WO2012105590A1 - Determination marker for non-alcoholic steatohepatitis, and method for diagnosing non-alcoholic steatohepatitis using the marker as measure - Google Patents
Determination marker for non-alcoholic steatohepatitis, and method for diagnosing non-alcoholic steatohepatitis using the marker as measure Download PDFInfo
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Definitions
- the present invention relates to a differentiation marker (diagnostic marker) for nonalcoholic steatohepatitis and a diagnostic method (detection method) for nonalcoholic steatohepatitis using the marker as an index.
- Fatty liver disease is a general term for diseases in which neutral fat is deposited in hepatocytes to cause liver disease.
- Fatty liver disease used to be alcohol-induced liver damage but it has been found that diabetes and obesity also cause similar liver damage.
- a liver disorder characterized mainly by large droplets of liver fat is called nonalcoholic fatty liver disease (NAFLD).
- NAFLD is further classified into simple fatty liver (simple steatosis) and nonalcoholic steatohepatitis (NASH), but the prognosis of the disease is greatly different.
- the pathology of simple fatty liver rarely progresses, but NASH is thought to progress to cirrhosis in 5-20% in 5-10 years.
- diagnosis of whether a patient has simple fatty liver or NASH has been made by liver biopsy tissue diagnosis, but this is extremely invasive and burdensome to the patient, and also causes liver bleeding. Because there are dangers such as accompanying, there are also facility requirements at the time of enforcement.
- a blood biochemical method is generally used as a simple test method, but NASH has only a slight increase in a blood biochemical liver function test such as a test for transaminase or ⁇ GTP. Differentiation of NASH is difficult.
- image diagnostic methods such as abdominal ultrasound, CT, and MRI are useful for the diagnosis of simple fatty liver, but it is difficult to distinguish NASH.
- NASH is a progressive disease, early detection is desired, but there is currently no means to replace liver biopsy in differential diagnosis.
- YKL-40 is a protein having a molecular weight of about 40 kDa discovered in 1988 and is also called glycoprotein-39 (gp-39), chitinase 3-like-protein-1 (CHI3L1), etc. (for example, Non-patent Document 1) reference). It has been reported that the amount of YKL-40 in blood is increased in patients with inflammatory diseases and cancers, suggesting that it can be a biomarker for asthma, rheumatoid arthritis, fibrosis and the like.
- Patent Document 1 discloses that YKL-40 is an inflammatory or degenerative joint disease (eg, rheumatoid arthritis), a disease related to the loss of connective tissue of organs and / or other tissues (eg, cirrhosis), metastatic It is described that it is useful for diagnosing cancer and the like.
- Patent Document 2 discloses that fibrosis stages F2, F3, and F4 of a patient infected with hepatitis C virus (HCV) can be distinguished from F0 and F1, YKL-40, HA rather than YKL-40 alone. , And a combination of three markers of ⁇ 2-MG is described as being useful.
- HCV hepatitis C virus
- Non-Patent Document 2 there was a correlation between the liver fibrosis score and the serum YKL-40 level in HCV-infected patients in the late stage of renal injury (Fig. 1a), but the correlation was higher than other indices It is described that it was not high (FIG. 2). Furthermore, Non-Patent Document 3 describes that the concentration of YKL-40 in the serum of patients with alcoholic liver disease increased in proportion to the severity of the patient. Non-patent document 4 shows that the amount of serum YKL-40 in alcoholic liver disease patients was significantly higher than that in healthy individuals (page 181, left column, lines 16 to 14), and simplicity. Patients without fatty liver and fibrosis had higher serum YKL-40 levels compared with healthy individuals, but lower levels than those with cirrhosis or alcoholic hepatitis with fibrosis (left column on page 182). 19-19 lines from the bottom).
- Non-Patent Document 5 there was no difference in the amount of YKL-40 in serum between patients with simple fatty liver (NAS score 1-3) and NASH patients (NAS score 4-8). (Page 566, Fig. 1c and left column, lines 4 to 6), and fibrosis markers (YKL-40, etc.) alone cannot distinguish simple fatty liver from NASH (right column, page 567, 3 to 5). Line).
- Diagnosis of nonalcoholic fatty liver disease is based on the presence or absence of alcohol consumption by excluding viral hepatitis and autoimmune liver disease by blood tests from patients with fatty liver and mild liver damage.
- the screening is to exclude alcoholic fatty liver and liver damage.
- hepatic biopsy histology with invasion is required (edited by the Japan Society of Liver Studies, “NASH / NAFLD Clinical Practice Guide”, Bunko Do, 2006, pp. 30-31). Because liver biopsy is a very invasive test, careful judgment is required to perform it.
- liver biopsy cannot be performed more than once in a short period of time due to the heavy burden on the patient.
- an object of the present invention is to distinguish between simple fatty liver and NASH in NAFLD patients, which is an alternative to a diagnostic method that places a heavy burden on the patient, such as liver biopsy. Furthermore, simple fatty liver and early NASH It is to provide a differentiation marker that can be used for differentiation.
- a method for detecting nonalcoholic steatohepatitis which comprises measuring the concentration of YKL-40 in a test sample;
- a method for discriminating between simple fatty liver and nonalcoholic steatohepatitis which comprises measuring the concentration of YKL-40 in a test sample;
- a method for confirming the therapeutic effect on nonalcoholic steatohepatitis comprising measuring the concentration of YKL-40 in a test sample;
- Detection of nonalcoholic steatohepatitis comprising the steps of collecting a test sample from a patient suspected of suffering from nonalcoholic steatohepatitis, and measuring the concentration of YKL-40 in the test sample Method;
- the present invention also provides: A step of collecting a test sample (for example, blood, serum, plasma) from a patient suspected of suffering from simple fatty liver or nonalcoholic steatohepatitis, and a step of measuring the concentration of YKL-40 in the test sample
- a method for diagnosing nonalcoholic steatohepatitis comprising: A method for in vitro or ex vivo diagnosis of nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample; In vitro or ex vivo discrimination method between simple fatty liver and non-alcoholic steatohepatitis, characterized by measuring the concentration of YKL-40 in a test sample; An in vitro or ex vivo confirmation method for therapeutic effects on nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample; A diagnostic kit for non-alcoholic steatohepatitis, comprising an anti-YKL-40 antibody or a probe or primer for measuring YK
- the results of microarrays using liver biopsy samples are summarized for each NASH stage using a box plot according to John Tukey's method.
- the significant difference test is based on Welch's t-test.
- the results of measuring the amount of YKL-40 in serum are summarized for each NASH stage using a box diagram according to John Tukey's method.
- Significant difference test is based on Welch's t test.
- the correlation between the microarray results using liver biopsy samples and the amount of serum YKL-40 is shown.
- the result of the ROC analysis regarding the discrimination of simple fatty liver and NASH using the amount of YKL-40 in serum is shown.
- 3 shows the results of ROC analysis regarding the differentiation between simple fatty liver and stage I NASH using serum YKL-40 amount.
- the present invention relates to a differential marker for nonalcoholic steatohepatitis (NASH), more specifically, a differential marker for distinguishing between simple fatty liver and NASH in nonalcoholic fatty liver disease (NAFLD), YKL-40 is used as a differentiation marker for distinguishing between fatty fatty liver and early NASH.
- NASH nonalcoholic steatohepatitis
- NAFLD nonalcoholic fatty liver disease
- YKL-40 is used as a differentiation marker for distinguishing between fatty fatty liver and early NASH.
- NAFLD is a liver disorder characterized mainly by large droplets of liver fat, similar to alcoholic liver disorder, although there is no clear drinking history, and simple fatty liver and NASH are categorized. Simple fatty liver is a case in which hepatocytes with lipid droplets are observed in 30% or more, and fat deposition disease is suspected in image diagnosis. However, it is often followed up because only fat deposits are allowed. NASH is a case of steatohepatitis with necrosis, inflammation, and fibrosis in liver tissue, and is considered a severe form of NAFLD. NASH is progressive and often follows the outcome of cirrhosis and liver cancer. Therefore, it is important to distinguish NASH from NAFLD.
- the most important index is fibrosis.
- the disease progression rarely progresses rapidly in cases with mild fibrosis, but there are cases in which advanced fibrosis cases progress to liver failure and liver cancer.
- the degree of fibrosis is mild, there is a high possibility that the progress of fibrosis can be delayed and measures to improve can be selected, but if advanced fibrosis leads to liver failure or liver cancer, eventually liver transplantation is applied. It becomes. Therefore, clarifying the degree of fibrosis (stage) is important for consideration of treatment prognosis, and in particular, detection and intervention during treatment with mild fibrosis is important for improving prognosis. . Brunt et al.
- Stage 1 Central leaflet (Zone 3)
- Stage 2 Central leaflet + portal vein area
- Stage 3 Bridge formation
- Stage 4 Liver cirrhosis (American Journal of Gastroenterology, 1999, 94, p2467-2474).
- test sample necessary for carrying out the present invention is collected from a patient suspected of suffering from NASH, for example.
- test sample for example, blood (for example, whole blood, serum, plasma), saliva, urine, and liver tissue can be used, and serum and plasma are particularly preferable.
- YKL-40 is a protein with a molecular weight of about 40 kDa, also called glycoprotein-39 (gp-39), chitinase-like protein-1 (CHI3L1), etc.
- YKL-40 is said to have no chitinolytic activity despite its high homology with chitinolytic enzymes. Moreover, it is said that it is released from activated neutrophils and macrophages and is involved in the remodeling of extracellular matrix, but the detailed function has not been fully elucidated. It is known that expression is increased in diseases mainly consisting of chronic inflammation such as rheumatoid arthritis, asthma and Crohn's disease. In recent years, it has been clarified that expression is increased in various cancers. There is a report on the relationship with liver disease in alcoholic hepatitis and viral hepatitis.
- the method for measuring the concentration or amount of YKL-40 in the test sample is not particularly limited.
- an immunological method using an anti-YKL-40 antibody eg, enzyme immunoassay, latex agglutination immunization
- Measurement method chemiluminescence immunoassay method, fluorescent antibody method, radioimmunoassay method, immunoprecipitation method, immunohistochemical staining, Western blot, etc.
- molecular biological method for measuring the amount of mRNA for measuring the amount of mRNA.
