行政院國家科學委員會專題研究計畫 成果報告
利用 B 型肝炎-昆蟲桿狀重組病毒以偵測急性感染時肝細胞
的基因表現
計畫類別: 個別型計畫 計畫編號: NSC93-2314-B-002-128- 執行期間: 93 年 08 月 01 日至 94 年 07 月 31 日 執行單位: 國立臺灣大學醫學院小兒科 計畫主持人: 倪衍玄 報告類型: 精簡報告 處理方式: 本計畫可公開查詢中 華 民 國 94 年 10 月 25 日
The Gene Expression Profiles of Hepatocytes when Hepatitis B Virus Genes are
Carried in by Baculovirus-A Possible Acute Infection Model
Yen-Hsuan Ni, Chun-Hsien Yu, Hong-Yuan Hsu, Huey-Ling Chen, Mei-Hwei Chang
Department of Pediatrics, College of Medicine and Hospital, National Taiwan
ABSTRACT
Background/Aim: The hepatocyte gene expression profile at the time they respond to
hepatitis B virus (HBV) acute infection was rarely studied. We aimed to describe
this profile, which may help to delineate signal transduction pathway when virus
enters inside the hepatocytes. Methods: We adapted baculo-HBV recombinant virus
system to transfect HBV into non-primate, mammalian hepatocytes. This system
was used to infect rat hepatocytes with HBV through portal vein injection of
baculo-HBV recombinant virus. The control was the rat undergoing the same
procedure but with the baculovirus that did not contain HBV. The hepatocyte RNAs
of both rats were extracted for microarray and made for comparison. An in vitro
study was done simultaneously. We also adapted the baculo-HBV recombinant virus
and the baculovirus without HBV insert as the control to infected HepG2 cell cultures.
The hepatoma cell RNAs of both cultures were also extracted for microarray and
compared. Results: There were three differently expressed genes commonly found
in both in vivo and in vitro studies: Stat1, Zinc finger protein 83 (ZNF83), and RNA
binding motif protein 3 (RBM3). Conclusion: In the future, these genes of interest
will become our foci to study their roles in the acute phase of HBV-hepatocyte
inside the hepatocytes. Based on these findings, we may develop antiviral therapies
by targeting these factors.
Key words: hepatitis B virus, baculo-HBV recombinant virus, microarray, Stat1, Zinc
INTRODUCTION
Hepatitis B virus (HBV) is a hepatotropic DNA virus that infects humans and
certain nonhuman primates. The major obstacles in the study of HBV have been the
inability of the virus to infect cells in vitro, and the lack of good animal model
systems due to a strict virus-host range. The chimpanzee system is very expensive
and difficult to handle, and is only available in few centers in the world. The duck
system and the woodchuck system are valuable models for studying HBV lifecycle;
however, many significant differences still existed between animal hepadanviruses
and human HBV.
To overcome the above problems, an HBV recombinant baculovirus system was
established (1). Baculovirus, Autographa californica multiple nuclear polyhedrosis
virus (AcMNPV),
is an insect cell virus and had been used widely for large-scale recombinant protein
production. It is unable to replicate in mammalian cells. However, some previous
reports showed this virus can express foreign genes under appropriate promoter
control in mammalian cells, especially interesting in its hepatotropic property (2,3).
The previous study showed that by way of its endogenous promoter, a 1.3x
genome of HBV construct inside a baculovirus could be successfully generated and
the previous cell lines: (i) High-level of HBV expression. (ii) HBV replication level
can be regulated over a wide range simply by changing the baculovirus multiplicity of
infection (MOI). (iii) HBV replication in this system is detectable one day after
infection and persisted for at least 11 days. (iv) The superinfection with this
baculovirus can enhance or extend the infection period of the cell line. For these
advantages, we decided to set up this novel system.
We have now established the HBV recombinant baculovirus system. A 1.5X
HBV genome was inserted into a baculovirus to construct this recombinant
baculovirus. This system was well demonstrated to be successful in terms of the
production of HBsAg, the dose-dependent manner of the HBsAg production, and the
sustained production of HBsAg by a superinfection of the recombinant virus. We
also know the temporal profile of HBsAg production in this system. In the future,
this system can be applied to: (i) The model for acute response to HBV infection in
hepatocytes; we may check the molecular profiles of hepatocytes before and after
infected by HBV recombinant baculovirus. (ii) The evaluation of efficacy of antiviral
therapies. (iii) The model of co-infection of HBV plus other viruses or superinfection
of other viruses upon an HBV-infected hepatocyte cell lines. In the current study, we
will focus on the first goal.
