Effects Of Low Does BDE-209Single Exposed And Combined With BDE-47on The Morphology And Proteomics Of Neural Stem Cells Of Neonatal Rats In Vitro

【Background】1.About Polybrominated diphenly ethers (PBDEs)PBDEs are a group of brominated flame retardant(BFR) chemicals，Because ofhigh efficiency of flame retardant with low cost that are often added to manufacturedproducts including resin, paints, polyurethane foam and other polystyrene polymersynthetic materials.The end products are widely used PBDEs for textiles, plasticproducts,electronic appliances and construction materials，etc.As early as in1960，people have began to produce and use more and more PBDEs，until1981the year thatSweden found PBDEs are a kind of environmental pollutants. Now the PBDEs havebecome ubiquitous environmental pollutants globally. Some data showed that about80%of the electronic waste were shipped to China, India and other Asian countries,which accounted for90%of electronic waste in China.PBDEs are similar to polychlorinated biphenyls (PCBs)，they have the propertyof low degradation, persist in the environment, high lipophilic,long-distance migrationand biological amplification functions, which can be transferd through food chain，through the food, atmosphere, breast milk and indoor dust bioaccumulate in the body,and finally can be harmful to human health. The main target organs of PBDEs arenervous system, reproductive system, adipose tissue and thyroid,etc. Some animalresearches also proved that PBDEs has a neural developmental toxicity,reproductivetoxici,immunotoxicity, endocrine toxicity and Potential carcinogenecity. Therefore low-brominated PBDEs have been produced and used restrictly in the United Statesand Europe. But some high-brominated PBDEs,such as BDE-209,is still widely usedbecause of its good flame-retardant, bio-toxicity of uncertainty. Understanding of thetoxicity of PBDEs is far less PCBs. The laboratory evidence of the toxicity of PBDEsis relatively more, while the research is mainly based on professional population, so itis lack of data. the distribution of PBDEs are not identical in different occupationalpopulations.The major PBDE congener in nonoccupationally exposed populationsoften is BDE-153in China，Netherlands, the United Kingdom and Sweden. BDE-209and BDE-183have been specifically related to occupational exposure.A variety ofpollutants are exists, and also co-play a role In the environment.2.About BDE-209and BDE-47BDE-209is a kind of high bromine PBDEs containing10bromine atoms, whichhas good thermal stability, less addition amount, cheap and other advantages. Ourcountry is the largest one to use， produce and export lots of PBDEs, especiallyBDE-209, so BDE-209has been the most environmental pollution in China. In theproduction, use and waste treatment processes,through a series of migration BDE-209can be transformed into the sediment, atmosphere, biological solid andorganisms.After that BDE-209can be broken down to the lower brominatedcongeners，polybrominated dibenzofurans，polybrominated dibenzo-p-dioxins, andinduce more toxicity. But the dispute about the PBDE-209hazard to the humanhaven’t stopped. Some research also proved that BDE-209has a neural developmentaltoxicity, Liver toxicity, thyroid toxicity，reproductive toxicity and carcinogenecity.However, some researchers also believe that the metabolism of BDE-209in the bodyis very fast, having low accumulation, and will not bring impact on the body undernormal exposure level. Due to the inconsistency of the results, researches on thebiological toxicity of BDE-209are becoming necessary.BDE-47is also one of the main PBDEs congeners.In recent years, BDE-47andBDE-209are the persistent environmental organic pollutants with rapid growth.Astudy reported that in the soil of pearl river delta states PBDEs pollution is serious,BDE-209is the main pollution content, BDE-47take the second place. BecauseBDE-209as high-PBDEs in the body,can broke down into BDE-47, so the ratio ofBDE-209and BDE-47can change in this conversion process. Also, the study foundthe people homework on BDE-209have high level of BDE-209and relatively low level of BDE-47.Stop exposure three years later, the level of BDE-209decreasedsignificantly， BDE-209content, but the level of BDE-47didn’t change obviously inthe blood samples.About the effect of this change on organisms is the focus of ourattention. Therefore, carry out the effects of BDE-209and BDE-47co-exposure isnecessary.3. About neural stem cells (NSCs)Neural stem cells (NSCs) is