JP2012115195A - Method for examining basedow disease, method for screening of preventative or therapeutic agent for basedow disease, and kit for examination of basedow disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and effectively examining recidivation and recurrence of Basedow disease.SOLUTION: The method for examining Basedow disease includes a step of measuring the expression amount of Siglec1 in a sample collected from a subject animal.

Description

  The present invention relates to a screening method for Graves' disease, a screening method for a prophylactic or therapeutic agent for Graves' disease, and a kit for testing Graves' disease.

  Graves' disease is an autoimmune disease, in which a TSH receptor antibody (TRAb) is produced in the body to stimulate the thyroid gland and cause excessive thyroid hormone secretion. The prevalence rate for those over the age of 20 is about 1 in 1000, the most common among thyroid diseases. Treatment methods include drugs, surgery, and radioiodine therapy. In Japan, drug therapy using antithyroid drugs is predominantly used. The remission rate due to pharmacotherapy is good at about 90%, but once it is in remission, the point at which pharmacotherapy should be discontinued is still a subject of debate and is a clinical problem. It has become. In fact, there is also a report that disease recurrence occurred in 42% of young adult patients and 34% of elderly patient groups after the end of drug treatment (Non-patent Document 1). Thus, a major drawback of antithyroid therapy is the high recurrence rate. Combined with other internal and external reports, the recurrence rate is 20-75%. The standard currently used is that drug therapy can be stopped if the TSH value is within the normal range and the TSH receptor antibody (TRAb) is negative for about one year with the minimum dose of antithyroid drug. However, even if drug therapy is discontinued on this basis, there are many patients who will later relapse or relapse. In the first place, the value of TRAb is a relatively weak marker for recurrence, and there is a report that hyperthyroidism recurred in 23% of patients who had normal values at the end of treatment (Non-patent Document 1). For this reason, the development of a test method capable of predicting recurrence and relapse of Graves' disease has been awaited in daily clinical practice.

  Siglec1 (Sialic acid-binding immunoglobulin-like lectin1) is a sialic acid-binding protein also called sialoadhesin, suggesting its relationship with coronary artery disease (Non-patent document 2) and systemic lupus erythematosus (Non-patent document 3). However, its association with Graves' disease is not known.

J Clin Endocrinol Metab 81: 2986-2993, (1996) Clin. Biochem. 42 (10-11), 1057-1063 (2009) Arthritis Rheum. 58 (4), 1136-1145 (2008)

  This invention makes it a subject to provide the method for test | inspecting recurrence, relapse, etc. of Graves' disease correctly.

  As a result of intensive studies to solve the above problems, the present inventors have obtained the knowledge that the expression level of Siglec1 in leukocytes of patients with Graves' disease is significantly high in patients who relapse or relapse, and the expression level of Siglec1 The present invention has been completed by finding it as a useful index for the examination of Graves' disease.

That is, the present invention is as follows.
(1) A method of measuring the expression level of Siglec1 in a sample collected from a test animal and examining Graves' disease based on the measured value.
(2) The method according to (1), wherein the recurrence or relapse of Graves' disease is examined.
(3) The method according to (1) or (2), wherein the test animal is a human.
(4) The method according to any one of (1) to (3), wherein the sample is a sample containing leukocytes.
(5) The method according to any one of (1) to (4), wherein the measurement of the expression level is a measurement of the amount of mRNA. (6) A screening method for a prophylactic or therapeutic agent for Graves 'disease, comprising the step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for Graves' disease and then measuring the expression level of Siglec1 in the subject. .
(7) A reagent for testing Graves' disease comprising a reagent capable of measuring the expression level of Siglec1.

  By measuring the expression level of Siglec1 in leukocytes of patients with Graves 'disease, relapse and recurrence of Graves' disease can be predicted with a high probability, and it is considered to be very useful in determining whether or not to continue antithyroid drugs.

The figure which shows the result of having compared the mRNA expression level of Siglec1 in the relapse / relapse group (R) of Graves' disease and the non-relapse / non-relapse group (non-R). The figure which shows the result of the flow cytometry using an anti- Siglec1 antibody.

Hereinafter, embodiments of the present invention will be described in detail.
(1) Method for examining Graves 'disease The method for examining Graves' disease of the present invention includes a step of measuring the expression level of Siglec1 in a sample collected from a test animal. Here, the test includes not only a test for relapse or recurrence of Graves 'disease, but also a test for predicting whether to develop Graves' disease. In addition, recurrence means that Grace's disease is completely cured, and Grace's disease relapses. Relapse of Graves 'disease means that Grace's disease worsens during the treatment of Graves' disease.