portal vascular system for infusion. By this method, we may easily infect the
hepatocytes by injecting the recombinant virus into the portal system. We can enhance
the virus dosage by repeating the injection. This technique was well established in
large animal models such as baboons, dogs, pigs, and rabbits (4-7). However, the
technique for catheterization of the portal vein in large animals requires surgical
expertise and a whole set of surgical equipment. Therefore, in order to perform more
extensive experiments, a simplified portal cannulation technique needs to be
developed for small-sized animals. Actually, we now have already set up a rat
model for the development of long-term cannulation in the main portal vein (detail in
the Method section). The portal cannula remained secure without perturbing portal
circulation and allowed for both noninvasive infusions of baculovirus.
Although the host range of HBV is thought to be controlled at the stage of viral
entry, the hepatic tropism of HBV is also due to tissue-specific viral gene expression
(8). In the past, the studies focusing on the hepatocyte gene expression during acute
phase were rarely seen. A woodchuck model showed the recovery from acute
hepatitis was preceded by a significantly greater hepatic expression of interferon-γand
CD3 (9). These cytokines can help the host to overcome HBV infection. In a
mouse model, mouse hepatitis virus-induced hepatitis could be protected by a
concentration of HNF-1αiscoincidentwith decreased HBV transcription in an HBV
transgenic mice (11,12). All the above results may help to elucidate the pathways
that regulate the viral life cycle and suggested additional approaches for the treatment
of chronic HBV infection.
In this study, we plan to use the HBV-recombinant baculovirus to infect human
hepatocytes in vitro and rat hepatocytes in vivo. The RNA will be extracted from the
human and rat hepatocytes after the HBV-recombinant baculovirus infection. The
RNA will then be subjected to microarray study (the strategy is listed below). Based
upon the microarray results, we are likely to find out some possible candidate genes
that may regulate the antiviral responses. It is also likely for us to find out possible
viral receptor(s) in the mammalian hepatocytes. As the duck model system, it was
shown the overexpression of gp180 (receptor) could enhance the duck hepatitis B
virus entry into the hepatocytes (13). The microarray possibly will find out
overexpression of candidate receptor(s) genes when the recombinant
HBV-baculovirus enters the hepatocytes, either in vitro or in vivo. By the
bioinformatics analysis, we might zero in on several candidates and do further
research on these ones.
Cell culture. Sf21 insect cells (kindly provided by Prof. Bor-Leung Chiang) were
maintained in complete TNM-FH medium (Grace’sinsectmedium supplemented with
10% fetal bovine serum and 1% PS) in non-humidified incubator at 27℃ without CO2.
Huh7 cells were maintained in Dulbecco's modified essential medium supplemented
with 10% fetal bovine serum and 1% PS and were grown in humidified incubator at
37℃ and 5% CO2. (All medium, serum, and antibiotic are from Gibco, Life
Technology).
Construction of Baculovirus Transfer Vector. A recombinant transfer vector was
constructed by excising a PvuII/PvuII fragment containing 1.5×HBV DNA (~5 kb)
from pGEM3Z4.8 (kindly provided by Dr. Hui-Ling Wu) and cloned into the SmaI
site of the multiple cloning region of pBlueBac4.5 (Invitrogen, Chatsworth, CA,
USA). The insertion and orientation of the recombinant transfer vector were then
confirmed by HindIII/KpnI enzyme digestion and DNA sequencing (Fig. 1).
Generation of Recombinant Baculovirus Containing 1.5× HBV DNA. Seed 2×106
Sf21 cellsin a60 mm dish,and gently wash twicewith Grace’sinsectmedium withoutFBS to removetheserum. Six μg ofthepurified recombinanttransfer vector,0.5μg oftheBac-N-BlueTM
DNA (linear AcMNPV baculovirus DNA), and 20μlofCellfectin®
reagent were then co-transfected into the Sf21 cells according to themanufacturer’sinstructions(BAC-N-BLUE transfection kit, Invitrogen). After
4-hour of incubation at room temperature, 1 ml of complete TNM-FH medium was
added. The dish was then sealed with paraffin and incubated at 27℃for 72 hours
before harvesting the recombinant baculovirus.
