| Cetaceans are at the top of the marine ecosystem and inhabit the marine ecosystem for a long time.Due to the interference of global climate change and human activities,the health and reproduction of cetaceans are seriously threatened.At the same time,due to species characteristics and legal and moral restrictions,it has been prohibited to conduct potentially harmful in vivo experiments,so it is urgent to build an in vitro research model based on cetaceans.First of all,through the establishment of in vitro research model,risk assessment of this species can be carried out to explore the environmental threshold of stress factors,which can provide strong scientific support for the health assessment and management of cetaceans.Secondly,through the establishment of different cetacean in vitro research models,different molecular response mechanisms of different marine mammals to stress factors are cross-compared,so that the protection work can be carried out pertinently.Finally,through the establishment of in vitro research models of different species,we can achieve the preservation of germplasm and gene resources of species and promote the protection of biodiversity.Based on the above reasons,the immortalized skin fibroblast cell line was established by using the skin tissue of Indo-Pacific finless porpoise as the experimental material,and the cell line was used as a research tool.The health risks of typical environmental pollutants to cetaceans were studied.On the one hand,the threshold of side effects of typical pollutants on cetaceans was explored by means of cell activity;on the other hand,through exposure to typical pollutants,the molecular mechanism of toxic side effects of typical pollutants such as PBDEs on different cetaceans was studied.The main results are as follows:(1)The immortalized cell line(FPT)of skin fibroblasts of Indo-Pacific finless porpoise was established by using fresh skin tissue of Indo-Pacific finless porpoise as experimental material.The cell line can still maintain the morphological characteristics of spindle-shaped fibroblasts and stably express SV40 T antigen in vitro.The growth rate of FPT was faster than that of primary cells,and the doubling time was 32.32 h,and the growth limitation of primary cells was completely overcome.Therefore,this study successfully established the skin fibroblast immortalized cell line of the Indo-Pacific finless porpoise.(2)Based on the established cetacean skin fibroblast cell lines,we compared the cytotoxicity of heavy metals(copper,methylmercury,cadmium)and persistent organic pollutants(PCB126,PCB153 and BDE47)to three species of marine mammals(IndoPacific finless porpoise,pygmy killer whale and pantropical spotted dolphin)and two species of terrestrial mammals(human and mice).Cell viability experiments revealed that Indo-Pacific finless porpoises were more sensitive to copper chloride(Cu Cl2)and methylmercury chloride(Me Hg)than other mammals.However,there were no significant differences in cell viability between mammals following exposure to persistent organic pollutants(e.g.,BDE47,PCB126,and PCB153).However,there was a significant positive correlation between the expression level of POPs metabolismrelated genes and exposure concentration after exposure to PCB126.In contrast,no significant change in m RNA expression of target genes was detected in cells exposed to BDE47 and PCB153.Therefore,this study assessed the potential risk of environmental pollutants by cell viability and biomarker genes,and proved that skin fibroblast cell line was a powerful tool to evaluate the toxic mechanism of cetaceans in vitro.(3)Using skin fibroblast cell line FPT of Indo-Pacific finless porpoises as experimental material,transcriptome analysis was conducted to investigate the toxicity mechanism of three important PBDEs congeners to Indo-Pacific finless porpoises,and to study the molecular action mechanism of POPs targeting a single species.Through transcriptome analysis,it was found that different PBDEs congeners had different effects on FPT,but they all affected the biological function of extracellular matrix of FPT cells.Different PBDEs congeners affected the composition and structure-related gene expression of extracellular matrix,and affected the expression of collagen,elastin and laminin-related genes.These differential genes were verified by q RT-PCR,the results showed that three PBDEs congeners could down-regulate the expression of these genes,indicating that PBDEs may inhibit the expression of extracellular matrixrelated proteins.The protein expression of type I collagen and type VI collagen was detected by Western blot and immune cell staining assay.The results showed that three kinds of PBDEs congeners could decrease the transcription and expression level of type I collagen and type VI collagen.In addition,PBDEs significantly inhibited the expression of p-Smad2,did not affect the change of Smad2/3,and inhibited the expression of α-SMA.The results showed that PBDEs significantly inhibited wound healing of FPT cells by inhibiting collagen synthesis and differentiation.(4)By comparing the effects of PBDEs congeners on collagen expression in skin fibroblasts of Indo-Pacific finless porpoise and pygmy killer whale,we found that under the action of BDE47,BDE100 and BDE209,the collagen I and VI of skin fibroblasts of Indo-Pacific finless porpoise decreased;the expression levels of type I and type VI collagen in skin fibroblasts of pygmy killer whale correlated with PBDEs species.Through the RNA-seq comparative analysis of the transcriptional groups of the two species,it was found that different PBDEs congeners showed distinct differences in the gene transcription of the skin fibroblasts of the two species of cetaceans,and the negative regulation of TGF-beta signaling pathway only appeared in the Indo-Pacific finless porpoise.The heat map and PCA results of PBDEs on the expression of extracellular matrix-related proteins showed that the effects of different PBDEs congeners on cetaceans were significantly different.PBDEs significantly reduced the expression levels of α-SMA and Smad2/3 induced by TGF-beta,as well as the p-Smad2.Compared with Indo-Pacific finless porpoise,PBDEs had no significant effect on the expression of α-SMA and Smad2/3 and the p-Smad2 induced by TGF-beta.On the one hand,the results of this study proved that different POPs congeners have different molecular mechanisms of stress to cetaceans;on the other hand,different cetaceans may have species specificity in response to POPs stress.Therefore,it is of great practical significance to establish in vitro research models of cetaceans with more species and more cell species to support the protection of different cetaceans. |
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