples from patients who have developed idiopathic interstitial pneumonia, in biological samples from patients receiving drugs, patients receiving radiation therapy, patients with collagen disease, or patients with infections, Periostin can be used as a marker for detecting or diagnosing non-idiopathic interstitial pneumonia. Therefore, the present invention includes a step of measuring the expression level of periostin gene or periostin protein in a test sample from a patient who has received a drug, a patient who has received radiation therapy, a collagen disease patient, or an infectious disease patient. A method for detecting idiopathic interstitial pneumonia is provided.

  Non-idiopathic interstitial pneumonia means interstitial pneumonia whose cause is known, and is interstitial pneumonia other than idiopathic interstitial pneumonia whose cause is unknown. And non-idiopathic interstitial pneumonia includes those that develop due to drug administration, those that develop due to radiation, those derived from patients with collagen disease, or those derived from patients with infectious diseases. Examples of symptoms that develop due to drug administration include interstitial pneumonia that develops after administration of an anticancer drug to cancer patients. In patients with collagen disease, fibrosis may occur in rheumatoid arthritis, systemic scleroderma, dermatomyositis, polymyositis, etc., and therefore interstitial pneumonia frequently occurs. Furthermore, the onset due to radiation irradiation includes onset due to weak radiation irradiation with a degree of image diagnosis and onset due to strong radiation irradiation such as cancer treatment. Therefore, in the present invention, as a test sample, a biological sample derived from a patient with drug-induced interstitial pneumonia caused by drug administration, a biological sample derived from a patient with interstitial pneumonia caused by radiation irradiation, a cause of collagen disease A biological sample derived from a patient with interstitial pneumonia that develops in 1) and a biological sample derived from a patient with interstitial pneumonia that develops due to an infection can be used.

  The detection method of the present invention includes, for example, a step of measuring the following expression level (i) and the following expression level (ii) and comparing them.

(i) Periostin gene or periostin using a test sample derived from a patient who received a drug, a patient who received radiation, a patient who received a combination of drug and radiation, a collagen disease patient or an infectious disease patient Protein expression level
(ii) As a control, expression level of periostin gene or periostin protein in a normal sample And by comparing these measurement results, it is determined whether the patient has developed idiopathic interstitial pneumonia .

  The expression level refers to the level of gene expression and protein expression, and means both the transcription activity level from DNA to mRNA and the translation activity level from mRNA to protein. Therefore, the process of the present invention includes a step of qualitatively or quantitatively measuring the gene expression level and / or protein expression level. The expression level is measured as the amount or presence / absence of periostin protein or the amount or presence / absence of nucleic acid (DNA, RNA, etc.) encoding periostin.

  As a biological sample used for the measurement of the expression level, for example, lung tissue, blood, sputum, exhaled breath condensate or alveolar lavage fluid collected from the above patient, or airway covering fluid collected using a bronchoscope or the like is used. It is preferable to use lung tissue or blood derived from the above patients. Methods for collecting lung tissue, blood, sputum, exhaled breath condensate, alveolar lavage fluid, airway covering fluid or the like are known to those skilled in the art.

  The normal sample is, for example, a sample derived from a person who is not suffering from non-idiopathic interstitial pneumonia or a healthy person, and the origin of the tissue or cells used as a comparison control is the tissue to be measured collected from the patient. Or it is preferable that it is the same kind as a cell. For example, when blood is used as a test sample, it is preferable to use blood as a normal sample. However, in the present specification, it does not mean that the test sample and the normal sample are limited to the same type.

  When the biological sample is lung tissue, sputum or the like, it is preferable to prepare, for example, a lysate or extract DNA or RNA (for example, mRNA) in order to use it for measurement of the expression level. Lysate preparation and RNA extraction can be performed using a known method or a commercially available kit. In addition, when the biological sample is in a liquid form such as blood and alveolar lavage fluid, it is preferably used for measurement of the amount of DNA or RNA encoding periostin after dilution with a buffer solution or the like.

The measurement of the expression level of periostin protein is preferably an immunoassay in terms of easy operation and high sensitivity. Examples of the immunoassay include radioimmunoassay (RIA), immunofluorescence assay (FIA), immunoluminescence assay, enzyme immunoassay (for example, Enzyme Immunoassay (EIA), Enzyme-linked Immunosorbent assay (ELISA)). , Immunohistochemical staining method, Western blot method and the like.
As such a detection method, for example, an antibody that binds to periostin protein can be used.

