| Background: The development of the nervous system,especially the brain,is a complex and precisely regulated process by multiple factors.Among these,Thyroid hormones play a crucial role in the development of normal brain.Studies have shown that a variety of causes of thyroid hormone deficiency during pregnancy will affect the neurodevelopment of offspring,resulting in irreversible damage to neural differentiation and migration.Cerebral cortex is involved in advanced cognition of human beings and animals(eg,language,learning,abstract thinking,emotion),and sensory function,autonomic motor function.Hypoplasia of offspring caused by lack of thyroid hormone,and dyskinesia related to cerebral cortex dysfunction.Thyroid hormone receptor(TR),divided into two subtypes: TRα1 and TRβ1,respectively,by the TRα and TRβ gene encoding.These two subtypes have different functions in regulating the development of the body.Among them,the early brain development is mainly regulated by TRα1.TRα1 began to express extensively in the early stage of brain development and accounted for 60% of all TR.TRβ1 was localized mainly at birth and accounted for only 30% of TR.The regulation of thyroid hormone on brain development has time and space specificity,and its mechanism is not clear at present.In brain tissue,the cerebral cortex is the earliest developed tissue.TRα1 begins to be expressed in the cerebral cortex starting from the 8th week of human pregnancy.TRα1 also appears in rat studies at 11.5 days of gestation(equivalent to the neural tube formation time of fetal rats),and then widely expressed in brain tissue,but the lack of thyroid hormone results Cerebral cortex damage mainly occurs in maternal mice 16 days after pregnancy,leading to a decrease in the number of fetal cortical neurons and neuronal maturation(dendrites and axons),neuronal migration disorders,but the neural tube formation,Proliferation of neural precursor cells in the cerebral cortex had no effect.Although previous studies focused on the effects of thyroid hormones on brain development,most of them focus on late pregnancy and after birth.The role of thyroid hormones in the early development of embryonic nerves remains unclear.Thyroid autoimmune(TAI)is prevalent among women of reproductinve age and is associated with premature delivery,miscarriage,infertility or other adverse pregnancy outcomes.In recent years,more and more studies begin to pay close attention to the effects of TAI on offspring’s development.Epidemiological investigation showed that children of women with TAI had significantly differences in intelligence score,motor score and extroversion expression.Therefore,factors affecting thyroid autoimmunity also play a key role in brain development of offspring.In recent years,the effect of iron on brain development has been more and more widely recognized.During the period of rapid brain development,iron uptake was also the highest in the brain.Iron deficiency(ID)itself can lead to irreversible damage to brain development,including abnormal emotional behavior,cognitive decline and attention deficit.Although there have been many studies on the effects of iron and thyroid hormones on brain development,it is still unclear whether iron interacts with thyroid hormones or thyroid autoantibodies,and can co-regulate brain development.COUP-TF,a member of the orphan nuclear receptor family in nuclear receptors,has two subtypes COUP-TF1 and COUP-TF2 Widely expressed in brain tissue.The study found: COUP-TF1 expression in the early brain tissue,widely expressed in neural precursor cells and nerve cells.At 7.5 days pregnant,COUP-TF1 was detected in the fetal rat brain tissue and was widely expressed in brain tissue.The expression of COUP-TF1 was highest in the cerebral cortex 13.5 days after pregnancy,and then decreased significantly.The high expression of COUP-TF1 in different brain regions early in brain development suggests that COUP-TF1 may play an important role in brain development.Mice with conditional knockout of COUP-TF1 in the cerebral cortex expanded the motor area of the cerebral cortex and sensory area decreased.That COUP-TF1 knockout will change the natural process of cerebral cortex development.It has been reported in the literature that thyroid hormone nuclear receptor alpha and COUP-TF1 nuclear receptors begin to appear in early pregnancy,suggesting that COUP-TF1 and thyroid hormone nuclear receptor alpha are likely to participate in the regulation of early brain development.Based on the above understanding,this study intends to establish a model of mouse embryonic stem cells in vitro induction of differentiation into pyramidal neurons to explore the thyroid hormone on embryonic stem cells to neuron differentiation process,and COUP-TF1 knockout C57 BL / 6N ES cell line using the technology of lentivirus transfection and CRISPR / CAS9 to investigate the effects of COUP-TF1 on neurodevelopment and synergistic effect of thyroid hormone and COUP-TF1 during the differentiation of embryonic stem cells into neurons.The interaction between iron and thyroid hormones or thyroid autoantibodies was investigated by cross-sectional study with large multicenter samples.Methods: In this study,mouse embryonic stem cells(ESC)were differentiated in vitro using cortical neuronal differentiation.Cortical neuron differentiation in mouse ESCs occurs in two stages,with each stage using a specific conditioned medium.Using this method,we were able to obtain a