JP2018153160A - Method for supporting detection of pre-disease condition of hepatic dysfunction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for supporting detection of a pre-disease condition of hepatic dysfunction.SOLUTION: With respect to body fluid sampled from a living body, an expression level of at least one gene represented by the following (1) and (2) is measured. (1) BAG6, FAM104A, GMPR, PIM1, RAD23A, RPIA, STOM, TESC and TSPAN5, (2) ARHGAP30, CAST, CALM2, CIRBP, CORO1A, DNAJB1, RSRP1, VIM, CCNDBP1, CD164, CD48, EIF2S1, IFIT2, IFIT3, IGSF6, ISCA1, RAB8B and SUPT4H1. An increase in the expression level of at least one gene selected from the group consisting of genes represented by (1) and/or a decrease in the expression level of at least one gene selected from the group consisting of genes represented by (2) indicates a pre-disease condition of hepatic dysfunction.SELECTED DRAWING: None

Description

  The present invention relates to a method for assisting in the detection of an unaffected liver dysfunction.

  “Not sick” is an intermediate state between illness and health that is not yet ill, but not healthy. If a non-disease can be detected, it becomes possible to take measures at the non-disease stage and prevent a disease. Therefore, the ability to detect non-disease is very important in preventive medicine.

  However, until now, no method for detecting non-disease has been known.

  On the other hand, mulberry leaves (hereinafter referred to as “mulberry leaves”) contain deoxynojirimycin, quercetin, kaempferol and the like, and have been taken over as healthy tea since the Kamakura period. Mulberry leaves have been reported to have lipid metabolism abnormality improvement effect, carbohydrate metabolism abnormality improvement effect, blood pressure suppression effect, fibrinolytic system activation effect, mutagenicity suppression effect, and antioxidant ability (Non-patent Document 1). And Non-Patent Document 2). Moreover, it is known that the value of various liver function markers such as AST, ALT, and γ-GTP is improved by ingesting mulberry leaves (Non-patent Document 3).

Kobayashi Y. et al., Biosci Biotechnol Biochem. 74, 2385-2395 (2010) Kobayashi Y. et al., Journal of Pharmacognosy & Natural Products (2016) Makiko Komine et al., NMCC Joint Use Research Results Report 12 (2004) pp.193-196

  An object of the present invention is to provide a method for assisting in the detection of non-disease of liver dysfunction.

  It has been confirmed that mulberry leaves exhibit the above effects, and it is also known that the values of various liver function markers are improved by ingesting mulberry leaves. The inventors of the present invention allowed rats to ingest a placebo that does not contain mulberry leaves or mulberry leaves, extracted genes that change expression only in the mulberry leaf intake group and genes that change expression only in the placebo intake group, and further conducted human intervention studies. In the experiment, 27 genes whose gene expression was remarkably varied by mulberry leaf intake were identified. Furthermore, the expression level of the above-mentioned gene in human blood whose numerical values of liver function markers hardly change was examined, and 20 types, particularly 8 types, of genes that increase and decrease when mulberry leaves are ingested were found. Then, the inventors have conceived that it is possible to detect a non-disease of liver dysfunction by measuring the expression level of any of these 27 types, 20 types or 8 types of genes, thereby completing the present invention.

That is, the present invention is a method for assisting detection of an unaffected liver dysfunction, comprising measuring the expression level of at least one gene shown in the following (1) and (2) in a body fluid collected from a living body. There,
(1) BAG6, FAM104A, GMPR, PIM1, RAD23A, RPIA, STOM, TESC and TSPAN5
(2) ARHGAP30, CAST, CALM2, CIRBP, CORO1A, DNAJB1, RSRP1, VIM, CCNDBP1, CD164, CD48, EIF2S1, IFIT2, IFIT3, IGSF6, ISCA1, RAB8B and SUPT4H1
The expression level of at least one gene selected from the group consisting of the gene shown in (1) above is increased, and / or at least one gene selected from the group consisting of the gene shown in (2) above. A method is provided wherein a reduced level of expression indicates that the liver is dysfunctional.

  The present invention provides for the first time a method for assisting in the detection of non-disease of liver dysfunction.

  As described above, the method of the present invention includes measuring the expression level of at least one gene shown in the above (1) and (2) in a body fluid separated from a living body. Preferred body fluids include blood (including whole blood, serum, and plasma). In addition, these genes are genes that are common to at least most mammals, and the “living body” is not particularly limited as long as it is an animal having these genes, but a human is preferable.

  The genes shown in (1) and (2) are all known genes, and their base sequences are also known. Tables 1 and 2 below show Entrez Gene ID and GeneBank Accession No. of human gene databases. In addition, the base sequence of cDNA of each human gene is described in the sequence listing, and the sequence number is also shown in the following Table 1 and Table 2.

  The expression level of each gene can be measured by measuring the amount of mRNA in the body fluid. Since the base sequence of each mRNA is known (also described in the sequence listing), mRNA can be easily quantified by a well-known method using a commercially available DNA array (see Examples below).