  The antibody used for this detection can be obtained using techniques well known to those skilled in the art. The antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody (Milstein C, et al., 1983, Nature 305 (5934): 537-40). For example, in the case of a polyclonal antibody against periostin protein, blood of a mammal sensitized with an antigen is taken out, and serum is separated from this blood by a known method. As the polyclonal antibody, serum containing the polyclonal antibody can be used. Alternatively, a fraction containing a polyclonal antibody can be further isolated from this serum as necessary. In order to obtain a monoclonal antibody, immune cells are collected from a mammal sensitized with the antigen and fused with myeloma cells. The thus obtained hybridoma can be cloned, and the antibody can be recovered from the culture to obtain a monoclonal antibody.

As a radioactive substance used for labeling by RIA, for example, ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, ³⁵ S, or ³² P can be used. As a fluorescent substance used for labeling by FIA, for example, a fluorescent substance such as Eu (europium), FITC, TMRITC, Cy3, PE, or Texas-Red can be used. As the luminescent substance used for labeling in the immunoluminescence measurement method, for example, luminol, luminol derivatives, luciferin, lucigenin and the like can be used. Examples of enzymes used for labeling in enzyme immunoassay include horseradish peroxidase (HRP), alkaline phosphatase (ALP), glucose oxidase (GO), and the like. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen to these labeling substances.

  Measurement of the expression level of DNA or RNA encoding periostin can be carried out by a known method, for example, PCR method, Northern blotting, etc. Quantification based on fluorescent labeling, expression product expression by agarose gel electrophoresis Visualization can be performed. In general, higher animal genes are frequently associated with polymorphisms. There are also many molecules that produce isoforms consisting of different amino acid sequences during the splicing process. Even if the gene has a mutation in the base sequence due to polymorphism or isoform, the gene having the same activity as the periostin gene and involved in non-idiopathic interstitial pneumonia is any gene targeted for detection of the present invention. include. The base sequence of the periostin gene is known.

  For example, Northern hybridization can be performed using a polynucleotide having the base sequence shown in SEQ ID NO: 1, 3 or 5 or a polynucleotide containing a part thereof as a probe.

  The probe or primer used in the test of the present invention can be set based on the base sequence of the periostin gene. Therefore, for example, a primer is designed from a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 or a nucleotide containing a part thereof, and a PCR method for amplifying DNA or RNA encoding periostin is performed. it can.

  As the primer or probe, a polynucleotide comprising the base sequence of the indicator gene or a polynucleotide comprising at least 15 nucleotides complementary to its complementary strand can be used. Here, the “complementary strand” refers to the other strand with respect to one strand of double-stranded DNA comprising A: T (U in the case of RNA) and G: C base pairs. In addition, “complementary” is not limited to the case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. As described above, more preferably 98% or more of homology on the base sequence is sufficient. The homology of the base sequence can be determined by an algorithm such as BLAST.

  Such a polynucleotide can be used as a probe for detecting the periostin gene and as a primer for amplifying the gene. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, DNA having at least a part or all of the periostin gene or its complementary strand and having a chain length of at least 15 bp is used. When used as a primer, the region on the 3 ′ side needs to be complementary, but a restriction enzyme recognition sequence or a tag can be added on the 5 ′ side.

  These polynucleotides may be synthesized or natural. In addition, a labeled DNA is usually used as the probe DNA used for hybridization. Any known method can be used as the labeling method. The term oligonucleotide means a polynucleotide having a relatively low degree of polymerization. Oligonucleotides are included in polynucleotides.

  The inspection using the hybridization technique can be performed using, for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, or the like. Furthermore, gene amplification techniques such as RT-PCR can be used. In the RT-PCR method, it is possible to perform more quantitative analysis of the expression of the gene of the present invention by using the PCR amplification monitoring method in the gene amplification process.

  And, for example, when the expression level of the periostin gene or periostin protein in the case of (i) is higher than the expression level of the periostin gene or periostin protein in the case of (ii) above, for example, about 20% or more, About 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 100% or more, about 150% or more, Or when it is about 200% or more, it can be judged that it is non-idiopathic interstitial pneumonia or non-idiopathic interstitial pneumonia is suspected.

