JP7374196B2 - ヘリコバクターピロリ連関疾患に対する遺伝子マーカー - Google Patents
ヘリコバクターピロリ連関疾患に対する遺伝子マーカー Download PDFInfo
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Description
本出願は、ヘリコバクターピロリの感染により発現が増加または減少する遺伝子の発現量を測定する製剤を含むヘリコバクターピロリ連関疾患の診断用組成物、及び前記組成物を含むヘリコバクターピロリ連関疾患の診断用キットを提供する。本出願の一具現形態において、前記ヘリコバクターピロリの感染により発現が増加する遺伝子は、FGF21(fibroblast growth factor 21.Gene ID:26291)、CTH(cystathionine g-lyase.Gene ID:1491)、CREBRF(cAMP responsive element binding protein.Gene ID:153222)、DLL4(delta-like 4.Gene ID:54567)、FGF18(fibroblast growth factor 18.Gene ID:8817)、FOS(Fos proto-oncogene、AP-1 transcription factor subunit.Gene ID:2353)、PDK1(phosphoinositide-dependent kinase-1、Gene ID:5170)、PTPRN(receptor-type tyrosine-protein phosphatase-like N、Gene ID:5798)、CLIC4(chloride intracellular channel 4.Gene ID:25932)、PTPRB(protein tyrosine phosphatase、receptor type B.Gene ID:5787)、PPP1R15A(protein phosphatase 1 regulatory subunit 15A.Gene ID:23645)、PTPRH(protein tyrosine phosphatase、receptor type H.Gene ID:5794)、DDIT3(DNA damage inducible transcript3.Gene ID:1649)、BIRC3(baculoviral IAP repeat containing3.Gene ID:330)、FOXO3(forkhead box O3.Gene ID:2309)、BCL2(BCL2、apoptosis regulator.Gene ID:596)、PRKCE(protein kinase C epsilon.Gene ID:5581)、CHMP4C(charged multivesicular body protein 4C.Gene ID:92421)、CLDN14(claudin 14.Gene ID:23562)、SLC7A11(solute carrier family 7 member 11.Gene ID:23657)、BOC(BOC cell adhesion associated、oncogene regulated.Gene ID:91653)、AJUBA(ajuba LIM protein.Gene ID:84962)、LMO7(LIM domain 7.Gene ID:4008)、MMP24(matrix metallopeptidase 24.Gene ID:10893)、C3orf58(divergent protein kinase domain 2A.Gene ID:205428)、KLF10(Kruppel like factor 10.Gene ID:7071)、CSGALNACT1(chondroitin sulfate N-acetylgalactosaminyltransferase 1.Gene ID:55790)、CEP120(centrosomal protein 120.Gene ID:153241)、CHAC1(ChaC glutathione specific gamma-glutamylcyclotransferase 1.Gene ID:79094)、ASNS(asparagine synthetase(glutamine-hydrolyzing).Gene ID:440)、NFE2L2(nuclear factor、erythroid 2 like 2.Gene ID:4780)、RIOK3(RIO kinase3.Gene ID:8780)、TXNIP(thioredoxin interacting protein.Gene ID:10628)、MTR(5-methyltetrahydrofolate-homocysteine methyltransferase.Gene ID:4548)、IFRD1(interferon related developmental regulator 1.Gene ID:3475)、及びこれらの組み合わせよりなる群から選択されてよいが、これに限定されるものではなく、前記遺伝子の発現の増加有無を確認することによりヘリコバクターピロリの感染有無を診断することができる。本出願の一具現形態において、前記ヘリコバクターピロリの感染により発現が増加する遺伝子は、FGF21、CTH、CREBRF、PTPRN及びこれらの組み合わせよりなる群から選択されてよいが、これに限定されるものではない。
