JP4752032B2 - Hepatocellular carcinoma marker and test method for hepatocellular carcinoma - Google Patents
Hepatocellular carcinoma marker and test method for hepatocellular carcinoma Download PDFInfo
- Publication number
- JP4752032B2 JP4752032B2 JP2006104554A JP2006104554A JP4752032B2 JP 4752032 B2 JP4752032 B2 JP 4752032B2 JP 2006104554 A JP2006104554 A JP 2006104554A JP 2006104554 A JP2006104554 A JP 2006104554A JP 4752032 B2 JP4752032 B2 JP 4752032B2
- Authority
- JP
- Japan
- Prior art keywords
- hepatocellular carcinoma
- sugar chain
- amount
- marker
- subject
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 206010073071 hepatocellular carcinoma Diseases 0.000 title claims description 168
- 231100000844 hepatocellular carcinoma Toxicity 0.000 title claims description 168
- 239000003550 marker Substances 0.000 title claims description 91
- 238000010998 test method Methods 0.000 title claims description 22
- 235000000346 sugar Nutrition 0.000 claims description 269
- 238000000034 method Methods 0.000 claims description 44
- 238000012360 testing method Methods 0.000 claims description 35
- 210000002966 serum Anatomy 0.000 claims description 27
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 26
- 238000010828 elution Methods 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 230000007423 decrease Effects 0.000 claims description 10
- 238000002372 labelling Methods 0.000 claims description 6
- 239000000377 silicon dioxide Substances 0.000 claims description 6
- 238000005349 anion exchange Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 description 49
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 15
- 206010016654 Fibrosis Diseases 0.000 description 13
- 230000007882 cirrhosis Effects 0.000 description 13
- 208000019425 cirrhosis of liver Diseases 0.000 description 13
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- 238000006345 epimerization reaction Methods 0.000 description 12
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 10
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 10
- 108090001090 Lectins Proteins 0.000 description 8
- 102000004856 Lectins Human genes 0.000 description 8
- 150000001408 amides Chemical class 0.000 description 8
- 230000008859 change Effects 0.000 description 8
- 239000002523 lectin Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000007689 inspection Methods 0.000 description 5
- 230000001575 pathological effect Effects 0.000 description 5
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 238000013507 mapping Methods 0.000 description 4
- 239000013610 patient sample Substances 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000007704 transition Effects 0.000 description 4
- 239000000439 tumor marker Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 3
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108010063628 acarboxyprothrombin Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 238000001215 fluorescent labelling Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012916 structural analysis Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 108010059712 Pronase Proteins 0.000 description 2
- 102000004338 Transferrin Human genes 0.000 description 2
- 108090000901 Transferrin Proteins 0.000 description 2
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003234 fluorescent labeling method Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- -1 octadecylsilyl group Chemical group 0.000 description 2
- OIPPWFOQEKKFEE-UHFFFAOYSA-N orcinol Chemical compound CC1=CC(O)=CC(O)=C1 OIPPWFOQEKKFEE-UHFFFAOYSA-N 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 125000005629 sialic acid group Chemical group 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012581 transferrin Substances 0.000 description 2
- MUKRVWAUDAPCIN-UHFFFAOYSA-N 2-(2-amino-1h-pyridin-2-yl)acetic acid Chemical compound OC(=O)CC1(N)NC=CC=C1 MUKRVWAUDAPCIN-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 235000011437 Amygdalus communis Nutrition 0.000 description 1
- 108010004032 Bromelains Proteins 0.000 description 1
- DCQFONCCLHVTGL-UHFFFAOYSA-N C(C)(=O)OC(C)=O.NC1=NC=CC=C1 Chemical compound C(C)(=O)OC(C)=O.NC1=NC=CC=C1 DCQFONCCLHVTGL-UHFFFAOYSA-N 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000220304 Prunus dulcis Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229910000085 borane Inorganic materials 0.000 description 1
- KHYAFFAGZNCWPT-UHFFFAOYSA-N boron;n,n-diethylaniline Chemical compound [B].CCN(CC)C1=CC=CC=C1 KHYAFFAGZNCWPT-UHFFFAOYSA-N 0.000 description 1
- RJTANRZEWTUVMA-UHFFFAOYSA-N boron;n-methylmethanamine Chemical compound [B].CNC RJTANRZEWTUVMA-UHFFFAOYSA-N 0.000 description 1
- NNTOJPXOCKCMKR-UHFFFAOYSA-N boron;pyridine Chemical compound [B].C1=CC=NC=C1 NNTOJPXOCKCMKR-UHFFFAOYSA-N 0.000 description 1
- 235000019835 bromelain Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000013100 final test Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 108010015750 fucose-binding lectin Proteins 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920002959 polymer blend Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- HAOKKJHTFUZWIM-UHFFFAOYSA-N pyridin-1-ium-2-amine;chloride Chemical compound Cl.NC1=CC=CC=N1 HAOKKJHTFUZWIM-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Description
本発明は肝細胞癌マーカー及び肝細胞癌の検査法に関する。 The present invention relates to a hepatocellular carcinoma marker and a method for examining hepatocellular carcinoma.
肝細胞癌（hepatocellular carcinoma）は肝細胞から発生する悪性腫瘍であり、原発性肝癌の90%以上を占める。肝細胞癌のほとんどはウイルス性肝炎から発生する。本邦においてはC型肝炎ウイルスによる感染が原因で肝細胞癌に至る場合が圧倒的に多い（約80%）。一般に、肝硬変の病態を経て肝細胞癌に至ることが多く、C型肝炎ウイルス感染者の場合は通常、慢性肝炎、肝硬変を経て肝細胞癌へと病態が進行する。
現在、肝細胞癌の腫瘍マーカー（診断マーカー）としてAFP、PIVKA−II、AFPレクチン分画が臨床診断で利用されている。これらの３種類の腫瘍マーカーにはそれぞれ特徴がある。AFPは肝炎、肝硬変、肝細胞癌で上昇することから、肝細胞癌の腫瘍マーカーとしての特異性に欠ける。PIVKA−IIはAFPとの相関性がなく、肝細胞癌に対する特異性の高い腫瘍マーカーといわれているが、肝硬変でも上昇することがある。AFPレクチンL3分画は、肝細胞癌と肝硬変とを鑑別することが唯一可能であるといわれるが、小肝細胞癌での陽性率は低いとされている。必要に応じてこれらの腫瘍マーカーを組み合わせて肝細胞癌の検出が試みられるが、現在の臨床検査では肝硬変から肝細胞癌への移行について、約２０%強を検出できていない。
Hepatocellular carcinoma is a malignant tumor arising from hepatocytes and accounts for more than 90% of primary liver cancer. Most hepatocellular carcinomas arise from viral hepatitis. In Japan, infection with hepatitis C virus leads to hepatocellular carcinoma (over 80%). In general, hepatocellular carcinoma often passes through a pathological condition of cirrhosis, and in the case of a hepatitis C virus-infected person, the pathological condition usually progresses to hepatocellular carcinoma through chronic hepatitis and cirrhosis.
Currently, AFP, PIVKA-II, and AFP lectin fractions are used in clinical diagnosis as tumor markers (diagnostic markers) for hepatocellular carcinoma. Each of these three types of tumor markers has its characteristics. Since AFP increases in hepatitis, cirrhosis, and hepatocellular carcinoma, it lacks specificity as a tumor marker for hepatocellular carcinoma. PIVKA-II has no correlation with AFP and is said to be a highly specific tumor marker for hepatocellular carcinoma, but it may also increase in cirrhosis. The AFP lectin L3 fraction is said to be the only one capable of differentiating hepatocellular carcinoma from cirrhosis, but is said to have a low positive rate in small hepatocellular carcinoma. Although detection of hepatocellular carcinoma is attempted by combining these tumor markers as necessary, current clinical tests have not detected about 20% or more of the transition from cirrhosis to hepatocellular carcinoma.
本発明は肝細胞癌の検出に有用な新規腫瘍マーカー（肝細胞癌マーカー）を提供すること、及びそれを利用することで的中率の高い肝細胞癌の判定が可能となる検査法を提供することを課題とする。 The present invention provides a novel tumor marker (hepatocellular carcinoma marker) useful for the detection of hepatocellular carcinoma, and provides a test method that makes it possible to determine hepatocellular carcinoma with high accuracy by using the same. The task is to do.
