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本帖最后由 风雨不动 于 2012-4-14 14:58 编辑
认识"胡黄莲"的成员请发表评论.
论文来自
stef2011.我们不知道究竟是谁写的论文.
http://www.res-medical.com/chinese-medicine/13274
Inhibitory Effects of Picrosides on Hepatitis B Virus Covalently
Closed Circular DNA in Vitro
Covalently closed circular DNA（cccDNA）,serving as the original
template of hepatitis B virus（HBV） replication,is the first
replicative intermediate emerging during the life cycle of
hepadnavirus,which indicates establishment of viral infection and
origination of the replication cycle.A stable pool of cccDNA,has been
found in each nucleus of persistently-infected hepatocyte,and is
thought to be one of the main factors responsible for reoccurrence of
chronic hepatitis B after withdrawal of antiviral agents.
Therefore,determination of HBV cccDNA does not only play an vital role
in the development and evaluation of aritiviral agents,but also may
provide a useful guide to the efficacy of antiviral treatment and the
likelihood of long-term response in patients with hepatitis B,and may
aid in the decision of when to cease therapy.However,poor efficacy has
been achieved aiming at clearance or inhibition of HBV cccDNA with
current anti-HBV therapies,and few visible progresses were made in
drug development and efficacy monitoring work against HBV cccDNA. In
this research,we aimed to confirm the effectiveness and
characteristics of picrosides inhibiting cccDNA in HepG2.2.15
cells,based on the specific and sensitive strategy developed earlier
by our laboratory to determine HBV cccDNA.This research was composed
of three parts:
Ⅰ.Comparative study on the effectiveness and characteristics in inhibiting HBV cccDNA in HepG2.2.15 cells: picrosides versus adefovir dipivoxil.
Ⅱ.Effect of picrosides on the supercoil configuration of HBV cccDNA molecules in HepG2.2.15 cells.
Ⅲ.Effect of picrosides on the transcription of OAS1,STAT2,ISGF3γ,MyD88 and MxA genes in HepG2.2.15 cells
PartⅠ
Inhibitory Effect of Pierosides on Hepatitis B Virus Covalently Closed Circular DNA in HepG2.2.15 Cells
Objectives
To observe the effect of picrosides on HBV cccDNA in HepG2.2.15 cells.
Materials and Methods
HepG2.2.15 cells were separately incubated with culture medium
containing 50μg/ml picrosides,100μg/ml picrosides or 5μg/ml adefovir
dipivoxil for 2 and 5 days.HBV DNA in supernatant,intracellular
cccDNA, intracellular relaxed circular DNA（rcDNA） and pregenomic
RNA（pgRNA） were quantified by specific real-time PCR or RT-PCR.
Results
①Treatment with 50μg/ml picrosides for 2 and 5 days could reduce the
production of HBV in the cell line,as shown by the decline of HBV DNA
in the supernatant（49.74%and 79.48%）. Intracellular synthesis of
cccDNA had also been markedly lowered by 43.55%and 56.43%,and
intracellular rcDNA decreased by 43.39%and 63.86%,as well as
intracellular pgRNA decreased by 54.72%and 56.08%.
②Treatment with 100μg/ml picrosides for 2 and 5 days resulted in
decline of HBV DNA（51.11%and 82.07%） in the supernatant,as well as
intracellular cccDNA（41.13%and 57.59%）,rcDNA（45.09% and 67.31%） and
pgRNA（52.68%and 52.47%）.
③Comparative treatment with adeforvir dipivoxil for 2 and 5 days
resulted in decline of HBV DNA（25.56%and 92.44%） in the
supernatant,cccDNA（18.54%and 47.19%）,rcDNA（21.20%and 71.47%） and
pgRNA（11.14%and 37.61%） in HepG2.2.15 cells.
Conclusions Our research indicated that picrosides can interfere with
the replication cycle of HBV, including the formation of cccDNA in
HepG2.2.15 cells.The mechanism of picrosides on cccDNA may differ from
adefovir dipivoxil’s in its earlier inhibition phase.
PartⅡ
Impact of Picrosides on the Distribution of Supercoils inside
Hepatitis B Virus cccDNA in HepG2.2.15 Cells
Objectives
To observe the distribution of superhelixes inside HBV cccDNA
molecules in HepG2.2.15 cells and the effect of picrosides or adefovir
dipivoxil on such distribution.
Materials and Methods
HepG2.2.15 cells were divided into 3 groups: Cells of normal control
group were incubated with fresh complete culture medium, while the
other two groups were incubated with culture medium containing 50μg/ml
picrosides or 5μg/ml adefovir dipivoxil respectively for 2 and 5
days.HBV cccDNA molecules were extracted from HepG2.2.15 cells by
methods based on Hirt-fractionation and then divided into different
groups according to the distinct number of innate superhelixes inside
each cccDNA molecule through special two-dimensional gel
electrophoresis process.Heterogeneous groups of cccDNA with different
superhelix number were detected with 32P-dCTP labeled cccDNA probe
after transfer from gels to nitrocellulose blotting membranes by the
Southern procedure. Integral gray values of different groups,named as
A（containing 0-5 supercoils）,B （containing 6-10
supercoils）,C（containing 11-15 supercoils） and D（containing 16-20
supercoils） were converted into percentage value and then analyzed
with nonparametric test.
Results
①The median percentage values of A to D distribution group in
picrosides group on day 2 were 14.94%,33.32%,35.96%and
14.05%,respectively; while in adefovir dipivoxil group they were
13.00%,20.04%,25.55%and 39.04%and 12.36%,21.04%,23.22%and 40.77%in
normal control group.The distribution of HBV cccDNA superhelixes in
picrosides group were significantly different from that in adefovir
dipivoxil group(Nemenyi Test,x1,22=8.28,P＜0.01) and normal control
group(x1,32=12.86,P＜0.01),while no significant difference was found
between adefovir dipivoxil group and normal control
group(x2,32=2.58,P＞0.25).
