Download 論文要旨・審査の要旨

学位論文の内容の要旨
Aw Wanping
論文申請者氏名
論文審査担当者
主査：村松
正明
副査：石川
俊平
副査：淺原
弘嗣
副査：北野 宏明（Sony CSL）
Next Generation Optimal Inflammatory Bowel Disease Management Using
論 文 題 目
SmartAmp
Genotyping
Technology
for
Personalized
Medicine
and
Multi-faceted Omics Evaluation of Nutraceuticals as a Dietary Intervention
in Colitic Mice
（論文内容の要旨）
Current treatment of Inflammatory Bowel Disease (IBD) involves thiopurine therapy where single
nucleotide polymorphisms (SNP) in genes encoding thiopurine-S-methyltransferase in Caucasian
populations and ATP-binding-cassette transporter 4 (ABCC4) in Japanese populations leads to
intracellular accumulation of 6-thioguanine nucleotides which culminates in treatment failure. Due to
adverse side effects in IBD therapy, non-pharmaceutical control is highly recommended. Firstly, I
established a rapid and accurate genotyping technology using the Smart Amplification Process
(SmartAmp) to identify patients who are genetically susceptible to adverse side effects of thiopurine
therapy. Next, I proposed Western Parsley (PAR, Petroselinum crispum) as nutraceutical intervention
in IBD using dextran sodium sulphate (DSS) induced colitic mice by multi-integrated-omics analysis.
SNP 2269G>A genotyping using SmartAmp for personalized medicine
SmartAmp amplification requires five different primers: turn-back primer (TP), folding primer (FP),
boost primer (BP), and outer primers (OP1 and OP2) which were selected based on primer length, free
energy of annealing and probability of non-paired state after extensive screening. Prior to genotyping,
genomic DNA samples were denatured at 98 °C for 3 mins. The reaction mixture (total volume of 25
µl) containing 2.0 µM FP, 2.0 µM TP, 1.0 µM BP, 0.25 µM of each (OP1 and OP2, 20 µM CP, 1.4
mM dNTPs, 0.04M betaine, 20 mM Tris-HCl (pH 8.0), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM
MgSO4, 0.1% (v/v) Tween20, 1/100,000-diluted SYBR Green I and 12 units of Aac DNA polymerase
into which 5μL of pretreated sample was mixed. Isothermal DNA amplification occurring at 60°C was
monitored via the fluorescence intensity of the SYBR Green I dye indicating DNA amplification in an
Mx3000P real-time PCR machine.
Genotyping of SNP 2269G>A in ABCC4 gene was examined by using genomic DNA samples from
Japanese IBD patients. I compared the SmartAmp results against those of direct sequencing and
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PCR-RFLP analyses. I have successfully completed and validated the SmartAmp primer set for
accurate detection and discrimination of all possible homozygotes and heterozygotes of the SNP
2269G>A in the ABCC4 gene within 40 minutes.
Multi-omics--The essential dynamic power behind comprehensive understanding of PAR in
DSS-colitic mice
7-week-old male C57BL/6J mice were divided equally into three groups (n=7) fed either 2% PAR or
control diet for 1 week after which colitis was induced by administering 1.5% (w/v) DSS-containing
drinking water for 9 days. Body weight, stool consistency and fecal blood were recorded daily to
calculate the Disease Activity Index (DAI). Colonic mRNA and hepatic mRNA were extracted and
subjected to microarray (Mouse Genome 230 2.0, Affymetrix). Liver protein was extracted for
quantitative Isobaric tag for relative and absolute quantitation (iTRAQ) proteome analysis and
metabolome was evaluated using Capillary Electrophoresis Time-of-Flight Mass Spectrometry
(CE-TOFMS) and Capillary Electrophoresis- triple quadrupole Mass Spectrometry. Plasma
metabolites were measured using CE-TOFMS.
PAR significantly suppressed colon length decrease, DAI from Day 8 and mitigated the loss of crypts
and tended to reduce the infiltration of inflammatory cells into the colonic mucosal wall as compared
to DSS. Global colon gene evaluation of DSSPAR mice revealed significant downregulation of
interleukin 6 signal transducer (Il6st) and its downstream Il6. Il6 plasma levels also tended to be lower
in DSSPAR mice. Expression of genes downstream of Il6-- CD163 antigen and matrix
metallopeptidase 10 (Mmp10) were also significantly reduced. Haptoglobin, a commonly elevated
marker in IBD, which is downstream of Il6st and Il6, was remarkably downregulated. Chemokine
(C-C motif) ligand 5, CCAAT/enhancer-binding protein beta and matrix metallopeptidase 3 which are
regulated by Mmp10 were also significantly dowregulated. Expression levels of tissue inhibitor of
metalloproteinase 1 (Timp1) and its downstream, interleukin 1 receptor 1 (Il1r1) were both
significantly decreased. Upstream and downstream regulators of Timp1: collagen, type IV, alpha 2 and
metallopeptidase 13 tended to be downregulated, thereby decreasing collagenous deposits in the colon
wall, which may improve colon shortening. Chemokine (C-X-C motif) ligand 9, the downstream of
Il1r1, also tended to be reduced. PAR supplementation suppressed colon cytokine signalling, thereby
ameliorating IBD.