本発明者らは血清中糖タンパク質の構成糖鎖群（非特許文献１）に注目し、その中から新規腫瘍マーカー（肝細胞癌マーカー）を見出すことを目的として鋭意検討した。具体的には、最終的に肝細胞癌と診断された患者について、肝細胞癌に至る前（即ち肝硬変のとき）と肝細胞癌に至った後にそれぞれ血清を採取し、血清中に存在する糖鎖のプロファイルを詳細に分析した。その結果、肝細胞癌の発症に伴って消失もしくは減少する糖鎖をトリシアリル糖鎖群の中に発見した。即ち、肝細胞癌の検出に有効な肝細胞癌マーカーを見出すことに成功し、当該肝細胞癌マーカーを指標とすれば的中率の高い肝細胞癌の検査法を確立できることが判明した。
本発明は主として上記知見ないし成果に基づくものであり、以下の検査法及びを提供する。
［１］以下の構造式で表されるトリシアリル糖鎖からなる肝細胞癌マーカー。
［３］被験者より採取された検体中の前記肝細胞癌マーカーの量と、該検体中に含まれ、肝細胞癌患者と健常者との間で量の差が小さい特定の糖鎖の量との比率を用いて検査結果を得ることを特徴とする、［２］に記載の肝細胞癌の検査法。
［４］前記特定の糖鎖が、前記検体中に含まれるトリシアリル糖鎖であることを特徴とする、［３］に記載の肝細胞癌の検査法。
［５］以下のステップ(1)及び(2)を含み、被験者より採取された検体中の前記肝細胞癌マーカーの量の増減が調べられることを特徴とする、［２］に記載の肝細胞癌の検査法、
(1)被験者より採取された検体中の前記肝細胞癌マーカーの量を測定するステップ、及び
(2)前記ステップで測定された前記肝細胞癌マーカーの量と、以前に同一の被験者より採取された検体中の前記肝細胞癌マーカーの量とを比較し、前記肝細胞癌マーカーの量の増減を評価するステップ。
［６］以下のステップ(1)及び(2)を含み、被験者より採取された検体中の前記肝細胞癌マーカーの量と、健常者より採取された検体中の前記肝細胞癌マーカーの量との差又は比が評価されることを特徴とする、［２］に記載の肝細胞癌の検査法、
(1)被験者より採取された検体中の前記肝細胞癌マーカーの量を測定するステップ、及び
(2)前記ステップで測定された前記肝細胞癌マーカーの量と、健常者より採取された検体中の前記肝細胞癌マーカーの量とを比較し、両者の差又は比を評価するステップ。
［７］
検査結果が、肝硬変から肝細胞癌へ進行したか否かの判定、又は肝硬変から肝細胞癌へ進行する徴候があるか否かの判定に利用されることを特徴とする、［２］〜［６］のいずれかに記載の肝細胞癌の検査法。
［８］前記ステップ(1)が以下のステップを含む、［５］〜［７］のいずれかに記載の肝細胞癌の検査法、
(1-1)被験者より採取された検体から糖鎖を調製するステップ、
(1-2)調製した糖鎖を標識化するステップ、
(1-3)標識化糖鎖を陰イオン交換カラムに供し、トリシアリル糖鎖画分を分取するステップ、及び
(1-4)分取したトリシアリル糖鎖画分を、ＯＤＳシリカカラムを使用した高速液体クロマトグラフィーに供し、該高速液体クロマトグラフィーの溶出パターンを分析して前記肝細胞癌マーカーの量を算出するステップ。
［９］前記検体が血清である、［２］〜［８］のいずれかに記載の肝細胞癌の検査法。
The present inventors paid attention to a constituent sugar chain group of serum glycoprotein (Non-patent Document 1), and conducted extensive studies for the purpose of finding a novel tumor marker (hepatocellular carcinoma marker). Specifically, for patients finally diagnosed with hepatocellular carcinoma, sera were collected before hepatocellular carcinoma (ie, cirrhosis) and after hepatocellular carcinoma respectively, and the sugars present in the serum were collected. The chain profile was analyzed in detail. As a result, a sugar chain disappearing or decreasing with the onset of hepatocellular carcinoma was discovered in the trisialyl sugar chain group. That is, it succeeded in finding a hepatocellular carcinoma marker effective for detection of hepatocellular carcinoma, and it became clear that a test method for hepatocellular carcinoma having a high accuracy could be established by using the hepatocellular carcinoma marker as an index.
The present invention is mainly based on the above findings or results, and provides the following inspection methods.
[1] A hepatocellular carcinoma marker comprising a trisialyl sugar chain represented by the following structural formula.
[3] The amount of the hepatocellular carcinoma marker in a sample collected from a subject, and the amount of a specific sugar chain contained in the sample and having a small difference in the amount between a hepatocellular carcinoma patient and a healthy person, The method for examining hepatocellular carcinoma according to [2], wherein a test result is obtained using a ratio of
[4] The method for testing hepatocellular carcinoma according to [3], wherein the specific sugar chain is a trisialyl sugar chain contained in the specimen.
[5] The hepatocyte according to [2], comprising the following steps (1) and (2), wherein the increase or decrease in the amount of the hepatocellular carcinoma marker in the sample collected from the subject is examined: Cancer testing,
(1) measuring the amount of the hepatocellular carcinoma marker in a sample collected from a subject; and
(2) The amount of the hepatocellular carcinoma marker measured in the step is compared with the amount of the hepatocellular carcinoma marker in a specimen previously collected from the same subject, and the amount of the hepatocellular carcinoma marker Step to evaluate the increase or decrease.
[6] Including the following steps (1) and (2), the amount of the hepatocellular carcinoma marker in the sample collected from the subject, and the amount of the hepatocellular carcinoma marker in the sample collected from the healthy person The method of testing for hepatocellular carcinoma according to [2], wherein the difference or ratio of
(1) measuring the amount of the hepatocellular carcinoma marker in a sample collected from a subject; and
(2) comparing the amount of the hepatocellular carcinoma marker measured in the step with the amount of the hepatocellular carcinoma marker in a sample collected from a healthy person, and evaluating the difference or ratio between the two.
[7]
[2] to [2], wherein the test result is used to determine whether or not there is a sign of progression from cirrhosis to hepatocellular carcinoma, or whether or not there is a sign of progression from cirrhosis to hepatocellular carcinoma. [6] The method for examining hepatocellular carcinoma according to any one of [6].
[8] The method for examining hepatocellular carcinoma according to any of [5] to [7], wherein the step (1) includes the following steps:
(1-1) preparing a sugar chain from a sample collected from a subject;
(1-2) labeling the prepared sugar chain,
(1-3) subjecting the labeled sugar chain to an anion exchange column and fractionating the trisialyl sugar chain fraction; and
(1-4) The fractionated trisialyl sugar chain fraction is subjected to high performance liquid chromatography using an ODS silica column, and the elution pattern of the high performance liquid chromatography is analyzed to calculate the amount of the hepatocellular carcinoma marker. Step.
[9] The method for testing hepatocellular carcinoma according to any one of [2] to [8], wherein the specimen is serum.
本発明の肝細胞癌マーカーはトリシアリル糖鎖であって、以下の構造式で表される。
後述の実施例に示すように、肝細胞患者由来の検体では非常に高い確率で当該糖鎖が消失又は減少していた。従って、当該糖鎖を利用すれば高い的中率で肝細胞癌の検査を行うことが可能といえる。このように、当該糖鎖は肝細胞癌の検査において非常に有用であり、単独で又は他の腫瘍マーカー（例えばAFPやPIVKA-II）と組み合わされて、肝細胞癌の検査に利用され得る。 As shown in Examples described later, in the specimen derived from a hepatocyte patient, the sugar chain disappeared or decreased with a very high probability. Therefore, it can be said that hepatocellular carcinoma can be examined at a high accuracy rate by using the sugar chain. Thus, the sugar chain is very useful in the examination of hepatocellular carcinoma, and can be used for the examination of hepatocellular carcinoma alone or in combination with other tumor markers (for example, AFP and PIVKA-II).
上記の糖鎖構造からわかるように、当該糖鎖は糖鎖根部にフコースを持たず且つシアル酸を３つ保持した構造を有し、現行の臨床検査で利用されているAFPレクチンL3分画に含まれる糖鎖とは明らかに異なる。また、AFPレクチンL3分画には糖鎖根部にフコースを持ったあらゆる糖鎖が含まれることから、AFPレクチンL3分画を利用した臨床検査では糖鎖群を指標とした検査が行われていることになる。これに対して本発明の糖鎖を利用すれば単独の糖鎖を指標とした検査を行うことができる。このように単独で肝細胞癌の検査に利用可能な糖鎖を見出したことは非常に大きな成果である。尚、AFPレクチンL3分画を用いた検査は、AFP分子上の糖鎖の癌性変化をレクチンとの親和性を利用して検出し、肝細胞癌由来AFPを分別測定する手法である。レクチン親和性の相違は糖鎖根部に結合するフコースの有無に関係していると考えられており、肝細胞癌ではフコース結合型AFPが増加するといわれている。 As can be seen from the above sugar chain structure, the sugar chain does not have fucose at the root of the sugar chain and has a structure that retains three sialic acids, and the AFP lectin L3 fraction used in current clinical tests It is clearly different from the sugar chain contained. In addition, since the AFP lectin L3 fraction contains all sugar chains with fucose at the root of the sugar chain, clinical tests using the AFP lectin L3 fraction are conducted using the sugar chain group as an index. It will be. On the other hand, if the sugar chain of the present invention is used, a test using a single sugar chain as an index can be performed. Thus, finding a sugar chain that can be used alone for the examination of hepatocellular carcinoma is a great achievement. The test using the AFP lectin L3 fraction is a technique for detecting the cancerous change of the sugar chain on the AFP molecule using the affinity with the lectin, and separately measuring AFP derived from hepatocellular carcinoma. The difference in lectin affinity is thought to be related to the presence or absence of fucose binding to the sugar chain root, and it is said that fucose-bound AFP increases in hepatocellular carcinoma.
本発明の肝細胞癌マーカーである糖鎖はトランスフェリン由来と考えられる。トランスフェリンは679個のアミノ酸と糖鎖を2個結合したタンパク質である。後述の実施例に示すようにタンパク質の精製操作を経ることなく血清から糖鎖を切り出しているにもかかわらず、本発明の肝細胞癌マーカーの量の評価が可能であった。この知見より、本発明の肝細胞癌マーカーは簡便な操作による肝細胞癌検査法を実現できるという点において有益であるといえる。一方、後述の実施例に示すように少量の血清を用いて本発明の肝細胞癌マーカーの量の評価が可能であった。このことは通常の生化学検査や血清検査のために用意された血清の一部を用いて本発明の肝細胞癌マーカーを指標とした検査が可能であることを意味する。従って、本発明の肝細胞癌マーカーを用いた検査法はルーチン化が容易で且つ被験者の負担の少ないもととなる。 The sugar chain which is a hepatocellular carcinoma marker of the present invention is considered to be derived from transferrin. Transferrin is a protein that combines 679 amino acids and two sugar chains. Although the sugar chain was cut out from the serum without undergoing a protein purification operation as shown in Examples described later, it was possible to evaluate the amount of the hepatocellular carcinoma marker of the present invention. From this finding, it can be said that the hepatocellular carcinoma marker of the present invention is beneficial in that it can realize a hepatocellular carcinoma test method by a simple operation. On the other hand, as shown in the Examples described later, it was possible to evaluate the amount of the hepatocellular carcinoma marker of the present invention using a small amount of serum. This means that a test using the hepatocellular carcinoma marker of the present invention as an index can be performed using a part of serum prepared for normal biochemical tests and serum tests. Therefore, the test method using the hepatocellular carcinoma marker of the present invention is easy to routineize and is less burdensome on the subject.