  In this specification, “Siglec1” is a sialic acid-binding protein also called sialoadhesin and may be Siglec1 derived from a test animal. Specifically, human-derived Siglec1 has the amino acid sequence of SEQ ID NO: 2. Examples are proteins. Furthermore, protein fragments, derivatives, homologues and mutants having the same function are also included.

  In the above test method, the “test animal” may be any animal as long as it has the possibility of causing Graves' disease. Specific examples include humans, monkeys, and rodents such as rats and mice. It is done. Of these, the method for examining Graves' disease of the present invention is particularly preferably carried out in humans suspected of having Graves' disease or humans after the onset of Graves' disease.

  The “sample” collected from the test animal is not particularly limited as long as it expresses Siglec1 and its concentration can be measured, but a sample containing leukocytes is preferable, and specifically, EDTA plasma, citrate Any of plasma such as plasma, serum, whole blood, and isolated leukocytes may be used, but among these, isolated leukocytes are preferably used.

Examples of the method for measuring the expression level of Siglec1 include a method for measuring the amount of Siglec1 mRNA and a method for measuring the amount of Siglec1 protein.
Specific examples of methods for measuring the amount of mRNA include quantitative RT-PCR using primers and probes specific for the Siglec1 gene, hybridization, and Northern blot. For example, a primer or probe can be designed based on the base sequence of SEQ ID NO: 1.
Specific examples of the method for measuring the amount of protein include ELISA, FACS, and Western blot using an antibody specific for the Siglec1 protein. FACS is preferred.
These measurement methods include, for example, the New Chemistry Laboratory (Edited by the Japanese Biochemical Society; Tokyo Kagaku Dojin), Molecular Cloning, A Laboratory Manual (T. Maniatis et al., Cold Spring Harbor Laboratory (1982)), Antibodies-A Laboratory. The method can be performed according to a method described in a general experiment such as Manual (E. Harlow, et al., Cold Spring Harbor Laboratory (1988)) or the like.

  In the test method of the present invention, Grace's disease can be tested using the expression level of Siglec1 in a sample collected from a test animal as an index. In addition, it is possible to test more accurately by combining TSH value and TRAb value, which are existing Graves' disease markers.

  When performing Graves' disease test using the expression level of Siglec1 as an index, an appropriate cutoff value between the recurrence (or relapse) group of Graves' disease and the non-relapse / non-relapse group of Graves' disease for the expression level of Siglec1 in the sample It may be a method of inspecting by specifying Here, the cut-off value refers to a value determined when a target disease group and a non-disease group are determined by paying attention to a certain substance. When determining the target disease and non-disease, it is negative if it is below the cut-off value, positive if it is above the cut-off value, or positive if it is below the cut-off value, and if it is above the cut-off value The disease can be determined as negative.

(2) Testing kit for Graves 'disease The present invention also includes a kit for use in testing Graves' disease. The kit includes a reagent for measuring the expression level of Siglec1. Examples of the reagent include primers and probes specific to the Siglec1 gene used for quantitative RT-PCR, hybridization, Northern blot, etc., when measuring the amount of Siglec1 mRNA. Further, when measuring the protein mass of Siglec1, an anti-Siglec1 antibody is exemplified. In addition, RT-PCR reagents, biological sample dilutions, reaction buffers, washings, labeled secondary antibodies or antibody fragments thereof, reagents for detecting labeled bodies, standard substances, and the like are also included as necessary.

(3) Screening method for preventive or therapeutic agent for Graves 'disease The third aspect of the present invention is that after contacting a subject with a candidate compound for preventive or therapeutic agent for Graves' disease, expression of Siglec1 in the subject A method for screening a preventive or therapeutic agent for Graves' disease, which comprises a step of measuring the amount.

  As the subject, Grace's disease model animal, leukocytes obtained therefrom, leukocyte cells or the like are used. Examples of Graves' disease model animals include TSH receptor-expressing adenovirus-introduced BALB / c mice (reference: Nagayama Y et al. J Immunol. 168: 2789-2794, 2002). Leukocyte cells are preferred, and Jurkat cells, K562 cells, and the like can be used.