  The expression level of at least one gene selected from the group consisting of the genes shown in (1) (Table 1) is increased and / or the group consisting of the genes shown in (2) (Table 2) A decrease in the expression level of at least one gene selected from the above indicates that there is a high possibility that the liver is dysfunctional. Here, “increase” and “decrease” are obtained by comparing measured values measured over time for the same subject. For example, the measurement with time can be performed again at a certain time point and after 2 to 24 weeks, particularly after 4 to 12 weeks, particularly after 6 to 10 weeks. During this time, it is possible to detect non-disease by examining whether the expression level of the gene is increased or decreased. “Increase” and “decrease” are, for example, measured values using a microarray in the same manner as in the following examples, “DFW (the Distribution Free Weighted method”) (reference: Bioinformatics. 23 (3): 321-327 (2007 )) And normalized by the WAD weighted SAM method (reference: Proc Natl Acad Sci US A. 98 (9): 5116-5121 (2001), Algorithms Mol Biol. 3: 8 (2008)) (0 w vs. 8 w) Perform a corresponding test, extract the top 1000 probe sets, perform statistical processing on the corresponding t-test for genes containing the probe set sequences, and statistically significant If there is a difference, it can be judged to be “increase” or “decrease.” In the paired t-test, whether or not there is a statistically significant difference is that the p-value is less than 0.05. It can be determined by being there.

  As specifically described in the Examples below, lactate dehydrogenase (LD), which is one of liver function markers in blood, is significantly reduced in the mulberry leaf intake group. Among those selected within the placebo group, the average change in LD within ± 2SD was 21 out of 27 genes (BAG6, FAM104A, GMPR, PIM1, RPIA, TESC, TSPAN5, ARHGAP30, CAST, CALM2, CIRBP, CORO1A, DNAJB1, RSRP1 , VIM, CD164, CD48, IGSF6, ISCA1, RAB8B, and SUPT4H1), 8 out of 27 genes (CAST, CIRBP, CORO1A, VIM, CD164, EIF2S1, ISCA1, and SUPT4H1) are extracted from those who fall within the mean ± SD It was done. Therefore, the 21 genes, particularly the 8 genes, can be used as particularly sensitive markers in the method of the present invention.

  Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

Materials and Methods <Human Test>
Test meal For mulberry leaves, mulberry leaves powder (Sagamihara Chamber of Commerce), brown rice powder (Marsan Nakanoen), and spinach powder (Mikasa Sangyo) were mixed in a ratio of 80: 15: 5. For placebo food, dextrin (Pinedex # 2 AG, Matsutani Chemical Industry), brown rice powder (Marsan Nakanoen), spinach powder (Mikasa Sangyo) were mixed at a ratio of 80: 15: 5. In each case, 2 g each was packaged at Marsan Nakanoen.

Human intervention study The human intervention study was conducted at Hokkaido Information University. A placebo-controlled randomized, double-blind, parallel group comparison study was conducted with 36 volunteer subjects. Divided into 2 groups, mulberry leaf food or placebo food was taken for 8 weeks. The amount of intake was 2 g 3 times a day, for a total of 6 g. Blood samples were collected before ingestion (0w) and after ingestion after 8 weeks (8w) to obtain blood components and blood gene expression analysis samples.

Serum biochemical parameter measurement Serum biochemical parameters were measured at the Sapporo Clinical Laboratory.

RNA extraction and DNA microarray experiments
RNA extracted from blood collected from PAXgene (trade name) Blood RNA tube (Nippon Becton Dickinson Company, Tokyo, Japan) according to a standard method, and prepared using GeneChip 3 'IVT PLUS Kit (Affymetrix, Santa Clara, CA, USA) The cRNA was subjected to GeneChip (trade name) Human Genome U133 PLUS 2.0 Array (Affymetrix, Santa Clara, CA, USA).

DNA microarray analysis All samples were normalized by the Distribution Free Weighted method (DFW) method, and corresponding tests were performed before and after intake (0 w vs. 8 w) by the WAD weighted SAM method. The top 1000 probe sets were defined as having significant expression variation and extracted. From here, before and after ingestion, genes that varied in expression only in the mulberry leaf ingestion group and genes that varied in expression only in the placebo ingestion group were extracted. The former is a gene whose expression is changed by ingestion of mulberry, and the latter is a gene whose expression is originally changed, but the expression is not changed by ingestion of mulberry, and the direction of the change indicates the opposite of the action by mulberry . Both were defined as mulberry leaf intake marker genes.

<Animal test>
The mulberry leaves used for the material test were presented by the Sagamihara Chamber of Commerce.

Test meal intake
7-week-old Wistar male rats were purchased from Japan SLC and bred at 21 ± 1 ° C, relative humidity 50 ± 10%, light period 08: 00-20: 00; dark period 20: 00-08: 00 . After acclimatization with normal diet CE-2 (Japan Claire), 10 animals were divided into 3 groups and raised for 8 weeks. Normal diet (CE-2) intake group, high fat diet (Quick fat, Japan Claire) intake group, high fat diet 1% mulberry leaf powder added diet group. All were 24 hours free intake. Blood was collected after fasting the night before dissection. The test was conducted in accordance with the animal experiment guidelines of Kanagawa Prefectural Institute of Health.