本発明の他の局面は、上記本発明の肝細胞癌マーカー（以下、「マーカー糖鎖」ともいう）を用いた肝細胞癌の検査法に関する。本発明の肝細胞癌の検査法では被験者より採取された検体中におけるマーカー糖鎖の量が指標として用いられる。
本発明では、肝細胞癌であるか否かの判定を必要とする者（被験者）に由来する検体が使用される。検体としては、被験者より採取された体液（血液、リンパ液、髄液等）が用いられる。好ましくは被験者より採取された血液を分離して得られる血清を検体とする。即ち本発明の好ましい一形態では被験者の血清中に存在するマーカー糖鎖の量が測定される。血清は調製が容易であるという利点を有する。ところで、他の多くの検査の検体として血清が利用されており、このような検査と同時に本発明の検査法が実施されることも想定される。このような場合、本発明の検査法の検体として血清を採用すれば本発明の実施のために改めて検体を調製する必要がなくなり、被験者及び検査する者の負担が軽減し、検査時間の短縮化も図られる。
Another aspect of the present invention relates to a method for examining hepatocellular carcinoma using the above-described hepatocellular carcinoma marker of the present invention (hereinafter also referred to as “marker sugar chain”). In the test method for hepatocellular carcinoma of the present invention, the amount of marker sugar chain in a sample collected from a subject is used as an index.
In the present invention, a specimen derived from a person (subject) who needs to determine whether or not hepatocellular carcinoma is used. As the specimen, body fluid (blood, lymph, spinal fluid, etc.) collected from the subject is used. Preferably, serum obtained by separating blood collected from a subject is used as a specimen. That is, in a preferred embodiment of the present invention, the amount of marker sugar chain present in the serum of a subject is measured. Serum has the advantage of being easy to prepare. By the way, serum is used as a sample for many other tests, and it is also assumed that the test method of the present invention is performed simultaneously with such tests. In such a case, if serum is used as the sample of the test method of the present invention, it is not necessary to prepare a sample again for the implementation of the present invention, reducing the burden on the subject and the person to be tested, and shortening the test time. Is also planned.
被験者は特に限定されない。即ち、肝細胞癌であるか否かの判定が必要な者に対して広く本発明を適用することができる。また、例えば、肝炎又は肝硬変と診断された患者に対して本発明の検査法を適用すれば肝細胞癌への進行の徴候を早期に把握でき、適切な治療法の選択及びそれに伴う患者のＱＯＬ（Quality of Life、生活の質）向上が可能となる。一方、他の検査法によって肝細胞癌であると判定された者（又は肝細胞癌のおそれが高いと判定された者）に対して本発明の検査法を適用することもできる。このように、他の検査法による判定結果を検証することに本発明の検査法を利用してもよい。 The subject is not particularly limited. That is, the present invention can be widely applied to those who need to determine whether or not hepatocellular carcinoma. Further, for example, if the test method of the present invention is applied to a patient diagnosed with hepatitis or cirrhosis, signs of progression to hepatocellular carcinoma can be grasped at an early stage, and selection of an appropriate treatment method and associated patient QOL (Quality of Life) can be improved. On the other hand, the test method of the present invention can be applied to a person who has been determined to have hepatocellular carcinoma by another test method (or a person who has been determined to have a high risk of hepatocellular carcinoma). As described above, the inspection method of the present invention may be used to verify the determination result obtained by another inspection method.
（マーカー糖鎖量の測定手法）
試料中のマーカー糖鎖量の測定には高速液体クロマトグラフィー（HPLC）、核磁気共鳴（NMR）、質量分析などを利用することができる。但し、分離能、定量性、及び簡便性などの観点から、逆相クロマトグラフィーカラムを使用した高速液体クロマトグラフィー（HPLC）を利用してマーカー糖鎖量を測定することが好ましい。
糖鎖の分離能に優れた逆相クロマトグラフィーカラムとしてODSシリカカラムが頻用されており、本発明においてもこれを利用することができる。ここで、ODSシリカカラムとは、担体としてのシリカゲルにオクタデシルシリル（Octadecylsilyl）基を結合させたカラムであり、例えばShim-pack HRC-ODS（島津製作所社製）、Union UK-C18（インタクト社製）、Cadenza CD-C18（インタクト社製）等の名称で市販されている。
(Measurement method of marker sugar chain amount)
High-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), mass spectrometry, and the like can be used to measure the amount of marker sugar chain in the sample. However, it is preferable to measure the amount of the marker sugar chain using high performance liquid chromatography (HPLC) using a reverse phase chromatography column from the viewpoints of resolution, quantification, and simplicity.
An ODS silica column is frequently used as a reverse-phase chromatography column excellent in the separation of sugar chains, and can be used in the present invention. Here, the ODS silica column is a column in which an octadecylsilyl group is bonded to silica gel as a carrier. For example, Shim-pack HRC-ODS (manufactured by Shimadzu Corporation), Union UK-C18 (manufactured by Intact Corporation) ) And Cadenza CD-C18 (manufactured by Intact).
HPLC分析を利用したマーカー糖鎖量の測定手順の一例を以下に詳述する。
（１）糖鎖の調製
まず、被験者より採取された検体から糖鎖を調製する。具体的には被験者より採血した後、血清を分離し、次いで糖鎖を遊離させる。採血及び血清の分離は常法で行うことができる。糖鎖の遊離法としては酵素を利用した方法（酵素化学的手法）と、無水ヒドラジンを利用した方法（化学的手法）が知られている。これらの中のいずれを採用することにしてもよいが操作に熟練を必要としないことや試薬の取り扱いの面から、酵素を利用した方法の方が好ましい。酵素を利用した糖鎖の遊離法の詳細については、糖蛋白質糖鎖研究法 生物化学実験法（高橋禮子著、学会出版センター）等を参照することができる。酵素を利用した糖鎖の遊離法では、グリコペプチダーゼA（グリコアミダーゼ、N-グリカナーゼなどとも呼称される、EC 3.5.1.52）（N.Takahashi、Biochemical and Biophysical Research Communications Volume 76,Issue 4,20 june 1977,1194-1201）が使用される。グリコペプチダーゼAは市販されており（例えば生化学工業社製のグリコペプチダーゼA；アーモンド）、容易に入手可能である。グリコペプチダーゼAの使用法は製品に添付された説明書又は上掲の文献などに従えばよい。但し、グリコペプチダーゼAを作用させる操作に先立って、試料に対してタンパク質分解酵素（好ましくはペプシン）を作用させることが好ましい。グリコペプチダーゼが作用し易くなり、糖鎖の遊離を効率的に行うことができるからである。尚、グリコペプチダーゼの作用を妨げない限りにおいて、タンパク質分解酵素をグリコペプチダーゼと同時に作用させてもよい。
糖鎖を遊離させた後、糖鎖を分離精製する。例えば、グリコペプチダーゼAを作用させた後の試料に対して蛋白分解酵素（プロナーゼ、ブロメライン（EC 3.4.22.）など）を更に作用させる。これによって残存するペプチドが断片化される。次に、試料をゲルろ過カラムに供し、分子量の差に基づいて糖鎖を分画する。
An example of the procedure for measuring the marker sugar chain amount using HPLC analysis will be described in detail below.
(1) Preparation of sugar chain First, a sugar chain is prepared from a sample collected from a subject. Specifically, after blood is collected from the subject, serum is separated, and then sugar chains are released. Blood sampling and serum separation can be performed by conventional methods. As a method for releasing a sugar chain, a method using an enzyme (enzymatic chemical method) and a method using an anhydrous hydrazine (chemical method) are known. Any of these may be adopted, but an enzyme-based method is preferred from the viewpoint of not requiring skill in operation and handling of reagents. For the details of the method for releasing sugar chains using enzymes, reference can be made to glycoprotein sugar chain research methods, biochemical experiment methods (authored by Atsuko Takahashi, Academic Publishing Center), and the like. Glycopeptidase A (also called glycoamidase, N-glycanase, etc., EC 3.5.1.52) (N. Takahashi, Biochemical and Biophysical Research Communications Volume 76,
After releasing the sugar chain, the sugar chain is separated and purified. For example, a proteolytic enzyme (pronase, bromelain (EC 3.4.22.), Etc.) is further allowed to act on the sample after glycopeptidase A is allowed to act. As a result, the remaining peptide is fragmented. Next, the sample is subjected to a gel filtration column, and sugar chains are fractionated based on the difference in molecular weight.
（２）標識化
糖鎖の標識法としては２−アミノピリジンで蛍光標識する方法（PA化法）と、トリチウムラベルで放射線標識する方法等が知られている。試薬が取り扱い易く、検出も高速液体クロマトグラフィーで行えて比較的容易である点などを考慮すれば、２−アミノピリジンによる蛍光標識法を採用することが好ましい。２−アミノピリジンによる蛍光標識法として、２−アミノピリジン塩酸溶液を蛍光標識剤として、水素化シアノホウ素ナトリウム（NABH3CH）を還元剤として使用する方法（S.Hase, et.al. Biochem. Biophys. Res. Commun. 85, 257-263 (1978)、S.Hase, T.Ibuki, T.Ikenaka,J.Biochem, Vol.95, 197-203(1984)）と、２−アミノピリジン無水酢酸溶液を蛍光標識剤として、ボランジメチルアミンコンプレックスを還元剤として使用する方法（Kondo,et.al. Agric. Biol. Chem. 54, 2169 〜2170 (1990)）が知られている。前者は使用する装置が少なく、簡便である。また、後者については専用の装置（GlycoTAG（登録商標）、タカラバイオ株式会社）が市販されており、当該装置を利用することもできる。PA化の際の還元剤として上記の還元剤の他、ボラン-N,N-ジエチルアニリンコンプレックス（C6H5N（C2H5）2・BH3）、ボラン−ピリジンコンプレックス（C5H5N・BH3）などを利用してもよい。
(2) Labeling As a method for labeling a sugar chain, a method of fluorescent labeling with 2-aminopyridine (PA method), a method of radiolabeling with a tritium label, and the like are known. Considering the fact that the reagent is easy to handle and the detection can be performed by high performance liquid chromatography and is relatively easy, it is preferable to adopt a fluorescent labeling method using 2-aminopyridine. As a fluorescent labeling method using 2-aminopyridine, a method using 2-aminopyridine hydrochloride solution as a fluorescent labeling agent and sodium cyanoborohydride (NABH 3 CH) as a reducing agent (S. Hase, et.al. Biochem. Biophys. Res. Commun. 85, 257-263 (1978), S. Hase, T. Ibuki, T. Ikenaka, J. Biochem, Vol. 95, 197-203 (1984)) and 2-aminopyridine acetic anhydride A method using a solution as a fluorescent labeling agent and borane dimethylamine complex as a reducing agent (Kondo, et.al. Agric. Biol. Chem. 54, 2169-2170 (1990)) is known. The former is simple because it uses few devices. For the latter, a dedicated device (GlycoTAG (registered trademark), Takara Bio Inc.) is commercially available, and the device can also be used. In addition to the above reducing agents, the borane-N, N-diethylaniline complex (C 6 H 5 N (C 2 H 5 ) 2 · BH 3 ), borane-pyridine complex (C 5 H) 5 N · BH 3 ) may be used.