- the anti-YKL-40 antibody may be a polyclonal antibody or a monoclonal antibody, and the antibody itself or an antibody fragment maintaining the reaction specificity [for example, Fab, Fab ′, F (ab ′) 2 , or Fv] Can be used.
- NASH in which fibrosis has been induced correlates with the stage of fibrosis and YKL- 40 dose is increased. Therefore, a patient in whom an increase in the concentration or amount of YKL-40 in the test sample can be determined as NASH. This determination is performed by collecting specimens from the subject patient and healthy subjects and comparing them, or by comparing the normal values of healthy subjects determined in advance with the measured values of patient specimens. However, as shown in Example 3 described later, it is preferable that the determination threshold is determined in advance.
- simple fatty liver and NASH can be distinguished, but simple fatty liver and early NASH (that is, stage 1) can also be distinguished.
- stage 1 simple fatty liver and early NASH (that is, stage 1) can also be distinguished.
- YKL-40 a correlation with YKL-40 was also reported in viral liver disease or alcoholic liver disease, but in all cases, a significant increase in YKL-40 was observed in severe cases.
- patients with early stage viral liver disease or alcoholic liver disease could not be distinguished from healthy individuals.
- the therapeutic effect on NASH can be confirmed by measuring the amount of YKL-40 after appropriately treating NASH patients.
- treatments for NASH patients include diet therapy, exercise therapy, drug therapy, antioxidant therapy, and surgical therapy (intragastric balloon therapy, laparoscopic gastric banding, liver transplantation, etc.), etc. If the amount of YKL-40 in the test sample collected from a patient who has undergone these treatments is reduced as compared to that before treatment, it can be determined that there was a therapeutic effect. On the other hand, if the level is the same as or higher than that before treatment, it can be determined that there is no therapeutic effect, and for example, it is possible to cope with selecting a new treatment method.
- Example 1 Comprehensive gene expression variation analysis of liver biopsy samples of NAFLD and NASH (1) Liver biopsy and NASH diagnosis As part of a medical practice independent of the study, liver biopsy was performed to confirm the diagnosis of NAFLD patients. The following tests were further conducted on patients whose blood samples were obtained and informed consent was obtained in writing. NASH liver histopathological diagnosis was performed using a scoring system by Brunt et al. As a result, it was found that among 122 subjects, 23 cases of simple fatty liver and 99 cases of NASH were found. Of those determined to be NASH, it was revealed that there were 61 stage I, 28 stage II and 10 stage III by fibrosis score.
- Example 2 Measurement of YKL-40 in NAFLD and NASH Serum Samples (1) Blood Collection and Serum Preparation The blood collected in Example 1 (1) above was coagulated at room temperature and centrifuged. Serum was prepared. Of these, 76 were able to be analyzed by ELISA, and the breakdown was for 20 simple fatty livers and 56 for NASH. Among those determined as NASH, there were 35 stages I, 16 stages II, and 5 stages III according to the fibrosis score.
- Example 3 ROC Analysis Using YKL-40 Amount in Serum Samples of NAFLD and NASH
- AUC was calculated to be 0.74.
- the cutoff value indicating the maximum efficiency was calculated to be 21.8 ng / mL.
- the sensitivity was calculated to be 67% and the specificity was 80% (FIG. 4).
- the AUC was calculated to be 0.69, and the cutoff value indicating the maximum efficiency was calculated to be 21.8 ng / mL.
- the sensitivity was calculated to be 63% and the specificity was 80% (FIG. 5).
- the present invention can be used in NAFLD patients to distinguish between simple fatty liver and NASH, and further to distinguish between simple fatty liver and early NASH.
- this invention was demonstrated along the specific aspect, the deformation
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Abstract
Provided is a determination marker which can be used for distinguishing between simple steatosis and NASH, particularly between simple steatosis and the initial stage of NASH, in an NAFLD patient. The determination marker comprises YKL-40.
Description
本発明は、非アルコール性脂肪肝炎の鑑別マーカー（診断マーカー）及び当該マーカーを指標とした非アルコール性脂肪肝炎の診断方法（検出方法）に関する。
The present invention relates to a differentiation marker (diagnostic marker) for nonalcoholic steatohepatitis and a diagnostic method (detection method) for nonalcoholic steatohepatitis using the marker as an index.
脂肪性肝疾患（fatty liver disease）は、肝細胞に中性脂肪が沈着して肝疾患をきたす疾患の総称である。脂肪性肝疾患は以前はアルコールによる肝障害が多かったが、糖尿病や肥満によっても同様な肝障害が生ずることがわかり、明らかな飲酒歴がないにもかかわらず、肝組織所見はアルコール性肝障害に類似した主に大滴性の肝脂肪沈着を特徴とする肝障害を、非アルコール性脂肪性肝疾患（nonalcoholic fatty liver disease；ＮＡＦＬＤ）と呼ぶ。
Fatty liver disease (fatty liver disease) is a general term for diseases in which neutral fat is deposited in hepatocytes to cause liver disease. Fatty liver disease used to be alcohol-induced liver damage, but it has been found that diabetes and obesity also cause similar liver damage. A liver disorder characterized mainly by large droplets of liver fat is called nonalcoholic fatty liver disease (NAFLD).
ＮＡＦＬＤは、さらに単純性脂肪肝（simple steatosis）と非アルコール性脂肪肝炎（nonalcoholic steatohepatitis；ＮＡＳＨ）に分類されるが、疾患の予後は大きく異なる。単純性脂肪肝は病態が進行することはほとんどないが、ＮＡＳＨは５～１０年で５～２０％が肝硬変に進行すると考えられている。従来、患者が単純性脂肪肝であるか、ＮＡＳＨであるかの診断は肝生検組織診断により行われているが、これは極めて侵襲的であり患者の負担が大きく、また、肝臓の出血を伴うなど危険性もあることから施行に際しては施設的な要件も伴う。一般的に簡便な検査方法としては血液生化学的な方法が挙げられるが、ＮＡＳＨは血液生化学的な肝機能検査、例えばトランスアミナーゼやγＧＴＰの検査では軽度な上昇しか認めず、単純性脂肪肝とＮＡＳＨの鑑別は困難である。また、腹部超音波、ＣＴおよびＭＲＩと言った画像診断法は単純性脂肪肝の診断には有用であるが、ＮＡＳＨを鑑別することは困難である。このように、ＮＡＳＨは進行性の疾患であるため早期発見が望まれるものの、鑑別診断においては肝生検に代わる手段が無いのが現状である。
NAFLD is further classified into simple fatty liver (simple steatosis) and nonalcoholic steatohepatitis (NASH), but the prognosis of the disease is greatly different. The pathology of simple fatty liver rarely progresses, but NASH is thought to progress to cirrhosis in 5-20% in 5-10 years. Conventionally, the diagnosis of whether a patient has simple fatty liver or NASH has been made by liver biopsy tissue diagnosis, but this is extremely invasive and burdensome to the patient, and also causes liver bleeding. Because there are dangers such as accompanying, there are also facility requirements at the time of enforcement. A blood biochemical method is generally used as a simple test method, but NASH has only a slight increase in a blood biochemical liver function test such as a test for transaminase or γGTP. Differentiation of NASH is difficult. In addition, image diagnostic methods such as abdominal ultrasound, CT, and MRI are useful for the diagnosis of simple fatty liver, but it is difficult to distinguish NASH. Thus, although NASH is a progressive disease, early detection is desired, but there is currently no means to replace liver biopsy in differential diagnosis.
ＹＫＬ－４０は１９８８年に発見された分子量約４０ｋＤａのタンパク質で、glycoprotein-39（ｇｐ－３９）、chitinase 3-like-protein-1（ＣＨＩ３Ｌ１）等とも呼ばれている（例えば、非特許文献１参照）。炎症性の疾患や癌の患者で血中ＹＫＬ－４０量が亢進することが報告されており、喘息や関節リウマチ、線維症などのバイオマーカーとなりうることが示唆されている。
YKL-40 is a protein having a molecular weight of about 40 kDa discovered in 1988 and is also called glycoprotein-39 (gp-39), chitinase 3-like-protein-1 (CHI3L1), etc. (for example, Non-patent Document 1) reference). It has been reported that the amount of YKL-40 in blood is increased in patients with inflammatory diseases and cancers, suggesting that it can be a biomarker for asthma, rheumatoid arthritis, fibrosis and the like.
例えば、特許文献１には、ＹＫＬ－４０は、炎症性又は退行性の関節疾患（例えば、関節リウマチ）、臓器の結合組織及び／又は他の組織の損失に関する疾患（例えば、肝硬変）、転移性癌などを診断するのに有用であることが記載されている。
また、特許文献２には、Ｃ型肝炎ウイルス（ＨＣＶ）に感染した患者の線維症段階Ｆ２、Ｆ３及びＦ４を、Ｆ０及びＦ１と区別しうること、ＹＫＬ－４０単独よりもＹＫＬ－４０、ＨＡ、及びα２－ＭＧの３種マーカーの組合せが有用であることが記載されている。非特許文献２には、腎障害後期のＨＣＶ感染患者における肝線維化スコアと血清中ＹＫＬ－４０量の間に相関関係が見られた（Ｆｉｇ．１ａ）が、他の指標と比べて相関は高くなかったこと（Ｆｉｇ．２）が記載されている。
更に、非特許文献３には、アルコール性肝疾患患者の血清中のＹＫＬ－４０濃度は患者の重症度に比例して上昇していたことが記載されている。非特許文献４には、アルコール性肝疾患患者の血清中ＹＫＬ－４０量は、健常人に比べて有意に上昇していたこと（１８１頁左欄下から１６～１４行）、また、単純性脂肪肝と線維化のない患者は、健常人と比べて血清中ＹＫＬ－４０量が高いが、線維化のある肝硬変又はアルコール性肝炎の患者と比べて低いレベルであったこと（１８２頁左欄下から１９～１２行）が記載されている。 For example,Patent Document 1 discloses that YKL-40 is an inflammatory or degenerative joint disease (eg, rheumatoid arthritis), a disease related to the loss of connective tissue of organs and / or other tissues (eg, cirrhosis), metastatic It is described that it is useful for diagnosing cancer and the like.