②The median percentage values of A to D in picrosides group on day 5
were 15.40%,33.48%, 34.47%and 15.07%,respectivel;while in adefovir
dipivoxil group they were 13.30%, 20.52%,23.22%and 42.12%and
13.02%,21.24%,22.91%and 40.61%in normal control group.The distribution
of HBV cccDNA superhelixes in picrosides group were significantly
different from those of adefovir dipivoxil group(Nemenyi
Test,x1,22=9.58, P＜0.01) and normal control
group(x1,32=11.61,P＜0.01),but no significant difference was found
between adefovir dipivoxil group and normal control group(x2,32=0.86,
P＞0.75).
③ The median percentage values of A to D in picrosides group on day 5
were 15.40%,33.48%, 34.47%and 15.07%,respectivel;while in adefovir
dipivoxil group they were 13.30%, 20.52%,23.22%and 42.12%and
13.02%,21.24%,22.91%and 40.61%in normal control group.The distribution
of HBV cccDNA superhelixes in picrosides group were significantly
different from those of adefovir dipivoxil group(Nemenyi
Test,x1,22=9.58, P＜0.01) and normal control
group(x1,32=11.61,P＜0.01),but no significant difference was found
between adefovir dipivoxil group and normal control group(x2,32=0.86,
P＞0.75).
③The distribution of superhelixes inside cccDNA molecules in
HepG2.2.15 cells was found as a heterogeneous population,with group D
showing the maximum percentage（about 40%）,group B and C a less
percentage（but both above 20%） and group A the least（less than 15%）.No
significant difference was found between data on day 2 and day
5（Mann-Whitney U test,U=0.321,P=0.748）.
Conclusions
The distribution of superhelixes numbers inside cccDNA molecules in
HepG2.2.15 cells exists as a heterogeneous population,treatment with
adefovir dipivoxil for 2 days and 5 days imposed no significant effect
on the distribution pattern.Treatment with picrosides reduced the
number of innate supercoils inside HBV cccDNA in HepG2.2.15 cells.
Such alteration may cause subsequent reduction of cccDNA molecular
stability which facilitates the clearance of cccDNA in nucleus.
PartⅢ
Effect of picrosides on the transcription of OAS1,STAT2,ISGF3γ, MyD88
and MxA genes in HepG2.2.15 cells
Objectives
To observe the effect of picrosides on the transcription of some host
genes and its relationship with picrosides’ anti-HBV activity in
HepG2.2.15 Cells.
Materials and Methods
①A group of host genes including 2′,5′-oligoadenylate synthetase
1（OAS1）,interferon-stimulated gene factor 3-γ（ISGF3γ）,signal
transducer and activator of transcription-2（STAT-2）,myeloid
differentiation primary response gene 88（MYD88） and myxovirus
resistance protein A（MxA） were selected as candidate genes according
to their reported activity of decreasing pgRNA.Their mRNA levels with
or without picrosides treatment were quantitated by realtime reverse
transcriptase PCR（RT-PCR） withβ-actin mRNA as internal reference.
②HepG2.2.15 cells were treated with picrosides,and effect of MyD88
gene being silenced by siRNA or not were assessed by quantifying
intracellular HBV pgRNA and MyD88 mRNA level as well as total HBV DNA
in supernatant by realtime PCR.
③MyD88 gene being silenced by siRNA or not,intracellular HBV cccDNA
and MyD88 mRNA level were quantitated by realtime PCR,while the
distribution of superhelixes inside HBV cccDNA was evaluated by
two-dimensional gel electrophoresis.
Results
①Ratio of those candidate gene mRNA levels to normal control were
1.17（OAS1）,0.87（STAT2）,0.93（ISGF3γ）, 5.14（MyD88） and 0.83（MxA）.The
transcription of MyD88 gene was significantly activated by picrosides’
treatment（P＜0.01）,while no significant difference were observed in the
transcription of other candidate genes between groups with or without
picrosides treatement（P＞0.05）.
②MyD88 siRNA can effectively silence MyD88 gene transcription with a
ratio up to 86.35%,while the inhibition rate of HBV DNA in supernatant
and intracellular pgRNA by picrosides declined to 5.02%and 6.67%
respectively on day 2,indicating the partial block of picrosides’
anti-HBV activity by MyD88 siRNA.
③The inhibition rate of intracellular cccDNA by 2 days’ picrosides
treatment in HepG2.2.15 cells remained as 46.24%in despite of silenced
MyD88 gene transcription（with a ratio up to 85.79%）,and the inhibition
rate of intracellular cccDNA in mock-transfection HepG2.2.15 cells was
52.73%.The A,B,C and D distribution pattern of cccDNA superhelixes in
MyD88 siRNA group were 17.08%,33.82%, 34.20%and 14.89%while in normal
control group they were 13.82%,20.44%,23.14% and
42.60%,respectively.The distribution of HBV cccDNA superhelixes in
MyD88 siRNA group were also significantly different from that of
normal control group (Nemenyi Test,x1,32=24.74,P＜0.01).
Conclusions
Treatment with picrosides can activate transcription of MyD88 gene and may subsequently induce the degradation of pgRNA through MyD88 pathway,but MyD88 gene might not participate in the rapid, early inhibition of HBV cccDNA by picrosides. The inhibition
effect of picrosides on HBV cccDNA in vitro and the reduction of superhelix number of cccDNA might rely on further activation of other host genes.
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发表于 2012-1-12 13:00
, 我认为是先进的，但不能充分履行科学文献翻译.