Hepatic transcriptome interpretation indicated significant upregulation of fatty acid metabolism
genes—fatty acid synthase, NADP-dependent malic enzyme, stearoyl-CoA desaturase-1 (Scd1) and
ELOVL family member 6, elongation of long chain fatty acids in DSSPAR mice where improved
body weight loss was also observed. Importantly, Scd1 converts stearoyl-coA to oleoyl-coA which
produces oleoyl lysophosphatidylcholine thereby muting the production of inflammatory cytokines in
the colon.
Metabolome analysis revealed that PAR induced significant decreases in plasma aa related to
-2-
oxaloacetate (Asn and Asp), pyruvate (Ala, Ser and Thr), succinyl coA (Met, Val and
Isoleucine)—elements of the citric cycle. PAR intervention improved concentrations of these elevated
metabolites indicating impaired glycolysis thereby improving inflammatory symptoms.
Evidently decreased levels of hepatic urea in the liver, and plasma orthinine and arginine (Arg) were
observed in DSSPAR. Arg is also a substrate for nitric oxide. The ameliorative properties of PAR in
IBD may be in part due to improved hepatic nitrogen contributing to reduced oxidative stress.
IBD inflammatory responses are accompanied cellular methylation reactions where the methyl donor
for these reactions is S-adenosylmethionine (SAM). Significant downregulation of methionine
adenosyltransferase II, alpha—the enzyme responsible for SAM production was observed.
Cystathionine, a metabolite downstream of SAM, and cystathionine gamma-lyase which coverts
cystathionine into cysteine, were significantly downregulated, indicating a shift to methylation
inhibition in DSSPAR. The indirect increased biosynthesis of cysteine indicates oxidative stress in
IBD gut. PAR may improve IBD by inhibiting methylation and reducing oxidative stress.
I engaged nutrigenomics to provide an evidence-based insight on the influence of PAR on IBD. The
nutrition-relevant colon transcriptome revealed a decrease in the expression of inflammatory
cytokines and fibrotic markers resulting in reduced inflammation and improved colon shortening. The
dietary signature of PAR on hepatic transcriptome and proteome complements that of the
metabolome, showed a decrease in expression of carcinogenic markers, thereby reducing cancer risk;
increase in expression of fatty acid synthesis related markers thereby improving body weight loss;
upregulation in molecules of the TCA and urea cycle and downregulaion of markers involved in the
methionine-recycling pathway thereby indicating improved oxidative phosphorylation and reduced
oxidative stress. The clues from each analysis complement each other, successfully leading to the
crucial findings of the present study.
Integrating SmartAmp and PAR dietary intervention actively promotes integrated omics nutrition
research for personalized healthcare, providing a novel perspective in optimal IBD management,
bringing forward a new evolution in nutritional science.
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学位論文の審査の要旨
論文申請者氏名
論文審査担当者
Aw Wanping（甲第4644号）
主査
村松
正明
副査
石川
俊平
副査
淺原
弘嗣
副査
北野
宏明（Sony CSL）
（論文審査の要旨）
論文は主に二つの内容に関するものである。前半はアザチオプリン投与量にゲノム薬理
学的な意義を持つヒト ABC トランスポーター遺伝子である ABCG の SNP（2269G>A）
を SmartAmp 法で検出するアッセイ系を作製したことである。Taqman 法と比べて、反
応が等温で進行することから利便性も良く、POC（point of care）に応用され得るレベル
まで反応系をブラッシュアップしたことは高く評価できる。
後半では炎症性腸炎のモデルマウスに対する機能性食品（パセリ）の効果を肝臓および
腸管における遺伝子発現、タンパク、メタボロームのパターンを比較している。パセリの
抗炎症効果が見られたが、その原因として旧来のものながら IL-6 サイトカインによる炎
症性パスウェイの関与が浮き彫りにされ得た。またクエン酸回路のエレメントの原料とな
るアミノ酸の低下、様々なメチル化のプロセスに関わる S-アデノシルメチオニン（SAM）
の合成経路の代謝物や遺伝子発現が低下していることを報告している。これらの反応によ
り炎症性サイトカインの低下など抗炎症作用が起こることが示唆されたことは、今後炎症
性腸炎の病態および治療反応に関する重要な知見であると考えられる。
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