（３）標識化糖鎖の精製
標識化操作に続いて標識化糖鎖の精製を行う。例えば、標識化後の試料をゲルろ過カラムに供し、分子量の差に基づく分画によって夾雑物を除去する。標識化糖鎖の精製方法としてアミノカラムを使用する方法も提案されており（特開平８−２２８７９５号公報）、当該方法を採用してもよい。
(3) Purification of labeled sugar chain The labeled sugar chain is purified following the labeling operation. For example, the labeled sample is applied to a gel filtration column, and impurities are removed by fractionation based on the difference in molecular weight. A method of using an amino column has been proposed as a method for purifying a labeled sugar chain (Japanese Patent Laid-Open No. 8-228795), and this method may be employed.
（４）トリシアリル糖鎖画分の分取
標識化糖鎖を、DEAE（diethlaminoethil）カラム等の陰イオン交換カラムに供し、シアル酸残基の数によって中性糖鎖、モノシアリル糖鎖、ジシアリル糖鎖、トリシアリル糖鎖を分離する。トリシアリル糖鎖を分画できる限り、使用する陰イオン交換カラムの種類は特に限定されない。DEAEカラムとしては、市販のTSK−GEL DEAE-5PW（トーソー社製）等を利用することができる。
（５）ODSシリカカラムを使用したHPLC分析
HPLC装置にODSカラムを装填し、常法に従い平衡化する。続いて、市販の標準PA化グルコース重合体混合物をカラムに流し、カラムの状態を確認するとともに、溶出位置の補正規格化を行う。これによって、目的の糖鎖（即ちマーカー糖鎖）の溶出時間（溶出位置）が決定される。次に、上記の方法で調製したトリシアリル糖鎖画分をカラムに流す。得られたHPLCの溶出パターン（チャート）から、溶出時間を指標として目的の糖鎖のピークを特定し、その面積を求める。得られた面積から、検体（血清）中のマーカー糖鎖の量を算出する。また、目的の糖鎖の面積と他の糖鎖の面積とを比較する。
本発明では、以上のようにして算出された検体中のマーカー糖鎖量を用いて肝細胞癌の判定に有用な情報を得る。
(4) Fractionation of trisialyl sugar chain fraction The labeled sugar chain is applied to an anion exchange column such as a DEAE (diethlaminoethil) column, and depending on the number of sialic acid residues, neutral sugar chain, monosialyl sugar chain, disialyl sugar chain Isolate the trisialyl sugar chain. As long as the trisialyl sugar chain can be fractionated, the type of anion exchange column to be used is not particularly limited. As the DEAE column, commercially available TSK-GEL DEAE-5PW (manufactured by Tosoh Corporation) or the like can be used.
(5) HPLC analysis using ODS silica column
The ODS column is loaded into the HPLC apparatus and equilibrated according to a conventional method. Subsequently, a commercially available standard PA-glucose polymer mixture is allowed to flow through the column, and the column state is confirmed and the elution position is corrected and normalized. Thereby, the elution time (elution position) of the target sugar chain (ie, marker sugar chain) is determined. Next, the trisialyl sugar chain fraction prepared by the above method is applied to the column. From the HPLC elution pattern (chart) obtained, the peak of the target sugar chain is identified using the elution time as an index, and the area is determined. From the obtained area, the amount of marker sugar chain in the specimen (serum) is calculated. Moreover, the area of the target sugar chain is compared with the areas of other sugar chains.
In the present invention, information useful for the determination of hepatocellular carcinoma is obtained using the marker sugar chain amount in the specimen calculated as described above.
血清を検体としたODS分析において実質的に検出されるトリシアリル糖鎖は、本発明のマーカー糖鎖を含めて主に３種類であり非常に少ないことから、ODS分析によって本発明の糖鎖を特定し、その量を算出することが可能である。このように、本発明の検査法ではODS分析までで検査結果を得ることができる。但し、さらに糖鎖分析（例えばアミド順相カラムによる分析）を実施し、その結果も利用して最終的な検査結果を導き出してもよい。 The trisialyl sugar chain that is substantially detected in the ODS analysis using serum as a specimen is mainly three, including the marker sugar chain of the present invention, and is very small. Therefore, the sugar chain of the present invention is specified by the ODS analysis. It is possible to calculate the amount. Thus, in the inspection method of the present invention, the inspection result can be obtained up to the ODS analysis. However, sugar chain analysis (for example, analysis using an amide normal phase column) may be further performed, and the result may be used to derive a final test result.
本発明の一態様では、被験者より採取された検体中のマーカー糖鎖量を、同一の検体中に含まれる特定の標準糖鎖（内部標準）の量を利用して評価する。ここでの標準糖鎖とは肝細胞癌患者と健常者との間で量の差が小さい糖鎖である。後述の実施例に示すように、血清を検体とした場合、ODSシリカカラムを用いたHPLC分析においてマーカー糖鎖の他に２種類のトリシアリル糖鎖を検出することが可能である。説明の便宜上、マーカー糖鎖を糖鎖bと呼び、他の２種類のトリシアリル糖鎖を糖鎖a及び糖鎖cと呼ぶことにする。糖鎖a及び糖鎖cの構造を以下に示す。 In one embodiment of the present invention, the amount of a marker sugar chain in a sample collected from a subject is evaluated using the amount of a specific standard sugar chain (internal standard) contained in the same sample. Here, the standard sugar chain is a sugar chain having a small difference in amount between a hepatocellular carcinoma patient and a healthy person. As shown in Examples described later, when serum is used as a specimen, two types of trisialyl sugar chains can be detected in addition to the marker sugar chain in HPLC analysis using an ODS silica column. For convenience of explanation, the marker sugar chain is called sugar chain b, and the other two types of trisialyl sugar chains are called sugar chain a and sugar chain c. The structures of sugar chain a and sugar chain c are shown below.
糖鎖aの構造
糖鎖cの構造
後述の実施例に示すように、糖鎖a及び糖鎖cの量については肝細胞癌との関連性が認められなかった。したがって、これら２種類のトリシアリル糖鎖のいずれか又は両者を特定の標準糖鎖として用いることができる。そこで、具体的には例えば以下の(1)〜(4)のいずれかで算出される値（マーカー糖鎖量の比率）を用いてマーカー糖鎖の量を評価することができる。
(1)糖鎖bの量／糖鎖aの量、(2)糖鎖bの量／糖鎖cの量、(3)糖鎖bの量／（糖鎖aの量＋糖鎖cの量）、(4)糖鎖bの量／（糖鎖aの量＋糖鎖bの量＋糖鎖cの量）
尚、各糖鎖の量としてはHPLC分析の溶出チャートのピーク面積（又はピーク面積比）を用いることができる。
As shown in Examples described later, the amount of sugar chain a and sugar chain c was not associated with hepatocellular carcinoma. Therefore, either or both of these two types of trisialyl sugar chains can be used as a specific standard sugar chain. Therefore, specifically, for example, the amount of marker sugar chain can be evaluated using the value (ratio of marker sugar chain amount) calculated in any of (1) to (4) below.
(1) Amount of sugar chain b / amount of sugar chain a, (2) Amount of sugar chain b / amount of sugar chain c, (3) Amount of sugar chain b / (amount of sugar chain a + sugar chain c Amount), (4) Amount of sugar chain b / (Amount of sugar chain a + Amount of sugar chain b + Amount of sugar chain c)
As the amount of each sugar chain, the peak area (or peak area ratio) of the elution chart of HPLC analysis can be used.
後述の実施例に示すように、PA化操作を伴うODS分析ではPA化の際に糖鎖のエピメル化が生じ、ODS分析の溶出チャートではエピメル化前後の糖鎖が別々のピークとして現れる。そこで、上記の式(1)〜(4)に用いる糖鎖の量として、エピメル化されていない糖鎖とエピメル化後の糖鎖の総量、エピメル化されていない糖鎖の量、又はエピメル化された糖鎖の量を用いることができるが、好ましくは前２者のいずれかを用い、更に好ましくはエピメル化されていない糖鎖とエピメル化後の糖鎖の総量を用いる。 As shown in Examples described later, in ODS analysis accompanied by PA conversion, sugar chains undergo epimerization during PA conversion, and in the elution chart of ODS analysis, sugar chains before and after epimerization appear as separate peaks. Therefore, as the amount of sugar chains used in the above formulas (1) to (4), the total amount of sugar chains that are not epimerized and the sugar chains after epimerization, the amount of sugar chains that are not epimerized, or epimerization The amount of the sugar chain formed can be used, but preferably one of the former two is used, and more preferably, the total amount of the sugar chain that is not epimerized and the sugar chain after epimerization is used.