  It is also possible to measure the expression level of the periostin gene or periostin protein by immunohistochemistry. Specifically, periostin or RNA encoding periostin is visualized by immunohistochemistry, and the amount of periostin protein or RNA is compared. Specific examples of the immunohistochemical staining method include an enzyme-labeled antibody method and a fluorescent antibody method.

  The enzyme-labeled antibody method is not particularly limited. Labeled antibody methods such as direct method, indirect method, avidin / biotinylated peroxidase complex method (ABC method), or streptavidin / biotinylated antibody method (SAB) method Or a non-labeled antibody method such as a peroxidase / anti-peroxidase method (PAP method).

  The fluorescent antibody method is also not particularly limited, and either a direct method or an indirect method can be employed. Examples of the enzyme or fluorescent substance used for labeling include those described above. When the expression level of the periostin gene or periostin protein in the case (i) is higher than the expression level of the periostin gene or periostin protein in the case (ii), for example, about 20% or more, about 30 % Or more, about 40% or more, about 50% or more, or about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 100% or more, about 150% or more, or When it is about 200% or more, it can be determined that the patient who is the subject of detection has non-idiopathic interstitial pneumonia or non-idiopathic interstitial pneumonia is suspected.

  Here, (i) in a test sample derived from a patient who has been administered and / or irradiated with a drug, the expression level of the periostin gene or periostin protein and (ii) the expression level of the periostin gene or periostin protein in a normal sample The difference is a critical value for detection or diagnosis of non-idiopathic interstitial pneumonia, and can be set as follows, for example.

  First, the expression level of the periostin gene or periostin protein in a biological sample derived from two or more patients with non-idiopathic interstitial pneumonia (for example, drug-induced idiopathic interstitial pneumonia) is measured, and the average value (A) is obtained. . At this time, the number of target patients is 2 or more, for example, 5 or more, 10 or more, 50 or more, or 100 or more.

  On the other hand, the expression level of the periostin gene or periostin protein in a biological sample derived from two or more healthy subjects is measured, and the average value (B) is obtained. At this time, the number of examples of the healthy subject is 2 or more, for example, 5 or more, 10 or more, 50 or more, or 100 or more.

  Then, [(A−B) / B] × 100 (%) is calculated from the obtained average values A and B, and the expression level of the periostin gene or periostin protein in the biological sample derived from the patient with non-idiopathic interstitial pneumonia The average value is determined by what percentage higher than the average value of the expression level of the periostin gene or periostin protein in the sample derived from a healthy person. The value thus obtained is set as a critical value for detection or diagnosis of non-idiopathic interstitial pneumonia. That is, when the expression level of the periostin gene or periostin protein in the case of (i) is higher than the difference obtained by comparison with the expression level of the periostin gene or periostin protein in the case of (ii) (for example, statistical The patient who is the subject of detection can be determined to be idiopathic interstitial pneumonia or suspected to be idiopathic interstitial pneumonia.

  In the detection method or diagnosis method of non-idiopathic interstitial pneumonia of the present invention, the expression level of the periostin gene or periostin protein may be measured using biological samples derived from a plurality of patients. It is preferable to incorporate the values into the average values (A) and (B) to increase the number of target patients and the number of healthy subjects. By increasing the number of cases, the accuracy of detection or diagnosis of non-idiopathic interstitial pneumonia can be increased. Moreover, the average value (B) of the expression level of the periostin gene or periostin protein in the sample derived from a healthy person may be used as the standard expression level used as a control for the test sample.

  The detection result of the non-idiopathic interstitial pneumonia can be used as, for example, main data or auxiliary data when performing a definitive diagnosis of non-idiopathic interstitial pneumonia. When performing a definitive diagnosis of idiopathic interstitial pneumonia, in addition to the above detection results, results of physical findings, results of serological tests (for example, serum KL-6, LDH or SC-A), A comprehensive determination may be made in combination with at least one selected from the group consisting of the result of the respiratory function test, the result of the chest X-ray image findings, and the result of the chest HRCT image findings. Since periostin serves as an index of fibrosis, measuring periostin is useful in that the severity of fibrosis and the state of clinical activation can be evaluated.