本出願の他の側面は、個体のサンプルから試料DNAを準備するステップ;前記試料DNAを本出願の組成物またはキットを用いて増幅するステップ;増幅結果からヘリコバクターピロリ感染により発現が増加または減少する遺伝子の発現の程度を確認するステップを含む、個体のヘリコバクターピロリ連関疾患の診断のために必要な情報を提供する方法を提供する。
<1-1> ヘリコバクターピロリ感染の胃癌細胞モデル
胃腺癌種(AGS)は、ATCC(Manassas,VA)で購入して保管しながら用いた。全ての細胞は、蘇生後6ヶ月以上用いなかった。AGS細胞は、RPMI-1640培養液(Gibco BRL,Gaithersburg,MD)及び10%FBS(fetal bovine serum,Gibco BRL)が含まれている培地で37℃、5%CO2で培養した。細胞を6ウェルプレートに105細胞/ml濃度で分株し、24時間吸着させた後、細胞にヘリコバクターピロリを50MOI(5005 CFU)で6時間露出させて感染させた。
キムチは、通常のキムチの製法により製造した。坑癌キムチ(以下、「CpKimchi」又は「CPK」で示す場合がある)は、一般の白菜ではないリコペン含量が高い白菜を主原料に用い、ロイコノストック・メセンテロイデスCJLM119及びラクトバチルス・プランタラムCJLP133の2種の乳酸菌を用いたことを除いては、一般キムチ(以下、「sKimchi」又は「SK」で示す場合がある)の製法と同一に製造した。前記ロイコノストック・メセンテロイデスCJLM119は、韓国生命工学研究院の遺伝子バンクに受託番号KCTC 13043BPとして寄託された微生物菌株であって、前記菌株の分離及び同定の方法に対しては韓国登録特許第10-1807995号公報に具体的に開示されている。また、前記ラクトバチルス・プランタラムCJLP133は、韓国生命工学研究院の遺伝子バンクに受託番号KCTC 11403BPとして寄託された微生物菌株であって、前記菌株の分離及び同定の方法に対しては韓国登録特許第10-1486999号公報に具体的に開示されているので、その具体的な記載を省略する。
前記実施例<1-2>で製造されたキムチを凍結乾燥した後、粉砕して粉末として準備した。ここに20倍重量のメタノールを加えた後、攪拌機を用いて12時間抽出した。抽出された上澄み液を濾過紙で濾過し、残った沈殿物は再びメタノールを追加して12時間抽出した。前記抽出物を濃縮器で約40℃の温度で加温濃縮した濃縮物を製造した後、これを4℃で貯蔵して追加的な実験に用いた。
試験管内実験には、実施例<1-3>で製造したキムチ抽出物の濃縮液5 mg/ml及び10mg/mlでそれぞれ用いた。生体内実験のためのキムチ摂取量は、次のように設定した:韓国人の通常のキムチ摂取量は、個人の趣向に応じて一日に約30から100gであり、坑癌キムチ抽出物の濃縮液は、動物モデルの食餌療法ペレットに1週間に1回、体重2倍、1.7g/kg/日、5.1g/kg/日の比率で混合し、一般的な韓国人のキムチ摂取量に換算して用いた。
実施例<1-1>で製造したヘリコバクターピロリに感染した胃癌細胞の培養培地を吸引して除去した後、前記細胞をダルベッコ(Dulbecco)のPBSで2回洗浄した。RiboEX(500μl,GeneAll,Seoul,Korea)を4℃で10分間培養したプレートに添加した。RiboEXを収去して1.5mlチューブに移した後、100μlのクロロホルムを入れてゆっくり混合した。氷で10分間静置した後、サンプルを10,000gで30分間遠心分離した。上澄み液を200μlのイソプロパノールと混合した後、前記混合物を4℃で1時間静置した。13,000gで30分間遠心分離した後、沈殿物を70%(vol/vol)のエタノールで洗浄した。エタノールを完全に蒸発させた後、沈殿物を100μlのDEPC(Diethylene pyrocarbonate)で処理された水(Invitrogen Life Technologies,Carlsbad,CA)に溶解させた。モロニーマウス白血病ウイルス(Promega,Madison,WI)から由来された逆転写酵素を用いてcDNAを製造した。PCRは、94℃で20秒、58℃で30秒、72℃で45秒の30サイクルを用いて行った。
SMARTer Stranded RNA-Seqキット(Clontech Laboratories,Inc.,USA)を用いて2μgの総RNAからライブラリを準備した。mRNAの分離は、Poly(A)RNA Selection Kit(LEXOGEN,Inc.,Austria)を用いて行った。分離されたmRNAは、cDNA合成及び切断に用いた。インデックスは、Illumina指数1-12を用い、PCRを用いて増幅した。次いで、Agilent 2100 bioanalyzer(DNA High Sensitivity Kit)を用いてライブラリを確認することで、平均断片の大きさを評価した。定量は、Step OneリアルタイムPCRシステム(Life Technologies,Inc.,USA)を用いてライブラリ定量キットを用いた。大量-高効率配列分析(high-throughput sequencing)は、HiSeq 2500(Illumina,Inc.,USA)を用いてペアエンド(paired-end)100配列分析で行った。