以上の方法で得られたマーカー糖鎖の量に関する情報は、肝疾患の診断に有用である。本発明の一態様では、予め基準量を設定しておき、マーカー糖鎖量が基準量より少ないという検査結果から、被験者に肝細胞癌のおそれがある又は被験者に肝細胞癌のおそれが高いなどと判定する。一方、マーカー糖鎖量が基準量より多いという検査結果を利用して、肝細胞癌のおそれはない又は肝細胞癌のおそれは低いなどと判定することができる。マーカー糖鎖量の範囲に対応させた複数の区分（例えば、(1)肝細胞癌のおそれはない、(2)肝細胞癌のおそれは低い、(3)肝細胞癌の徴候が認められる、及び(4)肝細胞癌のおそれが高い、からなる４区分）を設定し、検査結果がいずれの区分に属するかを判定してもよい。 Information on the amount of marker sugar chain obtained by the above method is useful for diagnosis of liver disease. In one embodiment of the present invention, a reference amount is set in advance, and the test result that the marker sugar chain amount is less than the reference amount indicates that the subject has a risk of hepatocellular carcinoma or the subject has a high risk of hepatocellular carcinoma. Is determined. On the other hand, using the test result that the marker sugar chain amount is larger than the reference amount, it can be determined that there is no risk of hepatocellular carcinoma or low risk of hepatocellular carcinoma. Multiple categories corresponding to the range of marker sugar chain amount (for example, (1) No risk of hepatocellular carcinoma, (2) Low risk of hepatocellular carcinoma, (3) Signs of hepatocellular carcinoma are observed, And (4) (4 categories comprising a high risk of hepatocellular carcinoma) may be set, and it may be determined to which category the test result belongs.
本発明の他の一態様では、被験者より採取された検体中のマーカー糖鎖量と、健常者より採取された検体中のマーカー糖鎖量とを比較する。即ち、この態様では、被験者より採取された検体中のマーカー糖鎖量と、健常者より採取された検体中のマーカー糖鎖量との差又は比を検査結果として与える。例えば、健常者の場合に比較して被験者ではマーカー糖鎖の量が少ないとの検査結果を利用して、被験者に肝細胞癌のおそれがある、又は被験者に肝細胞癌のおそれが高いなどと判定することができる。他方、健常者と被験者との間でマーカー糖鎖の量に顕著な差が認められないという検査結果を利用して、例えば、被験者に肝細胞癌のおそれはない、又は被験者に肝細胞癌のおそれは低いと判定することができる。
健常者の検体中のマーカー糖鎖量の測定は、被験者の検体中のマーカー糖鎖量の測定と同様の手順で行われる。また、予め健常者についてのマーカー糖鎖量が明らかになっている場合は、当該マーカー糖鎖量を比較対照として用いても良い。
この態様においても、上記の態様と同様に、同一の検体中に含まれる特定の標準糖鎖（内部標準）の量を利用してマーカー糖鎖量を評価することにしてもよい。即ち、以下の(1)〜(4)のいずれかで算出される値（マーカー糖鎖の比率）を用いてマーカー糖鎖の量を評価してもよい。この場合、エピメル化されていない糖鎖とエピメル化後の糖鎖の総量、エピメル化されていない糖鎖の量、又はエピメル化された糖鎖の量を用いることができる。
(1)糖鎖bの量／糖鎖aの量、(2)糖鎖bの量／糖鎖cの量、(3)糖鎖bの量／（糖鎖aの量＋糖鎖cの量）、(4)糖鎖bの量／（糖鎖aの量＋糖鎖bの量＋糖鎖cの量）
In another aspect of the present invention, the amount of marker sugar chains in a sample collected from a subject is compared with the amount of marker sugar chains in a sample collected from a healthy person. That is, in this embodiment, the difference or ratio between the amount of marker sugar chain in the sample collected from the subject and the amount of marker sugar chain in the sample collected from the healthy subject is given as the test result. For example, using the test result that the amount of marker sugar chain in the subject is small compared to the case of a healthy person, the subject has a risk of hepatocellular carcinoma, or the subject has a high risk of hepatocellular carcinoma, etc. Can be determined. On the other hand, using the test results that there is no significant difference in the amount of marker sugar chain between healthy subjects and subjects, for example, there is no risk of hepatocellular carcinoma in the subject, or hepatocellular carcinoma in the subject It can be determined that the fear is low.
The measurement of the amount of marker sugar chain in the sample of a healthy person is performed in the same procedure as the measurement of the amount of marker sugar chain in the sample of the subject. In addition, when the amount of marker sugar chain for a healthy person is known in advance, the amount of marker sugar chain may be used as a comparative control.
In this embodiment, similarly to the above embodiment, the amount of the marker sugar chain may be evaluated using the amount of a specific standard sugar chain (internal standard) contained in the same specimen. That is, the amount of marker sugar chain may be evaluated using the value (marker sugar chain ratio) calculated in any of the following (1) to (4). In this case, the total amount of sugar chains that are not epimerized and sugar chains after epimerization, the amount of sugar chains that are not epimerized, or the amount of sugar chains that are epimerized can be used.
(1) Amount of sugar chain b / amount of sugar chain a, (2) Amount of sugar chain b / amount of sugar chain c, (3) Amount of sugar chain b / (amount of sugar chain a + sugar chain c Amount), (4) Amount of sugar chain b / (Amount of sugar chain a + Amount of sugar chain b + Amount of sugar chain c)
本発明のさらに他の態様では、ある時点で測定された検体中のマーカー糖鎖量と、以前に同一の被験者より採取された検体中のマーカー糖鎖量とを比較し、マーカー糖鎖量の増減の有無及び／又は増減の程度を評価する。この態様での検査結果（マーカー糖鎖量の変化に関するデータ）は特に肝疾患の病態の進行をモニターするために有用な情報となる。即ち、マーカー糖鎖量の減少が認められたという検査結果を利用して、例えば、前回の検査から今回の検査までの間に肝硬変から肝細胞癌へと病態が進行した、又は肝硬変から肝細胞癌への病態の進行の徴候が認められる、と判定することができる。他方、マーカー糖鎖量の増加が認められたという検査結果を利用して、例えば、前回の検査から今回の検査までの間に病態が改善した、又は病態の改善の徴候が認められる、と判定することができる。さらには、マーカー糖鎖量の変動が認められないという検査結果を利用して、例えば、前回の検査から今回の検査までの間に病態は変化しないと判定することができる。
治療と並行して本発明の検査法を実施して病態の変化をモニターすれば、治療効果を確認することができ、的確な治療方針の決定を可能にする。このように本発明の方法は、肝疾患に罹患した患者に対する的確な治療方針の決定に有益な情報を与えるものであり、患者のQOL（生活の質）向上に多大な貢献をする。
尚、この態様での評価についても、上記の態様と同様に、同一の検体中に含まれる特定の標準糖鎖（内部標準）の量を利用して行うことにしてもよい。即ち、以下の(1)〜(4)のいずれかで算出される値（マーカー糖鎖の比率）を用いてマーカー糖鎖の量を評価してもよい。この場合、エピメル化されていない糖鎖とエピメル化後の糖鎖の総量、エピメル化されていない糖鎖の量、又はエピメル化された糖鎖の量を用いることができる。
(1)糖鎖bの量／糖鎖aの量、(2)糖鎖bの量／糖鎖cの量、(3)糖鎖bの量／（糖鎖aの量＋糖鎖cの量）、(4)糖鎖bの量／（糖鎖aの量＋糖鎖bの量＋糖鎖cの量）
In yet another aspect of the present invention, the amount of marker sugar chain in a sample measured at a certain point in time is compared with the amount of marker sugar chain in a sample previously collected from the same subject. Evaluate the presence or absence and / or degree of increase or decrease. The test result (data relating to the change in the amount of the marker sugar chain) in this embodiment is particularly useful information for monitoring the progression of the pathology of liver disease. That is, using the test result that a decrease in the amount of the marker sugar chain was observed, for example, the pathology progressed from cirrhosis to hepatocellular carcinoma between the previous test and the current test, or from cirrhosis to hepatocyte It can be determined that there are signs of progression of the condition to the cancer. On the other hand, using the test result that an increase in the amount of marker sugar chain was observed, for example, it was determined that the pathological condition improved from the previous test to the current test, or signs of improvement in the pathological condition were observed can do. Furthermore, using the test result that the change in the amount of the marker sugar chain is not recognized, for example, it can be determined that the pathological condition does not change between the previous test and the current test.
If the change of the disease state is monitored by carrying out the test method of the present invention in parallel with the treatment, the treatment effect can be confirmed, and an accurate treatment policy can be determined. As described above, the method of the present invention provides useful information for determining an accurate treatment policy for a patient suffering from liver disease, and greatly contributes to improving the QOL (quality of life) of the patient.
The evaluation in this aspect may also be performed using the amount of a specific standard sugar chain (internal standard) contained in the same specimen, as in the above aspect. That is, the amount of marker sugar chain may be evaluated using the value (marker sugar chain ratio) calculated in any of the following (1) to (4). In this case, the total amount of sugar chains that are not epimerized and sugar chains after epimerization, the amount of sugar chains that are not epimerized, or the amount of sugar chains that are epimerized can be used.
(1) Amount of sugar chain b / amount of sugar chain a, (2) Amount of sugar chain b / amount of sugar chain c, (3) Amount of sugar chain b / (amount of sugar chain a + sugar chain c Amount), (4) Amount of sugar chain b / (Amount of sugar chain a + Amount of sugar chain b + Amount of sugar chain c)
＜肝細胞癌マーカーの検索＞
新規な肝細胞癌マーカーを見出すことを目的として、肝細胞癌患者の血清の糖鎖プロファイリング（構造解析）を実施した。
１．実験方法
肝細胞癌と診断された合計９名の患者について、肝細胞癌発症前及び発症後の血清を用意した（９検体×２）。肝細胞癌発症前の検体は、肝細胞癌発症後よりも６〜８年前のものである。これらの患者は全てC型肝炎ウイルス（Hepatitis C virus,以下「HCV」と略記する）感染者であった。比較対照として健常者４名の血清を用いた。以上の１３名２２検体について、血清中のN結合型糖鎖の構造を解析した。構造解析には3-Dマッピング法（Trendes in Glycosciene and Glycotechnology Vol15 No.84 july2003)を利用した。3-Dマッピング法とは、糖タンパク質より切り出されたN-結合型糖鎖を蛍光ラベル化した後、３種類のカラム（DEAEカラム、ODSカラム、Amideカラム）を利用した高速液体クロマトグラフィー（HPLC；SHIMAZU LabSolutionを使用）で分離し、溶出位置から構造を決定する方法であり、GALAXY（Glycoanalysis by the three axes of MS and chromatography）としてインターネット上で公開されている（http://www.glycoanalysis.info/）。3-Dマッピング法では、各カラムでの溶出時間から糖鎖の構造を決定できる。
<Search for hepatocellular carcinoma marker>
In order to find a novel hepatocellular carcinoma marker, sugar chain profiling (structural analysis) was performed on the serum of hepatocellular carcinoma patients.
