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Establishment of Two Novel Cell Lines from Spotted Halibut (Verasper Variegates) and Barfin Flounder (Verasper Moseri), and Studies on the Cytotoxicities of Tributyltin Oxide to the Cell Lines

Author: GuoXueYang
Tutor: FanTingJun
School: Ocean University of China
Course: Cell Biology
Keywords: The round plaques Star flounder Streak Star flounder Fin cell line Gill cell line Tributyltin oxide
CLC: S917.4
Type: Master's thesis
Year: 2010
Downloads: 23
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Abstract

Recent years, due to the apathy of the rapid development of modern industry and agriculture and people to protect the environment awareness, a large number of hazardous substances, including organic tin compounds, including a number of ways by land-based sources of pollution and pollution of the sea into the seawater to the marine environment caused by the great destruction. Which, tributyltin oxide (TBTO) as widely used in the hull and nets in a biological anti attached paint, is considered to be the most toxic environmental pollutants and endocrine disruptors one of its major aquatic organisms Growth and body tissue and organ function resulting in a greater impact, leading to its a large number of deaths and the fecundity decline, a serious threat to aquatic populations of biological resources and the development of the aquaculture industry, and through the role of bio-rich human health caused great who threaten. Round spots Star flounder Verasper variegatus (Temminck et Schegel) streak Star flounder Verasper moseri (Jordan et Gilbert) is part of the bony fish the Gang (Osteicthys) Pleuronectiformes (Pleuronectiformes) Flounder Division (Pleuronectidae) Star flounder are (Verasper ), are large cold temperate and valuable benthic species. In recent years, the scale of farming has gradually expanded to create huge economic value, but equally well received by of TBTO, including a variety of pollutants poison. Fish cell line as a cellular toxicology, in vitro research tool, and is widely used in the detection of various pollutants in seawater and in vivo detection and fish vivo compared with the economy, sensitive, rapid, etc. there is an urgent need to establish the Star flounder the organization cell lines in vitro studies of cell toxicology. But so far, yet studies have reported the successful establishment of the Star flounder tissue cell lines. In order to establish the organization of Star flounder cell lines, respectively 0.5% -0.2% Ⅱ collagenase digestion hyaluronidase and 0.25% trypsin digestion of the tissue of the round spots Star flounder fin and streak Star flounder gill tissue processing and add have 50μg/mLN- acetyl glucosamine hydrochloride, 50μg/mL carboxymethyl chitooligosaccharides, 10ng/mL alkaline fibroblast-like growth factor (bFGF) and of 40ng/mL I insulin-like growth factor ( IGF-I) containing 20% ??fetal bovine serum (FBS), DMEM/F-12, Leibovitz's L-15 and M199 culture medium (pH7.2) at 18 ° C, respectively, at 22 ° C and 26 ℃ two organizations in vitro culture. The results show that organizations the round spots Star flounder fin and streak Star flounder gill tissue are optimal culture medium containing the the above additives 20? S-DMEM/F-12 (pH 7.2), the optimal incubation temperature are 22 ℃; to start primary cultured, the organization the round spots Star flounder fin and streak Star flounder gill tissue culture cells move out after 7d and 10d, cell morphology are into fiber, respectively, at the start in this under optimal conditions kind, good growth and division status, the formation of a confluent cell monolayer and 20d and 30d, respectively; After passage, the two kinds of tissue cells can successfully subcultured, round plaques Star flounder fin cells has now spread to 110 on behalf of, the streaks Star flounder gill cells has now spread to the 70th generation successfully established the the round spots Star flounder fin cells in continuous cell lines (SHF01) and the streak Star flounder gill cells continuous cell line (BFG01). The analysis showed that the growth characteristics of the cells of the two cell lines, Generation 60 spots on Star flounder fin cells and bars plaques Star flounder gill cell population doubling time 41.5h and 54.4h, that the growth of separatist state is still strong; chromosome analysis of the results of the two cell lines cells, cell lines chromosome number 60 on behalf of two Star flounder organization of aneuploidy, but its characteristic chromosome number are still 46. , The round spots Star flounder fin cell line cells have 46 chromosomes proportion of 57.7%, and has a typical diploid karyotype characteristic (46t) streak Star flounder gill cell line cells have 46 chromosomes proportion 68.3%, and the typical normal diploid karyotype characteristics (44t 2sm). These results suggest that both cell lines established indeed round spots Star flounder fin streak Star flounder gill continuous cell lines. Tributyltin oxide (TBTO) of the toxic effects of the two star flounder lineage cells, TBTO select 0.5-64 ng / mL, respectively, of the two cell lines were treated by dimethyl-thiazole diphenyltetrazolium bromide (MTT) assay and protein content assay detection TBTO on the toxic effects of the two cell lines. MTT assay and protein assay results, 0.5ng/mL TBTO can produce toxic effects of two types of cells, and showed a significant dose - response relationship. TBTO was calculated the round spots Star flounder fin cell for 48 hours 50% inhibitory concentration (48h-IC50 values) were 41.58 ng / mL and 44.49 ng / mL streak Star flounder gill cells 48h-IC50 values ??were 37.07 ng / mL and 39.48 ng / mL; TBTO treatment streak Star flounder gill cell activity assay results, 40ng/mL TBTO can cause streaks Star flounder gill cell superoxide dismutase (SOD), glutathione peptide-S-transferase (GST) and glutathione peroxidase (GSH-Px) activity was significantly changed. The above results indicate that TBTO is the toxic effects of the two cell lines in a concentration dependent manner, wherein the streak star flounder gill cells TBTO is more sensitive to higher than the circular spot of 5 flounder fin cell, more suitable for Toxicity of TBTO and SOD, GST and GSH-Px activity of TBTO toxic effects can be used as a detection and evaluation. In summary, this paper successfully established the of the round spots Star flounder fin and streaks Star flounder gill continuous cell lines, streaks Star flounder gill cells TBTO has a higher sensitivity, its SOD, GST and GSH-Px TBTO toxicity effect can be used as a testing and evaluation. The results of this paper TBTO Star flounder mechanism of cytotoxicity laid the foundation for further research, not only for environmental toxicology, in vitro studies have important theoretical significance, but also in line with national standards, rapid and sensitive organotin pollutant toxicity detection system the establishment also has important theoretical guidance value.

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