| Abstract |
The research of fish cell culture has developed rapidly since 1960s when
Wolf and Quimby established the first fish cell line - RTG-2 - in the world.
At the present time, fish cell culturing has become an essential research
technology which has been used extensively, ranged from virology,
environmental toxicology, cytobiology, oncology, genomics, genetics and
environmental protection. Cultured fish cells have more advantages than
live fish as the experimental material: 1. the materials are cheap and
easy to obtain; 2. the experimental condition could be controlled
accurately and the experiment could be repeated. In the present study, the
culture methods of fish embryonic cell had been researched deeply. The
results were as follows. 1. Three embryonic cell lines had been
established: Japanese flounder (Paralichthys olivaceus) embryonic cell
line (FEC), turbot embryonic cell line (TEC) and Southern Flounder
embryonic cell line (SFEC). At the same time, a liver cell line of half-
smooth tongue-sole (Cynoglossus semilaevis) (HTLC) was established. FEC
has been cultured for more than 100 passages and the same as the TEC. SFEC
has been cultured for more than 80 passages while HTLC has been cultured
for more than 30 passages. 2. The embryonic cells were cultured in DMEM
medium supplemented with antibiotics, fetal bovine serum (FBS), sea perch
serum (SPS), basic fibroblast growth factor (bFGF). The HTLC cells were
cultured in MEM medium supplemented with antibiotics, fetal bovine serum
(FBS), sea perch serum (SPS), basic fibroblast growth factor (bFGF). The
embryonic cells are small and polygon, rotundity. The liver cells of half-
smooth tongue-sole are fibriform. Effect of temperature, fetal bovine
serum (FBS) concentration and bFGF on the growth of cells was examined. To
some extent, the growth rate of cells depends on FBS concentration, and
can be restrained by higher or lower FBS concentration. Addition of bFGF
to the medium can significantly increase the growth rate of cells. The
cells grow well in at the temperature range of 24-30 Celsius, but have a
reduce growth rate at temperature below 18 Celsius or above 30 Celsius. 3.
Chromosome analysis reveals that FEC cells have a normal diploid karyotype
with 2n=48t; TEC cells have a normal diploid karyotype with 2n=4m+12st+28t;
SFEC cells have a normal diploid karyotype with 2n=6st+42t; HTLC cells
have a normal diploid karyotype with 2n=42t. Heterotypic sex chromosome
(ZW) was investigated on the metaphase chromosome of half-smooth tongue-
sole (female) and homotypic sex chromosome were investigated on the
metaphase chromosome of half-smooth tongue-sole (male). 4. GFP reporter
gene were transferred into SFEC and HTLC cells with Genejammer-mediated
and successfully expressed in two cell lines. The transformation
efficiency was about twenty percent. FEC cells were injected by Flounder
lymphocystis disease virus (LCV) and Turbot reddish body iridovirus (TRBV).
Virus replicated rapidly in FEC and the cytopathic effect (CPE) was
investigated,. The mRNA expression levels of Hepcidin gene in TEC after
the challenge of Pichia pastoris were measured by semi-quantitative RT-PCR.
Sex-specific bands located on the W sex chromosome of females of half-
smooth tongue-sole by fluorescence in situ hybridization (FISH) analysis.
In summary, the technology of establishing embryonic cell lines is founded.
it is the first time that FEC, TEC and SFEC were established in the world
while the HTLC was established by traditional method. Some research was
explored in the application of cell lines and laid foundation of using
cell lines to study theory and experiment.
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