Harvest the Recombinant Baculovirus by Plaque assay. Seed 5×106Sf21 cells to 100 mm plate and infect the cells with 102, 103, and 104dilutions of the transfection viral stock in TNM-FH medium at room temperature for one hour. The medium was
then aspirated and an agarose/medium/X-gal mixture, consisting of 2.5 ml of 2.5%
agarose solution (47℃), 2.5 ml of complete TNM-FH medium (47℃), and 5 ml of
complete TNM-FH medium with X-gal (concentration: 150μg/ml,room temperature)
was gently poured into the plate to overlay the virus-infected Sf21 cultured cells.
The plates were sealed and incubated at 27℃ for 5 days until the distinguishable blue
plaques are formed.
Select the Recombinant Baculovirus by PCR. The putative recombinant viruses
were amplified from blue plaques. 72 hrs after infection, 0.75 ml of the medium was
mixed with the same volume of 20%polyethylene/1M NaCl to precipitate the released
virions from the blue plaques. The medium were then collected and labelled as P-1
stocks and store at 4℃. Viral DNA was purified by the standard Proteinase K
digestion, phenol/chloroform extraction, and isopropanol precipitation. The DNA wasused astemplatesforpolymerasechain reaction (PCR)(PrimersP1:5’-TCA
CCA TAT TCT TGG GAA CAA GA-3’and P8:5’-TTA GGG TTT AAA TGT ATG
CCC-3’)to detectifthevirusisolatescontain the1.5× HBV DNA.
Preparation of High-Titer Viral Stocks. Seed 25 cm2flask with 2×106log-phase Sf21 cellsand add 20 μlofthe P-1 viral stock in 5 ml complete TNM-FH medium.
Incubate at 27℃for 7 days until all the cells were lysed. Centrifuge at 1000g for 20
minutes to remove the cell debris and store at 4℃as P-2 stock. Virus titres were
determined by the plaque assay using 106, 107, and 108dilutions of P-2 stock.
Analysis of Secreted Hepatitis B Surface Antigen (HBsAg). Detection of HBsAg
was performed by enzyme linked immunoassay kit (EIA, Abbott). The media from
Huh7 cells were collected at several time points after infection and stored at -80℃
until analysis.
Hepatocyte harvest from human liver fragment. A surgical specimen of liver (10 g)
was put in 50ml test tube containing 15 ml icy cold Ringer, and minimized the
ischemic time as short as possible. Then, the specimen will be transferred to a 6 cm
petri dish, hold the specimen with gauze, and find a best vessel to cannulate, and
perfusion at least 10 min with EGTA and collagenase. The perfusion will be
continued till the liver is pale yellow. After the perfusion, the liver specimens is
incubated at 37℃for 10 minutes. The hepatocytes are then filtered through 100-um
C, 3 min) to isolate the single hepatocytes. The viability of the isolated hepatocytes
will be counted by trypan blue dye exclusion. At least 80% viability should be
achieved and available to go further study.
Infection of HepG2 cell cultures with Recombinant Baculovirus. Seed 6-well
culture plates with 105HepG2 cells per well and the cells were grown for 16 to 24 hours before infection. On the day of infection, P-2 virus stock was diluted with
DMEM/FBS/PS according to their titers to achieve the desired multiplicity of
infection (moi) and adjust the final infection volume to1 ml. After 1 hour of
infection at 37℃, the inoculum was aspirated and the cells were gently washed with
HBSS (Gibco, Life Technology) for 3 times to remove all the virus-containing
supernatant. 2.5 ml of DMEM/FBS/PS was re-fed and maintained at 37℃incubator.
Animals and cannula insertion into portal vein. F-344 rats (160-200 gm) were fed
a laboratory diet with water and food ad libitum until use and were kept under
constant environmental conditions with a 12-hour light-dark cycle. The animals
received humane care in compliance with the guidelines of the National Science
Council (NSC 1997). Animals were maintained in separate cages. The cannula was
an 18 cm length of polyethylene tubing (PE-50, ID 0.58 mm, OD 0.96 mm; Becton
Dickinson, Sparks, MD). One end was sheathed with a 2.5 cm length of PE-160
were fused at one end with heat. The other end of the PE-50 cannula was beveled. A 3
mm section of a 20G A BD Vialon catheter (ID 1.1 mm; Becton Dickinson, Sandy,
Utah) was placed 7 mm from the tip of the bevel as a collar. The finished cannula was
sterilized with ethylene oxide. A novel spring-guide-wire introducer needle was
created by the modification of the spring-wire guide of the pediatric central venous
catheterization set (0.46 mm x 45 cm, Arrow International, Inc). The 25 GA
introducer needle was cut 6 cm from its point, and the cut edge was smoothed. This
needle was adapted to the straight-tip end of the spring-wire guide.
