JPH0972901A - Evaluation method of liver disease group - Google Patents

Abstract

PROBLEM TO BE SOLVED: To grasp a patient having high possibility of deteriorating liver disease in early stage by predicting a patient having high possibility of transition from chronic liver disease to cirrhosis and primary carcinoma of liver and taking countermeasures for suppressing deterioration of liver disease. SOLUTION: In the method for evaluating liver disease (e.g. determination of possibility of deteriorating liver disease), concentration of soluble Fas antigen in the blood is measured. The liver diseases include chronic hepatitis, cirrhosis and primary carcinoma.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to chronic hepatitis, cirrhosis,
The present invention relates to a method for detecting liver disease such as primary liver cancer. The present invention also relates to a detection method which is an index of the condition of liver disease when continuously measured. It also relates to a detection method for estimating the possibility of transition from chronic hepatitis to cirrhosis and primary liver cancer.

[0002]

2. Description of the Related Art In recent years, a mechanism of a suicide phenomenon of apoptosis called apoptosis has been clarified, and a protein called Fas antigen has been attracting attention in this mechanism. Fas antigen is expressed in lymphoid cells such as thymocytes and activated T cells, heart, liver, kidney and the like, and it is said that when the Fas antigen is presented on the cell surface, apoptosis occurs.

Recently, Fa has been reported in some hepatocyte deaths such as fulminant hepatitis.
Although the possibility that s-antigen is involved has been suggested in animal experiments, it is speculated that apoptosis is involved in hepatocyte death due to liver disease, and therefore Fa in liver cells was used as a test for detecting liver disease.
It is considered to measure s antigen.

On the other hand, in addition to the Fas antigen incorporated in the above cell membrane, a soluble one was discovered in 1994 (J. Ch.
eng et al. (1994) SCIENCE vol.263 p1759), soluble-Fas
Antigen (herein referred to as "soluble Fas antigen").

At present, a relationship between the serum concentration of this soluble Fas antigen and the activity of autoimmune diseases, particularly systemic lupus erythematosus, has been suggested. Further, a mechanism of inhibiting soluble Fas antigen has been speculated, and it is considered that the soluble Fas antigen has a completely different role from the membrane-bound Fas antigen in vivo. Therefore, there is no report that it is associated with liver disease.

By the way, liver diseases such as chronic hepatitis, cirrhosis and primary liver cancer are very serious diseases in which hepatocytes are destroyed. Chronic hepatitis is mainly caused by viral infections,
It is well known that heavy drinking of alcohol also helps.
It is known that about 25% of patients with chronic hepatitis shift to cirrhosis, and about 70% of them shift to primary liver cancer.

At present, there are many liver disease tests such as chronic hepatitis, cirrhosis and primary liver cancer, and biochemical liver function tests are roughly classified into metabolic tests of bile, pigment excretion, enzymes, plasma proteins and lipids. As a specific test for various liver diseases, virus-related tests, type IV collagen, PIIIP, proline hydroxylase, fibrosis markers such as hyaluronic acid, and AFP and P
Cancer markers such as IVKAII are mentioned. More direct methods include ultrasound echo, liver biopsy, and laparoscopic examination. Then, these tests are combined to diagnose liver disease. These diagnostic methods screen patients with the possibility of various liver diseases, chronic hepatitis to cirrhosis,
It is often used to monitor when liver disease worsens to primary liver cancer.

[0008] However, although there are many tests for liver diseases as described above, there is no detection test for predicting the possibility of worsening liver diseases.

[0009]

Therefore, in the present invention, in order to prevent the deterioration of liver disease by anticipating patients with a high possibility of transition from chronic liver disease to cirrhosis, primary liver cancer, and taking measures, The goal is to identify patients with a high possibility of worsening liver disease at an early stage.

[0010]

Means for Solving the Problems As a result of studies to solve the above problems, the present inventor has found that soluble Fa in serum is
The inventors have found a relationship between s-antigen concentration and liver disease, and completed the present invention. That is, the present invention is a method for evaluating a liver disease group characterized by measuring the concentration of soluble Fas antigen in blood. The liver disease group includes chronic hepatitis, liver cirrhosis and primary liver cancer.

The above-mentioned evaluation can be carried out by comparing the concentration of soluble Fas antigen in the blood of the subject to be evaluated with the concentration of soluble Fas antigen in the blood collected for patients with liver disease and / or normal subjects. it can.

