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Xiao-Fang
Liu, Sheng-Quan Zou, Fa-Zu Qiu, Department of General Surgery of
Tongji Hospital, Wuhan 430030, Hubei Province, China
Correspondence to: Dr.Xiao-Fang Liu, Department of Genneral
Surgery of Tongji Hospital, 1095 Jiefang Road, Wuhan 430030, Hubei
Province,China. Liu634@263.net
Telephone: +86-27-83662134
Received 2001-04-21 Accepted 2001-08-15
Abstract
AIM:
To
establish an experimental model for exploring the role of hepatitis
C virus (HCV) in the development of cholangiocarcinoma.
METHODS:
Recombinant
plasmid of HCV-core gene was constructed with molecular cloning
technique and transfected into QBC939 cells with lipofection.After
it was selected with G418, resistant colonies were obtained. The
colonies were analysed by immunocytochemistry and Western
blotting.The morphology was observed under transmission electron
microscope(TEM) and microscope.
RESULTS:
The
recombinant plasmid was proved to carry the target gene by PCR and
restriction enzymed mapping. Moreover, it could express HCV-C
protein efficiently in QBC939 cells. The HCV-like particles were
found in the cytoplasm by electron microscope, which were spherical
with a diameter of 50nm-80nm possessing outer membrane.The
transfected cells had lower differentiation and higher malignant
degree under microscope.
CONCLUSION:
Because HCV-core gene could express steadily in cholangiocarcinoma
cells,the transfected tumor cells(QBC939-HCVC) could be used to
study the effect of HCV in the development of cholangiocarcinoma.
Liu
XF, Zou SQ, Qiu FZ. Construction of HCV-core gene vector and its
expression in cholangiocarcinoma. World J Gastroenterol
2002;8(1):135-138
INTRODUCTION
Cholangiocar
cinoma is the second cancer of hepatobiliary system. The
incidence and mortality of cholangiocarcinoma are increasing
yearly.There are 3000 new patients in America each year. Recent
reports showing the expression of HCVRNA and HCV antigents in
cholangiocarcinoma[1-8],have provided new insights into
the pathogenesis of cholangiocarcinoma. Hepatitis C virus(HCV) is
recognized as a kind of serious infectious source to harm the health
of the humans.It could lead to cancer[9-17].Moreover,its
core protein could act as a transcriptional regulator of various
viral and cellular promoters to potentially disrupt normal cellular
functions[18-21] .Thus,the function of core protein is
important for carcinogenesis.We constructed the recombinant plasmid
of HCV core gene with molecular cloning technique and transfected
into cholangiocarcinoma cells with lipofection, and established an
experimental model for exploring the role of HCV in the development
of cholangiocarcinoma.
MATERIALS
AND METHODS
Materials
Plasmids and bactrium strain PBK-HCV encompassing the core and
envelope genomic regions of HCV,containing 330nt - 2020nt of HCV II
strain, was provided as a gift by Dr.Chen (Institute of Infectious
Disease,Zhejiang University),whose restriction sites were Pst Ⅰand
EcoR Ⅰ.The
prokaryotic expressing vector of PBK-CMV containing MCS(multiple
cloning site),the neomycin- and kanamycin-resistance gene, SV40
poly(A), was purchased from Stratagene Co. E.coliJM109 was
obtained from the collection kept in our research group.
Cells The QBC939 cells(a cholangiocarcinoma cell line)were a
generous gift from Dr. Wang Shuguang(Third Military Medical
University,China). QBC939 cells were cultured in RPMI 1640
supplemented with 100mL·L-1 FBS and incubated at 37℃
in a 50mL·L-1 CO2 atomosphere.
Reagents
Restriction
enzymes( Sal Ⅰand
BamH Ⅰ),
Taq DNA polymerase,T 4DNA ligase,were purchased from Hua Mei Co.
Lipofection was provided by Boehringer Mannheim Co. Mouse anti-HCV C
protein monoclonal antibody was purchased from Chemicon Co. House
anti-mouse IgG(mouse)-AP,NBT/BICP were purchased from Zhong Shan Co.
Biotinylated-conjugated sheep anti-mouse IgG was purchased from
Boster Co.
PCR
primers
Two primers were designed according to the sequence of HCV core
genomic regions and synthesized by the Shanghai GeneCore Bio
Technologies Co. Primer 1:5’-CTCGTCGACCATGAGCACAAATCCTAA-3’;
Primer 2:5’-CTCGGATCCTAAGCGGAAGC- TGGGATG-3’. Primer 1 was
5’primer containing Sal Ⅰ
site and Primer 2 was 5’ primer containing BamH Ⅰsite.