Rats were anesthetized intramuscularly with 75 mg/kg ketamine hydrochloride and 5
mg/kg xylazine. A midline laparotomy was performed, and the portal vein was
exposed (14). One stitch was made through the superficial part of the main portal
venous wall with a 7-0 silk suture armed with a BV-1 needle. The spring-guide-wire
introducer needle was introduced through the cannula. Using this introducer needle,
we punctured the portal vein about 1 mm down-stream from the stitch, advanced the
needle 5 mm into the vein, and slid the cannula through the guide-wire into the portal
vein up to the Vialon collar. The cannula was secured with a triple knot over the
Violon collar with another triple knot just behind the collar. The guide-wire introducer
needle was then withdrawn. After free blood returned into the cannula, the cannula
23 GA needle. Ampicillin (40 mg) and gentamicin (4 mg) were administered
intraperitoneally. The abdominal wall incision was closed with 4-0 chromic surgical
guts. The cannula was led through the subcutaneous tunnel to the interscapular
incision (Fig. 2). The distal end of the cannula was anchored to the interscapula by
suturing the cannula around the PE-160 tubing sheath to the skin. The patency of the
cannula was reconfirmed and checked twice a week. The rats were allowed to
recover from the surgery, and each was maintained in a separate cage for at least one
week before use in the experiments.
RNA extraction from the cultured hepatocytes and rat liver. From cell culture: The
tissue cultured cells lysate will be collected 48-72 hours after recombinant virus
infection. Use a cell scraper or trypsin to free cells from flask and transfer the cell
suspension to a centrifuge tube. Spin the cells for 10 minuets at 1000rpm at 10°C.
Discard as much supernatant as possible. Store immediately on ice. Resuspend cells in
equal volume of PBS and spin again at the same speed and temperature as above. Again,
discard as much supernatant as possible. Return sample to ice.
From tissues: The rats will be infused the recombinant virus through the portal cannula.
In the meantime, the other rats will be infused with the wild type baculovirus to serve as
the control group. The rats, both the experimental group and the control group will be
collected through superior vena cava and sent for HBsAg assay (see section below) and
a piece of liver tissue will be sent for histologic examination; the specimens were
serially sectioned in 10-µm slices and stained with H&E to define the analyzed regions.
Then, we will use a mini plastic tissue homogenizer to homogenize the tissues. Ensure
that no large pieces are visible. Pipetting up and down to disrupt any clumps and
incubate at room temp for 2-3 min. It is best that the samples are kept on ice as much as
possible while working with Trizol (Qiagen, Chatsworth, CA, USA). Add 20%
volume of chloroform. A milk-like liquid should be seen. Spin in a microcentrifuge at
4°C, max speed, for 15 min to get several layers. While storing samples on ice,
immediately remove the top supernatant ensuring that the pipet does not touch the
lower layers. Transfer small amounts at a time to a fresh 1.5ml tube. Add equal volume
of room temperature 70% ethanol. Mix and transfer immediately to the mini spin
column. Spin at 10,000rpm for 1 min. If any liquid left on the top of the filter, spin again.
Discard flow-through and save the collection tube for the next step. Pipet 700µl Buffers
three times and then pour onto the RNeasy column. At 20-25˚C,centrifugefor2 min at
max speed to dry the RNeasy membrane. Transfer the column to a 1.5 ml tube and add
30 µl of DEPC water to elute RNA with 1 min spin at 10,000rpm. Repeat two more
Microarray to identify genes responsible for acute phase viral interaction. The
overall study protocol is listed in Figure 2. The preparation of probes, hybridization,
and scanning was performed as described previously (15). The fluorescence intensities
of Cy5 (nontumor) and Cy3 (tumor) for each target spot were adjusted so that the mean
Cy5 and Cy3 intensities of 52 housekeeping genes for each slide were equal. The
relative expression of each gene (Cy3:Cy5 intensity ratio) into one of four categories:
up-regulated (ratio, >2.0), down-regulated (ratio, <0.5), unchanged (ratio, between 0.5
and 2.0), and not expressed (or slight expression but under the cutoff level for
detection). To detect differentially expressed genes, we recorded the number of
samples in each category within each subgroup, for each gene. Then we calculated the
U values of Mann-Whitney tests, which measured how the sample distributions
between subgroups overlap. The number of samples within each group is counted and,
according to the order of the category, the number of overlapped samples is
incorporated into the U value. A small U shows that the sample distribution of the two
groups is clearly separated, e.g., commonly up-regulated in the recombinant group or
down-regulated in the control group.