Patent Document 2 discloses that fibrosis stages F2, F3, and F4 of a patient infected with hepatitis C virus (HCV) can be distinguished from F0 and F1, YKL-40, HA rather than YKL-40 alone. , And a combination of three markers of α2-MG is described as being useful. In Non-Patent Document 2, there was a correlation between the liver fibrosis score and the serum YKL-40 level in HCV-infected patients in the late stage of renal injury (Fig. 1a), but the correlation was higher than other indices It is described that it was not high (FIG. 2).
Furthermore,Non-Patent Document 3 describes that the concentration of YKL-40 in the serum of patients with alcoholic liver disease increased in proportion to the severity of the patient. Non-patent document 4 shows that the amount of serum YKL-40 in alcoholic liver disease patients was significantly higher than that in healthy individuals (page 181, left column, lines 16 to 14), and simplicity. Patients without fatty liver and fibrosis had higher serum YKL-40 levels compared with healthy individuals, but lower levels than those with cirrhosis or alcoholic hepatitis with fibrosis (left column on page 182). 19-19 lines from the bottom).
また、特許文献２には、Ｃ型肝炎ウイルス（ＨＣＶ）に感染した患者の線維症段階Ｆ２、Ｆ３及びＦ４を、Ｆ０及びＦ１と区別しうること、ＹＫＬ－４０単独よりもＹＫＬ－４０、ＨＡ、及びα２－ＭＧの３種マーカーの組合せが有用であることが記載されている。非特許文献２には、腎障害後期のＨＣＶ感染患者における肝線維化スコアと血清中ＹＫＬ－４０量の間に相関関係が見られた（Ｆｉｇ．１ａ）が、他の指標と比べて相関は高くなかったこと（Ｆｉｇ．２）が記載されている。
更に、非特許文献３には、アルコール性肝疾患患者の血清中のＹＫＬ－４０濃度は患者の重症度に比例して上昇していたことが記載されている。非特許文献４には、アルコール性肝疾患患者の血清中ＹＫＬ－４０量は、健常人に比べて有意に上昇していたこと（１８１頁左欄下から１６～１４行）、また、単純性脂肪肝と線維化のない患者は、健常人と比べて血清中ＹＫＬ－４０量が高いが、線維化のある肝硬変又はアルコール性肝炎の患者と比べて低いレベルであったこと（１８２頁左欄下から１９～１２行）が記載されている。 For example,
Furthermore,
このように、ウイルス性肝疾患又はアルコール性肝疾患では、線維化等の重症度とＹＫＬ－４０との関連性が報告されていたが、非アルコール性脂肪肝炎（ＮＡＳＨ）の鑑別マーカーとして有用であるとの知見は全くなかった。例えば、非特許文献５には、単純性脂肪肝患者（ＮＡＳ ｓｃｏｒｅ １－３）とＮＡＳＨ患者（ＮＡＳ ｓｃｏｒｅ ４－８）との間で、血清中のＹＫＬ－４０量に違いは見られなかったこと（５６６頁Ｆｉｇ．１ｃ及び同頁左欄４～６行）、また、線維化マーカー（ＹＫＬ－４０等）単独では、単純性脂肪肝とＮＡＳＨを識別できないこと（５６７頁右欄３～５行）が記載されている。
Thus, in viral liver disease or alcoholic liver disease, the relationship between the severity of fibrosis and YKL-40 has been reported, but it is useful as a differential marker for nonalcoholic steatohepatitis (NASH). There was no finding that there was. For example, in Non-Patent Document 5, there was no difference in the amount of YKL-40 in serum between patients with simple fatty liver (NAS score 1-3) and NASH patients (NAS score 4-8). (Page 566, Fig. 1c and left column, lines 4 to 6), and fibrosis markers (YKL-40, etc.) alone cannot distinguish simple fatty liver from NASH (right column, page 567, 3 to 5). Line).
非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）の診断は、脂肪肝、軽度肝障害が認められる患者から、血液検査によりウイルス性肝炎や自己免疫性肝疾患を除外し、更に飲酒歴の有無に基づいてアルコール性脂肪肝、肝障害を除外するというスクリーニングで行われる。ＮＡＦＬＤが単純性脂肪肝であるか、ＮＡＳＨであるかを鑑別するには、侵襲を伴う肝生検組織診断が必要とされる（日本肝臓学会編、「ＮＡＳＨ・ＮＡＦＬＤの診療ガイド」、文光堂、２００６年、３０～３１頁）。肝生検は甚だ侵襲的な検査であるため、その施行には慎重な判断が求められる。このため、事前の血液生化学的な肝機能検査、例えばトランスアミナーゼやγＧＴＰなどの検査で異常が認められなければ、肝生検の施行を判断することはより難しくなる。一方で、先述のように初期のＮＡＳＨは血液生化学的な肝機能検査では軽度な異常しか認められないことから、進行性の疾患として早期の発見が望まれるにもかかわらず特に初期のＮＡＳＨでは肝生検を施行する機会を逸する可能性が高く、結果として確定診断、治療介入が遅れることとなる。従って、非侵襲的にＮＡＳＨを鑑別できる方法の開発は、早期の治療介入を可能にし生命予後の改善に大きく寄与するものと考えられる。また、肝生検は患者への負担が大きいために短期間のうちに複数回施行することができない。このため、ＮＡＳＨが疑われる経過観察中の患者に対してその病勢の変化をタイミング良くとらえるためや、ＮＡＳＨ患者への治療介入の効果を判定する際に肝生検を多用することは極めて難しい。このようなことからも、負担の少ない血液検査マーカーの開発が急務である。
従って、本発明の課題は、肝生検のように患者への負担が大きい診断方法に代わる、ＮＡＦＬＤ患者において、単純性脂肪肝とＮＡＳＨとの鑑別、更には、単純性脂肪肝と初期のＮＡＳＨとの鑑別に使用できる鑑別マーカーを提供することにある。 Diagnosis of nonalcoholic fatty liver disease (NAFLD) is based on the presence or absence of alcohol consumption by excluding viral hepatitis and autoimmune liver disease by blood tests from patients with fatty liver and mild liver damage. The screening is to exclude alcoholic fatty liver and liver damage. In order to distinguish whether NAFLD is simple fatty liver or NASH, hepatic biopsy histology with invasion is required (edited by the Japan Society of Liver Studies, “NASH / NAFLD Clinical Practice Guide”, Bunko Do, 2006, pp. 30-31). Because liver biopsy is a very invasive test, careful judgment is required to perform it. For this reason, it is more difficult to determine whether or not to perform liver biopsy unless abnormalities are observed in a prior blood biochemical liver function test, for example, a test such as transaminase or γGTP. On the other hand, as described above, since early NASH has only minor abnormalities in blood biochemical liver function tests, early NASH is particularly desirable even though early detection is desired as a progressive disease. Opportunities to perform liver biopsy are likely to be missed, resulting in delays in definitive diagnosis and therapeutic intervention. Therefore, development of a method that can distinguish NASH non-invasively is considered to enable early therapeutic intervention and contribute greatly to the improvement of life prognosis. In addition, liver biopsy cannot be performed more than once in a short period of time due to the heavy burden on the patient. For this reason, it is extremely difficult to frequently use liver biopsy in order to grasp the change in the disease state in a timely manner for patients undergoing follow-up with suspected NASH, or to determine the effect of therapeutic intervention on NASH patients. For this reason, it is urgent to develop a blood test marker with less burden.
Therefore, an object of the present invention is to distinguish between simple fatty liver and NASH in NAFLD patients, which is an alternative to a diagnostic method that places a heavy burden on the patient, such as liver biopsy. Furthermore, simple fatty liver and early NASH It is to provide a differentiation marker that can be used for differentiation.
従って、本発明の課題は、肝生検のように患者への負担が大きい診断方法に代わる、ＮＡＦＬＤ患者において、単純性脂肪肝とＮＡＳＨとの鑑別、更には、単純性脂肪肝と初期のＮＡＳＨとの鑑別に使用できる鑑別マーカーを提供することにある。 Diagnosis of nonalcoholic fatty liver disease (NAFLD) is based on the presence or absence of alcohol consumption by excluding viral hepatitis and autoimmune liver disease by blood tests from patients with fatty liver and mild liver damage. The screening is to exclude alcoholic fatty liver and liver damage. In order to distinguish whether NAFLD is simple fatty liver or NASH, hepatic biopsy histology with invasion is required (edited by the Japan Society of Liver Studies, “NASH / NAFLD Clinical Practice Guide”, Bunko Do, 2006, pp. 30-31). Because liver biopsy is a very invasive test, careful judgment is required to perform it. For this reason, it is more difficult to determine whether or not to perform liver biopsy unless abnormalities are observed in a prior blood biochemical liver function test, for example, a test such as transaminase or γGTP. On the other hand, as described above, since early NASH has only minor abnormalities in blood biochemical liver function tests, early NASH is particularly desirable even though early detection is desired as a progressive disease. Opportunities to perform liver biopsy are likely to be missed, resulting in delays in definitive diagnosis and therapeutic intervention. Therefore, development of a method that can distinguish NASH non-invasively is considered to enable early therapeutic intervention and contribute greatly to the improvement of life prognosis. In addition, liver biopsy cannot be performed more than once in a short period of time due to the heavy burden on the patient. For this reason, it is extremely difficult to frequently use liver biopsy in order to grasp the change in the disease state in a timely manner for patients undergoing follow-up with suspected NASH, or to determine the effect of therapeutic intervention on NASH patients. For this reason, it is urgent to develop a blood test marker with less burden.
Therefore, an object of the present invention is to distinguish between simple fatty liver and NASH in NAFLD patients, which is an alternative to a diagnostic method that places a heavy burden on the patient, such as liver biopsy. Furthermore, simple fatty liver and early NASH It is to provide a differentiation marker that can be used for differentiation.