Methods
PCR amplification
PCR amplification was done using the PBK-HCV as a template and
primers 1 and 2 as primers.The reaction was performed according to
the parameters of 94℃
3min,94℃
1min，55℃
30s and 72℃
1min for 35 cycles.Then it was extended 10min at 72℃.The
amplified fragment (approx 600bp)was analyzed by 9 g·L-1
agarose-gel electrophoresis.The product was purified and used for
DNA recombinction.
DNA
recombination DNA
recombination was performed according to the methods described in
reference[22]. A 0.6kb fragment,containing HCV core
gene,was obtained from the PCR product after digestion with Sal Ⅰand
BamH Ⅰ.The
fragment was recombinated into plasmid PBK-CMV and the resulted
recombinant plasmid was designated as PBK-HCVC (Figure 1).
Transfection
of cells
Transfection was performed with lipofection.The constructed vector
and control plasmid were used to transfect QBC939 cells in culture.
Seven-two h after transfection, they were selected with G418. Then
the cells were harvested and used for the detections.
Immunocytochemistry
for HCV core protein Detection
of HCV core protein expression was performed using a mouse anti-HCV
core protein monoclonal antibody(1:50) on cell sections of
transfected QBC939 and control plasmid.
Figure
1(PDF)
Construction of plasmid PBK-HCVC.
Western blotting Detection of HCV core protein by
immunoblotting was performed. Briefly,cells (1×106)were
scraped,centrifuged briefly,and lysed for 30min on ice in 50mmol·L-1
Tris-cl( pH 7.5), 150mmol·L-1 Nacl,0.2mmol·L-1
EDTA,1mmol·L-1 PMSF and 10g·L-1 NP-40.The
samples were cleared by centrifugation(140000r·min-1
,30min,4℃),and
assessed for protein concentration.SDS-PAGE was performed,and
proteins were electroblotted onto nitrocellulose membranes.After 1h
incubation in blocking solution,the membrane was exposed to the
primary antiobody 2h at 4℃.After
washing in PBS,the secondary AP-labeled antibody was added for 2h at
room temperature.The proteins were visualized with NBT-BICP.
Transmission electron microscope A pellet of the transfected
cells was fixed in 2.5g·L-1 glutraraldehyde,postfixed
with 10g·L-1 osmium tetroxide,treated with 20g·L-1
uramyl acctate,dehydrated in ethanol, infiltrated with propylene
oxide,and embedded in Epon mixture.Ultrathin sections were observed
under Opton EM 10C(German).
Microscope
After
selected with G418, the cell sections were stained with HE.The cell
sections were observed under microscope.
RESULTS
Identification
of reconstructed plasmid by PCR and restriction enzymes
The reconstructed plasmid was amplified by PCR, using the PBK-HCVc
as a template and primers 1 and 2 as primers, the reaction was
performed according to the same parameters. PCR product was approx
600bp(Figure2). Then it was identified by the digestion with
restriction enzymes( Sal Ⅰand
BamH Ⅰ).
Fragments of 4500bp and 600bp were produced from the digestion. It
was proved to carry the target gene(Figure 3).
Figure
2 Electrophoretic
analysis of PCR product
M:Marker DL2000; 1:Negative control;2:PCR product
Figure
3 The
restriction mapping of PBK-HCVC
M:Maker;λDNA/HindⅢ;1:PBK-CMV;2:PBK-HCVC;3:PBK-HCVC
restricted by Sal Ⅰ
and BamH Ⅰ
Expression
of the HCV core protein
Cell sections of transfected with PBK-HCVC and control
plasmid(PBK-CMV) were stained for HCV core protein. Staining for HCV
core protein was seen in the PBK-HCVC transfected QBC939
cells.Positive staining was located to the cytoplasm(Figure 4).
Figure
4 Detection
of expressed HCV C antigen by immunocytochemistry. ×200
The expressed products in the supernatant which were
transfected with PBK-HCVC and control plasmid(PBK-CMV) were
identified by Western blotting. A appox 21ku band,similar to the
size of HCV core protein,was observed in the supernatant of
transfection with PBK-HCVC(Figure 5). These results showed that the
QBC939 cells transfected with PBK-HCVC could express HCV core
protein.