mRNA-Seq分析は、整列ファイルを得るためにTopHatソフトウェア(Cole Trapnell et al.,2009)ツールを用いた。差別的に発現された遺伝子は、Bedtools(Quinlan AR,2010)の適用範囲を用いて固有及び多重整列の計数に基づいて決定した。RT(Read Count)データは、Bioconductor(Gentleman et al.,2004)を用いて統計プログラムR(R development Core Team,2016)のEdgeRを用いるQuantile正規化法に基づいて処理した。Cufflinksを用いて転写体を組み立て、存在量を推定し、発現及び調節を分析しており、遺伝子またはアイソ(iso)フォームの差別的な発現を検出するのに用いた。遺伝子領域の発現水準を決定する方法として、FPKM(fragments per kilobase of exon per million fragments)を用いた。遺伝子の分類は、DAVID(http://david.abcc.ncifcrf.gov/)で行った検索を基盤とした。
<2-1> RNAseqを介した遺伝子の確認
ヘリコバクターピロリ感染による標的遺伝子を探すためにRNAseq分析を行い、遺伝子中、ヘリコバクターピロリ感染で発現が増加したがCpKimchiの併用処理で正常化された遺伝子126個を確認した。これらの遺伝子は、ERストレス遺伝子、酸化ストレス遺伝子、組織再生遺伝子、血管新生遺伝子などで表れた(図1)。
RNAseqのヒートマップ分析結果をRT-PCRで検証した。血管新生遺伝子のうち、DLL4(delta-like 4)及びFGF18(fibroblast growth factor 18)がCpKimchi 併用処理後にその発現が有意に減少し、ERストレス遺伝子のうちFGF21(fibroblast growth factor 21)、CTH(cystathionine-lyase)及びCREBPF(cAMP responsive element binding protein)と、酸化的ストレス遺伝子のうちFOS、PDK1(phosphoinositide-dependent kinase-1)及びPTPRN(receptor-type tyrosine-protein phosphatase-like N)もまた、CpKimchi併用処理後にその発現が有意に減少した(図2)。
<3-1> RNAseqを介した遺伝子の確認
遺伝子中、ヘリコバクターピロリ感染で発現が減少したがCpKimchiの処理で正常化され発現が増加する遺伝子を分析した。総262個の遺伝子が同定された。ヘリコバクターピロリ感染により発現が減少したがCpKimchiの併用治療で正常化された遺伝子を分類してみると、細胞防御反応遺伝子、組織再生遺伝子、抗酸化遺伝子、細胞結合遺伝子などである(図3)。
RNAseqのヒートマップ分析結果をRT-PCRで検証し、ALB、NEO1(neogenin precursor1)、CLDN8(claudin 8)、KLRG1(killer cell lectin-like receptor subfamily G member 1)及び IGFBP1(insulin-like growth factor=binding protein 1)の場合、CpKimchi処理後に減少した遺伝子の発現が正常化されたことを確認した(図4)。
ヘリコバクターピロリ感染動物モデル実験において、RNAseq結果と同様にERストレス遺伝子の発現を確認した。ヘリコバクターピロリに感染したグループ2でERストレス遺伝子の発現が有意に増加しており、これはRNAseqの結果と一致した。ERストレスに関与するこれらの遺伝子は、CpKimchiが投与されたグループ3とグループ4で有意に改善され、やはりRNAseq結果と一致した(図5)。
ヘリコバクターピロリ感染動物モデル実験において、RNAseq結果と同様に抗酸化関連遺伝子の発現を確認した。抗酸化遺伝子は、ヘリコバクターピロリに感染したグループ2で有意に減少したが、CpKimchiが投与されたグループ3とグループ4で全て有意に発現が増加して回復された(図6)。
Claims (11)
- FGF21(fibroblast growth factor 21.Gene ID:26291)遺伝子の発現量を測定する製剤を含む、ヘリコバクターピロリの感染による胃癌の診断用組成物。
- CTH(cystathionine g-lyase.Gene ID:1491)またはCREBRF(cAMP responsive element binding protein.Gene ID:153222)遺伝子の発現量を測定する製剤を追加で含む、請求項1に記載のヘリコバクターピロリの感染による胃癌の診断用組成物。
- DLL4(delta-like 4.Gene ID:54567)、FGF18(fibroblast growth factor 18.Gene ID:8817)、FOS(Fos proto-oncogene、AP-1 transcription factor subunit.Gene ID:2353)、PDK1(phosphoinositide-dependent kinase-1、Gene ID:5170)、PTPRN(receptor-type tyrosine-protein phosphatase-like N、Gene ID:5798)、CLIC4(chloride intracellular channel 4.Gene ID:25932)、PTPRB(protein tyrosine phosphatase、receptor type B.Gene ID:5787)、PPP1R15A(protein phosphatase 1 regulatory subunit 15A.Gene