1. Experimental Method Serum before and after the onset of hepatocellular carcinoma was prepared for a total of 9 patients diagnosed with hepatocellular carcinoma (9 specimens × 2). The specimen before the onset of hepatocellular carcinoma is from 6 to 8 years before the onset of hepatocellular carcinoma. All of these patients were infected with hepatitis C virus (hereinafter abbreviated as “HCV”). As a comparative control, sera from 4 healthy subjects were used. The structure of N-linked sugar chains in serum was analyzed for the above 13 subjects and 22 samples. The 3-D mapping method (Trendes in Glycosciene and Glycotechnology Vol15 No.84 july2003) was used for the structural analysis. The 3-D mapping method refers to high-performance liquid chromatography (HPLC) using three types of columns (DEAE column, ODS column, Amide column) after fluorescently labeling N-linked glycans excised from glycoproteins. ; Using SHIMAZU LabSolution) and determining the structure from the elution position, GALAXY (Glycoanalysis by the three axes of MS and chromatography) is published on the Internet (http: //www.glycoanalysis. info /). In the 3-D mapping method, the sugar chain structure can be determined from the elution time in each column.
肝細胞癌患者から採取された検体（血清、300μl）について、まず、安全性を確保するために100℃で10分のウイルス不活化をした後、減圧乾燥させた。この条件でタンパク変性を行ったことにもなる。健常者から採取された検体についても同様の操作を行った。次に以下の手順で糖鎖の遊離（糖タンパク質からの切り出し）を行った。まず、上記操作後の各試料に0.5Mクエン酸緩衝液（pH4.0）を110μl、ペプシン（シグマ社）430μg、グリコアミダーゼA（生化学工業社）0.5mUを添加後、37℃で一晩反応させてN結合型糖鎖を遊離させた。混在しているペプチドをアミノ酸にまで分解するためにプロナーゼ50μｇ、1Mトリス塩酸緩衝液（pH8.1）90μlを添加後、37℃一晩反応させた。次に糖鎖とペプチド（アミノ酸）を分離するため、試料をP-2バイオゲル（ゲル濾過カラム, Bio-Rad社）に供した。280nmの吸光度をモニタリングしながら超純水（MQ水）で溶出し（DETECTOR UV-2000及びFRACTION COLLECTOR PC-1500を使用）、オルシノール硫酸反応を利用して糖鎖を分取した後、一晩減圧乾燥させた。尚、オルシノール硫酸反応とは、糖鎖が存在すると紫色に発色するというものであり、各分画の50μlを乾固した後、5μlのMQ水に溶解してTLCプレートに添加、乾燥し、続いて2％オルシノール／50%硫酸を噴霧した後、加熱・発色させた。 A specimen (serum, 300 μl) collected from a patient with hepatocellular carcinoma was first inactivated at 100 ° C. for 10 minutes and then dried under reduced pressure to ensure safety. This also means that protein denaturation was performed under these conditions. The same operation was performed on samples collected from healthy individuals. Next, the sugar chain was released (cut out from the glycoprotein) by the following procedure. First, 110 μl of 0.5 M citrate buffer (pH 4.0), 430 μg of pepsin (Sigma) and 0.5 mU of glycoamidase A (Seikagaku) were added to each sample after the above operation, and overnight at 37 ° C. The N-linked sugar chain was released by reaction. In order to decompose the mixed peptide into amino acids, 50 μg of pronase and 90 μl of 1M Tris-HCl buffer (pH 8.1) were added, followed by reaction at 37 ° C. overnight. Next, in order to separate the sugar chain and the peptide (amino acid), the sample was subjected to P-2 biogel (gel filtration column, Bio-Rad). Elution with ultrapure water (MQ water) while monitoring absorbance at 280 nm (using DETECTOR UV-2000 and FRACTION COLLECTOR PC-1500), separating sugar chains using orcinol-sulfuric acid reaction, and then reducing the pressure overnight Dried. The orcinol-sulfuric acid reaction is to develop a purple color when sugar chains are present. After drying 50 μl of each fraction, dissolve in 5 μl of MQ water, add to the TLC plate, dry, After spraying with 2% orcinol / 50% sulfuric acid, the mixture was heated and colored.
続いて糖鎖の還元末端を蛍光ラベル化した。即ち、試料に２−アミノピリジン酢酸溶液を添加して90℃で60分間反応させた後、ジメチルアミン・ボラン酢酸水溶液を添加して80℃で35分間反応させた。反応終了後の試料をSephadex G-15カラム（Amersham Bioscience社）に供し、PA化糖鎖を含む画分を分取し、一晩減圧乾燥させた。このようにして得られた試料を、DEAEカラムに使用するA液（以下に示す）100μlに溶解した後、15μl分をDEAEカラム（TSK-Gel、DEAE−5PW 75×7.5mm、Tosoh）を用いたHPLCで分画した。その結果得られた中性糖鎖画分、モノシアリル糖鎖画分、ジシアリル糖鎖画分、トリシアリル糖鎖画分、及びテトラシアリル糖鎖画分を一晩減圧乾燥した。
Subsequently, the reducing end of the sugar chain was fluorescently labeled. That is, a 2-aminopyridine acetic acid solution was added to the sample and reacted at 90 ° C. for 60 minutes, and then an aqueous dimethylamine / borane acetic acid solution was added and reacted at 80 ° C. for 35 minutes. The sample after completion of the reaction was applied to a Sephadex G-15 column (Amersham Bioscience), and a fraction containing a PA sugar chain was collected and dried overnight under reduced pressure. The sample obtained in this way is dissolved in 100 μl of solution A used for the DEAE column (shown below), and then 15 μl is used for the DEAE column (TSK-Gel, DEAE-
次に、上記の方法で得られた各試料をMQ水50μｌに溶解した後、中性糖鎖を含む試料は25μlを、モノシアリル糖鎖を含む試料は5μlを、ジシアリル糖鎖を含む試料は1μlを、トリシアリル糖鎖を含む試料は5μlをODS逆相カラム（Shim−pack HRC-ODS 6.0×150mm、島津製作所）に供した。ODS逆相カラムを用いたHPLCの溶出チャートから、トリシアリル糖鎖群の中に肝細胞癌発症前後で存在量の異なる糖鎖が認められた。糖鎖構造の決定のため、当該糖鎖を含む溶出画分をAmideカラム（TSK-Gel AMIDE−80 4.6×250mm）を用いたHPLCに供した。ODS逆相カラムの溶出結果と、Amideカラムの溶出結果から目的の糖鎖のグルコースユニットを計算し、3-Dマッピング法（GALAXY、http://www.glycoanalysis.info/）で構造を検索し、候補の糖鎖を絞り込んだ。次に、選ばれた候補の標準品と試料（目的の糖鎖を含む画分）をODSカラムに共打ちし、目的の糖鎖の構造の同定を試みた。
以下に上記の各HPLC分析の条件を記載する。DEAEカラムを用いたHPLC分析、ODS逆相カラムを用いたHPLC分析、Amideカラムを用いたHPLC分析の条件を記載する。
(1)DEAEカラムを用いたHPLC分析
カラム温度：30℃
溶出条件：B液0%（5min）〜20％（45min）の連続勾配
A液：10％アセトニトリル含有0.01％トリエチルアミン水溶液
B液：10％アセトニトリル含有7.4％トリエチルアミン水溶液（pH7.25〜7.34）
(2)ODS逆相カラムを用いたHPLC分析
カラム温度55℃
溶出条件：B液20%（0min）〜B液50%（60min）の連続勾配、流速1ml/min
検出条件：励起波長320nm、蛍光波長400nm
A液：0.01Mリン酸緩衝液(pH3.8)
B液：ブタノールを終濃度0.5％でA液に添加して得られた溶液
(3)Amideカラムを用いたHPLC分析
カラム温度：40℃
溶出条件： B液0%（0min）〜B液60%（30min）の連続勾配、流速1ml/min
検出条件：励起波長320nm、蛍光波長400nm
A液：10％アセトニトリル含有7.4％トリエチルアミン水溶液（pH7.25〜7.34）とアセトニトリルを３５：５５の比率で混合した溶液
B液：10％アセトニトリル含有7.4％トリエチルアミン水溶液（pH7.25〜7.34）とアセトニトリルを50:40の比率で混合した溶液
Next, after each sample obtained by the above method is dissolved in 50 μl of MQ water, the sample containing neutral sugar chains is 25 μl, the sample containing monosialyl sugar chains is 5 μl, and the sample containing disialyl sugar chains is 1 μl. 5 μl of the sample containing the trisialyl sugar chain was applied to an ODS reverse phase column (Shim-pack HRC-ODS 6.0 × 150 mm, Shimadzu Corporation). From the HPLC elution chart using an ODS reversed phase column, sugar chains with different abundances were observed in the trisialyl sugar chain group before and after the onset of hepatocellular carcinoma. In order to determine the sugar chain structure, the eluted fraction containing the sugar chain was subjected to HPLC using an Amide column (TSK-Gel AMIDE-80 4.6 × 250 mm). The glucose unit of the target sugar chain is calculated from the elution results of the ODS reverse phase column and the Amide column, and the structure is searched by the 3-D mapping method (GALAXY, http://www.glycoanalysis.info/). , Narrowed down the candidate sugar chains. Next, the selected candidate standard and the sample (fraction containing the target sugar chain) were co-imposed on the ODS column to attempt to identify the structure of the target sugar chain.
The conditions for each of the above HPLC analyzes are described below. The conditions for HPLC analysis using a DEAE column, HPLC analysis using an ODS reverse phase column, and HPLC analysis using an Amide column are described.