RESULTS
Containing 1.5× HBV DNA. The baculo-HBV recombinant virus was constructed
and harvested (Fig 3) as described in the methods and the viral titer was determined
after we proved that it could produce HBsAg (Fig 4).
Microarray by RNA extraction from the cultured hepatocytes and rat liver. The
cellular RNAs extracted from in vitro cell cultures of either baculo-HBV recombinant
virus infection or the control baculovirus infection were extracted to process for
microarray. Totally we found 55 differentially expressed genes between the control
and the experimental settings (Table 1). As for the in vivo study, we extracted the
RNA from the rat liver which was infected the baculo-HBV recombinant virus
through the portal vein and the RNA from the control rat liver which was infected
with the baculovirus, also through the portal vein injection. We also performed the
microarray study to compare the differentially expressed RNAs and found 1823 genes
with either 2 folds up-regulated (n=758) or two-folds down regulated (n=1065) (Fig.
5). We then compared the 55 and 1823 genes and found only three genes were
overlapped in both settings. These three genes are: Signal transducer and activator
of transcription 1 (Stat1), Zinc finger protein 83 (ZNF83), and RNA binding
motif protein 3 (RBM3).
This study utilized a baculo-HBV recombinant virus system to deliver the HBV
DNA into the hepatocytes. By way of comparing with the control baculovirus, we
performed the microarray to detect the differentially expressed genes. The
transfection routes were through the portal vein in rats and directly infected the
cultured HepG2 cells in vitro.
This study design indeed contains several default drawbacks: (1) this is not a natural
route of infection, either in vivo or in vitro. Thus, it hardly simulated the real
condition in acute HBV infection. (2) The microarray gene chip used here is actually a
mouse liver library, and very likely is different from the human and rat ones.
However, because HBV has very limited natural hosts and is hard to replicate in vitro,
this model is an option for the study of hepatocyte gene expression profile.
We summarized the results and picked up the three genes that were differentially
expressed in both in vitro and in vivo experiments. These three genes: Stat1, ZNF83,
and RBM3, are actually important molecules in the signal transduction pathway.
They may be not the specific viral receptor or the very first several ligands for viral
entry, but they may play some roles in the successive steps. Initially, we aimed to
find out any viral receptors or acute phase reatants of HBV infection by this study.
All these three molecules may not be the very first receptor(s) existing in the cell
messengers.
When cytokines and growth factors combining with their respective receptors in
cell surface and transmit the stimulation across the cell membrane, Stat1 can be
activated (16). We believed that STAT1 was also playing the same role when
hepatocytes were infected with the virus, even though this virus may not be
transfected through the natural receptor. After the transfection, HBV likely induces
some cytokine reaction intracellularly and triggers the activation of STAT1. STAT1
protein was shown to be essential for cell growth suppression in response to
interferon-γ(IFN-γ). The molecular mechanism mediating this growth inhibition
involves the regulation of the gene encoding the cyclin-dependent kinase inhibitor
p21 WAF1/CIP1 by STAT1 (17). In summary, STAT1 is important in the signal
pathway inside hepatocytes after HBV enters into the cells, but it seems not a receptor
for virus.
Our laboratory had already demonstrated that RBMY gene, a RNA binding motif
gene on Y chromosome had an oncogenic implication and related to HBV-associated
hepatocellular carcinoma (18). This study also showed the RBM3, a family of RNA
binding motif gene, had a differential expression between the baculo-HBV
recombinant virus infected hepatocytes and the control group in vivo and in vitro.
reaction rather than the receptors themselves.
A previous report revealed that most of the liver genes up-regulated by HBxAg
expression can be clustered into three major groups, including genes encoding
ribosomal proteins, transcription factors with zinc-finger motifs, and proteins
associated with protein degradation pathways (19). These results led to the
hypothesis that HBxAg may function as a major regulator in a common cellular
pathway that, in turn, regulates protein synthesis, gene transcription, and protein
degradation. In this study, we did not specify the role of HBxAg, instead, we cloned
the whole HBV genome and transfected to the hepatocytes. We may divided the
four open reading frames of HBV genes and check their respective differential
expressions by this model.