本発明は、
［１］ＹＫＬ－４０を含むことを特徴とする、患者が罹患している疾患が単純性脂肪肝であるか、又は非アルコール性脂肪肝炎であるかを判別することができる鑑別マーカー；
［２］被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎の検出方法；
［３］被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、単純性脂肪肝と非アルコール性脂肪肝炎とを判別する方法；
［４］被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎に対する治療効果の確認方法；
［５］非アルコール性脂肪肝炎への罹患が疑われる患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎の検出方法；
［６］前記患者が非アルコール性脂肪性肝疾患患者である、［５］の方法；
［７］患者が罹患している疾患が単純性脂肪肝であるか、又は非アルコール性脂肪肝炎であるかを判別する方法であって、該患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、方法；
［８］前記患者が非アルコール性脂肪性肝疾患患者である、［７］の方法；
［９］非アルコール性脂肪肝炎の治療を受けた患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎に対する治療効果の確認方法;
に関する。 The present invention
[1] A differential marker capable of discriminating whether a disease affected by a patient is simple fatty liver or non-alcoholic steatohepatitis, comprising YKL-40;
[2] A method for detecting nonalcoholic steatohepatitis, which comprises measuring the concentration of YKL-40 in a test sample;
[3] A method for discriminating between simple fatty liver and nonalcoholic steatohepatitis, which comprises measuring the concentration of YKL-40 in a test sample;
[4] A method for confirming the therapeutic effect on nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample;
[5] Detection of nonalcoholic steatohepatitis, comprising the steps of collecting a test sample from a patient suspected of suffering from nonalcoholic steatohepatitis, and measuring the concentration of YKL-40 in the test sample Method;
[6] The method of [5], wherein the patient is a patient with nonalcoholic fatty liver disease;
[7] A method for determining whether a disease affecting a patient is simple fatty liver or non-alcoholic steatohepatitis, the method of collecting a test sample from the patient, and the subject Measuring the concentration of YKL-40 in the test sample;
[8] The method of [7], wherein the patient is a patient with nonalcoholic fatty liver disease;
[9] Therapeutic effect on nonalcoholic steatohepatitis, comprising the steps of collecting a test sample from a patient treated for nonalcoholic steatohepatitis and measuring the concentration of YKL-40 in the test sample Confirmation method;
About.
［１］ＹＫＬ－４０を含むことを特徴とする、患者が罹患している疾患が単純性脂肪肝であるか、又は非アルコール性脂肪肝炎であるかを判別することができる鑑別マーカー；
［２］被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎の検出方法；
［３］被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、単純性脂肪肝と非アルコール性脂肪肝炎とを判別する方法；
［４］被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎に対する治療効果の確認方法；
［５］非アルコール性脂肪肝炎への罹患が疑われる患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎の検出方法；
［６］前記患者が非アルコール性脂肪性肝疾患患者である、［５］の方法；
［７］患者が罹患している疾患が単純性脂肪肝であるか、又は非アルコール性脂肪肝炎であるかを判別する方法であって、該患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、方法；
［８］前記患者が非アルコール性脂肪性肝疾患患者である、［７］の方法；
［９］非アルコール性脂肪肝炎の治療を受けた患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎に対する治療効果の確認方法;
に関する。 The present invention
[1] A differential marker capable of discriminating whether a disease affected by a patient is simple fatty liver or non-alcoholic steatohepatitis, comprising YKL-40;
[2] A method for detecting nonalcoholic steatohepatitis, which comprises measuring the concentration of YKL-40 in a test sample;
[3] A method for discriminating between simple fatty liver and nonalcoholic steatohepatitis, which comprises measuring the concentration of YKL-40 in a test sample;
[4] A method for confirming the therapeutic effect on nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample;
[5] Detection of nonalcoholic steatohepatitis, comprising the steps of collecting a test sample from a patient suspected of suffering from nonalcoholic steatohepatitis, and measuring the concentration of YKL-40 in the test sample Method;
[6] The method of [5], wherein the patient is a patient with nonalcoholic fatty liver disease;
[7] A method for determining whether a disease affecting a patient is simple fatty liver or non-alcoholic steatohepatitis, the method of collecting a test sample from the patient, and the subject Measuring the concentration of YKL-40 in the test sample;
[8] The method of [7], wherein the patient is a patient with nonalcoholic fatty liver disease;
[9] Therapeutic effect on nonalcoholic steatohepatitis, comprising the steps of collecting a test sample from a patient treated for nonalcoholic steatohepatitis and measuring the concentration of YKL-40 in the test sample Confirmation method;
About.
また、本発明は、
単純性脂肪肝又は非アルコール性脂肪肝炎への罹患が疑われる患者から被検試料（例えば、血液、血清、血漿）を採取する工程、前記被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎の診断方法；
被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎の体外（in vitro or ex vivo）診断方法；
被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、単純性脂肪肝と非アルコール性脂肪肝炎との体外（in vitro or ex vivo）判別方法；
被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎に対する治療効果の体外（in vitro or ex vivo）確認方法；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、非アルコール性脂肪肝炎の診断キット；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、単純性脂肪肝と非アルコール性脂肪肝炎との判別用キット；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、非アルコール性脂肪肝炎に対する治療効果の確認用キット；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、非アルコール性脂肪肝炎の診断薬の製造における使用；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、単純性脂肪肝と非アルコール性脂肪肝炎の判別薬の製造における使用；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、非アルコール性脂肪肝炎の治療効果の確認薬の製造における使用；
に関する。 The present invention also provides:
A step of collecting a test sample (for example, blood, serum, plasma) from a patient suspected of suffering from simple fatty liver or nonalcoholic steatohepatitis, and a step of measuring the concentration of YKL-40 in the test sample A method for diagnosing nonalcoholic steatohepatitis, comprising:
A method for in vitro or ex vivo diagnosis of nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample;
In vitro or ex vivo discrimination method between simple fatty liver and non-alcoholic steatohepatitis, characterized by measuring the concentration of YKL-40 in a test sample;
An in vitro or ex vivo confirmation method for therapeutic effects on nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample;
A diagnostic kit for non-alcoholic steatohepatitis, comprising an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA, and an instruction manual describing the relationship between YKL-40 and non-alcoholic steatohepatitis;
Simple fatty liver and nonalcoholic steatohepatitis, including an anti-YKL-40 antibody, a probe or primer for measuring YKL-40 mRNA, and an instruction manual describing the relationship between YKL-40 and nonalcoholic steatohepatitis And discrimination kit;
Confirmation of therapeutic effect on non-alcoholic steatohepatitis, including anti-YKL-40 antibody, probe or primer for measuring YKL-40 mRNA, and instruction manual describing the relationship between YKL-40 and non-alcoholic steatohepatitis Kit for;
Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a diagnostic agent for nonalcoholic steatohepatitis;
Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a discrimination agent for simple fatty liver and nonalcoholic steatohepatitis;
Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a confirmation drug for the therapeutic effect of nonalcoholic steatohepatitis;
About.
単純性脂肪肝又は非アルコール性脂肪肝炎への罹患が疑われる患者から被検試料（例えば、血液、血清、血漿）を採取する工程、前記被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎の診断方法；
被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎の体外（in vitro or ex vivo）診断方法；
被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、単純性脂肪肝と非アルコール性脂肪肝炎との体外（in vitro or ex vivo）判別方法；
被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎に対する治療効果の体外（in vitro or ex vivo）確認方法；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、非アルコール性脂肪肝炎の診断キット；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、単純性脂肪肝と非アルコール性脂肪肝炎との判別用キット；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、非アルコール性脂肪肝炎に対する治療効果の確認用キット；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、非アルコール性脂肪肝炎の診断薬の製造における使用；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、単純性脂肪肝と非アルコール性脂肪肝炎の判別薬の製造における使用；
抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、非アルコール性脂肪肝炎の治療効果の確認薬の製造における使用；
に関する。 The present invention also provides:
A step of collecting a test sample (for example, blood, serum, plasma) from a patient suspected of suffering from simple fatty liver or nonalcoholic steatohepatitis, and a step of measuring the concentration of YKL-40 in the test sample A method for diagnosing nonalcoholic steatohepatitis, comprising:
A method for in vitro or ex vivo diagnosis of nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample;
In vitro or ex vivo discrimination method between simple fatty liver and non-alcoholic steatohepatitis, characterized by measuring the concentration of YKL-40 in a test sample;
An in vitro or ex vivo confirmation method for therapeutic effects on nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample;
A diagnostic kit for non-alcoholic steatohepatitis, comprising an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA, and an instruction manual describing the relationship between YKL-40 and non-alcoholic steatohepatitis;
Simple fatty liver and nonalcoholic steatohepatitis, including an anti-YKL-40 antibody, a probe or primer for measuring YKL-40 mRNA, and an instruction manual describing the relationship between YKL-40 and nonalcoholic steatohepatitis And discrimination kit;
Confirmation of therapeutic effect on non-alcoholic steatohepatitis, including anti-YKL-40 antibody, probe or primer for measuring YKL-40 mRNA, and instruction manual describing the relationship between YKL-40 and non-alcoholic steatohepatitis Kit for;
Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a diagnostic agent for nonalcoholic steatohepatitis;
Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a discrimination agent for simple fatty liver and nonalcoholic steatohepatitis;
Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a confirmation drug for the therapeutic effect of nonalcoholic steatohepatitis;
About.
本発明によれば、ＮＡＦＬＤ患者において、単純性脂肪肝とＮＡＳＨとの鑑別、更には、単純性脂肪肝と初期のＮＡＳＨとの鑑別を行うことができる。
According to the present invention, it is possible to distinguish between simple fatty liver and NASH, and further, between simple fatty liver and initial NASH in NAFLD patients.
本発明は、非アルコール性脂肪肝炎（ＮＡＳＨ）の鑑別マーカー、より具体的には、非アルコール性脂肪性肝疾患（ＮＡＦＬＤ）における単純性脂肪肝とＮＡＳＨとを鑑別する鑑別マーカー、更には、単純性脂肪肝と初期のＮＡＳＨとを鑑別する鑑別マーカーとして、ＹＫＬ－４０を使用することを特徴とする。
The present invention relates to a differential marker for nonalcoholic steatohepatitis (NASH), more specifically, a differential marker for distinguishing between simple fatty liver and NASH in nonalcoholic fatty liver disease (NAFLD), YKL-40 is used as a differentiation marker for distinguishing between fatty fatty liver and early NASH.