(1) HPLC analysis using DEAE column Column temperature: 30 ° C
Elution condition: B liquid 0% (5min) to 20% (45min) continuous gradient
Liquid A: 0.01% triethylamine aqueous solution containing 10% acetonitrile
Liquid B: 7.4% aqueous triethylamine solution containing 10% acetonitrile (pH 7.25 to 7.34)
(2) HPLC analysis using ODS reverse phase
Elution condition: Continuous gradient from 20% B solution (0min) to 50% B solution (60min), flow rate 1ml / min
Detection conditions: excitation wavelength 320 nm, fluorescence wavelength 400 nm
A solution: 0.01M phosphate buffer (pH3.8)
Liquid B: A solution obtained by adding butanol to Liquid A at a final concentration of 0.5%
(3) HPLC analysis using Amide column Column temperature: 40 ° C
Elution conditions: Continuous gradient from
Detection conditions: excitation wavelength 320 nm, fluorescence wavelength 400 nm
Solution A: A solution prepared by mixing 7.4% triethylamine aqueous solution (pH 7.25 to 7.34) containing 10% acetonitrile and acetonitrile in a ratio of 35:55
Liquid B: A solution in which 7.4% triethylamine aqueous solution (pH 7.25 to 7.34) containing 10% acetonitrile and acetonitrile are mixed at a ratio of 50:40
２．結果
今回の分析で確認できる全てのトリシアリル糖鎖の構造を図１の表に示す。一方、肝細胞癌患者検体のODS分析結果（溶出チャート）の代表例を図２（肝細胞癌発症前）及び図３（肝細胞癌発症後）に示す。肝細胞癌発症前の溶出チャート（図２）では、10min〜15minの間に３種類の糖鎖（Ta、Tb、Tc）が認められる。他方、肝細胞癌発症後の溶出チャート（図３）ではTa及びTcのピークは認められるものの、Tbのピークが消失している。このように、ODS逆相カラムを用いたHPLC分析の結果、血清中のトリシアリル糖鎖群の中に肝細胞癌発症前後で量が顕著に変動する糖鎖を認めた。Amideカラムを用いたHPLC分析、及びその後の候補標準品との共打ち試験による構造解析の結果、当該トリシアリル糖鎖（ピーク番号Tb）が以下の構造からなり、Code No 3A2-300.8で表される糖鎖であることが判明した。
尚、説明の便宜上、当該糖鎖を「糖鎖b」と呼称し、同様にピークTa及びTcに対応する糖鎖をそれぞれ「糖鎖a」及び「糖鎖c」と呼称する。 For convenience of explanation, the sugar chain is referred to as “sugar chain b”, and the sugar chains corresponding to the peaks Ta and Tc are also referred to as “sugar chain a” and “sugar chain c”, respectively.
Taよりも早い溶出のピーク（Ta’、Tb’、Tc’）は３種類の糖鎖（糖鎖a、糖鎖b、糖鎖c）がエピメル化されたものであることがわかった。エピメル化された糖鎖は、エピメル化前の糖鎖よりもODS分析における溶出時間が早いことが知られている。従って、エピメル化糖鎖のグルコースユニット値はエピメル化前の糖鎖のそれと異なる。一方で、エピメル化前後で分子量は変化しないため、Amide分析ではエピメル化された糖鎖とエピメル化前の糖鎖の間でグルコースユニット値は変わらない。 It was found that the elution peaks (Ta ′, Tb ′, Tc ′) earlier than Ta were epimerized three types of sugar chains (sugar chain a, sugar chain b, sugar chain c). It is known that epimerized sugar chains have a faster elution time in ODS analysis than sugar chains before epimerization. Therefore, the glucose unit value of the epimerized sugar chain is different from that of the sugar chain before epimerization. On the other hand, since the molecular weight does not change before and after epimerization, Amide analysis does not change the glucose unit value between the epimerized sugar chain and the sugar chain before epimerization.
各検体のODS分析の結果を図４の表にまとめた。この表において1A〜9Bは患者検体、10〜13は健常者検体を表す。患者検体の検体No.については、肝細胞癌発症前に採取された検体であることを表すために数字の次にAを付し、肝細胞癌発症後に採取された検体であることを表すために同様にBを付した。
表の左欄には、各糖鎖のピーク面積（TA、TB、TC）及びトリシアリル糖鎖全体のピーク面積（Tri-Total）を示した。尚、TAはTaのピーク面積値とTa'のピーク面積値を合算したものであり、エピメル化された分も含めた糖鎖aの量に対応する。同様に、TBはTbのピーク面積値とTb'のピーク面積値を合算したもの、TCはTcのピーク面積値とTc'のピーク面積値を合算したものである。一方、表の右欄にはピーク面積比を示した。
The results of ODS analysis for each specimen are summarized in the table of FIG. In this table, 1A to 9B represent patient specimens, and 10 to 13 represent healthy subject specimens. In order to indicate that the sample number of the patient sample is a sample collected before the onset of hepatocellular carcinoma, an A is added after the number to indicate that the sample was collected after the onset of hepatocellular carcinoma. In the same way, B was attached.
The left column of the table shows the peak area (TA, TB, TC) of each sugar chain and the peak area (Tri-Total) of the entire trisialyl sugar chain. TA is the sum of the peak area value of Ta and the peak area value of Ta ′, and corresponds to the amount of sugar chain a including the amount of epimerization. Similarly, TB is the sum of the peak area value of Tb and the peak area value of Tb ′, and TC is the sum of the peak area value of Tc and the peak area value of Tc ′. On the other hand, the peak area ratio is shown in the right column of the table.
表の左欄から明らかなように、肝細胞癌患者９名中４名では肝細胞癌後の検体（1B、2B、3B、4B）で糖鎖bの消失が認められる。また肝細胞癌患者中１名（7A,7B）では肝細胞癌前後で糖鎖bの量の顕著な減少を認めた。さらに、肝細胞癌患者中２名（5A,5B、9A,9B）では肝細胞癌前後で糖鎖bの量が減少する傾向が認められる。このように、肝細胞癌前後の検体を比較したとき、肝細胞癌後の検体において糖鎖bが消失するか又肝細胞癌前の検体に比較して糖鎖bの量が減少する割合は約８０％であった。この結果より、糖鎖bの量に注目すれば、肝細胞癌への移行（即ち病態の悪化）を高い的中率で判定可能であるといえる。
一方、肝細胞癌患者９名中６名の肝細胞癌発症後の検体（1B、2B、3B、4B、5B、7B）では糖鎖bの量（TBのピーク面積値）が健常者の糖鎖bの量（平均値）よりも少ない。即ち、肝細胞癌発症後の検体では糖鎖bの量が健常者に比較して少ない傾向にある。このことから、肝細胞癌の判定の指標として糖鎖bの量が有用であるといえる。
As is clear from the left column of the table, in 4 of 9 patients with hepatocellular carcinoma, the loss of sugar chain b is observed in specimens after hepatocellular carcinoma (1B, 2B, 3B, 4B). In one hepatocellular carcinoma patient (7A, 7B), a significant decrease in the amount of sugar chain b was observed before and after hepatocellular carcinoma. Furthermore, in 2 patients with hepatocellular carcinoma (5A, 5B, 9A, 9B), the amount of sugar chain b tends to decrease before and after hepatocellular carcinoma. Thus, when comparing specimens before and after hepatocellular carcinoma, the rate at which sugar chain b disappears in the specimen after hepatocellular carcinoma or the amount of sugar chain b decreases compared to the specimen before hepatocellular carcinoma is About 80%. From this result, if attention is paid to the amount of sugar chain b, it can be said that the transition to hepatocellular carcinoma (that is, the deterioration of the disease state) can be determined with a high predictive value.
On the other hand, in the specimens (1B, 2B, 3B, 4B, 5B, 7B) after the onset of hepatocellular carcinoma in 9 out of 9 hepatocellular carcinoma patients, the amount of sugar chain b (TB peak area value) is a healthy sugar. Less than the amount of chain b (average value). That is, in the specimen after hepatocellular carcinoma onset, the amount of sugar chain b tends to be smaller than that in healthy subjects. From this, it can be said that the amount of sugar chain b is useful as an index for determination of hepatocellular carcinoma.
ここで糖鎖a及び糖鎖cに注目すると当該糖鎖の量は、糖鎖bの場合に比較して、肝細胞癌発症前後で変動が小さい。このことから、肝細胞癌との顕著な関連性が認められる糖鎖bの量を評価するための対照（内部標準）として糖鎖aの量及び糖鎖cの量が有用であるといえる。そこで、肝細胞癌発症後の検体について糖鎖aの量及び／又は糖鎖cの量に対する糖鎖bの量の比率（TB/TAやTB/TCなどの面積比、表の右欄を参照）を求め、健常者の対応する値と比較してみると、一部の例外を除いて、両者の間に顕著な差が認められた。また、トリシアリル糖鎖全体の量に対する糖鎖bの量の比率（TB/Tri total）についても同様の比較を行ったところ同じ傾向が認められた。尚、糖鎖aや糖鎖cについてはこのような傾向は認められない。以上のように、糖鎖a、糖鎖c又はトリシアリル糖鎖全体の量に対する糖鎖bの量の比率が肝細胞癌の判定に有用であることが示された。
一方、肝細胞癌発症前後で糖鎖bの量の比率（TB/TA、TB/TC、TB/Tri total）を比較すれば、一部の例外を除いて、肝細胞癌発症後の方がその値が小さい。このように、糖鎖bの量の比率が減少することと、肝細胞癌への移行との間に高い相関が認められた。換言すれば、糖鎖bの量の比率は、肝細胞癌の移行を判定するための有効な指標であることが示された。
Here, when attention is paid to sugar chain a and sugar chain c, the amount of the sugar chain is smaller before and after the onset of hepatocellular carcinoma than in the case of sugar chain b. From this, it can be said that the amount of sugar chain a and the amount of sugar chain c are useful as a control (internal standard) for evaluating the amount of sugar chain b, which is remarkably related to hepatocellular carcinoma. Therefore, the ratio of the amount of sugar chain a to the amount of sugar chain a and / or the amount of sugar chain c for the specimen after the onset of hepatocellular carcinoma (area ratio of TB / TA, TB / TC, etc., see the right column of the table) ) And compared with the corresponding values of healthy individuals, with some exceptions, there was a marked difference between the two. Moreover, the same tendency was recognized when the same comparison was performed also about the ratio (TB / Tri total) of the quantity of the sugar chain b with respect to the quantity of the whole trisialyl sugar chain. Note that such a tendency is not observed for sugar chain a and sugar chain c. As described above, it was shown that the ratio of the amount of sugar chain b to the total amount of sugar chain a, sugar chain c, or trisialyl sugar chain is useful for the determination of hepatocellular carcinoma.
