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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (7): 751-756.doi: 10.3736/jcim20120705

• Original Clinical Research • Previous Articles     Next Articles

Analysis of differential gene expression profile in peripheral blood of patients with chronic hepatitis B and syndromes of dual deficiency of liver and kidney yin and accumulation of dampness heat

Yan Guan1, Hui Zhang1, Wei Zhang2, Shi-bing Su1()   

  1. 1. Research Center for Traditional Chinese Medicine Complexity System, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    2. Department of Liver Diseases, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Received:2011-10-17 Accepted:2011-11-20 Online:2012-07-20 Published:2018-07-15
  • Contact: Su Shi-bing E-mail:shibingsu@yahoo.com

Objective: To investigate the differential gene expression profile in two typical traditional Chinese medicine (TCM) syndromes of patients with chronic hepatitis B (CHB), and to find the relationship between TCM syndromes and gene expressions.
Methods: Patients with CHB were collected from Department of Liver Diseases of Longhua Hospital in Shanghai and diagnosed as dual deficiency of liver and kidney yin syndrome (n=3) or accumulation of dampness heat syndrome (n=3). Three healthy volunteers were used as a control. Blood samples were collected before treatment. After total RNA was isolated from leukocytes, the gene expression profiles were detected by microarray. For confirming the gene expressions, a further 10 patients with dual deficiency of liver and kidney yin syndrome or accumulation of dampness heat syndrome and 10 healthy volunteers were enrolled. Their peripheral blood was used to test the gene expression by real-time reverse transcription-polymerase chain reaction (RT-PCR) which was differently expressed by microarray.
Results: Microarray analysis identified that there were significant differences in gene expression between patients with dual deficiency of liver and kidney yin syndrome and patients with accumulation of dampness heat syndrome, as well as between CHB patients and healthy volunteers. Patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome were fully distinguished by clustering of 403 differentially expressed genes (P<0.05). A total of 239 genes were significantly differentially expressed (absolute value of foldchange≥2, P<0.05), among which 142 were up-regulated and 97 were down-regulated. Genes specially regulated by the dual deficiency of liver and kidney yin syndrome were mainly related to peroxide activity and stem cell maintenance; genes specially regulated by the accumulation of dampness heat syndrome were mainly related to cytokines, immune and inflammatory responses. ATP-binding cassette, sub-family C (CFTR/MRP), member 3, peroxisome proliferator-activated receptor α and phosphatidic acid phosphatase type 2A mRNA expressions detected by RT-PCR were similar to the results of microarray.
Conclusion: Differential gene expressions are observed in CHB patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome, indicating that there is molecular foundation in the classification of TCM syndrome. The preliminary findings of this study suggest that TCM syndrome types are related to gene expression.

Key words: hepatitis B, chronic, syndromes, liver-kidney yin deficiency, dampness-heat, gene, gene expression profiling, nontherapeutic human experimentation

Table 1

Checked genes and their primer sequences by real-time RT-PCR"

Gene
symbol
GenBank number Forward primer Reverse primer Product
length
Tm
ABCC3 NM_003786 5′-TGACTCAGGCCAGTG
TGTCTCTGA-3′
5′-CCCAGGTGAAGGTGC
CACTGTG-3′
135 59.04/59.60
PPARA NM_001001928 5′-ACACTGTGTATGGCT
GAGAAGA-3′
5′-GACGGTCTCCACTGA
CGTG-3′
117 60.80/62.00
PPAP2A NM_176895 5′-GGCTTCCATGCCTA
TGGCTGTTC-3′
5′-ATGGGCAAGCCAACC
CCCACTA-3′
150 58.04/60.11

Table 2

Differentially expressed genes between CHB patients with dual deficiency of liver and kidney yin syndrome and accumulation of dampness heat syndrome"

Probe ID Gene symbol Fold change (D/E) Result
NM_030915 LBH 15.373 0 Up-regulated
BC051300 PHYHD1 11.317 7 Up-regulated
NM_005098 MSC 9.840 3 Up-regulated
NM_003786 ABCC3 8.759 2 Up-regulated
NM_018071 FLJ10357 7.532 8 Up-regulated
NM_001001928 PPARA 3.921 1 Up-regulated
NM_176895 PPAP2A 3.319 2 Up-regulated
AK055816 PODXL 0.161 1 Down-regulated
XM_933718 LOC646576 0.148 1 Down-regulated
NM_016588 NRN1 0.141 5 Down-regulated
NM_012338 TSPAN12 0.139 3 Down-regulated
NM_002142 HOXA9 0.119 3 Down-regulated
NM_016215 EGFL7 0.115 5 Down-regulated
BC007600 HOXA10 0.110 6 Down-regulated
BC041772 SPNS2 0.104 6 Down-regulated

Table 3

Differentially expressed genes between CHB patients with dual deficiency of liver and kidney yin syndrome and healthy controls"

Probe ID Gene symbol Fold change (D/N) Result
NM_005561 LAMP1 8.219 3 Up-regulated
BC041396 RANGAP1 7.027 1 Up-regulated
NM_003943 STBD1 5.860 3 Up-regulated
XM_933162 LOC150776 5.738 0 Up-regulated
NM_006351 TIMM44 5.641 1 Up-regulated
BC016333 CCNL2 5.488 0 Up-regulated
BC043150 IQCE 5.476 1 Up-regulated
NM_001033052 TBCD 5.350 4 Up-regulated
BC037870 C10orf81 0.117 6 Down-regulated
XM_934733 LOC400927 0.109 8 Down-regulated
NM_001001875 TPD52L3 0.108 1 Down-regulated
NM_003537 HIST1H3B 0.100 4 Down-regulated
NM_207491 KIAA1680 0.094 9 Down-regulated
XM_372592 LOC145814 0.085 0 Down-regulated
NM_000761 CYP1A2 0.072 4 Down-regulated

Table 4

Differentially expressed genes between CHB patients with accumulation of dampness heat syndrome and healthy controls"

Probe ID Gene symbol Fold change (E/N) Result
NM_001711 BGN 24.735 7 Up-regulated
NM_001312 CRIP2 19.541 1 Up-regulated
NM_003246 THBS1 17.055 4 Up-regulated
BC007659 NQO1 15.312 2 Up-regulated
NM_080668 CDCA5 14.662 3 Up-regulated
NM_012323 MAFF 13.630 3 Up-regulated
NM_004104 FASN 13.549 4 Up-regulated
NM_002142 HOXA9 13.101 5 Up-regulated
XM_372543 IGHV1OR15-5 0.051 8 Down-regulated
XM_925817 FAM138F 0.047 9 Down-regulated
BC070352 IGLV3-21 0.046 6 Down-regulated
AY341951 FAM138D 0.045 6 Down-regulated
X57812 IGLJ3 0.035 9 Down-regulated
BC089418 IGLV3-25 0.021 0 Down-regulated
BC018749 IGLV2-14 0.015 9 Down-regulated

Table 5

Expressions of ABCC3, PPARA and PPAP2A mRNAs (x±s)"

Group n ABCC3 PPARA PPAP2A
D 10 0.013 6±0.015 8 0.010 0±0.016 0 0.031 7±0.077 8
E 10 0.002 0±0.001 8 0.000 9±0.000 6 0.000 1±0.000 1
N 10 0.006 8±0.005 1 0.002 0±0.001 6 0.001 1±0.001 3
P (D vs E) 0.03 0.09 0.22
P (D vs N) 0.21 0.13 0.23
P (E vs N) 0.01 0.07 0.05
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