ＮＡＦＬＤは、明らかな飲酒歴がないにもかかわらず、肝組織所見はアルコール性肝障害に類似した主に大滴性の肝脂肪沈着を特徴とする肝障害であり、更に単純性脂肪肝とＮＡＳＨに分類される。
単純性脂肪肝は、脂肪滴を伴う肝細胞が３０％以上に認められる症例であり、画像診断でも脂肪沈着症が疑われるものである。しかし、脂肪沈着のみを認めるものであることからしばしば経過観察となる。
ＮＡＳＨは、肝組織で壊死・炎症・線維化を伴う脂肪性肝炎を認める症例であり、ＮＡＦＬＤの重症型と考えられている。ＮＡＳＨは進行性であり、しばしば肝硬変、肝癌という転帰を辿る。従って、ＮＡＦＬＤからＮＡＳＨを鑑別することは重要である。ＮＡＳＨの病態や予後を考察する上で、最も重要な指標は線維化である。ＮＡＳＨの中でも軽度線維化例では急速にその病勢が進行することは稀であるが、高度線維化例では肝不全、肝癌へと進行する症例が存在する。線維化の程度が軽度であれば、線維化の進行を遅延させ、改善するための方策を選択できる可能性が高いが、高度線維化で肝不全や肝癌となると最終的には肝移植の適用となる。従って、線維化の程度（ステージ）を明らかにすることは治療予後の考察に重要であり、特に線維化の程度が軽度なうちに発見し治療介入することは予後を改善させる上で重要である。Bruntらは、ＮＡＳＨの進展経過を線維化の程度により、ステージ１：小葉中心部（Zone 3）、ステージ２：小葉中心部+門脈域、ステージ３：架橋形成、ステージ４：肝硬変の４段階に分類する（American Journal of Gastroenterology, 1999, 94, p2467-2474）。 NAFLD is a liver disorder characterized mainly by large droplets of liver fat, similar to alcoholic liver disorder, although there is no clear drinking history, and simple fatty liver and NASH are categorized.
Simple fatty liver is a case in which hepatocytes with lipid droplets are observed in 30% or more, and fat deposition disease is suspected in image diagnosis. However, it is often followed up because only fat deposits are allowed.
NASH is a case of steatohepatitis with necrosis, inflammation, and fibrosis in liver tissue, and is considered a severe form of NAFLD. NASH is progressive and often follows the outcome of cirrhosis and liver cancer. Therefore, it is important to distinguish NASH from NAFLD. When considering the pathology and prognosis of NASH, the most important index is fibrosis. Among the NASH cases, the disease progression rarely progresses rapidly in cases with mild fibrosis, but there are cases in which advanced fibrosis cases progress to liver failure and liver cancer. If the degree of fibrosis is mild, there is a high possibility that the progress of fibrosis can be delayed and measures to improve can be selected, but if advanced fibrosis leads to liver failure or liver cancer, eventually liver transplantation is applied. It becomes. Therefore, clarifying the degree of fibrosis (stage) is important for consideration of treatment prognosis, and in particular, detection and intervention during treatment with mild fibrosis is important for improving prognosis. . Brunt et al. Described the progress of NASH depending on the degree of fibrosis: Stage 1: Central leaflet (Zone 3), Stage 2: Central leaflet + portal vein area, Stage 3: Bridge formation, Stage 4: Liver cirrhosis (American Journal of Gastroenterology, 1999, 94, p2467-2474).
単純性脂肪肝は、脂肪滴を伴う肝細胞が３０％以上に認められる症例であり、画像診断でも脂肪沈着症が疑われるものである。しかし、脂肪沈着のみを認めるものであることからしばしば経過観察となる。
ＮＡＳＨは、肝組織で壊死・炎症・線維化を伴う脂肪性肝炎を認める症例であり、ＮＡＦＬＤの重症型と考えられている。ＮＡＳＨは進行性であり、しばしば肝硬変、肝癌という転帰を辿る。従って、ＮＡＦＬＤからＮＡＳＨを鑑別することは重要である。ＮＡＳＨの病態や予後を考察する上で、最も重要な指標は線維化である。ＮＡＳＨの中でも軽度線維化例では急速にその病勢が進行することは稀であるが、高度線維化例では肝不全、肝癌へと進行する症例が存在する。線維化の程度が軽度であれば、線維化の進行を遅延させ、改善するための方策を選択できる可能性が高いが、高度線維化で肝不全や肝癌となると最終的には肝移植の適用となる。従って、線維化の程度（ステージ）を明らかにすることは治療予後の考察に重要であり、特に線維化の程度が軽度なうちに発見し治療介入することは予後を改善させる上で重要である。Bruntらは、ＮＡＳＨの進展経過を線維化の程度により、ステージ１：小葉中心部（Zone 3）、ステージ２：小葉中心部+門脈域、ステージ３：架橋形成、ステージ４：肝硬変の４段階に分類する（American Journal of Gastroenterology, 1999, 94, p2467-2474）。 NAFLD is a liver disorder characterized mainly by large droplets of liver fat, similar to alcoholic liver disorder, although there is no clear drinking history, and simple fatty liver and NASH are categorized.
Simple fatty liver is a case in which hepatocytes with lipid droplets are observed in 30% or more, and fat deposition disease is suspected in image diagnosis. However, it is often followed up because only fat deposits are allowed.
NASH is a case of steatohepatitis with necrosis, inflammation, and fibrosis in liver tissue, and is considered a severe form of NAFLD. NASH is progressive and often follows the outcome of cirrhosis and liver cancer. Therefore, it is important to distinguish NASH from NAFLD. When considering the pathology and prognosis of NASH, the most important index is fibrosis. Among the NASH cases, the disease progression rarely progresses rapidly in cases with mild fibrosis, but there are cases in which advanced fibrosis cases progress to liver failure and liver cancer. If the degree of fibrosis is mild, there is a high possibility that the progress of fibrosis can be delayed and measures to improve can be selected, but if advanced fibrosis leads to liver failure or liver cancer, eventually liver transplantation is applied. It becomes. Therefore, clarifying the degree of fibrosis (stage) is important for consideration of treatment prognosis, and in particular, detection and intervention during treatment with mild fibrosis is important for improving prognosis. . Brunt et al. Described the progress of NASH depending on the degree of fibrosis: Stage 1: Central leaflet (Zone 3), Stage 2: Central leaflet + portal vein area, Stage 3: Bridge formation, Stage 4: Liver cirrhosis (American Journal of Gastroenterology, 1999, 94, p2467-2474).
本発明を実施するために必要とする被検試料は、例えば、ＮＡＳＨの罹患が疑われる患者から採取する。被検試料としては、例えば、血液（例えば、全血、血清、血漿）、唾液、尿及び肝臓組織を用いることができ、特には血清、血漿が好ましい。
The test sample necessary for carrying out the present invention is collected from a patient suspected of suffering from NASH, for example. As the test sample, for example, blood (for example, whole blood, serum, plasma), saliva, urine, and liver tissue can be used, and serum and plasma are particularly preferable.
ＹＫＬ－４０は、glycoprotein-39（ｇｐ－３９）、chitinase 3-like-protein-1（ＣＨＩ３Ｌ１）等とも呼ばれる分子量約４０ｋＤａのタンパク質である。ＹＫＬ－４０はキチン分解酵素と相同性が高いにもかかわらず、キチン分解活性は無いとされている。また、活性化した好中球やマクロファージから放出され細胞外基質のリモデリングに関与しているとされているが，詳細な機能はまだ十分に解明されていない。慢性関節リウマチ、喘息、Crohn病など慢性炎症を主体とする疾患で発現亢進することが知られている。近年では様々な癌においても発現亢進していることが明らかになっている。肝臓疾患との関連性についても、アルコール性肝炎やウイルス性肝炎において報告がある。
YKL-40 is a protein with a molecular weight of about 40 kDa, also called glycoprotein-39 (gp-39), chitinase-like protein-1 (CHI3L1), etc. YKL-40 is said to have no chitinolytic activity despite its high homology with chitinolytic enzymes. Moreover, it is said that it is released from activated neutrophils and macrophages and is involved in the remodeling of extracellular matrix, but the detailed function has not been fully elucidated. It is known that expression is increased in diseases mainly consisting of chronic inflammation such as rheumatoid arthritis, asthma and Crohn's disease. In recent years, it has been clarified that expression is increased in various cancers. There is a report on the relationship with liver disease in alcoholic hepatitis and viral hepatitis.
被検試料中のＹＫＬ－４０の濃度又は量を測定する方法は、特に限定されるものではなく、例えば、抗ＹＫＬ－４０抗体を用いる免疫学的手法（例えば、酵素免疫測定法、ラテックス凝集免疫測定法、化学発光免疫測定法、蛍光抗体法、放射免疫測定法、免疫沈降法、免疫組織染色、又はウエスタンブロット等）、又はｍＲＮＡ量を測定する分子生物学的手法などを挙げることができる。前記抗ＹＫＬ－４０抗体としては、ポリクローナル抗体又はモノクローナル抗体を問わず、抗体それ自体、あるいは、反応特異性を維持した抗体断片〔例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、又はＦｖ〕を用いることができる。
The method for measuring the concentration or amount of YKL-40 in the test sample is not particularly limited. For example, an immunological method using an anti-YKL-40 antibody (eg, enzyme immunoassay, latex agglutination immunization) Measurement method, chemiluminescence immunoassay method, fluorescent antibody method, radioimmunoassay method, immunoprecipitation method, immunohistochemical staining, Western blot, etc.) or molecular biological method for measuring the amount of mRNA. The anti-YKL-40 antibody may be a polyclonal antibody or a monoclonal antibody, and the antibody itself or an antibody fragment maintaining the reaction specificity [for example, Fab, Fab ′, F (ab ′) 2 , or Fv] Can be used.
後述の実施例に具体的な実験データで示すように、単純性脂肪肝に比して、線維化が惹起されているＮＡＳＨにおいては、線維化のステージに相関して被検試料中のＹＫＬ－４０量が亢進する。従って、被検試料中のＹＫＬ－４０濃度又は量の上昇が認められた患者は、ＮＡＳＨであると判断することができる。この判定は、被検患者と健常者からそれぞれ検体を採取し、それらを比較することにより、あるいは、予め決定した健常者の正常値と、患者検体の測定値とを比較することにより、実施することもできるが、後述の実施例３に示すように、判定用閾値を予め決定しておくことが好ましい。
As shown by specific experimental data in the examples described later, in comparison with simple fatty liver, NASH in which fibrosis has been induced correlates with the stage of fibrosis and YKL- 40 dose is increased. Therefore, a patient in whom an increase in the concentration or amount of YKL-40 in the test sample can be determined as NASH. This determination is performed by collecting specimens from the subject patient and healthy subjects and comparing them, or by comparing the normal values of healthy subjects determined in advance with the measured values of patient specimens. However, as shown in Example 3 described later, it is preferable that the determination threshold is determined in advance.