ID:23645)、PTPRH(protein tyrosine phosphatase、receptor type H.Gene ID:5794)、DDIT3(DNA damage inducible transcript3.Gene ID:1649)、BIRC3(baculoviral IAP repeat containing3.Gene ID:330)、FOXO3(forkhead box O3.Gene ID:2309)、BCL2(BCL2、apoptosis regulator.Gene ID:596)、PRKCE(protein kinase C epsilon.Gene ID:5581)、CHMP4C(charged multivesicular body protein 4C.Gene ID:92421)、CLDN14(claudin 14.Gene ID:23562)、SLC7A11(solute carrier family 7 member 11.Gene ID:23657)、BOC(BOC cell adhesion associated、oncogene regulated.Gene ID:91653)、AJUBA(ajuba LIM protein.Gene ID:84962)、LMO7(LIM domain 7.Gene ID:4008)、MMP24(matrix metallopeptidase 24.Gene ID:10893)、C3orf58(divergent protein kinase domain 2A.Gene ID:205428)、KLF10(Kruppel like factor 10.Gene ID:7071)、CSGALNACT1(chondroitin sulfate N-acetylgalactosaminyltransferase 1.Gene ID:55790)、CEP120(centrosomal protein 120.Gene ID:153241)、CHAC1(ChaC glutathione specific gamma-glutamylcyclotransferase 1.Gene ID:79094)、ASNS(asparagine synthetase(glutamine-hydrolyzing).Gene ID:440)、NFE2L2(nuclear factor、erythroid 2 like 2.Gene ID:4780)、RIOK3(RIO kinase3.Gene ID:8780)、TXNIP(thioredoxin interacting protein.Gene ID:10628)、MTR(5-methyltetrahydrofolate-homocysteine methyltransferase.Gene ID:4548)、IFRD1(interferon related developmental regulator 1.Gene ID:3475)、ADGRA2(adhesion G protein-coupled receptor A2.Gene ID:25960)、TBX4(T-box 4.Gene ID:9496)、OVOL2(ovo like zinc finger 2.Gene ID:58495)、ACVRL1(activin A receptor like type 1.Gene ID:94)、ALB(albumin.Gene ID:213)、JADE1(jade family PHD finger 1.Gene ID:79960)、MLLT11(MLLT11、transcription factor 7 cofactor.Gene ID:10962)、ADORA1(adenosine A1 receptor.Gene ID:134)、TNFRSF8(TNF receptor superfamily member 8.Gene ID:943)、NLRP12(NLR family pyrin domain containing 12.Gene ID:91662)、CASP14(caspase 14.Gene ID:23581)、LTA(lymphotoxin alpha.Gene ID:4049)、SMAGP(small cell adhesion glycoprotein.Gene ID:57228)、NEO1(neogenin precursor1.Gene ID:4756)、MTSS1(MTSS1、I-BAR domain containing.Gene ID:9788)、TSPAN32(tetraspanin32.Gene ID:10077)、CLDN8(claudin 8.Gene ID:9073)、MYBPH(myosin binding protein H.Gene ID:4608)、SGCE(sarcoglycan epsilon.Gene ID:8910)、CDH15(cadherin 15.Gene ID:1013)、ARHGAP6(Rho GTPase activating protein 6.Gene ID:395)、ZFP36L2(ZFP36 ring finger protein like 2.Gene ID:678)、EHF(ETS homologous factor.Gene ID:26298)、SLAMF6(SLAM family member 6.Gene ID:114836)、HLA-DPB1(major histocompatibility complex、class II、DP beta 1.Gene ID:3115)、SSC5D(scavenger