| Abstract |
The establishment of fish cell lines plays an important role in aquatic
animal virology, environmental toxicology, fish resource protection and
genetic breeding, and fish endocrinology. Since the establishment of the
world's first fish cell line, the rainbow trout (Oncorhynchus mykiss)
gonadal cell line RTG-2, more than 280 cell lines of different tissue
origin including freshwater fish and marine fish have been established so
far, but the gonad origin somatic cell lines are relatively rare. Fish
gonadal cells include granulosa cells, sheath cells, interstitial cells of
the ovary, and Sertoli cells and Leydig cells of the testis. They play a
decisive role in the proliferation, differentiation and development of
germ cells, especially in recent years The focus of stem cell research
makes the cultivation of gonadal cells and the establishment of cell lines
particularly necessary. Studies have shown that using zebrafish (Danio
rerio) gonadal cells as a feeder layer, their reproductive stem cells can
be cultured in vitro for months. The southern catfish (Silurus meridionalis
Chen) belongs to the genus Polygonaceae and is a valuable economic fish
mainly distributed in the middle and upper reaches of the Yangtze River in
China. It has fast growth speed, tender meat, wide temperature range, and
strong disease resistance. It has become one of the important farmed fish
in China. Under natural conditions, the sex ratio of southern catfish
is about 1:1. Interestingly, the offspring of southern catfish are
almost all females under artificial breeding conditions. Although this has
been explored for many years now, its specific mechanism remains to be
further studied. The establishment of the southern catfish gonadal cell
line is expected to provide a powerful tool for the study of the culture,
proliferation and differentiation of germ cells, especially germ stem
cells, the effects of functional genes and environmental endocrine
disruptors.
On the other hand, in recent years, more and more reports show that
environmental estrogen pollution in waters around the world is increasing,
which has significant effects on human and animal reproductive
developmental abnormalities, malignant tumors, and metabolic disorders.
Real-time and efficient monitoring is a prerequisite for preventing and
controlling its pollution. There are many types of environmental estrogen
substances, large quantities and small amounts, and their biological
detection has an irreplaceable status and role. It has been reported that
the pEGFP-ERa, a eukaryotic expression vector capable of stably expressing
mammalian estrogen receptor (Estrogen Receptora, ERa) and the true
expression vector containing four tandem estrogen receptor response
elements (ERE) The nuclear expression plasmid pGL3-ERE4-luc was co-
transfected into mammalian cells to monitor the estrogen activity of
environmental estrogens. This method has the advantages of high
efficiency, speed, and high sensitivity. However, the expression of the
environment and related molecules in fish cells is significantly different
from that of mammalian cells, and the sensitivity to environmental
estrogen is also significantly different. Therefore, can the above-
mentioned detection system of mammalian cells reflect the effects of
environmental estrogen on fish, For further study. In view of this, this
study mainly carried out two aspects of work. On the one hand, the
southern catfish ovary and testis somatic cell lines were established and
their biological characteristics were identified; on the other hand, the
channel catfish (Ictalurus punctatus) ERa was established. The eukaryotic
expression vectors pEGFP-ccERa and pGL3-ERE4-luc were co-transformed into
mammalian cells and fish gonadal cell lines, respectively, and compared
and analyzed in order to establish a highly sensitive detection system for
the effects of environmental estrogens on fish. details as follows:
(I) Establishment and biological characteristics of the southern catfish
ovary and testis somatic cell line
(1) Southern catfish ovary somatic cell line About 3 months old Southern
catfish ovary tissue was digested with collagenase IV and trypsin-EDTA,
and contained 15% fetal bovine Serum (FBS), 25 mM Hepes, 1% non-essential
amino acids, 0.5 mM beta-mercaptoethanol, 10 ng/mL basic fibroblast growth
factor (bFGF), 1 ng/mL leukemia inhibitory factor (LIF), 500 U/mL L-15