[0012] The above-mentioned evaluation can be carried out as a determination of a morbidity risk rate of liver disease or a determination of a risk rate of progression of liver disease. As shown in Examples below, when the concentration of soluble Fas antigen in serum was measured for liver disease patients, 25% in chronic hepatitis, 33% in liver cirrhosis, and 50% in patients with primary liver cancer showed significantly higher levels of soluble Fas antigen. The blood level was high. Therefore, by measuring the soluble Fas antigen in blood, it is possible to determine the risk of developing liver disease. It is also possible to determine the risk of progression of liver disease. Progression of liver disease includes transition from chronic hepatitis to cirrhosis, chronic hepatitis to primary liver cancer, and liver cirrhosis to primary liver cancer.

[0013]

Next, an embodiment of the present invention will be described. The method of the present invention is carried out by measuring the concentration of soluble Fas antigen in blood. As a sample, serum,
Plasma, whole blood, filter paper blood, or saliva is used, but a fraction containing a soluble substance may be used.

The method for measuring the concentration of soluble Fas antigen is as follows:
It is not particularly limited, and may be measured as an absolute value or a relative value with respect to a standard substance. Specifically, soluble Fas
RIA (radioimmunoassay) using antibody against antigen, E
IA (enzyme immunoassay), FIA (trend immunoassay), CL
The usual immunoassay methods such as IA (chemiluminescent immunoassay), CLEIA (chemiluminescent enzyme immunoassay) or immunoagglutination method can be mentioned.

The above-mentioned antibody can be obtained as an antiserum from these animals by immunizing animals such as mice, rats, rabbits, goats, guinea pigs and sheep with the soluble Fas antigen. The obtained serum may be further purified to immunoglobulin or IgG, and further may be affinity-purified using soluble Fas or the like. The soluble Fas antigen used for immunization may be purified from human serum or the like, but can be obtained from Escherichia coli or cultured cells by using a gene recombination technique, or a synthetic peptide containing a part or all of the amino acid sequence can be used Is.

Alternatively, a monoclonal antibody may be prepared as the antibody. For example, spleen cells of a mouse immunized with soluble Fas are fused with myeloma cells to prepare a hybridoma, and the obtained hybridoma contains soluble Fas.
When a stock company that produces an antibody that binds to the s antigen is selected and the obtained strain is cultured in a suitable medium, a monoclonal antibody against the soluble Fas antigen is produced in the culture solution.
When the sandwich method is used as the immunoassay method, a monoclonal antibody and a polyclonal antibody are used in combination, or two types of monoclonal antibodies that recognize different epitopes are prepared. As such an antibody, an anti-Fas antibody commercially available from Institute of Medical Biology can also be used.

The data obtained by measuring the soluble Fas antigen concentration in the above-described patients with liver disease and normal subjects, and the soluble Fas antigen concentration in the blood of the subject to be evaluated are shown below. Compare and evaluate.

[0018]

[Examples] As subjects, sera of 12 cases of chronic hepatitis, 15 cases of cirrhosis, 34 cases of primary liver cancer, and 40 cases of healthy subjects were used.

The soluble Fas antigen in serum was measured by a sandwich ELISA method using two kinds of monoclonal antibodies against different epitopes of the extracellular domain of Fas antigen. Soluble Fas antigen concentration is
C into which an expression vector incorporating s antigen cDNA was introduced
It was determined by preparing a standard straight line of soluble Fas antigen from the culture supernatant of OS-7 cells.

[0020]

[Measurement Results] The results are shown in Table 1. Mann-W
In hitney's U-test, no significant difference was observed between the liver disease groups, but a significant difference (p <0.001) was observed between the healthy subjects and each liver disease. Next, when the positive rate of the soluble antigen was determined, the cases where the average was ± 3 SD (= 0.97 ng / ml) in healthy subjects were found in Table 2.
It was as follows. From these results, the positive rate of soluble Fas antigen was particularly high in primary liver cancer.

[0021]

[Table 1]

[0022]

[Table 2]

[0023]

As described above, in chronic hepatitis, liver cirrhosis, and primary liver cancer, soluble Fas antigen in blood was significantly increased from that in healthy subjects, and in particular, 50% of primary liver cancers were positive for soluble Fas antigen. Elevated levels of soluble Fas antigen in blood have been reported in leukemia patients, and tumor cells and mutant cells are
It is considered that the soluble Fas antigen is functioning as one of the mechanisms for evading the immunological surveillance mechanism of as ligand-positive cells. Therefore, the measurement of soluble Fas antigen in blood in various liver diseases is useful for the early diagnosis of primary liver cancer or the detection of the risk group of liver disease progression, and the significance of the measurement is clinically important.

Claims (3)

[Claims] 1. A method for evaluating a liver disease group, which comprises measuring the concentration of soluble Fas antigen in blood. 2. The method according to claim 1, wherein the liver disease group is chronic hepatitis, liver cirrhosis, or primary liver cancer. 3. The method according to claim 1, wherein the evaluation is determination of the risk of progression of liver disease.