4). Screening method The expression of periostin gene is increased in tissues or cells derived from patients with non-idiopathic interstitial pneumonia. Accordingly, a compound or a salt thereof that decreases the expression level of the periostin gene or periostin protein in a patient tissue or cell, for example, a compound that inhibits the expression or a salt thereof is a prophylactic and / or therapeutic agent for non-idiopathic interstitial pneumonia Can be used as

  Accordingly, the present invention provides a periostin gene or periostin when a cell or tissue having the ability to produce periostin is subjected to a microbial infection treatment, a drug treatment or a radiation treatment, or a combination thereof, and contacted with a candidate substance. Provided is a method for screening a preventive or therapeutic agent for non-idiopathic interstitial pneumonia, which comprises the step of measuring the expression level of the protein.

  The screening method of the present invention aims to obtain a substance that can be used as a preventive or therapeutic agent for non-idiopathic interstitial pneumonia. Therefore, in the above expression level measurement system, the cause of non-idiopathic interstitial pneumonia is It is preferable to set the following conditions. Therefore, the measurement system is preferably carried out in an environment exposed to microorganisms or drugs, or an environment irradiated with radiation.

  The present invention includes, for example, a step of measuring the following expression level (i) and the following expression level (ii) and comparing them.

(i) Expression level of periostin gene or periostin protein when a cell or tissue capable of producing periostin is treated with microbial infection, drug treatment and / or radiation, and brought into contact with a candidate substance
(ii) Expression level of periostin gene or periostin protein when cells or tissues capable of producing periostin are treated with microbial infection, drug treatment and / or radiation but not contacted with a candidate substance. The expression level of the periostin gene or periostin protein in cases i) and (ii) is measured, and the levels of both are compared. In a preferred embodiment of the present invention, the amount of periostin protein or the amount of mRNA encoding periostin may be measured and compared.

The cells or tissues to be subjected to microbial infection treatment, drug treatment and radiation treatment may be in vitro systems or in vivo systems. Accordingly, examples of the cells or tissues include cultured cells, cultured tissues, and cells or tissues in experimental animals (for example, mice and rats).
As a drug treatment method, an anticancer agent such as bleomycin, a Chinese medicine such as Sho-saiko-to, interferon, antibiotics, etc. is added to a cell culture system or tissue culture system at an appropriate dose.

In the case of radiation irradiation treatment, the treatment may be performed with radiation having the intensity when performing image diagnosis or the intensity when performing radiotherapy for cancer. However, the intensity of the radiation and the exposure time can be changed as appropriate.
Examples of the microbial treatment include bacterial infection treatment such as mycoplasma, virus infection treatment, and the like, and the amount and time for treatment can be appropriately set.
When drug treatment and radiation treatment are performed on animals, the drug usage / dose, the radiation dose, and the degree of microbial infection can be appropriately set. When the drug and radiation are used in combination, it may be less than the single use.

  “Contact” means that a cell or tissue producing periostin and a candidate substance are present in the same reaction system or culture system. Specifically, adding a candidate substance to the incubator containing the cell or tissue, mixing the cell or tissue and the candidate substance, culturing the cell or tissue in the presence of the candidate substance, or Candidate substances are administered to transgenic animals that express the periostin gene, disease model animals that express the periostin gene (eg, collagen disease model animals), or animals in which interstitial pneumonia has been induced by drug treatment, microbial infection treatment, or radiation treatment To do. When culturing a cell or tissue in the presence of a candidate substance, the culture conditions and the number of culture days of the cell or tissue can be appropriately selected according to the cell or tissue to be cultured.

  The drug treatment and the radiation treatment may be performed simultaneously, and one may be performed first and the other may be performed later. These treatments may be performed before, during, or after the addition of the candidate substance regardless of whether the candidate substance is present.

  The term “cell having the ability to produce periostin” means a cell capable of expressing periostin naturally or genetically, for example, a cell established from lung tissue derived from a patient with non-idiopathic interstitial pneumonia, Cells derived from experimental animals that have developed non-idiopathic interstitial pneumonia due to microbial infection, drug administration, or irradiation, transformants transformed with vectors containing periostin-encoding DNA (transformed cells, Tissue or transgenic experimental animals).

  Examples of host cells to be transformed include E. coli and yeast, and animal cells such as CHO cells, HEK293 cells, and 293 cells. Examples of transgenic experimental animals into which the beliostin gene has been introduced include transgenic mice and transgenic rats. Construction of vectors, transformation, culture of transformed cells, and production of transgenic animals can be performed by known methods (eg, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd ed., (Cold Spring Harbor Laboratory Press (1989)).