a kind of stem cells and undifferentiated cells existin the brain and spinal cord.The same as other stem cells, neural stem cells also havethe biological characteristics:self-renewal, proliferate functional and differentiatedpotential.NSCs can differentiate into neurons, astrocytes and oligodendrocytes,belongto professional stem cells. The discovery of neural stem cells provides a new way forthe reconstruction of the central nerve and nerve regeneration and a new perspectivefor the study of learning and memory mechanism. In the period of mammalianembryo, neural stem cells are mainly distributed in the hippocampus, olfactory bulb，cerebellum，large annoying cortex, SVZ (subventficalar zone. SVZ) and hippocampusdentate gyrus (dentategyrus, DG), and in the hippocampus，striatum, Ventricularzone，SVZ and olfactory bulb in the adult. The regulation of NSC proliferation anddifferentiation is the core of recent researches on NSCs. Recent studies reveal thatproliferation and differentiation of NSCs are co-regulated by a variety of factors,exogenous factors (cytokines and micro-environment) and endogenous factor (gene)co-regulate, which plays a decisive role is the endogenous factors.The individual development process is stem cells differentiate into functionalcells, and the development of nervous system is embryonic stem cells constantlydifferentiate into neural stem cells，then differentiate into nerve cells. Any links of thedifferentiation process affected can reduce the number of neurons, and cause nervoussystem function defects. The brain is the consequence of NSCs proliferation anddifferentiation. NSCs self-renewal by symmetrical divisions and maintain cellpopulations, cell differentiation through the asymmetric split.But exogenouschemicals can affect self-renew and the direction of differentiation.4.About proteomicsProteome is refers to the complete set of proteins expressed in the cells or thegenetic material of an organism.Proteomics is a term in the study of genetics whichrefers to all the proteins expressed by a genome，study the composition of proteins and the changing rules at cells level and the overall level, and understanding of thepathophysiology of organisms process deeply.The word proteomics derives from thecombination of protein and genomics, means " the whole set of proteins expressed bya genome," which includes all the protein expression of a kind of cell or a organism.Proteomics means that research characteristics of protein in large-scale, involvesprotein expression level, modification after translation and interaction of proteins.Thus comprehensively understand the processes of diseases and cell metabolism atprotein level. Proteomics defined firstly by Marc Wilkins in1995. Therefore,proteomics research is not only to explore the mysteries of life，but also bring greatbenefits for human health. Proteomics is the characteristic of life into postgenomic era.Because there are many proteins and cytokines participate and disappear in theNSCs differentiation process，and explore the function and mechanism of the processis extremely complex and huge.However proteomics has the advantages of large scaleand high throughput, which penetrate and complement with genomics andbioinformatics. PBDEs can affect the proliferation and differentiation of NSCs, butthe mechanism has not yet been fully understood,so we cultured neonatal rathippocampal neural stem cells in vitro, single and co-exposed to low concentrationsof BDE-209and BDE-47, study the morphology and proteomics of NSCs, so that toexplore the role of BDE-209and BDE-47on neural stem cells and to explore theeffect of environmental factors on neuro-developmental toxicity and to explore therelationship between PBDEs and neurodevelopmental deficits, provide the Theoreticalbasis on effective detection and prevention of neurodevelopmental deficits induced byPBDEs in the clinical. this issue is divided into the following two parts for thespecific experiments.Part IEffects of low does BDE-209Single Exposed and Combined withBDE-47on the morphology of neural stem cells in vitro【Objective】The hippocampal NSCs of24-hour old neonatal SD rats （CL grade） weresubcultured in serum-free DMEM, identificated the purity of NSCs, measured thediameter and the number of neurospheres in each group,tested the effect ofBDE-209single exposed and combined with BDE-47on the morphology of NSCs in vitro.