receptor cysteine rich family member with 5 domains.Gene ID:284297)、CCR4(C-C motif chemokine receptor 4.Gene ID:1233)、CCR7(C-C motif chemokine receptor 7.Gene ID:1236)、KLRG1(killer cell lectin-like receptor subfamily G member 1.Gene ID:10219)、BLNK(B cell linker.Gene ID:29760)、IGFBP1(insulin-like growth factor=binding protein 1.Gene ID:3484)、GIF(MIF、macrophage migration inhibitory factor.Gene ID:4282)遺伝子、及びこれらの組み合わせよりなる群から選択される遺伝子の発現量を測定する製剤を追加で含む、請求項2に記載のヘリコバクターピロリの感染による胃癌の診断用組成物。
- 前記遺伝子の発現量を測定する製剤は、前記遺伝子のmRNAの量を測定する製剤、前記遺伝子から発現されたタンパク質の量を測定する製剤、またはこれら2つ全てである、請求項1に記載のヘリコバクターピロリの感染による胃癌の診断用組成物。
- 前記遺伝子のmRNAの量を測定する製剤は、前記遺伝子のmRNAまたはcDNAに特異的に結合するプライマーまたはプローブを含む、請求項4に記載のヘリコバクターピロリの感染による胃癌の診断用組成物。
- 前記遺伝子から発現されたタンパク質の量を測定する製剤は、前記遺伝子から発現されたタンパク質に特異的な抗体またはアプタマーを含む、請求項4に記載のヘリコバクターピロリの感染による胃癌の診断用組成物。
- 請求項1から6の何れか一項に記載の組成物を含むヘリコバクターピロリの感染による胃癌の診断用キット。
- ヘリコバクターピロリに感染した患者のサンプルから試料DNAを準備するステップ;
前記試料DNAを請求項1から6の何れか一項に記載の組成物を用いて増幅するステップ;及び
前記増幅の結果からヘリコバクターピロリ感染により発現が増加または減少する遺伝子の発現の程度を確認するステップ;
を含むヘリコバクターピロリに感染した患者においてヘリコバクターピロリの感染による胃癌の診断のために必要な情報を提供する方法。 - 前記遺伝子の発現の程度を確認するステップは、前記遺伝子のmRNAの量、前記遺伝子から発現されたタンパク質の量、またはこれら2つ全てを測定するものである、請求項8に記載のヘリコバクターピロリに感染した患者においてヘリコバクターピロリの感染による胃癌の診断のために必要な情報を提供する方法。
- 前記遺伝子のmRNAの量を測定することは、前記遺伝子のmRNAまたはcDNAに特異的に結合するプライマーまたはプローブを用いるものである、請求項9に記載のヘリコバクターピロリに感染した患者においてヘリコバクターピロリの感染による胃癌の診断のために必要な情報を提供する方法。
- 前記遺伝子から発現されたタンパク質の量を測定することは、前記遺伝子から発現されたタンパク質に特異的な抗体またはアプタマーを用いるものである、請求項9に記載のヘリコバクターピロリに感染した患者においてヘリコバクターピロリの感染による胃癌の診断のために必要な情報を提供する方法。
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JP2014518640A (ja) | 2011-06-06 | 2014-08-07 | アムジエン・インコーポレーテツド | β−クロトおよびFGF受容体を含む複合体に結合するヒト抗原結合タンパク質 |
JP2018526988A (ja) | 2015-08-03 | 2018-09-20 | ノバルティス アーゲー | Fgf21関連障害を処置する方法 |
JP2018057362A (ja) | 2016-09-30 | 2018-04-12 | 国立研究開発法人国立がん研究センター | 胃癌発症リスクの診断補助方法ならびに当該方法に利用される人工dnaおよび胃癌発症リスク診断用キット |
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JP2022516117A (ja) | 2022-02-24 |
AU2019416610A1 (en) | 2021-07-22 |
KR102637908B1 (ko) | 2024-02-20 |
US20220064734A1 (en) | 2022-03-03 |
SG11202107005YA (en) | 2021-07-29 |
WO2020139021A1 (ko) | 2020-07-02 |
KR20200081879A (ko) | 2020-07-08 |
AU2019416610B2 (en) | 2023-06-01 |
TW202039861A (zh) | 2020-11-01 |
EP3904534A4 (en) | 2022-09-14 |
CA3125106A1 (en) | 2020-07-02 |
EP3904534A1 (en) | 2021-11-03 |
CN113490752A (zh) | 2021-10-08 |
TWI754201B (zh) | 2022-02-01 |
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