| Objects With the application of neurons differentiation model from ITSFn selection(Insulin-Transferrin Bovine-Sodium Selenite-Fibronectin), the developmental neurotoxicity of PBDEs and the sensitive stage have been investigated to expand the developmental neurotoxicity research.Methods Firstly, to set up the differentiation model along these following 5 stages. Stagel:In vitro culture and maintain the undifferentiated mouse embryonic stem cells(mESCs). Stage2: Embryoid bodies (EBs) culture from hanging drops (3d) and another 2 days to attach to dishes. Stage3:To gather neural precursor cells (NPCs) from ITSFn selection. Stage4:NPC proliferation. Stage5:Dopaminergic neurons and glial cells differentiating from NPCs. Secondly, the model has been verified by following techniques. The neuron-specific genes (MAP2, NESTIN, THY1, TH) have been detected by Real Time-PCR. The neuron-specific proteins (Nesin, Tyrosine Hydroxylase) have been marked by immunohistochemistry. The existence of mature dopaminergic neurons has been ensured by the exposure of dopaminergic neuron-specific toxic MPTP. Moreover, to define exposure doses of PBDEs and thyroxine based on inhibitory concentration 50%(IC50) resulting from MTT or CCK8 tests. Finally, the relating genes expression (Nanog, Sox2, Oct4, Protein Kinase C-a, Homeodomain, Basic helix-loop-helix, Pax6, Netrin-1, Olig2, Nkx6-1) have been investigated by RT-PCR. The cytotoxicity and oxidative stress changes (Na--K+ATPase, Ca2+-Mg2+ATPase, Sialic acid, SH, GSH, GSSG, ROS, SOD, GSH-px, CAT) have been tested by ELISA and Colorimetry. The apoptosis situation has been analyzed by Flow Cytometry. The synaptic stretch and synaptic contacts (length changes of dendrites and axons, the expression of synapsin-1 and PSD-95) have been detected and analyzed by Immunofluorescence and Image Pro Plus 6.0 software.Results PBDEs have inhibition influence on the expression of Sox2, Oct4 in initial differentiation and on the expression of protein kinase C-a, Basic helix-loop-heli, Netrin-1, Olig2, Nkx6.1 in later differentiation. As for netrin-1 and olig2 genes expression, thyroxine has suppression. PBDEs have increased the oxidative stress like GSH, SH, GSSG, ROS, which were more obvious in later differentiation. Thyroxine has suppression on this increase as well. PBDEs have reduced the antioxidant capacity like SOD, GSH-px, CAT which were more evident in embryonic period (stage 1,2), NPCs period (stage 4) and neurons period (stage 5). Thyroxine has suppression on the GSH-PX reduction. PBDEs have increased the apoptosis incidence, which were distinct during nestin+selection, NPCs stage. Thyroxine has no inhibition on this apoptosis induction of PBDEs. PBDEs have cytotoxicity as the decline of Na+/K+ATPase and Ca2+/Mg2+ATPase activity, which were more evident in the nestin+ selection, NPCs stage. Thyroxine has suppression effect on Ca2+-Mg2+ATPase reduction. PBDEs have decreased the dendrites and axons length, which were more significant in the NPCs stage and the neurons growth period. And thyroxine has inhibition on this reduction. PBDEs have suppressed the synapsin-1 protein expression and thyroxine has inhibition on this reduction.Conclusion Through the culture process, matured neurons could be developed and gained. Compared with other in vitro models, in this model the neuron-specific genes, proteins and dopamine secretion function could reappear on which could make dynamic assessment or reflection of neurotoxicity like PBDEs along time factor. After the application of this neuron development model, PBDEs have developmental neurotoxicity. And the influence focused on differentiation related genes expression, oxidative stress, antioxidant capacity, cytotoxicity, initial apoptosis induction, synaptic stretch ability. The PBDEs affect period focused on the embryonic stage, NPCs and neuron growth stages. Thyroxine have inhibition on the PBDEs developmental neurotoxicity and crucial points are NPCs and neuron growth stages. |
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