medium of penicillin, streptomycin, and 5% southern catfish serum was
cultured at 28 Celsius. The chromosome karyotype analysis, RT-PCR, gene
transfection and other methods were used to identify its biological
characteristics. The results showed that: after 2-3 days of isolation and
culture, fibroblast-like growth began. After about 15 days, a monolayer
was grown, digested with 0.25% trypsin-EDTA, and subcultured at 1:2. The
initial cell growth was slower after passage, with an average of 7-passage
on 10 days, after the passage to the 9th passage, the cell growth is
accelerated, and the passage is performed on average 3-6 days; the current
cells have been stably passaged to the 90th passage after 18 months of
subculture, and named SCO1. SCO1 in different media: (DMEM, L-15,
DMEM/F12), different blood concentration (5%, 15%, 25%) and culture at
different temperature conditions (18, 28, 37 Celsius). The results showed
that the culture was performed at 28 Celsius in L-15 medium of 15% fetal
bovine serum. SCO1 optimal growth: RT-test results showed the PCR, the
expression SCO1 Foxl2, Sf1 and Wt1b, do not express Vasa, Wt1a, Cyp19a and
Dmrt1 (d) The 70th generation SCO1 karyotype analysis results show that
the chromosome number of more than 80% of the cells is 50-65, of which the
division phase of 2n=58 accounts for 62%, and shows a normal diploid
karyotype. The hrGFP-1a plasmid was transfected with SCO1 with TransIT(@)-
2020. The expression of green fluorescent protein was observed 24 hours
later. By 48 hours, the expression of the target protein was visible in
about 30% of the cells.
(2) Southern catfish testis cell line The same method as above was used to
establish the southern catfish testis cell line. The cell morphology is
fibroblast-like. It has been cultured for 15 months and has been stably
passed to the 56th generation. It is named SCT1. Analysis of the
chromosome number and karyotype of SCT1 at the 19th and 43th generation
showed that the chromosome number of more than 70% of the cells was 50-65,
and the division phase of 2n=58 accounted for 52%, showing a normal
diploid karyotype; RT-PCR The analysis showed that SCT1 expressed Sf1 and
Foxl2, but not Vasa, Wt1a, Wt1b, Cyp19a, and Dmrt1; pIRES-hrGFP-1a plasmid
was transfected with SCT1, and about 25% of cells expressed green
fluorescent protein 48 hours later.
(II) Establishment of environmental estrogen detection system (a) RT-PCR
cloning and obtaining the full-length cDNA sequence of the estrogen a
receptor of Ictalurus punctatus, and the eukaryotic expression vector
pEGFP-ccERa was successfully constructed. (b) pEGFP-ccERa and pGL3-ERE4-
luc were co-transformed into HEK293 and SCO1, and compared with the human
estrogen receptor expression vector pEGFP-hERa. The results show that the
detection limit of E2 in HEK293 cells for pEGFP-hERa is 10-10 mmol/L, 10-9
mmol/L in SCO1, and pEGFP-ccERa is just the opposite, that is, the
detection limit of E2 in HEK293 is 10-9 mmol/L, and 10-10 mmol/L in SCO1.
Therefore, it was shown that pEGFP-ccERa has higher sensitivity to detect
estrogen luciferase activity in fish cells SCO1 than pEGFP-hERa. (c) 17a-
methyltestosterone (MT), 17a-ethinylestradiol (EE2), estradiol (E2),
bisphenol propane with different estrogen-like activity levels at the same
concentration (10-5 mmol/L) (BPA), nonylphenol (NP), ethylestradiol (DES)
and octylphenol (OP) treated cells. After 48 h, the relative activity of
luciferase was measured. The results showed that the relative activity of
environmental estrogen luciferase The size is consistent with its
corresponding estrogen activity (ie EE2> E2> DES> BPA> OP> NP> MT), which
indicates that pEGFP-ccERa can accurately detect the estrogen activity of
environmental estrogens.
To sum up, this study successfully established the southern catfish ovary
and testis somatic cell lines, namely SCO1 and SCT1, and successfully
constructed the P. punctatus estrogen a receptor expression vector pEGFP-
ccERa, which was co-transformed with pGL3-ERE4-luc into SCO1, thus
establishing an environmental estrogen detection system. The research not
only provided the necessary platform and tools for the culture of germ
cells, especially germline stem cells, and functional genes, etc., but
also laid an important foundation for the detection of environmental
estrogens.
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