本発明では、単純性脂肪肝とＮＡＳＨとを鑑別することができるが、単純性脂肪肝と初期のＮＡＳＨ（すなわち、ステージ１）とを鑑別することもできる。背景技術欄で先述したとおり、ウイルス性肝疾患またはアルコール性肝疾患においても、ＹＫＬ－４０との相関性が報告されていたが、いずれも、重症例において有意なＹＫＬ－４０上昇が認められるものの、初期段階のウイルス性肝疾患またはアルコール性肝疾患の患者では健常者と区別することができなかった。本発明ではＮＡＳＨの肝臓組織中ではＹＫＬ－４０遺伝子の発現が亢進しており、かつ血液中でのＹＫＬ－４０量がこれと高い相関を示すことを明らかにし、血液中でのＹＫＬ－４０量の亢進が単なる偶発的なものではなく肝臓組織の状態を反映したものであることを見出した。よって、本発明者らが見出した本知見は、極めて意外性があり、早期段階でＮＡＳＨの鑑別が可能である点で、極めて顕著な有用性を示すものである。
In the present invention, simple fatty liver and NASH can be distinguished, but simple fatty liver and early NASH (that is, stage 1) can also be distinguished. As described above in the Background Art section, a correlation with YKL-40 was also reported in viral liver disease or alcoholic liver disease, but in all cases, a significant increase in YKL-40 was observed in severe cases. However, patients with early stage viral liver disease or alcoholic liver disease could not be distinguished from healthy individuals. In the present invention, it has been clarified that the expression of the YKL-40 gene is increased in the liver tissue of NASH, and that the amount of YKL-40 in the blood is highly correlated with this, and the amount of YKL-40 in the blood is revealed. It was found that the increase in the blood pressure was not merely accidental but reflected the state of the liver tissue. Therefore, the present findings found by the present inventors are extremely surprising and show extremely remarkable utility in that NASH can be distinguished at an early stage.
また、本発明によれば、ＮＡＳＨ患者に対して適当な治療を行った後、ＹＫＬ－４０量を測定することにより、ＮＡＳＨに対する治療効果を確認することができる。ＮＡＳＨ患者に対する治療法としては、例えば、食事療法、運動療法、薬物療法、抗酸化療法および外科的療法（胃内留置バルーン療法、腹腔鏡下胃バンディング、肝移植等）等を挙げることができ、これらの治療を施した患者から採取した被検試料中のＹＫＬ－４０量が、治療前と比較して減少していれば、治療効果があったものと判断することができる。一方、治療前と比較して同程度、または上昇していれば、治療効果がないものと判断することができ、例えば、別の治療法を新たに選択するとの対応が可能である。
In addition, according to the present invention, the therapeutic effect on NASH can be confirmed by measuring the amount of YKL-40 after appropriately treating NASH patients. Examples of treatments for NASH patients include diet therapy, exercise therapy, drug therapy, antioxidant therapy, and surgical therapy (intragastric balloon therapy, laparoscopic gastric banding, liver transplantation, etc.), etc. If the amount of YKL-40 in the test sample collected from a patient who has undergone these treatments is reduced as compared to that before treatment, it can be determined that there was a therapeutic effect. On the other hand, if the level is the same as or higher than that before treatment, it can be determined that there is no therapeutic effect, and for example, it is possible to cope with selecting a new treatment method.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。また、市販の試薬やキットを用いる場合には市販品の指示書に従って実施可能である。
Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention. Moreover, when using a commercially available reagent or kit, it can be carried out in accordance with the instructions for the commercially available product.
《実施例１》ＮＡＦＬＤおよびＮＡＳＨの肝生検サンプルの網羅的遺伝子発現変動解析
（１）肝生検およびＮＡＳＨの診断
研究から独立した医療行為の一環として、ＮＡＦＬＤ患者の診断確定のために肝生検及び採血を行い、文書にてインフォームドコンセントが得られた患者に関してさらに以下の試験を実施した。Ｂｒｕｎｔ等によるスコアリングシステムを利用してＮＡＳＨ肝病理組織診断をおこなった。その結果、対象者１２２名のうち、単純性脂肪肝が２３名、ＮＡＳＨが９９名であると判明した。ＮＡＳＨと判定されたもののうち線維化のスコア別にはステージＩが６１名、ステージIIが２８名およびステージIIIが１０名であることが明らかとなった。 << Example 1 >> Comprehensive gene expression variation analysis of liver biopsy samples of NAFLD and NASH (1) Liver biopsy and NASH diagnosis As part of a medical practice independent of the study, liver biopsy was performed to confirm the diagnosis of NAFLD patients. The following tests were further conducted on patients whose blood samples were obtained and informed consent was obtained in writing. NASH liver histopathological diagnosis was performed using a scoring system by Brunt et al. As a result, it was found that among 122 subjects, 23 cases of simple fatty liver and 99 cases of NASH were found. Of those determined to be NASH, it was revealed that there were 61 stage I, 28 stage II and 10 stage III by fibrosis score.
（１）肝生検およびＮＡＳＨの診断
研究から独立した医療行為の一環として、ＮＡＦＬＤ患者の診断確定のために肝生検及び採血を行い、文書にてインフォームドコンセントが得られた患者に関してさらに以下の試験を実施した。Ｂｒｕｎｔ等によるスコアリングシステムを利用してＮＡＳＨ肝病理組織診断をおこなった。その結果、対象者１２２名のうち、単純性脂肪肝が２３名、ＮＡＳＨが９９名であると判明した。ＮＡＳＨと判定されたもののうち線維化のスコア別にはステージＩが６１名、ステージIIが２８名およびステージIIIが１０名であることが明らかとなった。 << Example 1 >> Comprehensive gene expression variation analysis of liver biopsy samples of NAFLD and NASH (1) Liver biopsy and NASH diagnosis As part of a medical practice independent of the study, liver biopsy was performed to confirm the diagnosis of NAFLD patients. The following tests were further conducted on patients whose blood samples were obtained and informed consent was obtained in writing. NASH liver histopathological diagnosis was performed using a scoring system by Brunt et al. As a result, it was found that among 122 subjects, 23 cases of simple fatty liver and 99 cases of NASH were found. Of those determined to be NASH, it was revealed that there were 61 stage I, 28 stage II and 10 stage III by fibrosis score.
（２）肝生検サンプルからの全ＲＮＡの抽出
上述の実施例１（１）で採取された肝生検サンプルの一部から、アレイ解析可能であったもの（対象９８名、うち単純性脂肪肝１６名、ＮＡＳＨ８２名。ＮＡＳＨと判定されたうち線維化のスコア別にはステージＩ ５３名、ステージII ２１名およびステージIII ８名）について、RNeasy mini kit（Qiagen社）を用いて全ＲＮＡを調製した。調製した全ＲＮＡは分光光度計ND-1000（NanoDrop Technologies）にて定量し、その１ｎｇを用いて2100 Bioanalyzer及びRNA 6000 pico kit（Agilent technologies社、以下アジレント社）にて品質確認を行い、すべてのサンプルがマイクロアレイ解析が可能な品質であることを確認した。 (2) Extraction of total RNA from liver biopsy sample Some of the liver biopsy samples collected in Example 1 (1) above were capable of array analysis (98 subjects, including simple fat 16 livers, 82 NASHs, and total RNA was prepared using RNeasy mini kit (Qiagen) for 53 fibrosis scores, 53 stage I, 21 stage II, and 8 stage III, according to NASH did. The prepared total RNA was quantified with a spectrophotometer ND-1000 (NanoDrop Technologies), and 1ng was used to check the quality with 2100 Bioanalyzer and RNA 6000 pico kit (Agilent technologies, hereinafter Agilent). The sample was confirmed to be of a quality that allows microarray analysis.
上述の実施例１（１）で採取された肝生検サンプルの一部から、アレイ解析可能であったもの（対象９８名、うち単純性脂肪肝１６名、ＮＡＳＨ８２名。ＮＡＳＨと判定されたうち線維化のスコア別にはステージＩ ５３名、ステージII ２１名およびステージIII ８名）について、RNeasy mini kit（Qiagen社）を用いて全ＲＮＡを調製した。調製した全ＲＮＡは分光光度計ND-1000（NanoDrop Technologies）にて定量し、その１ｎｇを用いて2100 Bioanalyzer及びRNA 6000 pico kit（Agilent technologies社、以下アジレント社）にて品質確認を行い、すべてのサンプルがマイクロアレイ解析が可能な品質であることを確認した。 (2) Extraction of total RNA from liver biopsy sample Some of the liver biopsy samples collected in Example 1 (1) above were capable of array analysis (98 subjects, including simple fat 16 livers, 82 NASHs, and total RNA was prepared using RNeasy mini kit (Qiagen) for 53 fibrosis scores, 53 stage I, 21 stage II, and 8 stage III, according to NASH did. The prepared total RNA was quantified with a spectrophotometer ND-1000 (NanoDrop Technologies), and 1ng was used to check the quality with 2100 Bioanalyzer and RNA 6000 pico kit (Agilent technologies, hereinafter Agilent). The sample was confirmed to be of a quality that allows microarray analysis.