  Candidate substances include, for example, compounds derived from natural or synthetic low-molecular compound libraries, gene library expression products (peptides, proteins, etc.), natural or synthetic oligonucleic acids, peptides derived from natural or synthetic peptide libraries , Antibodies, bacteria-derived substances (substances released by metabolism from bacteria, etc.), microorganisms, plant cell extracts, animal cell extracts, culture solutions (culture supernatants of microorganisms, plant cells, animal cells, etc.) or culture solutions The compound derived from, the compound in soil, the compound contained in a random phage peptide display library, etc. are mentioned.

  These compounds may be novel compounds or known compounds. Further, candidate compounds that have been appropriately modified or mutated by chemical, physical, or biochemical means can be used. Further, the candidate compound may be a compound identified by a pharmacophore search or a structure comparison program using a computer. Furthermore, the candidate compound may form a salt or a hydrate.

  The expression level of periostin protein can be measured by, for example, the above-described immunoassay method or protein chip method. Measurement of the amount of DNA or RNA can be performed using a known method, for example, the PCR method, Northern hybridization, DNA chip or the like. And, for example, a candidate substance that reduces the expression level of the periostin gene or periostin protein in the case of (i) as compared with the expression level of the periostin gene or periostin protein in the case of (ii), for example, about 20% More than about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% or more The substance can be selected as a compound that inhibits expression of the periostin gene or a salt thereof.

  Furthermore, in the present invention, a transcriptional regulatory region of the periostin gene of the present invention can be obtained to construct a reporter assay system. The reporter assay system refers to an assay system for screening a transcriptional regulatory factor that acts on a transcriptional regulatory region using the expression level of a reporter gene arranged downstream of the transcriptional regulatory region as an index. A reporter gene is a marker gene that is incorporated into DNA to examine the transcriptional activity of promoters and enhancers, and is not particularly limited as long as it can measure the expression level thereof. Those that can be quantified are preferred. Examples of such reporter genes include luciferase gene, CAT (chloramphenicol acetyltransferase) gene, β-Gal (β-galactosidase) gene, GFP (green fluorescent protein) gene, and GUS (β-glucuronidase) gene. Etc.

  An assay using a reporter gene can be appropriately performed by those skilled in the art. For example, a transcriptional regulatory region of the periostin gene and a cell introduced with a vector containing a reporter gene expressed under the control of the transcriptional regulatory region are contacted with a candidate substance, and the activity of the reporter gene is measured and compared with a control. To select a compound that reduces the expression level of the reporter gene.

5). Kit The present invention provides a kit for screening the method for detecting non-idiopathic interstitial pneumonia of the present invention or the therapeutic or prophylactic agent for non-idiopathic interstitial pneumonia of the present invention. The kit of the present invention includes an antibody against periostin protein, a probe for detecting a periostin gene designed from the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 and further for amplifying the periostin gene for use in the above detection method. Primers can be included. Further, for use in the screening method of the present invention, a primer set for amplifying the periostin gene designed from the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 is included.

  The kit of the present invention includes a cell or tissue or animal having the ability to produce periostin used in the detection method or screening method, reporter gene, gene expression product detection reagent, reaction container, optical analysis And a written instruction describing the assay protocol can be included as appropriate. Moreover, a preservative and preservative can be added to a reagent as needed.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Production of Rabbit Polyclonal Anti-Periostin Antibody Recombinant protein in which a V5 epitope / His tag is added to periostin (nucleotide sequence: SEQ ID NO: 1, Accession No. D13666; amino acid sequence: SEQ ID NO: 2, Accession No. BAA02837) is an insect cell It was expressed in S2 cells and purified.

Specifically, S2 cell transformants were prepared as follows. A cDNA encoding the above-mentioned part of periostin was inserted into pMT / Bip / V5-HisA plasmid (Invitrogen, Carlsbad, Calif.), and this was designated as pMT / Bip / V5-HisA-periostin. PCoHygro (Invitrogen), a plasmid expressing pMT / Bip / V5-HisA-periostin and a hygromycin resistance gene, was co-introduced and transformed into S2 cells by a known method. A transformant was selected with hygromycin to obtain a stable transformant.
And in the transformant of S2 cells, periostin to which a V5 epitope / His tag was bound was expressed.