  The expression level of the Siglec1 gene may be measured directly by measuring the amount of mRNA or the amount of protein, but can also be indirectly measured using a reporter gene linked to the promoter of the Siglec1 gene. Here, examples of the reporter gene include a luciferase gene, a GFP gene, and a chloramphenicol acetyltransferase gene. These reporter genes are linked to the promoter of the Siglec1 gene, incorporated into a plasmid used for introducing the gene into mammalian cells, and transfected into cells by a conventional method such as lipofection.

A drug candidate substance is added to the above-mentioned Graves' disease model animal, a cell expressing Siglec1, or a cell into which a reporter gene has been introduced, and the expression level of the Siglec1 protein, Siglec1 gene or reporter gene is measured. There is no restriction | limiting in particular as a pharmaceutical candidate substance, For example, a low molecular synthetic compound may be sufficient and the compound contained in a natural product may be sufficient. Moreover, a peptide may be sufficient. Although individual test substances may be used for screening, a compound library containing these substances may be used. By selecting a candidate substance that reduces the expression level of the Siglec1 protein, Siglec1 gene or reporter gene from candidate substances, a candidate substance for a prophylactic or therapeutic drug for Graves' disease can be obtained.

  The expression level of Siglec1 can be measured by methods such as quantitative RT-PCR, hybridization, Northern blot, FACS, ELISA, Western blotting. Although the expression level of the reporter gene depends on the type of the reporter gene, it can be measured by fluorescence intensity or the like.

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

RNA was extracted from leukocytes from patients with Graves' disease who relapsed and relapsed, and patients who had been in remission for a long time after drug therapy was discontinued, and the difference in gene expression between the two was examined using a DNA microarray. Among them, we found that the expression of Silicc1 gene, which is a cell adhesion molecule, was significantly increased in relapsed patients. Therefore, with the consent of the Medical School Ethics Committee, we obtained written consent from a patient undergoing Graves' disease in the Department of Endocrinology and Diabetes Mellitus of the Hospital, and reverse transcription of Siglec1 mRNA in leukocytes using Taqman PCR ^TM (Applied Biosystems) Quantified by PCR. As a result, as shown in FIG. 1, the relapse / relapse group (R) significantly increased the Siglec1 gene expression level as compared to the remission (non-relapse / non-relapse) group (non-R). Furthermore, when the patients were divided into the Sialic1 gene low expression group (less than 262 copies) and the high expression group (262 copies or more), and the history of relapse and recurrence was examined, it became as shown in Table 1A. A significant correlation was observed.
On the other hand, when the binding rate to TRAb was measured by a radioreceptor assay and classified into a high-value group and a low-value group of TRAb (Table 1B), no correlation was found between relapse and recurrence history (not significant; ns). These data indicate that the Siglec1 gene expression (mRNA) level in leukocytes is significantly correlated with relapse and recurrence of Graves' disease, and the expression is significantly increased in the relapse and relapse group.

  Since Siglec1 is a cell surface marker, a detection method using flow cytometry (FACS) and an anti-Siglec1 antibody (Siglec-1 (Hsn7D2): sc-53442, Santa Cruz Biotechnology, INC) is also conceivable. When FACS using normal human leukocytes was compared with the control antibody (IgG), Siglec1-positive cells were detected in about 40% of leukocytes (FIG. 2). This indicates that Siglec1, a cell surface protein, can be sufficiently identified by FACS.

  According to the method of the present invention, an accurate examination of Graves' disease can be performed, which is useful in the medical and diagnostic fields. Further, according to the method of the present invention, a therapeutic / prophylactic agent for Graves' disease can be screened, which is also useful in the pharmaceutical field.

Claims (7)

Siglec1 (Sialic acid-binding immunoglobulin-like) in samples collected from test animals
A method of measuring the expression level of lectin1) and examining Graves' disease based on the measured value.   The method according to claim 1, wherein the recurrence or relapse of Graves' disease is examined.   The method according to claim 1 or 2, wherein the test animal is a human.   The method according to any one of claims 1 to 3, wherein the sample is a sample containing leukocytes.  The method according to any one of claims 1 to 4, wherein the expression level is measured by measuring the amount of mRNA.   A method for screening a prophylactic or therapeutic agent for Graves 'disease, comprising the step of contacting a subject with a candidate compound for a prophylactic or therapeutic agent for Graves' disease and then measuring the expression level of Siglec1 in the subject.   A reagent for testing Graves' disease comprising a reagent capable of measuring the expression level of Siglec1.