KR100566337B1 - A kit for diagnosing liver fibrosis through identifying an upregulated gene expression of NOV - Google Patents

Abstract

The present invention is a kit for diagnosing hepatic fibrosis by detecting the expression of NOV gene (neoblastoma overexpressed gene) that is upregulated in activated hepatic stellate cells inducing hepatic fibrosis and liver fibrosis using NOV gene. It relates to a method for screening a therapeutic agent.

Liver fibrosis diagnostic kit according to the present invention is useful for preventing liver cirrhosis and liver cancer by diagnosing liver fibrosis, which is a stage before progression to cirrhosis.

Liver fibrosis, hepatic stellate cells (HSC), NOV

Description

A kit for diagnosing liver fibrosis through identifying an upregulated gene expression of NOV}             

1 is a diagram confirming the Northern blot analysis that NOV mRNA expression is upregulated in proportion to the culture period of hepatic stellate cells.

Figure 2 is a diagram confirming the expression of NOV mRNA in the liver treated CCl ₄ , and also through the RT-PCR method that the expression of NOV mRNA increases over time.

3 shows that the expression of NOV is increased in liver tissues of hepatitis B virus-induced liver cirrhosis, alcoholic cirrhosis, biliary atresia, and primary biliary cirrhosis compared to normal liver tissue. The figure confirmed through the PCR method.

4A is a diagram confirming that the expression of NOV mRNA increases with time when the dexamethasone is treated to hepatic stellate cells by RT-PCR method. Figure 4b is confirmed by Western blot analysis that the increase in the secretion of NOV protein in the cell culture when treated with dexamethasone to hepatic stellate cells.

Figure 5a is a diagram confirming the increase in the expression of NOV mRNA when treated with hepatic stellate TGF-β by RT-PCR method. Figure 5b is confirmed by Western blot analysis that the increase in the secretion of NOV protein in the cell culture when treated with hepatic stellate TGF-β.

FIG. 6 shows that NOV mRNA expression increases when hepatic stellate cells are treated with bile acids such as cholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The figure confirmed by the RT-PCR method.

7 shows the NCBI database search for sequences obtained from clones estimated to be NOV genes among sequences with increased gene transcription in activated hepatic stellate cells.

The present invention relates to a kit for diagnosing liver fibrosis by detecting upregulated expression of the NOV gene.

Chronic liver disease develops from liver or cirrhosis to liver cirrhosis in cases of chronic hepatitis after viral or alcoholic hepatitis, and some of them develop hepatocellular carcinoma. When hepatitis is damaged by hepatitis, hepatic fibrosis due to tissue reaction during wound regeneration after necrosis of hepatocytes eventually develops into cirrhosis. Hepatic stellate cells play the most important role in liver fibrosis among the four cells that make up the liver: hepatocytes, sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells (HSC). Known.

 Hepatic fibrosis is detected by Kupffer cells when the hepatocytes are damaged and secrete several cytokines, which proliferate and activate hepatic stellate cells (J. Gastroenterol. Hepatol., 14: 618-). 33, 1999). Activated hepatic stellate cells synthesize collagen and accumulate in extracellular matrix, and liver fibrosis is achieved by collagen accumulated continuously in extracellular matrix.

Hepatic stellate cells make up about 15% of all liver cells and are the main cells that store the retinoids, a precursor of vitamin A. After hepatic damage, hepatic stellate cells are activated, and these cells differentiate into myofibroblasts with proliferative and fibroblast-producing ability, whereby collagen is secreted and fibrosis proceeds.

Hepatic stellate cells also increase the production of macroglobulin (macroglobulin) and TIMP (Tissue inhibitor of metalloproteinase), which are inhibitors of collagen degrading enzymes, and thus play a central role in liver fibrosis. In addition, the damaged basement membrane is initially replaced with collagen and fibronectin, and thus, the exchange of substance between isovascular cells and hepatocytes is not facilitated, thereby degrading hepatocyte function.

Acetaldehyde, ascorbic acid, transforming growth factor (TGF-β), and carbon tetrachloride have been reported to activate hepatic stellate cells and stimulate collagen genes. In addition, changes in extracellular matrix, ie, destruction of normal extracellular matrix and collagen and Increased fibronectin and oxidative stress caused by iron and alcohol are important factors for hepatic stellate cell activation.