Recombinant protein was purified as follows. Recombinant protein expression was induced by adding copper sulfate to the medium of periostin gene stable transformant S2 cells. Thereby, the recombinant protein was expressed and secreted into the culture supernatant. The culture supernatant was dialyzed against PBS and then mixed with nickel resin (Ni-NTA Agarose, Qiagen, Hilden, Germany) to bind the recombinant protein to the resin. The resin was washed to remove impurities, and the recombinant protein was eluted with an imidazole-containing buffer. The eluted recombinant protein was dialyzed against PBS and used as an animal immunogen.
Next, these recombinant proteins were mixed with CFA (Complete Freund's Adjuvant) (Freund's Adjuvant, Complete from Sigma) to immunize rabbits, and then serum was collected.

The specific procedure is as follows. As a rabbit, New Zealand White rabbit, female, body weight 1.5-2 kg (KB Tea Oriental, Tosu) was used.
These rabbits were immunized by injecting a mixture of recombinant periostin and CFA, which are immunogens, into several rabbits on the back of the rabbit.
Five to seven weeks after immunization, blood was collected from the ear vein of the immunized rabbit using a needle and a syringe to obtain serum.
Finally, IgG was purified from the serum as follows. That is, a double amount of 50 mM sodium acetate (pH 4.0) was added to the collected serum, and then the pH was adjusted to 4.0 with hydrochloric acid. While stirring the serum, 1/15 volume of caprylic acid was added and stirred for 30 minutes, followed by centrifugation at 9200 g for 10 minutes. The supernatant was dialyzed against PBS to obtain purified IgG.

Tissue immunostaining method The tissue immunostaining method was performed by a general ABC method.
Paraffin-embedded lung tissue was used as a biological sample. Paraffin-embedded lung tissue was obtained from 4 patients with bleomycin-induced interstitial pneumonia, or 4 patients with gefitinib-induced interstitial pneumonia, and normal (non-interstitial pneumonia unaffected) The lung tissue was collected from each person and prepared. This was subjected to treatments such as deparaffinization, inhibition of endogenous peroxidase activity, and blocking. Specifically, these operations were performed as follows. The tissue was deparaffinized and then treated with methanol + 3% H ₂ O ₂ for 15-30 minutes and treated with Protein Block Serum-Free (DAKO, Glostrup, Denmark) for 5-10 minutes.

  Rabbit polyclonal anti-periostin antiserum was added as a primary antibody at a 1000-fold dilution. Thereafter, an antibody against the HRP-labeled primary antibody (Envision, purchased from DAKO) was added as a secondary antibody and developed with DAB (diaminobenzidine) (Sigma). Furthermore, operations such as nuclear staining, dehydration, and encapsulation were performed.

Specifically, these operations were performed as follows. 100 μl of the primary antibody was placed on the tissue and allowed to stand at room temperature for 60 minutes. After washing the primary antibody, 100 μl of the secondary antibody was placed on the tissue and allowed to stand at room temperature for 60 minutes. After washing the secondary antibody, the tissue slide was immersed in DAB substrate solution and allowed to develop for 1-20 minutes. After nuclear staining with hematoxylin, it was dehydrated and encapsulated by a known method.
The results are shown in FIG. FIG. 1 is a diagram showing the expression of periostin. The part dyed brown is the periostin expression site. Periostin expression is strong in the lung tissue of patients with drug-induced lung injury, whereas almost no periostin is observed in normal lung tissue.

In this example, bleomycin-administered mice were prepared, the lungs of the mice were removed, and immunostaining was performed using an anti-periostin antibody. Specifically, it was performed as follows.
10 mg / kg bleomycin (Nippon Kayaku Co., Ltd., Tokyo, Japan) was dissolved in 100 μl of physiological saline in an 8-week-old Balb / c mouse (female) and intratracheally administered under anesthesia with trifluoroethane. As a control, 100 μl of physiological saline was administered. Four weeks later, the lungs were taken out and fixed in formalin to produce paraffin-embedded tissue slices.

Hereinafter, the tissue immunostaining method using the rabbit polyclonal anti-periostin antiserum is the same as described in Example 1.
The results are shown in FIG. FIG. 2 shows periostin expression in lung tissue exposed to a drug (bleomycin). The part stained with brown is the periostin expression site, and in the lung tissue of mice administered bleomycin, the expression of periostin increases with fibrosis.