Plasma biochemical parameters Plasma alanine aminotransferase (ALT) was measured with Fuji DRI-CHEM 7000 system (Fujifilm, Tokyo, Japan). Statistical processing was performed by Tukey's test.

RNA extraction and DNA microarray experiment Blood RNA was performed according to the standard method of TRIzol LS reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) and purified with RNeasy Mini Kit (Qiagen KK, Tokyo, Japan). The aRNA prepared by GeneChip 3 'IVT Express Kit (Affymetrix, Santa Clara, CA, USA) was applied to GeneChip (registered trademark) Rat genome 230 2.0 Array (Affymetrix, Santa Clara, CA, USA).

DNA microarray analysis Four individuals in each group were selected at random, normalized by the Distribution Free Weighted method (DFW) method, and compared between two groups by the rank products method. A probe set determined to have false discovery rate (FDR) <0.05 is defined as having significant fluctuations. Although there was no change between the diet and the high fat diet, the gene that changed between the high fat diet and the mulberry leaf diet was defined as the mulberry leaf intake marker gene.

<Selection of marker gene>
The mulberry leaf intake marker genes extracted in human tests and animal tests were compared by gene name, and genes showing common fluctuations were extracted. The human test consists of genes that change expression only in the mulberry leaf intake group and genes that change expression only in the placebo group, but the former and animal tests have the same direction of change, and the latter is the opposite This was the extraction condition.

<Evaluation of the effectiveness of genetic markers>
Some of the markers extracted by mulberry leaf ingestion had a strong response to mulberry leaf ingestion, and some of them markedly changed compared to the liver function marker. Calculate the mean ± SD and mean ± 2SD of LD change in the placebo group, select subjects in the mulberry leaf group and placebo group that fall within this range, respectively, and normalize only by the DFW method for the selecters, respectively. A corresponding test was performed before and after intake (0 w vs. 8 w) by the WAD weighted SAM method to compare whether 27 genes are included in the top 1000 probe sets.

Results <human test>
Serum biochemical parameter measurement
As a result of comparing the parameters of 0 w and 8 w, it was found that lactate dehydrogenase (LD), one of the liver function markers in blood, was significantly decreased in the mulberry leaf ingestion group.

DNA microarray analysis The number of genes whose expression was changed only in the mulberry leaf intake group was 527, and the number of genes whose expression was changed only in the placebo intake group was 564.

<Animal test>
Measurement of plasma biochemical parameters Plasma ALT was significantly higher in the high fat diet group than in the normal group, and was significantly lower in the mulberry leaf group than in the high fat diet group.

DNA microarray analysis There were 256 mulberry leaf intake marker genes.

<Gene marker>
The gene markers extracted in a common direction comparing the human test and the animal test were 27 genes shown in Tables 1 and 2 above. All of these 27 genes were extracted by the DFW and SAM methods described above, and statistically significant differences were observed in the corresponding t-test using DFW normalized values.

<Evaluation of the effectiveness of genetic markers>
Among the selecters who fell within the mean ± SD of LD change in the placebo group, 8 genes out of 27 genes were extracted, and among the selecters who fell within the mean ± 2SD, 21 genes out of 27 genes were extracted. From here, the extracted 27 genetic markers were significantly more responsive than the existing liver function markers, and were defined as unaffected markers.

Claims (6)

A method for assisting detection of an unaffected liver dysfunction, comprising measuring the expression level of at least one gene shown in (1) and (2) below in a body fluid collected from a living body,
(1) BAG6, FAM104A, GMPR, PIM1, RAD23A, RPIA, STOM, TESC and TSPAN5
(2) ARHGAP30, CAST, CALM2, CIRBP, CORO1A, DNAJB1, RSRP1, VIM, CCNDBP1, CD164, CD48, EIF2S1, IFIT2, IFIT3, IGSF6, ISCA1, RAB8B and SUPT4H1
The expression level of at least one gene selected from the group consisting of the gene shown in (1) above is increased, and / or at least one gene selected from the group consisting of the gene shown in (2) above. A method wherein the decreased expression level indicates that the liver is dysfunctional.   At least one selected from the group consisting of BAG6, FAM104A, GMPR, PIM1, RPIA, TESC, TSPAN5, ARHGAP30, CAST, CALM2, CIRBP, CORO1A, DNAJB1, RSRP1, VIM, CD164, CD48, IGSF6, ISCA1, RAB8B and SUPT4H1 The method according to claim 1, comprising measuring the expression level of the gene.   The method according to claim 1, comprising measuring the expression level of at least one gene selected from the group consisting of CAST, CIRBP, CORO1A, VIM, CD164, EIF2S1, ISCA1 and SUPT4H1.   The method according to claim 1, wherein the body fluid is blood.   The method according to any one of claims 1 to 4, wherein the gene expression level is measured by measuring the amount of mRNA.   The method according to any one of claims 1 to 5, wherein the body fluid is derived from a human.