On the other hand, if the ratio of the amount of sugar chain b (TB / TA, TB / TC, TB / Tri total) is compared before and after the onset of hepatocellular carcinoma, with the exception of some exceptions, The value is small. Thus, a high correlation was observed between the decrease in the ratio of the amount of sugar chain b and the transition to hepatocellular carcinoma. In other words, it was shown that the ratio of the amount of sugar chain b is an effective index for determining the transition of hepatocellular carcinoma.
本発明の肝細胞癌マーカーは肝細胞癌の検査に利用され得る。本発明の肝細胞癌マーカーを利用した検査法（本発明の検査法）は、従来法よりも見逃しの少ない、確度の高い肝細胞癌の診断を可能とする。本発明の検査法は、肝細胞癌の判定、患者の病態の変化（例えば肝硬変から肝細胞癌への進行）をモニターすること、肝細胞癌に対する治療の効果の検証等に利用することができる。
本発明の肝細胞癌マーカーは肝癌患者の血清中に見出され、少量の血清を用いてその量の評価を行うことが可能である。従って、本発明の肝細胞癌マーカーを指標とすれば、通常の生化学検査や血清検査のために採血されたわずかな検体（血清）の一部を用いた糖鎖分析によって肝細胞癌の検査・診断が可能となる。
一方、本発明の肝細胞癌マーカーを研究目的での使用に供してもよい。例えば、肝細胞癌の発症メカニズムの研究に本発明の肝細胞癌マーカーを利用することができる。
The hepatocellular carcinoma marker of the present invention can be used for examination of hepatocellular carcinoma. The test method using the hepatocellular carcinoma marker of the present invention (the test method of the present invention) makes it possible to diagnose hepatocellular carcinoma with high accuracy and fewer misses than the conventional method. The test method of the present invention can be used for determination of hepatocellular carcinoma, monitoring changes in patient pathology (eg, progression from cirrhosis to hepatocellular carcinoma), verification of the effect of treatment on hepatocellular carcinoma, and the like. .
The hepatocellular carcinoma marker of the present invention is found in the serum of liver cancer patients, and the amount can be evaluated using a small amount of serum. Accordingly, if the hepatocellular carcinoma marker of the present invention is used as an index, hepatocellular carcinoma is examined by sugar chain analysis using a small portion of the sample (serum) collected for normal biochemical examination or serum examination.・ Diagnosis is possible.
On the other hand, the hepatocellular carcinoma marker of the present invention may be used for research purposes. For example, the hepatocellular carcinoma marker of the present invention can be used to study the onset mechanism of hepatocellular carcinoma.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
Claims (8)
(1)被験者より採取された検体中の前記肝細胞癌マーカーの量を測定するステップ、及び
(2)前記ステップで測定された前記肝細胞癌マーカーの量と、以前に同一の被験者より採取された検体中の前記肝細胞癌マーカーの量とを比較し、前記肝細胞癌マーカーの量の増減を評価するステップ。 The test for hepatocellular carcinoma according to claim 2, comprising the following steps (1) and (2), wherein an increase or decrease in the amount of the hepatocellular carcinoma marker in a sample collected from a subject is examined: Law,
(1) measuring the amount of the hepatocellular carcinoma marker in a sample collected from a subject; and
(2) The amount of the hepatocellular carcinoma marker measured in the step is compared with the amount of the hepatocellular carcinoma marker in a specimen previously collected from the same subject, and the amount of the hepatocellular carcinoma marker Step to evaluate the increase or decrease.
(1)被験者より採取された検体中の前記肝細胞癌マーカーの量を測定するステップ、及び
(2)前記ステップで測定された前記肝細胞癌マーカーの量と、健常者より採取された検体中の前記肝細胞癌マーカーの量とを比較し、両者の差又は比を評価するステップ。 A difference between the amount of the hepatocellular carcinoma marker in a sample collected from a subject and the amount of the hepatocellular carcinoma marker in a sample collected from a healthy person, comprising the following steps (1) and (2): The method for testing hepatocellular carcinoma according to claim 2, characterized in that the ratio is evaluated,
(1) measuring the amount of the hepatocellular carcinoma marker in a sample collected from a subject; and
(2) comparing the amount of the hepatocellular carcinoma marker measured in the step with the amount of the hepatocellular carcinoma marker in a sample collected from a healthy person, and evaluating the difference or ratio between the two.
(1-1)被験者より採取された検体から糖鎖を調製するステップ、
(1-2)調製した糖鎖を標識化するステップ、
(1-3)標識化糖鎖を陰イオン交換カラムに供し、トリシアリル糖鎖画分を分取するステップ、及び
(1-4)分取したトリシアリル糖鎖画分を、ＯＤＳシリカカラムを使用した高速液体クロマトグラフィーに供し、該高速液体クロマトグラフィーの溶出パターンを分析して前記肝細胞癌マーカーの量を算出するステップ。 The test method for hepatocellular carcinoma according to claim 5 or 6 , wherein the step (1) comprises the following steps:
(1-1) preparing a sugar chain from a sample collected from a subject;
(1-2) labeling the prepared sugar chain,
(1-3) subjecting the labeled sugar chain to an anion exchange column and fractionating the trisialyl sugar chain fraction; and
(1-4) The fractionated trisialyl sugar chain fraction is subjected to high performance liquid chromatography using an ODS silica column, and the elution pattern of the high performance liquid chromatography is analyzed to calculate the amount of the hepatocellular carcinoma marker. Step.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006104554A JP4752032B2 (en) | 2006-04-05 | 2006-04-05 | Hepatocellular carcinoma marker and test method for hepatocellular carcinoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006104554A JP4752032B2 (en) | 2006-04-05 | 2006-04-05 | Hepatocellular carcinoma marker and test method for hepatocellular carcinoma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2007278803A JP2007278803A (en) | 2007-10-25 |
| JP4752032B2 true JP4752032B2 (en) | 2011-08-17 |
Family
ID=38680397
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2006104554A Expired - Fee Related JP4752032B2 (en) | 2006-04-05 | 2006-04-05 | Hepatocellular carcinoma marker and test method for hepatocellular carcinoma |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4752032B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100301203A1 (en) * | 2007-11-30 | 2010-12-02 | Nishimura Shin-Ichiro | Method of diagnostic heparic disease by sugar chain analysis |
| WO2010058605A1 (en) * | 2008-11-21 | 2010-05-27 | 国立大学法人 北海道大学 | Method for evaluating state of cells |
| US8557602B2 (en) | 2009-03-05 | 2013-10-15 | National Institute Of Advanced Industrial Science And Technology | Method for detecting and distinguishing intrahepatic cholangiocarcinoma |
| EP2479577A1 (en) * | 2009-09-18 | 2012-07-25 | Mitsubishi Chemical Corporation | Hepatocellular carcinoma marker |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3547879B2 (en) * | 1995-12-06 | 2004-07-28 | 株式会社中埜酢店 | Biochemical test method using sialyl sugar chain in fibrinogen and method for purifying fibrinogen |
| US20040147033A1 (en) * | 2002-12-20 | 2004-07-29 | Zachary Shriver | Glycan markers for diagnosing and monitoring disease |
| US20050221397A1 (en) * | 2004-03-30 | 2005-10-06 | Northern Advancement Center For Science & Technology | RM2 antigen (beta1,4-GalNAc-disialyl-Lc4) as prostate cancer-associated antigen |
-
2006
- 2006-04-05 JP JP2006104554A patent/JP4752032B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2007278803A (en) | 2007-10-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8003392B2 (en) | Diagnostic test for the detection of early stage liver cancer | |
| JP6415547B2 (en) | Pancreatic cancer diagnostic composition and pancreatic cancer diagnostic method using the same | |
| US20070292869A1 (en) | Compositions and Methods for Analyzing Renal Cancer | |
| JP5737761B2 (en) | Hepatocellular carcinoma marker | |
| AU2006304605A1 (en) | Tissue-and serum-derived glycoproteins and methods of their use | |
| JP6612414B2 (en) | SRM assay for PD-L1 | |
| JP2009168470A (en) | Pancreas cancer marker and examination method of pancreas cancer | |
| KR101219519B1 (en) | A method for the diagnosis using lectin | |
| US20100055723A1 (en) | Galectin-3-Binding Protein as a Biomarker of Cardiovascular Disease | |
| CN109425739B (en) | Application of a group of proteins as tumor markers in preparation of malignant tumor diagnosis reagent and kit | |
| JP4752032B2 (en) | Hepatocellular carcinoma marker and test method for hepatocellular carcinoma | |
| EP2333552B1 (en) | Novel biomarkers for nonalcoholic fatty liver disease and methods for detecting nonalcoholic fatty liver disease using biomarker | |
| EP4414708A1 (en) | Biomarker for cancer diagnosis and use thereof | |
| CN101576495A (en) | Method for detecting N-glycome log (P9/P4) in serum and detecting system and application thereof | |
| JP6311608B2 (en) | Method for detecting liver cancer and method for detecting cirrhosis | |
| WO2011088026A2 (en) | Biomarkers of liver injury | |
| KR20150020392A (en) | Method for screening cancer marker based on de-glycosylation of glycoproteins and marker for HCC | |
| Bakry et al. | Recent advances in capillary electrophoresis for biomarker discovery | |
| JP2011512149A (en) | Cancer detection methods and techniques | |
| JP6294118B2 (en) | Colorectal cancer marker and method for detecting colorectal cancer | |
| US20080319678A1 (en) | Mass Tagging for Quantitative Analysis of Biomolecules using 13C Labeled Phenylisocyanate | |
| CN112782297A (en) | Liver cirrhosis related biomarker and screening method and application thereof | |
| JP6160085B2 (en) | Method for detecting hepatocellular carcinoma | |
| Hood et al. | Development of High-Throughput Mass Spectrometry–Based Approaches for Cancer Biomarker Discovery and Implementation | |
| WO2010071120A1 (en) | Method for diagnosing lung cancer by sugar chain analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20070719 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20070723 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20081031 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20081031 |
|
| RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20090123 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20101101 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20101124 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20101221 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20110111 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20110111 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20110419 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110422 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140603 Year of fee payment: 3 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |