Marine Biology Institute, College of Science Shantou University, Shantou, Guangdong, PR China; Faculty of Veterinary and Animal Sciences, Lasbela University of Agriculture, Water and Marine Sciences, Uthal, Balochistan, Pakistan.
Marine Biology Institute, College of Science Shantou University, Shantou, Guangdong, PR China.
Aquat Toxicol. 2018 Oct;203:1-9. doi: 10.1016/j.aquatox.2018.07.017. Epub 2018 Jul 25.
The presence of polybrominated diphenyl ethers (PBDEs) in the aquatic environment is an issue of major concern which may be a cause of increasing prevalence and severity of diseases in marine mammals. Although, cell culture model development and in vitro investigation approach is a prime need of time to progress immunotoxic research on aquatic mammals. In this study, we stablished fibroblast cell line (pantropic spotted dolphin) to assess the potential effects of PBDEs on cytokines response. Cells were grown in 6 well cell culture plate and complete media (DMEM and Ham's F12 nutrient mixture, fetal bovine serum, antibiotic and essential amino acids) was provided. The primary culture of (PSP-LWH) cells identification was achieved by vimentin (gene and protein) expressions. Karyotyping revealed pantropic spotted dolphin chromosomes 20 pairs with XX. Transfection was achieved by SV40 LT antigen and transfected cells were expended for passages. Stability of cell line was confirmed at various passages intervals using RT-PCR, western blotting and immunofluorescence methods. After confirmation, cell line was exposed to BDE-47 (250 ng/ml), BDE-100 (250 ng/ml) and BDE-209 (1000 ng/ml), with control group (PBS), positive control DMSO (0.1%) and negative control LPS (500 ng/ml) for 24 h. The ELISA results showed significant increase in IL-6 in BDE- 100 and BDE-209 while IL-1β and IL-8 were found higher in BDE-47 and BDE-100. TNFα and IL-10 secretion was noted higher in control and positive control groups. Altogether, these results emphasize importance of transfected (PSP-LWHT) cell line in aquatic research and potential effects of PBDEs on fibroblast provides evident to understand immune modulating effects of PBDEs in marine mammals. The impact of PBDEs on dolphin's fibroblast cells immune response and altered cytokine response have been presented for the first time.
多溴二苯醚（PBDEs）在水生环境中的存在是一个主要关注的问题，这可能是导致海洋哺乳动物疾病的发病率和严重程度不断增加的原因。尽管如此，开发细胞培养模型和体外研究方法是当前推进水生哺乳动物免疫毒性研究的当务之急。在这项研究中，我们建立了（宽吻海豚）成纤维细胞系，以评估 PBDEs 对细胞因子反应的潜在影响。细胞在 6 孔细胞培养板中生长，并提供完全培养基（DMEM 和 Ham's F12 营养混合物、胎牛血清、抗生素和必需氨基酸）。通过波形蛋白（基因和蛋白）表达实现了（PSP-LWH）细胞的原代培养鉴定。染色体组型分析显示，宽吻海豚有 20 对染色体，XX 为性染色体。通过 SV40 LT 抗原进行转染，转染细胞进行传代扩增。通过 RT-PCR、western blot 和免疫荧光方法在不同的传代间隔时间确认细胞系的稳定性。在确认后，用 BDE-47（250ng/ml）、BDE-100（250ng/ml）和 BDE-209（1000ng/ml）处理细胞系，对照组（PBS）、阳性对照 DMSO（0.1%）和阴性对照 LPS（500ng/ml）孵育 24 小时。ELISA 结果显示，BDE-100 和 BDE-209 中 IL-6 显著增加，而 BDE-47 和 BDE-100 中 IL-1β 和 IL-8 水平较高。TNFα 和 IL-10 的分泌在对照组和阳性对照组中较高。总之，这些结果强调了转染（PSP-LWHT）细胞系在水生研究中的重要性，以及 PBDEs 对成纤维细胞的潜在影响，为理解 PBDEs 对海洋哺乳动物的免疫调节作用提供了依据。首次报道了 PBDEs 对海豚成纤维细胞免疫反应和细胞因子反应的改变。