Figure
5
Western blotting detection of the expressed HCV-C protein
1:Transfected QBC939 cells with PBK-CMV；2:Transfected
QBC939 cells with PBK-HCVC
Figure
6
Morphology of QBC939-HCVC cells by TEM. ×6500
Figure
7 Morphology
of QBC939-HCVC cells by TEM. ×60 000
The
morphologic alteration of the transfected cells
On day 15 after QBC939 cells were transfected with PBK-HCVC, the HCV-like
particles were found in the cytoplasm under TEM,which were spherical
with a diameter of 50nm-80nm possessing outer membrane(Figures
6,7).The structures were absent in the negative control.The results
suggested that the recombinent plasmid could express HCV core
protein efficiently in cholangiocarcinoma cell lines.The splits of
nuclei were increased significantly in transfected QBC939 cells with
PBK-HCVC than PBK-CMV and QBC939 cells (Figure 8). It suggested that
the exent of the differentiation was reduced and malignant degree
was enhanced after QBC939 cells were transfected with PBK-HCVC.
Figure
8 The
morphologic alteration of the transfected cells HE. ×200
DISCUSSION
The
pathogenesis of cholangiocarcinoma is not clear.
Cholelithiasis,cystic dilation of the biliary system,ulcerative
colitis and primary sclerosing cholangitis are thought to be the
risk factors for cholangiocarcinoma.Recently, laboratory and
epidemiological studies found and epidemic found the infection of
HCV was related to the development of cholangiocarcinoma[1-8,23-25].But
the mechanism of how they are related has not been studied deeply.
HCV infection is an important cause of morbidity and mortality
worldwide,causing a spectrum of liver diseases ranging from an
asymptomative carrier state to end-stage liver disease.The most
important feature of persistent HCV infection is the development of
chronic hepatitis in half of the infected individuals and the
potential for disease progression to hepatocellular carcinoma.In our
country there are 30-40 million of HCV-carriers patients[26-28].
HCV
are hepatotropic viruses.Viral replication and cellar injury are
largely confined to the liver.Recent studies,however,have suggested
that HCV may replicate in tissues other than in hepatocyte only.
Many scholars have reported the presence of HCVRNA and HCV-antigens
in lymph nodes, pancreas, ovary, kidney, heart and bile duct
epithelial cells[29-35]. The infection of HCV could lead
to bile duct damage and loss. Moreover,bile duct injury is more
commonly associated with HCV than either HBV or autoimmune
hepatitis,which is the characteristic histologic feature of HCV. The
bile duct damage and loss are defined as variable
epithelial,steatosis,lymphocytic infiltration of bile ducts[36-38].The
more injury of HCV infection in bile duct provides new evidence that
bile duct epithelial cells could be an important reservoir of HCV
and might lead to the pathogenesis of cholangiocarcinoma. An HCV
genome contains a linear,positive-strand RNA molecule of 9500
nucleotides encoding a single ployprotein precursor of 3000 amino
acids.The polyprotein is cleaved by viral proteases to generate
three putative structural proteins(C, E1 and E2) and at least six
nonstructural proteins(NS2,NS3,NS4A, NS4B, NS5A and NS5B)[39,40].
The genomic region encoding the putative C protein is also called
core protein. The HCV core protein could act as a transcriptional
regulator of various viral and cellular promoters to potentially
disrupt normal cellular functions[18-21]. The core
protein may cooperate with ras oncogene and transform primary rat
embryo fibroblasts to a tumorigenic phenotype[41].It may
also cause anti-apoptosis by ressive of tumor suppressor gene-p53
and activation of NF-κB and implicate a mechanism by which HCV
may evade the host immune surveillance leading to viral persistence
and possibly to carcinogenesis[42-51]. Thus,the HCV core
protein plays a major role in the malignant transformation of cells.
It is important to explore the role of HCV in the development of
cholangiocarcinoma by establishing an experimential model which
expresses efficiently HCV core protein in cholangiocarcinoma cell
lines.
In
our study, recombinant plasmid of HCV-core gene was constructed with
molecular cloning technique,it was identified by restriction
enzymes.Then it was transfected into QBC939 cells with
lipofection.After it was selected with G418 made by the presence of
the necomycin and kanamycin resistance gene, resistant colonies were
obtained. The colonies were analysed by immunocytochemistry and
Western blotting. The results suggest that the recombinant plasmid
was proved to carry the target gene by PCR and restricted enzyme
map, and it could express HCV core protein efficiently in QBC939
cells.The morphology was observed under transmission electron
microscopy(TEM), and the HCV-like particles in the cytoplasm, were
found spherical with a diameter of 50nm-80nm possessing outer
membranes. Moreover, we also found that the splits of nuclei were
increased in transfected QBC939 cells with PBK-HCVC, and the exent
of differentiation was reduced. It suggested that the malignant
degree was enhanced after QBC939 cells were transfected with
PBK-HCVC. Because HCV-core gene could express steadily in
cholangiocarcinoma cells,the transfected tumor cells(QBC939-HCVC)
could be used to study the effect of HCV in the development of
cholangiocarcinoma.
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