In this example, 7-10 week old periostin-deficient Balb / c mice (provided by Dr. Conway, Indiana University, Periostin null mice exhibit dwarfism, incisor enamel defects, and an early-onset periodontal disease-like phenotypes, Mol Cell Biol , vol.25, 11131-11144, 2005) and their littermates (wild type) were used in the experiment.
Specifically, 10 mg / kg bleomycin (see Experimental Example 2) was dissolved in 100 μl of physiological saline in the mouse and administered intratracheally under anesthesia with trifluoroethane. Observation was performed for 4 weeks, and the survival rate was measured.
The results are shown in FIG. FIG. 3 is a view showing a survival curve of a mouse. In wild type mice administered with bleomycin, all mice died after 8 days of administration. In contrast, most periostin-deficient mice that received bleomycin survived. Therefore, it has been shown that suppression of periostin activity does not cause drug disorders such as bleomycin.

It is a figure which shows the expression of periostin. It is a figure which shows the expression of periostin. It is a figure which shows the survival curve of a mouse | mouth.

Claims (13)

  Non-idiopathic interstitial pneumonia, including the step of measuring the expression level of the periostin gene or periostin protein in a test sample from a patient receiving a drug, a patient receiving radiation, a collagen disease patient or an infectious disease patient Detection method.   (i) expression level of periostin gene or periostin protein in a test sample derived from a patient receiving a drug, a patient receiving radiation, a collagen disease patient or an infectious disease patient; and (ii) a periostin gene or periostin in a normal sample. The method of claim 1, comprising comparing the expression level of the protein.   (i) The expression level of periostin gene or periostin protein in a test sample derived from a patient receiving a drug, a patient receiving radiation, a collagen disease patient or an infectious disease patient is (ii) the periostin gene or periostin in a normal sample 4. The method of claim 3, comprising determining that the patient has non-idiopathic interstitial pneumonia or suspected non-idiopathic interstitial pneumonia when the protein expression level is higher.   The non-idiopathic interstitial pneumonia is at least one selected from the group consisting of drug-induced interstitial pneumonia and radiation-induced interstitial pneumonia, according to any one of claims 1 to 3. Method.   The method according to any one of claims 1 to 4, wherein the test sample is lung tissue and / or blood.   The method according to any one of claims 1 to 5, wherein the measurement is based on an immunoassay.   Non-idiopathic, comprising the step of measuring the expression level of a periostin gene or periostin protein when a cell or tissue capable of producing periostin is treated with a microbial infection, drug treatment and / or radiation and contacted with a candidate substance A screening method for a prophylactic or therapeutic drug for interstitial pneumonia.   (i) expression level of periostin gene or periostin protein when a cell or tissue capable of producing periostin is treated with microbial infection, drug treatment and / or radiation and contacted with a candidate substance; and (ii) periostin Comparing the expression level of the periostin gene or periostin protein when a cell or tissue capable of producing is treated with a microbial infection, drug and / or radiation but not contacted with a candidate substance. Method.   (i) expression level of periostin gene or periostin protein when a cell or tissue capable of producing periostin is treated with microbial infection, drug and / or radiation and contacted with a candidate substance; (ii) produces periostin Non-idiopathic interstitial properties of a candidate substance when it is lower than the expression level of the periostin gene or periostin protein in the case where cells or tissues having the ability to be treated with a microbial infection, drug and / or radiation but not contacted with the candidate substance The method according to claim 8, comprising a step of selecting as a preventive or therapeutic agent for pneumonia.   Non-idiopathic interstitial pneumonia is selected from the group consisting of drug-induced interstitial pneumonia, radiation-based interstitial pneumonia, interstitial pneumonia based on collagen disease, and interstitial pneumonia based on infection 10. The method according to any one of claims 7 to 9, wherein the number is one.   The method according to any one of claims 7 to 10, wherein the biological sample is lung tissue and / or blood.   The method according to any one of claims 7 to 11, wherein the measurement is performed by an immunoassay.   The detection of any one of Claims 1-6 containing the antibody for periostin, or the probe for periostin gene detection designed from the base sequence shown by sequence number 1, 3, or 5 or the primer set for amplification. The kit for screening methods according to any one of claims 7 to 12.