（３） 網羅的遺伝子発現変動解析
上述の実施例１（２）で作成した全ＲＮＡ ２００ｎｇをQuick Amp Labeling kit（アジレント社）を用いてＣｙ－３ラベル化し、ＤＮＡマイクロアレイ（G4845A Human Whole Genome Ver.2.0、アジレント社）とハイブリダイズさせた。数値化はマイクロアレイスキャナ（G2565BA、アジレント社）および数値化アプリケーション（Feature Extraction、アジレント社）を用いてプロトコールに従って行った。アレイ間の補正にはグローバルノーマライゼーションを行った。その結果、図１に示すとおり、単純性脂肪肝のサンプルにおけるＹＫＬ－４０のｍＲＮＡ発現量の平均値を１．０とすると、ステージＩ ＮＡＳＨのサンプルの平均値は２．６であり、ステージIIでは３．４であった。このことから、ＹＫＬ－４０はＮＡＳＨの肝臓において発現亢進しており、線維化のステージに伴って発現亢進していることが明らかとなった。 (3) Comprehensive geneexpression variation analysis 200 ng of total RNA prepared in Example 1 (2) above was labeled with Cy-3 using Quick Amp Labeling kit (Agilent) and DNA microarray (G4845A Human Whole Genome Ver. 2.0, Agilent). Digitization was performed according to the protocol using a microarray scanner (G2565BA, Agilent) and a digitization application (Feature Extraction, Agilent). Global normalization was performed for correction between arrays. As a result, as shown in FIG. 1, assuming that the average expression level of YKL-40 mRNA in simple fatty liver samples is 1.0, the average value of stage I NASH samples is 2.6, and stage II It was 3.4. This revealed that YKL-40 is upregulated in the liver of NASH and is upregulated with the fibrosis stage.
上述の実施例１（２）で作成した全ＲＮＡ ２００ｎｇをQuick Amp Labeling kit（アジレント社）を用いてＣｙ－３ラベル化し、ＤＮＡマイクロアレイ（G4845A Human Whole Genome Ver.2.0、アジレント社）とハイブリダイズさせた。数値化はマイクロアレイスキャナ（G2565BA、アジレント社）および数値化アプリケーション（Feature Extraction、アジレント社）を用いてプロトコールに従って行った。アレイ間の補正にはグローバルノーマライゼーションを行った。その結果、図１に示すとおり、単純性脂肪肝のサンプルにおけるＹＫＬ－４０のｍＲＮＡ発現量の平均値を１．０とすると、ステージＩ ＮＡＳＨのサンプルの平均値は２．６であり、ステージIIでは３．４であった。このことから、ＹＫＬ－４０はＮＡＳＨの肝臓において発現亢進しており、線維化のステージに伴って発現亢進していることが明らかとなった。 (3) Comprehensive gene
《実施例２》ＮＡＦＬＤおよびＮＡＳＨの血清サンプル中のＹＫＬ－４０量の測定
（１）採血および血清の調製
上述の実施例１（１）で採血されたものを室温下で凝固させ、遠心分離して血清を調製した。このうちＥＬＩＳＡ解析可能であったものは７６名分であり、内訳は単純性脂肪肝２０名分、ＮＡＳＨ５６名分であった。ＮＡＳＨと判定されたもののうち線維化のスコア別にはステージＩ ３５名分、ステージII １６名分およびステージIII ５名分であった。 Example 2 Measurement of YKL-40 in NAFLD and NASH Serum Samples (1) Blood Collection and Serum Preparation The blood collected in Example 1 (1) above was coagulated at room temperature and centrifuged. Serum was prepared. Of these, 76 were able to be analyzed by ELISA, and the breakdown was for 20 simple fatty livers and 56 for NASH. Among those determined as NASH, there were 35 stages I, 16 stages II, and 5 stages III according to the fibrosis score.
（１）採血および血清の調製
上述の実施例１（１）で採血されたものを室温下で凝固させ、遠心分離して血清を調製した。このうちＥＬＩＳＡ解析可能であったものは７６名分であり、内訳は単純性脂肪肝２０名分、ＮＡＳＨ５６名分であった。ＮＡＳＨと判定されたもののうち線維化のスコア別にはステージＩ ３５名分、ステージII １６名分およびステージIII ５名分であった。 Example 2 Measurement of YKL-40 in NAFLD and NASH Serum Samples (1) Blood Collection and Serum Preparation The blood collected in Example 1 (1) above was coagulated at room temperature and centrifuged. Serum was prepared. Of these, 76 were able to be analyzed by ELISA, and the breakdown was for 20 simple fatty livers and 56 for NASH. Among those determined as NASH, there were 35 stages I, 16 stages II, and 5 stages III according to the fibrosis score.
（２）ＹＫＬ－４０量のＥＬＩＳＡによる測定
上述の実施例２（１）で調製した血清サンプル中のＹＫＬ－４０量をＥＬＩＳＡキット（Human Chitinase 3-like 1 Quantikine ELISA Kit、R&D Systems社）を用いて測定した。その結果、図２に示すとおり、血清中ＹＫＬ－４０は単純性脂肪肝では１７．１±１．９ｎｇ／ｍＬ（平均値±標準誤差、以下同様）であったのに対し、ステージＩでは２９．９±４．０ｎｇ／ｍＬ、ステージIIでは３４．８±４．８ｎｇ／ｍＬ、ステージIIIでは１３８．８±４３．３ｎｇ／ｍＬであった。単純性脂肪肝との有意差を統計学的に検定（ウェルチのｔ検定）したところ、ステージＩとはｐ＜０．０１、ステージIIとはｐ＜０．０１、ステージIIIとはｐ＜０．０５であり、共に有意差をもって発現亢進していることが明らかとなった。 (2) Measurement of YKL-40 by ELISA Using the ELISA kit (Human Chitinase 3-like 1 Quantikine ELISA Kit, R & D Systems) for the amount of YKL-40 in the serum sample prepared in Example 2 (1) above. Measured. As a result, as shown in FIG. 2, serum YKL-40 was 17.1 ± 1.9 ng / mL (mean value ± standard error, the same applies hereinafter) in simple fatty liver, whereas it was 29 in stage I. It was 3.9 ± 4.0 ng / mL, 34.8 ± 4.8 ng / mL in Stage II, and 138.8 ± 43.3 ng / mL in Stage III. When statistically tested for significant difference from simple fatty liver (Welch's t test), p <0.01 for stage I, p <0.01 for stage II, and p <0 for stage III. It was revealed that the expression was enhanced with a significant difference.
上述の実施例２（１）で調製した血清サンプル中のＹＫＬ－４０量をＥＬＩＳＡキット（Human Chitinase 3-like 1 Quantikine ELISA Kit、R&D Systems社）を用いて測定した。その結果、図２に示すとおり、血清中ＹＫＬ－４０は単純性脂肪肝では１７．１±１．９ｎｇ／ｍＬ（平均値±標準誤差、以下同様）であったのに対し、ステージＩでは２９．９±４．０ｎｇ／ｍＬ、ステージIIでは３４．８±４．８ｎｇ／ｍＬ、ステージIIIでは１３８．８±４３．３ｎｇ／ｍＬであった。単純性脂肪肝との有意差を統計学的に検定（ウェルチのｔ検定）したところ、ステージＩとはｐ＜０．０１、ステージIIとはｐ＜０．０１、ステージIIIとはｐ＜０．０５であり、共に有意差をもって発現亢進していることが明らかとなった。 (2) Measurement of YKL-40 by ELISA Using the ELISA kit (Human Chitinase 3-like 1 Quantikine ELISA Kit, R & D Systems) for the amount of YKL-40 in the serum sample prepared in Example 2 (1) above. Measured. As a result, as shown in FIG. 2, serum YKL-40 was 17.1 ± 1.9 ng / mL (mean value ± standard error, the same applies hereinafter) in simple fatty liver, whereas it was 29 in stage I. It was 3.9 ± 4.0 ng / mL, 34.8 ± 4.8 ng / mL in Stage II, and 138.8 ± 43.3 ng / mL in Stage III. When statistically tested for significant difference from simple fatty liver (Welch's t test), p <0.01 for stage I, p <0.01 for stage II, and p <0 for stage III. It was revealed that the expression was enhanced with a significant difference.
（３）肝臓中のＹＫＬ－４０ ｍＲＮＡ発現量と血中ＹＫＬ－４０量の相関解析
上述の実施例１（３）および実施例２（２）で得られたデータの相関解析を行ったところ、図３に示すとおりであり、相関係数はｒ＝０．８７と計算された。このことから、血中ＹＫＬ－４０量の亢進は肝臓でのｍＲＮＡレベルでの発現亢進に由来すると考えられ、ＮＡＳＨの状態を反映していることが明らかになった。従って、血中ＹＫＬ－４０量を測定することでＮＡＳＨを鑑別することができる。 (3) Correlation analysis between the expression level of YKL-40 mRNA in the liver and the amount of YKL-40 in the blood The correlation analysis of the data obtained in Example 1 (3) and Example 2 (2) described above was performed. As shown in FIG. 3, the correlation coefficient was calculated as r = 0.87. From this, it was considered that the increase in the amount of YKL-40 in the blood was derived from the increased expression at the mRNA level in the liver and reflected the state of NASH. Therefore, NASH can be identified by measuring the blood YKL-40 level.
上述の実施例１（３）および実施例２（２）で得られたデータの相関解析を行ったところ、図３に示すとおりであり、相関係数はｒ＝０．８７と計算された。このことから、血中ＹＫＬ－４０量の亢進は肝臓でのｍＲＮＡレベルでの発現亢進に由来すると考えられ、ＮＡＳＨの状態を反映していることが明らかになった。従って、血中ＹＫＬ－４０量を測定することでＮＡＳＨを鑑別することができる。 (3) Correlation analysis between the expression level of YKL-40 mRNA in the liver and the amount of YKL-40 in the blood The correlation analysis of the data obtained in Example 1 (3) and Example 2 (2) described above was performed. As shown in FIG. 3, the correlation coefficient was calculated as r = 0.87. From this, it was considered that the increase in the amount of YKL-40 in the blood was derived from the increased expression at the mRNA level in the liver and reflected the state of NASH. Therefore, NASH can be identified by measuring the blood YKL-40 level.