【Materials and Methods】1. The hippocampal NSCs of24-hour old neonatal SD rats were subcultured invitro.2. Identification of NSCs and the purity of NSCs.3. Subcultured3-4generations of NSCs、dissipated into single cells suspensionand exposed to BDE-209and BDE-47, grouped as follows:（1）control group（DMSO）；（2）0.6ug/ml BDE-209group；（3）1.0ug/mlBDE-209group；（4）6.0ug/mlBDE-209group；（5）3.2ug/ml BDE-47group；（6）0.6ug/mlBDE-209+3.2ug/mlBDE-47group；（7）1.0ug/mlBDE-209+3.2ug/mlBDE-47group；（8）6.0ug/ml BDE-209+3.2ug/ml BDE-47group. Each section willparallel five. The control group joined with the1‰DMSO medium.4. When exposed to BDE-209and BDE-47after72h, we observed themorphology of NSCs under microscope and measured the number and thediameter of neurospheres in each group by image analysis software.【Results】1. Identification of NSCs: the NSCs formed neurospheres cultured in vitro after3-5days, the neurospheres is gradually bigger, the newly neurospheres showedNestin-positive by immunocytochemistry that suggesting that the cells cultured invitro are neural stem cells.2. The3-4generations of NSCs were observed under inverted microscope, tens tohundreds of cells aggregated to form neurospheres, refractive strong halo,percussed into a single cell suspension and identificated the purity of NSCs byimmunocytochemistry.we observed that the scattered neural stem cells accountedfor over90%.3. We measured the number and the diameter of neurospheres in each group byimage analysis software: In the low dose group（0.6ug/ml PBDE-209treatedgroup)，the number of neurospheres was as normal as that in the controlgroup．As the dose increasing，the number of neurospheres decreasing．And in thelow dose groups（0.6ug/ml PBDE-209and1.0ug/ml BDE-209treated groups)，the diameter of neurospheres had no significant difference from that in the controlgroup.The interaction between BDE-209and BDE-47on morphology of neurospheres presented in the number of neurospheres test(P<0.05)．【Conclusion】The NSCs which were isolated from hippocampus of neonatal rats and culturedin serum-free medium have the ability of self-renewal and proliferation，cells formedneurospheres suspended in the medium，they are NSCs by immunocytochemicalidentification，and BDE-209and BDE-47could change the morphology of NSCs.Aninteraction has been seen between BDE-209and BDE-47in NSCs in the presentpaper． Part IIEffects of low does BDE-209Single Exposed and Combined withBDE-47on the proteomics of neural stem cells in vitro【Objective】Using proteomic technique to research the effects of BDE-209and BDE-47on NSCsproteins and further explore the neurodevelopmental toxicity mechanism of PBDEs.【Materials and Methods】1. Subcultured3-4generations of NSCs and exposed to BDE-209and BDE-47,grouped as follows （:1）control group（DMSO）；（2）6.0ug/ml BDE-209group；（3）3.2ug/mlBDE-47group；（4）6.0ug/ml BDE-209+3.2ug/ml BDE-47group.Each section will parallel five.The control group joined with the1‰DMSOmedium.2. When exposed to BDE-209and BDE-47after72h， collect cells bycentrifugation, extract the total protein, determine the concentration of proteinsamples by Bradford method.3. Identified the differentially expressed proteins by two-dimensionalelectrophoresis（2-DE）.4. Staining：the gels for analysis were silver stained，and the gels for MS werestained with Coomassie Brilliant Blue G-250.5. After silver staining，Powerlook1100scanner was applied to get imagines of 2-DE,and Image Master2D platinum5.0software was applied to analyze the2-DE images and the different expression protein spots were identified.6. Different expression protein spots more than1.8times were digested in the geland MALDI-TOF-MS detection．After peptide mass fingerprintings were got，these identification of differentially expressed proteins were searched byapplication of BioTools search software in the NCBI nr database.7. Western blot was used to determine the expression levels of the two proteins.【Results】We established2-DE maps of total proteins from NSCs exposed to BDE-209andBDE-47．A total of39differential protein spots were found，and19differentialexpression proteins were identified by MALDI-TOF-MS．W estern blot showed thatcofilin1，vimentin were differential expression proteins in four groups，which wasconsistent with the results of the proteomic analysis.【Conclusion】Our results suggest that BDE-209and BDE-47may alter the expression of totalproteins of neural stem cells.Nineteen proteins can be identified byMALDI-TOF-MS.The19differential expression proteins are useful for studying theNeurodevelopmental toxicity mechanism of PBDEs，and provide evidence for furtherstudies of PBDEs neurotoxicity mechanism.