Basic Research
Copyright ©The Author(s) 2004.
World J Gastroenterol. Jun 1, 2004; 10(11): 1600-1607
Published online Jun 1, 2004. doi: 10.3748/wjg.v10.i11.1600
Table 1 Primer sequences for RT-PCR
Gene namePrimer sequenceExpected product size
Betaine homocysteineLeft 5’–GCC TAT AGC GGC TAC CAT GT-3’399 bp
methyltransferase (BHMT)Right 5’-CTC TGC AAT GGC CCT GAT GT-3’
Defender against cellLeft 5’-TTC GGC TAC TGT CTC CTC GT -3’197 bp
death protein 1 (DAD1)Right 5’-ACG AGG TGC AGG ATA GTG CT-3’
Annexin VILeft 5’-TAC CCC GGA GTA TTT TGC TG-3’220 bp
Neural cadherinLeft 5’-ATA CAG TGT CAC TGG GCC AG- 3’499 bp
(N-Cadherin)Right 5’-CGT AAG TGG GAT TGC CTT CC- 3’
Macrophage migrationLeft 5’-TTT TAG TGG CAC GAG CGA CC-3’199 bp
inhibitory factor (MIF)Right 5’-AAG CGA AGG TGG AAC CGT TC- 3’
Table 2 Part of the previously reported up-regulated genes in activation of HSC
GenBank accession numberGene nameRatio (Cy5/Cy3)Potential gene function
M73741Alpha-B2-crystallin, complete cds155.619Small heat shock protein gene, an early marker for HSC activation
X52046Procollagen, type III, alpha 1119.092Extracellular matrix (ECM), over- expressed in hepatic fibrosis
M18194Fibronectin mRNA29.958ECM, over expressed in hepatic fibrosis
J02870Laminin receptor mRNA6.62ECM receptor (integrin), cell adhesion
L08115CD9 antigen6.094Cell membrane glycoprotein, involved in cell activation and adhesion
U16163Prolyl 4-hydroxylase alpha (II) - subunit mRNA, complete cds4.555An enzyme which is essential for the collagen synthesis in HSC
X62622TIMP-2 mRNA for tissue inhibitor of metalloproteinase, type 23.732Inhibition for ECM degradation
X04017mRNA for cysteine-rich glycoprotein SPARC3.435Regulation of cell shape, adhesion, migration and proliferation
AF070470SPARC-related protein (SRG) mRNA, complete cds3.181Regulation of cell shape, adhesion, migration
M21495Cytoskeletal gamma-actin mRNA,3.092Relate skeleton structure of cell
AF188297TGF-beta receptor binding protein (Trip1) mRNA, complete cds2.976TGF-beta mediated signaling pathway
AJ245923alpha-tubulin 8 (Tuba 8 gene) mRNA2.901Relate skeleton structure of cell
AF053454Tetraspan TM4SF (Tspan-6) mRNA complete cds,2.896Cell development, differentiation, motility
AF013262Lumican gene, complete cds2.862Small, leucine-rich proteoglycan which involves in the regulation of collagen figril assembly
L02526Protein kinase (MEK) mRNA2.714Signaling molecule in MAPK family
Y00769mRNA for integrin beta subunit2.521Signaling molecular in cell adhesion, proliferation and migration
Table 3 A portion of up-regulated genes in activated HSC
GenBank accession numberGene nameRatio (Cy5/Cy3)Potential gene function
AF061017UDP-glucose dehydrogenase, mRNA complete cds15.56Glycosaminoglycan , hyaluronan and heparin sulfate biosynthesis
X65553mRNA for poly (A) binding protein7.405A regulator of translation initiation
AF029844Elongation factor 1-beta homology mRNA, complete cds6.305Translation factor and has multi-functions
NM_010798Macrophage migration inhibitory factor (Mif), mRNA5.847Proinflammatory peptide and a mediator of growth factor dependent ERK MAP kinase activation and cell cycle progression
M31131Neural cadherin (N-cadherin) mRNA5.559Cell adhesion molecule
AB029930mRNA for caveolin-1 beta isoform4.945Inhibit the eNOS activity
D12618mRNA for nucleosome assembly protein-1, complete cds4.528Histone H2A-H2B shutting protein that promotes histone deposition, and is important for maintaining chromatin structure
M22432Protein synthesis elongation factor Tu (eEF-Yu, eEf-1-1alpha) mRNA, complete cds4.344Translation factor involved in the protein biosynthesis
D31717mRNA for ribophorin4.319Glycoprotein synthesis
NM_012052Ribosomal protein S3 (Rps3), mRNA4.297Protein biosynthesis
X72711mRNA for replication factor C (140 Kda), long subunit4.245DNA synthesis and repair, and a regulator of NF-kappa B
X13460Annexin VI, p68 (Mouse mRNA for p68 protein of the lipocortin family)4.218Calcium binding protein
NM_011290Ribosomal protein L6 (Rp16), mRNA4.089Protein biosynthesis
U12273Apurinic/apyrimidinic endonuclease (APEX) gene, complete cds3.488A DNA repair enzyme and an activator of several transcription factors
D42048mRNA for squalene epoxidase3.298Rate-limiting enzyme of cholesterol biosynthesis
AJ250491mRNA for receptor activity modifying protein 3 (Ramp3) gene3.255Complex with the calcitonin receptor-like receptor, establishing a functional receptor for adrenomedullin
