| With environmental pollution becoming more and more serious,the harm to human health caused by it has drawn more and more attention.In particular,some eye diseases such as abnormal eyesight development and amblyopia of children caused by environmental pollution have become a research hotspot in recent years.Studies have shown that from the embryonic stage to the age of 6 is a critical period for the development of children’s retina development and visual function.Environmental pollution may adversely affect the normal development of the visual system during this period,resulting in abnormal visual development,color blindness,and vision loss.Polychlorinated biphenyls(PCBs),as a common global environmental pollutant,are extremely stable in their physicochemical properties,can exist in our environment for a long time and are extremely difficult to degrade.As PCBs are extremely fat-soluble,they are easily absorbed by human skin and accumulated in the human body.If children are exposed to excessive exposure to PCBs for a long period of time,they will have serious adverse consequences on retinal development.The results of previous research in our group have shown that exposure of polychlorinated biphenyls(PB)causes reduced and disordered retinal cell populations in zebrafish,confirming that they may cause abnormal retinal development.At the same time,through the optokinetic response study of zebrafish juveniles,it was found that the zebrafish juveniles exhibited reverse swimming and hesitancy after exposure to polychlorinated biphenyls,and the retinal-related specific expression genes decreased,confirming PCBs.Exposure can cause decreased vision in zebrafish and affect the development of visual function.However,further research is needed to clarify the mechanism of environmental retinal abnormalities caused by environmental pollution,and to provide new ideas and theoretical basis for corresponding prevention and control work.In recent years,with the development of molecular biology and the maturity of biosequencing technology,peptidomics has become a hot topic.An important methodfor the study of the peptidomics is mainly enzymatic polypeptides,which use biodegradation methods to obtain endogenous polypeptides for further functional studies.There have been reports in some literatures that endogenous polypeptides can participate in the development and repair of the retina.Therefore,in this study,endogenous peptides were used as the entry point,and the peptide polymorphism was first examined by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS)to study the endogenous polypeptide expression of zebrafish exposed to PCBs.Spectral effects,further analysis of physicochemical properties and biological information of differentially expressed polypeptides,searching for polypeptides closely related to retinal development in endogenous polypeptides that are differentially expressed after exposure,and selecting endogenous and well-organized endogenous polypeptides A preliminary study on the role and function of the polypeptide "CKASKGKL" in retinal development revealed that it could promote the proliferation of retinal ganglion cells,and through the whole transcriptome sequencing technology,further found its target genes and verified it.The results of this study provide new ideas for further research on the impact of environmental pollution on retinal development and the adoption of relevant prevention and control measures.PartⅠ:Analysis of zebrafish endogenous peptides after exposure to PCB and screening of peptides involved in retinal developmentObjective:To investigate the expression changes of endogenous polypeptides in zebrafish embryos after PCB exposure and to screen for endogenous polypeptides closely related to retinal development.Methods:(1)Using zebrafish as a model animal,48-hour zebrafish embryos were collected through an established zebrafish PCB exposure model,and a control group was set up.LC-MS/MS combined with tandem mass spectrometry(TMT)was used for detection.Differential expression of endogenous polypeptides was screened for expression of endogenous polypeptides between the two groups;(2)analysis of differentially expressed endogenous polypeptides for physicochemical properties,restriction sites,and precursor proteins,using IPA analysis,GO analysis and other bioinformatics analysis methods screen for endogenous polypeptides associated with retinal development.Results:(1)A total of 7900 endogenous peptides were screened by mass spectrometry.There were 90 differentially expressed peptides(P<0.05,multiples of difference>2),of which 29 were expressed after PCB exposure.Up-regulated,61 were down-regulated after PCB exposure.Physical and chemical properties analysis revealed that the molecular weight of multiple molecules was below 3000 Da.The isoelectric point was mainly composed of weak acid and weak alkalinity,with no neutrality.(2)Through Gene Ontology(GO)analysis,it was found that differential polypeptides mainly involve the cell development process of organisms,are closely related to the cell membrane,and the main molecular function is binding.Ingenuity Pathway Analysis(IPA)analysis found that differential peptide enrichment signaling pathways include neuron-mediated axon guidance pathways,Slit/Robo-mediated axon guidance pathways,and Wnt signaling pathways.Both are closely related to thedevelopment of the retina.By studying the regulatory relationship between different peptides and their relationship in the development of the retina and optic nerve,pick out peptides related to retinal development were finally selected.Conclusion: Polychlorinated biphenyl exposure can affect the expression of endogenous polypeptides in zebrafish.Endogenous polypeptides are involved in many life processes and are closely related to retinal development.Part Ⅱ : The role of retinal endogenous polypeptide "CKASKGKL" in retinal development and its mechanismObjective:To verify the peptide mass-spectrometry screening results,to explore the role and mechanism of retinal endogenous polypeptide "CKASKGKL" in retinal development.Methods:(1)Using zebrafish as a model animal,HE staining was used to verify the effect of endogenous peptide on zebrafish retina development;(2)Retinal ganglion cell(RGC)was used as a tool to investigate endogenous peptides.Effects on cell proliferation,migration,apoptosis,and cycle;(3)Screening the downstream target genes of the polypeptide “CKASKGKL” by sequencing and verifying the target genes to explore their role in the retina development.Results:(1)After injection of endogenous polypeptides,zebrafish retinal ganglion cells increase,promote proliferation and migration of retinal ganglion cells,have no effect on apoptosis,and can promote polypeptide precursor protein Ntn-1 and cell-specific gene Brn-3 expressions.(2)A total of 207 downstream target genes(differentiation multiples greater than 2 times P<0.05)were screened by whole-transcriptome sequencing(RNA-seq),of which 69 were down-regulated genes after injection and 138 were up-regulated after injection.Further bioinformatics analysis found 24 neurodevelopmental and retinal development related.Through real-time quantitative PCR,it was found that the results of four gene repeats of Bcl-6,Apaf1,Scrt2,and Srcc5 were stable,and the functional results of Bcl-6 were consistent with the current results.Polypeptide may promote cell proliferation by reducing the expression of Bcl-6.Conclusion: The polypeptide "CKASKGKL" can promote the proliferation and migration of retinal ganglion cells and promote the expression of cell precursor proteins and cell-specific genes.Bcl-6 may be a target gene for its regulation. |
PDF Full Text Request