In general, hepatic fibrosis, unlike cirrhosis, is reversible, consists of thin fibres, is known to have nodule formation, and normal recovery may be possible when the cause of liver damage is lost. Repeated hepatic fibrosis processes increase crosslinking between the extracellular matrix, forming thick fibrils and progressing to irreversible cirrhosis with nodules.

Therefore, in order to enable normal recovery by diagnosing liver fibrosis before progressing to cirrhosis, a diagnostic method has been developed to easily recognize early intrahepatic fibrosis. Currently commercially available tests for this purpose include immunologically active subunits of procollagen type III peptide (P-III-P), TIMP, and prolyl hydroxylase. ), 7S domains of type IV collagen, triple helix domains, and laminin. However, when comparing these tests with liver biopsy results, it is recognized that there is a correlation between hepatic fibrosis and these tests, but the clinical use is not universalized because its sensitivity and specificity are not satisfactory yet. .

Therefore, the present inventors have conducted a lot of research to find a new method for detecting activated hepatic stellate cells, and as a result, the expression of nephroblastoma overexpressed gene (NOV gene) in activated hepatic stellate cells is reduced by mRNA and protein levels. The present invention was completed by confirming that all of the upregulated and confirming the diagnosis of liver fibrosis through the upregulation of the NOV gene expression.

 It is an object of the present invention to provide a kit for diagnosing hepatic fibrosis through detection of upregulated expression of NOV gene.

Still another object of the present invention is to provide a method for screening a substance for inhibiting upregulated expression of NOV gene of hepatic stellate cells.

In order to achieve the above object, the present invention provides a nucleic acid sequence that hybridizes with mRNA transcribed from NOV gene; b) a fragment of said NOV gene; c) a peptide expressed by said NOV gene or fragment of NOV gene; And d) a kit for diagnosing hepatic fibrosis through detection of upregulated expression of the NOV gene in hepatic stellate cells, characterized in that it comprises any one selected from the group consisting of antibodies specifically binding to the peptide. to provide.

In the present invention, the NOV gene may be characterized in that the registration number AY082381 in the NCBI GenBank.

The present invention also comprises the steps of: a) providing a hepatic stellate cell line upregulated NOV gene; b) culturing the cells and contacting them with any chemicals; And c) selecting a substance that inhibits upregulated expression of the NOV gene.

In the present invention, the expressions "upregulated", "upregulated" and "upregulation" refer to specific genes by intracellular transcription or gene translation in activated cells as compared to normal tissue cells. This means that the expression level of the mRNA or the expression of the protein is significantly increased.

In the present invention, the expression NOV gene fragment is defined as encompassing a probe capable of detecting the NOV gene and a primer derived from the NOV gene.

In the present invention, the gene expression patterns of activated hepatic stellate cells (HSC) and stationary hepatic stellate cells were compared using the mass sequencing method of the cDNA library. Newly regulated, it was confirmed that both mRNA and protein expression of the NOV gene is upregulated in cultured activated hepatic stellate cells.

On the other hand, mRNA, DNA fragments, peptides, antibodies, etc. used to detect the upregulated expression of the NOV gene is prepared using conventional methods in the art based on the gene sequence of No. AY082381 registered to NCBI GenBank And detectable up-regulated expression at the mRNA or protein level of the NOV gene using the same or commonly known in the art.

For example, detection of NOV genes using mRNA or DNA fragments may be performed by attaching radioisotopes or fluorescent substances to the DNA fragments used as the probes in a conventional manner, and then separating them from hepatic stellate cells. The expression of NOV gene can be measured by using radiographs, fluorescence microscopy, or the like. At this time, the sequence of the probes need not be particularly limited as long as it is a sequence capable of binding to NOV mRNA, the probe sequence with high binding efficiency with NOV mRNA can be easily selected through repeated experiments.

Detection of upregulated expression of the NOV gene using a PCR primer can be performed by reverse transcriptase PCR (RT-PCR) method, using mRNA isolated from hepatic stellate cells as a template. At this time, the selection of the PCR primer is not particularly limited as long as it can specifically amplify only the NOV gene, the sequence can be easily selected by conventional methods in the art.

In addition, peptide sequences, recombinant fusion proteins, and fusion proteins expressed from all or part of the NOV gene sequence can be used to prepare polyclonal and monoclonal antibodies for detecting NOV at the protein level. Polyclonal and monoclonal antibodies can be prepared using antigen-antibody reactions such as enzyme immunoassay (ELISA), Western blot, immunohistochemistry, etc., to detect NOV proteins isolated from hepatic stellate cells. It can be used for known conventional methods of detecting proteins.

The present invention has been shown to increase the expression level of the NOV gene in patients with advanced liver fibrosis or cirrhosis of the cirrhosis, these results are a target gene for the development or screening of therapeutic agents for liver fibrosis and cirrhosis, NOV characterized by the present invention Genes can be used. That is, according to the present invention, a screening method for treating liver fibrosis or cirrhosis is treated with hepatic stellate cells or liver tissues isolated from patients with liver fibrosis or cirrhosis, and the amount of NOV gene expressed in these tissues is expressed in normal tissues. By comparing with the amount of expression, a therapeutic agent for liver fibrosis or cirrhosis can be screened. Such screening methods are commonly known in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Reference Example 1 Isolation and Culture of Hepatic Stellate Cells

The method described in the prior art (Lee et al., BBRC, 303: 954-61, 2003) and the method of another prior art (Iwamoto H., J Hepatol, 32: 762-70, 2000), 450- Rat hepatic stellate cells were isolated from pathogen-free male Sprague-Dawley rats weighing 500 g. Buoyants containing hepatic stellate cells were obtained by centrifugation using a bilayer (17%, 11.5%) metrizamide solution (Sigma, St. Louis, USA). After centrifugation at 1,700 g for 15 minutes, hepatic stellate cells are collected from the upper layer, and then for 3 hours in a WME (Williams Medium E (Sigma)) culture medium in plastic containing 10% Fat bovine serum (FBS). Incubated. The cultures were then washed with phosphate-buffered solution (PBS) to remove dead cells and cell debris.

In the following, attached cells were named quiescent hepatic stellate cells, and attached cells cultured for 14 days were named activated hepatic stellate cells in vitro.

Reference Example 2 Fiber Formation Model Experiment

Liver fibrosis was induced in 200-250 g of sterile male Sprague-Dawley rats. The mice were cared for according to the guidelines of the National Institute of Health (NIH). Carbon tetrachloride (CCl ₄ ) was induced by cirrhosis by treatment with the dose regimen mentioned in the prior art (Lee et al., Arch. Pharm. Res., 23: 613-9, 2000).

Human liver tissue was provided from Yonsei University Hospital and tested. Normal tissue, hepatic cirrhosis caused by hepatitis B virus, alcoholic cirrhosis, biliary atresia, and primary biliary cirrhosis were examined.

Example 1 Library Preparation and Sequencing

Using the methods described in the prior art (Okubo, K. et al., DNA seq., 2: 137-44, 1991 and Lee et al., Arch. Pharm. Res., 23: 613-9, 2000), CDNA libraries were constructed from stationary hepatic stellate cells and in vitro activated hepatic stellate cells, followed by transformation to E. coli. Sequences with increased gene transcription in in vitro activated hepatic stellate cells compared to stationary hepatic stellate cells were selected, and the finally obtained sequences were compared with each gene sequence by NCBI database search.

As a result, these expression patterns included upregulation of collagen 1 (I), collagen 1 (III), fibronectin, thrombospondin 1, and B-crystallin genes, and NOV was activated. Also newly revealed (see FIG. 7).

Example 2 Northern Blot Analysis

Total RNA of hepatic stellate cells was isolated using an RNA isolation kit (Rneasy Mini Kit; Qiagen, Valencia, Calif.), And quantified by measuring absorbance at 260 nm using a UV spectrometer. Electrophoresis was performed on 1 g of agarose gel containing 5.4% formaldehyde for 20 g of total RNA, followed by transfer to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech, Uppsala, Sweden) and UV irradiation. Fixed.

The immobilized RNA was hybridized with probes of [32P] -cDNA-specific NOV and GAPDH produced by random primers. Pretreatment hybridization and hybrid formation were performed according to the methods described in the prior art (Vitca, GD et al., BioTechniques, 8: 370-1, 1990). Reactive bands were detected by radiographic exposure to X-ray film (Kodak XAR5; Kodak, Rochester, NY). The results are shown in FIG. As shown in Figure 1, the major band at 2.5 kb size, the minor band appeared at 2.0kb size. These results indicate that the expression of NOV mRNA increases during the process of hepatic stellate cell activation.

Example 3 Preparation of NOV Antibody

To obtain anti-NOV antibodies, rabbits were immunized with a peptide consisting of 21 amino acids consisting of amino acids 334-354 of the amino acid sequence of the NOV protein. Serum immunoglobulins were purified using IgG purification kit (Pierce). Experiments with the produced antibodies are shown in Figures 4 and 5. In the group treated with dexamethasone and TGF-β, the secretion into the culture was confirmed by Western blot analysis using the NOV antibody.

Example 4 Regulation of NOV Expression by Dexamethasone, TGF-β, and Bile Acids

Hepatic stellate cells are grown in WME containing 10% FBS for 3-4 days, followed by 24 hours in WME containing 0.01% FBS. The control group does not add dexamethasone, and the experimental group adds dexamethasone to 10 M, and then, after 1, 6, 10, and 12 hours, the cells are collected and the total RNA is collected, and the cultures are collected and used to confirm that the NOV protein is secreted into the culture. As shown in Figure 4, the dexamethasone treated group was found to increase the expression of mRNA and protein of NOV for 12 hours.

The control group did not add TGF-β, and the experimental group added TGF-β to a concentration of 10 ng / ml, and after 1, 4, 8, and 12 hours, cells were collected and total RNA was collected, and the culture solution was collected to collect NOV protein into the culture medium. Used to confirm secretion. As shown in FIG. 5, the expression of TGF-β was treated at 4 hours and the expression gradually decreased until 12 hours.

Hepatic stellate cells were cultured in WME containing 10% FBS for 3-4 days, followed by 24 hours in WME containing 0.4% FBS. The control group did not add bile acids, and the experimental group added 50 M of cholic acid, chenodeoxycholic acid, and ursodeoxycholic acid to 50 M, respectively, and then 30, 60, and 90 Minutes later, the cells are harvested to extract the total RNA. As a result, the expression of NOV increased time-dependently when bile acid was added as shown in FIG. 6.

Example 5 Western Blot Analysis

Western blot analysis was performed to confirm whether NOV protein was secreted into the culture solution collected in Example 4. The collected culture solution was centrifuged at 2,000 g for 5 minutes, the supernatant was removed, molecular weight of 10 kD or less in the supernatant was removed, and concentrated using centricon (Chemicon) to obtain only 10 kD or more. 15 g of protein sample were loaded on each lane into a standard 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membrane using a transblot apparatus. The obtained nitrocellulose membrane was room temperature with a monoclonal antibody against smooth muscle-actin (diluted with Sigma: 1 / 1,000) or an antibody against rabbit anti-NOV prepared in Example 3 above (diluted with 1/250) as the primary antibody. Incubated for 2 hours at. The nitrocellulose membrane was washed and then reacted with horse radish peroxidase-bound anti-rabbit immunoglobulin antibody (Sigma; diluted to 1/500). After the nitrocellulose membrane was washed, a band was obtained by exposing it to an X-ray film using an ECL kit (Amersham), which is a chemical fluorescence detection kit.

Example 6 Reverse Transcription Polymerase Chain Polymerization (RT-PCR)

The experiment was carried out with the whole RNA isolated from the liver of the normal rat, the liver of the rat treated with carbon tetrachloride (CCl ₄ ) for 6 weeks, and the total RNA obtained from Example 4. Reverse transcription was performed using an oligo dT (15-18mer) primer and 5 g of each isolated RNA as a template.

Primers used to detect rat NOV were forward with the sequence of 5-ctg aga tga gac cct gcg ac-3 (SEQ ID NO: 1) corresponding to nucleotide positions 235-254 of the nucleotide sequence set forth in NCBI Accession No. NM030868. A reverse primer having a forward primer and a sequence of 5-gtg caa ttc ttg aac tgt agg tgg-3 (SEQ ID NO: 2) corresponding to nucleotide positions 830-853 in the same sequence were used. PCR reaction conditions were repeated 35 cycles of 30 minutes at 94 ℃, 30 seconds at 57 ℃, 1 minute at 72 ℃ in the presence of 1.5 mM MgCl ₂ and finally carried out a polymerization of 10 minutes at 72 ℃.

Primers used to detect human NOV are the same as the forward primer having the sequence of 5-ctg tgt gtt cga tgg ggt ca-3 (SEQ ID NO: 3) corresponding to nucleotide positions 399-418 of the nucleotide sequence described in NCBI Accession No. AY082381. A reverse primer having a sequence of 5-cac agc tct tgg agc atg c-3 (SEQ ID NO: 4) corresponding to nucleotide position 715-733 in the sequence was used. PCR reaction conditions were repeated 40 cycles of 30 seconds at 94 ℃, 30 seconds at 56 ℃, 30 seconds at 72 ℃ in the presence of 1.5 mM MgCl ₂ and finally, the polymerization was carried out for 10 minutes at 72 ℃.

Rat egr2 (Lynda, M. et al., Biochem. J., 316: 765-9, 1996), which is known as a gene that increases expression in hepatic stellate cells by bile acids, was used as a positive control. Primers used to detect rat egr2 are the same as the forward primer having the sequence of 5-tct gcc tcc ccc aac cca ct-3 (SEQ ID NO: 5) corresponding to nucleotide positions 755-774 of the nucleotide sequence set forth in NCBI Accession No. NM053633. A reverse primer having a sequence of 5-tgg tcc tcc aat ggc gct gt-3 (SEQ ID NO: 6) corresponding to nucleotide positions 1701-1720 in the sequence was used. PCR reaction conditions were repeated 30 cycles of 30 seconds at 94 ℃, 30 seconds at 60 ℃, 30 seconds at 72 ℃ in the presence of 1.5 mM MgCl ₂ and finally, polymerization was carried out for 10 minutes at 72 ℃.

The result of RT-PCR was a control primer for PCR amplification of the GAPDH gene (NCBI Accession No. AF106860) as 5-ccc ctt cat tga cct caa cta c-3 (SEQ ID NO: 7) corresponding to nucleotide positions 948-969 in the sequence AF106860. A forward primer having a sequence of) and a reverse primer having a sequence of 5-cat ggt gaa gac gcc ag-3 (SEQ ID NO: 8) corresponding to nucleotide positions 1139-1158 in the same sequence were used. The product amplified by the PCR reaction was separated by agarose gel electrophoresis and visualized by EtBr staining (ethidium bromide staining).

As described in detail above, the present invention has an effect of providing a kit for diagnosing liver fibrosis by detecting activated hepatic stellate cells through confirming upregulation of the NOV gene in hepatic stellate cells.

In another aspect, the present invention has the effect of providing a method for finding a therapeutic agent for liver fibrosis by screening a substance that inhibits upregulated expression of the NOV gene of hepatic stellate cells.

<110> Halim co. <120> A kit for diagnosing liver fibrosis through identifying an          upregulated gene expression of NOV <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of rat NOV <400> 1 ctgagatgag accctgcgac 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rat NOV <400> 2 gtgcaattct tgaactgtag gtgg 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human NOV <400> 3 ctgtgtgttc gatggggtca 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for human NOV <400> 4 cacagctctt ggagcatgc 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Rat egr2 <400> 5 tctgcctccc ccaacccact 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Rat egr 2 <400> 6 tggtcctcca atggcgctgt 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for human GAPDH <400> 7 ccccttcatt gacctcaact ac 22 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for human GAPDH <400> 8 catggtgaag acgccag 17

Claims (5)

NOV (Nephroblastoma Overexpressed Gene) gene marker for diagnosing liver fibrosis. The kit for diagnosing liver fibrosis comprising the NOV gene of claim 1, a fragment of the gene, an expression product thereof or an antibody thereof. [Claim 2] The kit for diagnosing liver fibrosis according to claim 1, wherein the NOV gene has a sequence of NCBI GenBank Accession No. AY082381. i) preparing hepatic stellate cells whose expression of the NOV gene is upregulated; ii) treating the cell with any compound; And, iii) selecting a substance that inhibits upregulated expression of said NOV gene. 5. The method of claim 4, wherein the NOV gene has a sequence of NCBI GenBank Accession No. AY082381. 6.

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