high proportion of nestin positive progenitor cells in the first phase and cortical neurons in the second phase of differentiation.Immunofluorescence and electrophysiological experiments confirmed that the differentiated cells were pyramidal neurons.In the process of embryonic stem cells differentiating into neurons,the cells were treated with gradient concentration of T3,Real-time PCR and immunofluorescence cytochemistry were used to detect the effect of T3 on the early differentiation,proliferation and late differentiation of neurons,and the differentiation of neurons Touch the growth of the situation,to explore the embryonic stem cell T3 during neural development.Real time PCR was used to detect m RNA expression of TRα and COUP-TF1 in differentiation of embryonic stem cells to neurons.The COUP-TF1 knockout C57 BL / 6N ES cell line was obtained using the CRISPR / CAS9 and lentivirus transfection technique.The effect of COUP-TF1 on cortical neuronal differentiation was analyzed by Real time PCR,immunofluorescence cytochemistry and RNA Seq.Gradient concentrations of T3 were given during the differentiation of COUP-TF1 knock-out embryonic stem cells into neurons and the co-effect of COUP-TF1 and T3 on cortical neuronal differentiation was analyzed.Cross-sectional data analysis of 7463 pregnant women during the first trimester of pregnancy and 2185 non-pregnant women of child bearing age nested within the Subclinical Hypothyroid in Early Pregnancy(SHEP)study,a prospective collection of pregnant and non-pregnant women’s data,was conducted in Liaoning province of China between 2012 and 2015.Serum thyrotropin(TSH),free thyroxine(f T4),thyroid peroxidase antibodies(TPOAbs),thyroglobulin antibodies(Tg Abs),serum ferritin,and urinary iodine were measured.Results: 1.Immunofluorescence staining and electrophysiological electrophysiological experiments to verify the differentiation of embryonic stem cells in vitro is functional pyramidal neurons.2.After the gradient concentration of T3 was given on the second day of each stage of neural progenitor cell differentiation,proliferation and neuron differentiation.Nerve precursor cells: There was no morphological change,the expression of Nestin,Hes1,Hes5,Hairless,but there was no significant difference in the expression of Nestin,Hes1,Hes5,Hairless,-tubulin III m RNA was not statistically different.In the late neurons,the number of neurons and the synaptic connections were increased.There was a significant difference of Tbr1 and Camk4 m RNA(p <0.05).The time window of thyroid hormone regulation by Tbr1 was also observed.3.TRα1 m RNA expression began in early culture,and then constantly expressed;COUP-TF1 m RNA began to express early in vitro culture and maximum expression was on the 18 th day,then began to decline.4.In vitro directed differentiation of embryonic stem cells using the adherent method revealed increased numbers of neurons and synaptic connections on the 22 nd day of T3 treatment.After COUP-TF1 was knocked out by lentiviral transfection technique,the cells were treated with physiological dose of T3.It was found that COUP-TF1 and T3 cooperate to regulate the expression of thyroid hormone target genes,Tbr1 and RC3,during the differentiation of neural precursor cells into neurons.5.COUP-TF1 knockout using CRISPR/CAS9 promoted the differentiation of neural precursor cells,The expression of Nestin was higher than that of normal cells and had statistical difference(p<0.05),and caused the apoptosised of neural progenitor cells in the middle and late stages.At the end of this phase,the expression of Nestin in the cell knockout of COUP-TF1 was significantly lower than that in the normal group.6.COUP-TF1 knockout also promoted the premature differentiation of neurons,and the expression of β-tubulin III was higher than that of the normal group and had statistical difference(p<0.05).But at the late of this stage,the expression of β-tubulin III in this stage was significantly lower than that in the normal group,p<0.05.7.In the stage of neural precursor cell differentiation into neurons,RNA Seq detection was performed on the fourth day.It was found that rbl2 was down-regulated in the PI3K/Akt signaling pathway,this case was also for Actin.8.The prevalence of isolated TPOAb-positive was markedly higher in women with ID than those without ID,in both pregnant and non-pregnant women.9.Serum free T4 levels were significantly lower in both pregnant and non-pregnant women with ID compared with those without.Conclusions: 1.In the technique of in vitro differentiation of embryonic stem cells using the embryoid body method,physiological concentrations of T3 have no significant effect on the differentiation of embryonic stem cells into neural precursor cells and the proliferation of neural precursor cells.But in the late stage of neuronal differentiation,it promotes the differentiation of neurons and the growth of synapses.2.TRα1 and COUP-TF1 synergistically regulate the expression of thyroid hormone target genes Trb1 and RC3 in the late stage of neuronal differentiation.3.COUP-TF1 can maintain the stability of cell cycle and cytoskeletal proteins.Knocked out COUP-TF1 can cause cells detached from the G1 phase of the cell cycle to differentiate into immature cells with apoptosis.4.ID was associated with TAI and isolated hypothyroxinemia in both pregnant women during the first trimester of pregnancy and non-pregnant women of child bearing age. |
PDF Full Text Request