In summary, HBV and hepatocytes may interact with each other through several
pathways (Fig . Our study demonstrated three of them. These three may be the
most pivotal factors in the later development of chronic infection events, like the
fibrogenesis and cancer development. We have to dissect the genes into detail to
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Table 1. The 55 differentially expressed genes in the HepG2 cells detected by
microarray
Gene Description
PIK3R1 Phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha) FH Fumarate hydratase
UPP1 Uridine phosphorylase 1 LIPE Lipase, hormone-sensitive
SCO1 SCO cytochrome oxidase deficient homolog 1 (yeast)
Homo sapiens gastric-associated differentially-expressed protein YA61P (YA61) mRNA, complete cds.
GART Phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase
DCK Deoxycytidine kinase
TOP2A Topoisomerase (DNA) II alpha 170kDa
TOMM70A Translocase of outer mitochondrial membrane 70 homolog A (yeast) ZNF83 Zinc finger protein 83 (HPF1)
NT5M 5',3'-nucleotidase, mitochondrial
Homo sapiens mRNA; cDNA DKFZp434I1820 (from clone DKFZp434I1820); partial cds. RBM3 RNA binding motif (RNP1, RRM) protein 3
PPIG Peptidyl-prolyl isomerase G (cyclophilin G) HSPD1 Heat shock 60kDa protein 1 (chaperonin)
ROCK1 Rho-associated, coiled-coil containing protein kinase 1 PRKDC Protein kinase, DNA-activated, catalytic polypeptide TTK TTK protein kinase
TRIP12 Thyroid hormone receptor interactor 12 UBQLN2 Ubiquilin 2
C20orf6 Chromosome 20 open reading frame 6 LOC400966 RAN-binding protein 2-like 1 short isoform
PIK3C2A Phosphoinositide-3-kinase, class 2, alpha polypeptide ERBP Estrogen receptor binding protein
FLJ39207 C219-reactive peptide
IL16 Interleukin 16 (lymphocyte chemoattractant factor) HSPCA Heat shock 90kDa protein 1, alpha
ch-TOG KIAA0097 gene product
TIPARP TCDD-inducible poly(ADP-ribose) polymerase RBBP9 Retinoblastoma binding protein 9
SMC2L1 SMC2 structural maintenance of chromosomes 2-like 1 (yeast) SCC-112 SCC-112 protein
CUL4B Cullin 4B
ANLN Anillin, actin binding protein (scraps homolog, Drosophila) ATF7IP Activating transcription factor 7 interacting protein
ITGAV Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) APOB Apolipoprotein B (including Ag(x) antigen)
APOB Apolipoprotein B (including Ag(x) antigen) CD2AP CD2-associated protein
RGS13 Regulator of G-protein signalling 13 IPO7 Importin 7
STAT1 Signal transducer and activator of transcription 1, 91kDa CLTC Clathrin, heavy polypeptide (Hc)
MATR3 Matrin 3
FAM51A1 Family with sequence similarity 51, member A1 FLJ16517 FLJ16517 protein
BTBD9 BTB (POZ) domain containing 9
CDNA FLJ10095 fis, clone HEMBA1002430 C14orf143 Chromosome 14 open reading frame 143
NTera2D1 cell line mRNA containing L1 retroposon, clone P2 Clone IMAGE:110218 mRNA sequence
Hypothetical gene supported by BC040718
FIGURE LEGENDS
Figure 1. The construction of baculo-HBV recombinant virus. The control virus
contains the same backbone except the 1.5X HBV insert.
Figure 2. The overview of the study flow chart.
Figure 3. Successful recombinant baculovirus yield blue plaques.
Figure 4. Analysis of HBsAg in the medium of Huh7 cells infected with HBV
baculovirus.
Figure 5. The illustration of microarray results in the in vivo experiment. We
picked up total 1823 genes, 758 of which are 2 folds up-regulated while 1065 of them
are 2 folds down-regulated.
EcorⅠ EcorⅠ H in d Ⅲ P v u Ⅱ Re c o m b in a tio n s e q u e n c e s ColE 1 Amp R pBlueBac4.5 HBV 1.5* Construct EcorⅠK p n Ⅰ P v u Ⅱ Recom binatio n Sequen ces (5’ lacZfragm ent)
Bioinformatics to select genes Microarray
Extract RNA from hepatocytes
infect in vitro Primary Human Hepatocyte culture
Bioinformatics to select genes# Microarray*
Extract RNA from liver tissue* Histologic examination
Check serum HBsAg Infect rats through portal vein Baculo-HBV recombinant virus
0 5 10 15 20 25 0 2 4 6 8 Days H B sA g T it er negative moi=50