《実施例３》ＮＡＦＬＤおよびＮＡＳＨの血清サンプル中のＹＫＬ－４０量を用いたＲＯＣ解析
ＲＯＣ解析により単純性脂肪肝とＮＡＳＨとの判別に際しては、ＡＵＣは０．７４と計算された。このとき最大効率を示すカットオフ値は２１．８ｎｇ／ｍＬと計算された。このカットオフ値を用いて今回のサンプル群を診断すると感度６７％、特異性８０％と計算された（図４）。
また、単純性脂肪肝と初期のＮＡＳＨであるステージＩとの判別に際しても同様に計算すると、ＡＵＣは０．６９、最大効率を示すカットオフ値は２１．８ｎｇ／ｍＬと計算された。このカットオフ値を用いて今回のサンプル群を診断すると感度６３％、特異性８０％と計算された（図５）。 Example 3 ROC Analysis Using YKL-40 Amount in Serum Samples of NAFLD and NASH When discriminating between simple fatty liver and NASH by ROC analysis, AUC was calculated to be 0.74. At this time, the cutoff value indicating the maximum efficiency was calculated to be 21.8 ng / mL. When this sample group was diagnosed using this cut-off value, the sensitivity was calculated to be 67% and the specificity was 80% (FIG. 4).
Further, when the same calculation was made for discrimination between simple fatty liver and stage I as the initial NASH, the AUC was calculated to be 0.69, and the cutoff value indicating the maximum efficiency was calculated to be 21.8 ng / mL. When this sample group was diagnosed using this cutoff value, the sensitivity was calculated to be 63% and the specificity was 80% (FIG. 5).
ＲＯＣ解析により単純性脂肪肝とＮＡＳＨとの判別に際しては、ＡＵＣは０．７４と計算された。このとき最大効率を示すカットオフ値は２１．８ｎｇ／ｍＬと計算された。このカットオフ値を用いて今回のサンプル群を診断すると感度６７％、特異性８０％と計算された（図４）。
また、単純性脂肪肝と初期のＮＡＳＨであるステージＩとの判別に際しても同様に計算すると、ＡＵＣは０．６９、最大効率を示すカットオフ値は２１．８ｎｇ／ｍＬと計算された。このカットオフ値を用いて今回のサンプル群を診断すると感度６３％、特異性８０％と計算された（図５）。 Example 3 ROC Analysis Using YKL-40 Amount in Serum Samples of NAFLD and NASH When discriminating between simple fatty liver and NASH by ROC analysis, AUC was calculated to be 0.74. At this time, the cutoff value indicating the maximum efficiency was calculated to be 21.8 ng / mL. When this sample group was diagnosed using this cut-off value, the sensitivity was calculated to be 67% and the specificity was 80% (FIG. 4).
Further, when the same calculation was made for discrimination between simple fatty liver and stage I as the initial NASH, the AUC was calculated to be 0.69, and the cutoff value indicating the maximum efficiency was calculated to be 21.8 ng / mL. When this sample group was diagnosed using this cutoff value, the sensitivity was calculated to be 63% and the specificity was 80% (FIG. 5).
従来の方法では単純性脂肪肝とＮＡＳＨのステージＩを簡便に鑑別することが難しく、また、客観的にその診断を支持するような臨床指示物質についてはこれまで明らかではなかった。ＮＡＳＨは肝生検され病理組織学的によって確定診断されるが、本発明者らは、単純性脂肪肝に比して線維化が惹起されているＮＡＳＨにおいて血中ＹＫＬ－４０量が亢進することを見出し、血中ＹＫＬ－４０量が単独の指標としてＮＡＳＨ診断に有用であることを明らかにした。
本発明のＹＫＬ－４０血中濃度測定により、単純性脂肪肝とＮＡＳＨ、さらには線維化の程度が初期のＮＡＳＨを鑑別できることを見出した。 In conventional methods, it is difficult to easily distinguish between simple fatty liver and NASH stage I, and clinical indicators that support the diagnosis objectively have not been clarified so far. Although NASH is biopsied in the liver and is diagnosed by histopathology, the present inventors show that the amount of YKL-40 in blood is increased in NASH in which fibrosis is induced compared to simple fatty liver. The amount of YKL-40 in blood was found to be useful for NASH diagnosis as a single indicator.
By measuring the blood concentration of YKL-40 of the present invention, it was found that simple fatty liver and NASH, and further, NASH with an initial fibrosis degree can be distinguished.
本発明のＹＫＬ－４０血中濃度測定により、単純性脂肪肝とＮＡＳＨ、さらには線維化の程度が初期のＮＡＳＨを鑑別できることを見出した。 In conventional methods, it is difficult to easily distinguish between simple fatty liver and NASH stage I, and clinical indicators that support the diagnosis objectively have not been clarified so far. Although NASH is biopsied in the liver and is diagnosed by histopathology, the present inventors show that the amount of YKL-40 in blood is increased in NASH in which fibrosis is induced compared to simple fatty liver. The amount of YKL-40 in blood was found to be useful for NASH diagnosis as a single indicator.
By measuring the blood concentration of YKL-40 of the present invention, it was found that simple fatty liver and NASH, and further, NASH with an initial fibrosis degree can be distinguished.
本発明は、ＮＡＦＬＤ患者において、単純性脂肪肝とＮＡＳＨとの鑑別、更には、単純性脂肪肝と初期のＮＡＳＨとの鑑別に使用することができる。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。 INDUSTRIAL APPLICABILITY The present invention can be used in NAFLD patients to distinguish between simple fatty liver and NASH, and further to distinguish between simple fatty liver and early NASH.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。 INDUSTRIAL APPLICABILITY The present invention can be used in NAFLD patients to distinguish between simple fatty liver and NASH, and further to distinguish between simple fatty liver and early NASH.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.
Claims (18)
- ＹＫＬ－４０を含むことを特徴とする、患者が罹患している疾患が単純性脂肪肝であるか、又は非アルコール性脂肪肝炎であるかを判別することができる鑑別マーカー。 A differential marker capable of discriminating whether a disease affecting a patient is simple fatty liver or non-alcoholic steatohepatitis, characterized by comprising YKL-40.
- 被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎の検出方法。 A method for detecting nonalcoholic steatohepatitis, which comprises measuring the concentration of YKL-40 in a test sample.
- 被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、単純性脂肪肝と非アルコール性脂肪肝炎とを判別する方法。 A method for discriminating between simple fatty liver and non-alcoholic steatohepatitis, characterized by measuring the concentration of YKL-40 in a test sample.
- 被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎に対する治療効果の確認方法。 A method for confirming the therapeutic effect on nonalcoholic steatohepatitis, comprising measuring the concentration of YKL-40 in a test sample.
- 非アルコール性脂肪肝炎への罹患が疑われる患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎の検出方法。 A method for detecting nonalcoholic steatohepatitis, comprising a step of collecting a test sample from a patient suspected of suffering from nonalcoholic steatohepatitis, and a step of measuring the concentration of YKL-40 in the test sample.
- 前記患者が非アルコール性脂肪性肝疾患患者である、請求項５に記載の方法。 The method according to claim 5, wherein the patient is a non-alcoholic fatty liver disease patient.
- 患者が罹患している疾患が単純性脂肪肝であるか、又は非アルコール性脂肪肝炎であるかを判別する方法であって、該患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、方法。 A method for discriminating whether a disease affecting a patient is simple fatty liver or non-alcoholic steatohepatitis, the step of collecting a test sample from the patient, and in the test sample Measuring the concentration of YKL-40.
- 前記患者が非アルコール性脂肪性肝疾患患者である、請求項７に記載の方法。 The method according to claim 7, wherein the patient is a non-alcoholic fatty liver disease patient.
- 非アルコール性脂肪肝炎の治療を受けた患者から被検試料を採取する工程、及び該被検試料中のＹＫＬ－４０の濃度を測定する工程を含む、非アルコール性脂肪肝炎に対する治療効果の確認方法。 A method for confirming the therapeutic effect on nonalcoholic steatohepatitis, comprising the steps of collecting a test sample from a patient who has been treated for nonalcoholic steatohepatitis, and measuring the concentration of YKL-40 in the test sample .
- 被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎の体外（in vitro or ex vivo）診断方法。 A method for in vitro diagnosis of non-alcoholic steatohepatitis characterized by measuring the concentration of YKL-40 in a test sample.
- 被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、単純性脂肪肝と非アルコール性脂肪肝炎との体外（in vitro or ex vivo）判別方法。 A method for discriminating between simple fatty liver and non-alcoholic steatohepatitis (in vitro) or non-alcoholic steatohepatitis, characterized by measuring the concentration of YKL-40 in a test sample.
- 被検試料中のＹＫＬ－４０の濃度を測定することを特徴とする、非アルコール性脂肪肝炎に対する治療効果の体外（in vitro or ex vivo）確認方法。 A method for confirming the therapeutic effect of non-alcoholic steatohepatitis in vitro (in vitro, or ex vivo), comprising measuring the concentration of YKL-40 in a test sample.
- 抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、非アルコール性脂肪肝炎の診断キット。 A non-alcoholic steatohepatitis diagnostic kit comprising an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA, and an instruction manual describing the relationship between YKL-40 and nonalcoholic steatohepatitis.
- 抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、単純性脂肪肝と非アルコール性脂肪肝炎との判別用キット。 Simple fatty liver and nonalcoholic steatohepatitis, including an anti-YKL-40 antibody, a probe or primer for measuring YKL-40 mRNA, and an instruction manual describing the relationship between YKL-40 and nonalcoholic steatohepatitis The kit for discrimination.
- 抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマー、及びＹＫＬ－４０と非アルコール性脂肪肝炎との関連性を記載した取扱説明書を含む、非アルコール性脂肪肝炎に対する治療効果の確認用キット。 Confirmation of therapeutic effect on non-alcoholic steatohepatitis, including anti-YKL-40 antibody, probe or primer for measuring YKL-40 mRNA, and instruction manual describing the relationship between YKL-40 and non-alcoholic steatohepatitis For kit.
- 抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、非アルコール性脂肪肝炎の診断薬の製造における使用。 Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the production of a diagnostic agent for nonalcoholic steatohepatitis.
- 抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、単純性脂肪肝と非アルコール性脂肪肝炎の判別薬の製造における使用。 Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the production of a discrimination agent for simple fatty liver and nonalcoholic steatohepatitis.
- 抗ＹＫＬ－４０抗体、又はＹＫＬ－４０ｍＲＮＡの測定用プローブ若しくはプライマーの、非アルコール性脂肪肝炎の治療効果の確認薬の製造における使用。 Use of an anti-YKL-40 antibody or a probe or primer for measuring YKL-40 mRNA in the manufacture of a drug for confirming the therapeutic effect of nonalcoholic steatohepatitis.
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