L04280Ribosomal protein (RPL12) mRNA3.172Protein biosynthesis
K02927Ribonucleotide reductase subunit M1 mRNA, complete cds3.156Rate-limiting enzyme in DNA synthesis and repair
AF087568Palmitoyl-protein thioesterase precursor mRNA, complete cds3.059Cell survival signaling
AF020039NADP-dependent isocitrate dehydrogenase (Idh) mRNA, complete cds3.038A key enzyme of the tricarboxylic acid cycle
X57960Ribosomal protein L72.962Protein biosynthesis
U09816GM2 activator protein (Gm2a) mRNA2.888Enzymatic hydrolysis of GM2
AB018575Cdc 7 mRNA, complete cds2.816Essential for G1/S transition
AF041054E1B19K/Bcl-2 binding protein homology (Nip3) mRNA, nuclear gene encoding mitochondria protein, complete cds2.813Nips is a pro-apoptotic mitochondria protein
L04128Ribosomal protein L18 (rpL18) mRNA2.751Protein biosynthesis
Z31554(129/SV) Cctd mRNA for CCT (chaperonin containing TCP-1) delta subunit2.743Stabilize the cytoskeletal protein actin and tubulin
D63784mRNA for MIDA1, complete cds2.703Regulate cell growth
AF026481eIF-1A (eIF-1A) mRNA, complete cds2.682Translation initiation factor
U35249CDK-activating kinase assembly factor p36/MAT1, complete cds2.645An assembly factor and a targeting subunit of cyclin-dependent kinase-activating kinase that is involved in cell cycle control, transcription and DNA repair
M76131Elongation factor (ef-2) mRNA, 3’end2.614Protein biosynthesis
X64713mRNA for cyclin B12.576Cell cycle regulatory protein
D26091mRNA for mCDC47, complete cds2.570A member of the minichromosome maintenance (Mcm) family involve in the DNA replication licensing system
U78085Ribosomal protein S5 mRNA2.507Protein synthesis
AF141322Caveolin-2 mRNA, complete cds2.458Inhibit the eNOS activity
M33934IMP dehydrogenase mRNA, complete cds2.429An enzyme involved in de novo synthesis of guanine nucleotides
U83628Defender against cell death protein 1 (DAD1) mRNA, complete cds2.372Apoptotic suppressor gene
Table 4 Part of down-regulated genes in the activated HSC
GenBank accession numberGene nameRatio (Cy5/Cy3)Potential gene function
D49949mRNA for IGIF precursor poly-peptide in (Interleukin 18), complete cds0.065In the development of Th1 cells and tissue injury inflammatory reaction
U63146Retinal binding protein (RBP) mRNA0.125Retinoid storage and metabolism
AF035644Potentially prenylated protein tyrosine phosphatase mPRL-2 mRNA, complete cds0.226Cellular regulation
X58287mR-PTPu gene for protein tyrosine phosphatase, receptor-type M0.280Cellular regulation
AF033381Betaine homocysteine methyl transferase (BHMT) mRNA, complete cds0.299A key liver enzyme that is important for homocysteine homeostasis
M75720Alpha-1 serine protease inhibitor 3 mRNA, complete cds0.308Anti-inflammatory effect
U20497P19 protein mRNA, complete cds0.324CDK4&CDK6 inhibitor, cell cycle inhibitor
D85596AMP deaminase H-type, complete cds0.339Involved in the biosynthesis, inter-conversion and degradation of purine compounds
Y10138Gene encoding prostaglandin D synthase0.361A PGD producing enzyme and a retinoid transporter
NM_013498CAMP response element modulator (CREM) mRNA0.366Down-regulator of CAMP-induced transcription
U67187G protein signaling regulator RGS2 (rgs2) mRNA, complete cds0.369Negatively regulate G-coupled receptor function
AF023919PK-120 precursor (itih-4) mRNA, complete cds0.421Inter-alpha-trypsin-inhibitor H4, is a potential regulator for ECM proteins
AF077950Protein inhibitor of activated STAT protein PIAS 1 mRNA, complete cds0.433Inhibition of STAT-1-mediated gene activation
M75718Alpha-1 protease inhibitor 4 mRNA, complete cds0.437Anti-inflammatory activity
D38046mRNA for type II DNA topoisomerase beta isoform, complete cds0.445An essential enzyme that alters DNA topology which is important for cell survive and apoptosis
AF073996myotubularin (Mtm1) mRNA complete cds0.464Subfamily of protein tyrosine phosphatases
Table 5 Semi-quantitative analysis of RT-PCR results
GroupsMean ± SDNp value
(quotient of the detected gene/GAPDH)
MIF from quiescent HSC0.269 ± 0.0165
MIF from activated HSC0.759 ± 0.0465< 0.05
N-Cadherin from quiescent HSC0.177 ± 0.0635
N-Cadherin from activated HSC0.776 ± 0.0875< 0.05
Annexin VI from quiescent HSC0.137 ± 0.045
Annexin VI from activated HSC0.478 ± 0.0255< 0.05
DAD1 from quiescent HSC0.174 ± 0.0165
DAD1 from activated HSC0.593 ± 0.0475< 0.05
BHMT from quiescent HSC0.602 ± 0.0835
BHMT from activated HSC0.134 ± 0.0595< 0.05