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Objective To investigate the regulation effects of Ngn2 gene transfection on retinal neuron differnetion in three-dimentional optic vesicle (OV) of mice.Methods OV was cultured in vitro using mouse induced pluripotent stem cells (iPSC) under specific conditions.During OV culture,it was transfected multiple times by lentivirus-mediated Ngn2 gene and then it was induced after maturation.The cells were specificly differentiated toward retinal nerve cells in OV.Using the green fluorescent protein (EGFP) gene as control,the differentiation of retinal nerve cells in OV was detected by immunohistochemistry.Reverse transcription PCR and Western blot were used to quantitatively detect the expressions of retinal neuron-specific proteins Pax6,Islet1 and Brn3b.Results The mouse iPS-derived OV was successfully cultured.The number of neural cells in the OV transfected with the Ngn2 gene was increased by the lentiviral-mediated lentivirus.The expressions of PAX6,Islet1 and Brn3b in the Ngn2 transfection group were significantly higher at the gene and protein levels than those in the control group,with significant differences between the two groups (P＜0.05).Conclusions The Ngn2 gene can effectively increase the number of retinal neuron differentiation in OV and make in vitro cultured OV more mature and form a more perfect retinal cell neural circuit.
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The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and glial cells. Glutamate, the main excitatory neurotransmitter, mediates information transfer from photoreceptors, bipolar cells, and ganglion cells, whereas interneurons, mainly amacrine and horizontal cells, use γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter. In this review we place an emphasis on glutamate and GABA transporters as highly regulated molecules that play fundamental roles in neurotransmitter clearance, neurotransmitter release, and oxidative stress. We pharmacologically characterized glutamate transporters in chicken retina cells and identified two glutamate transporters: one Na+-dependent transporter and one Na+-independent transporter. The Na+-dependent uptake system presented characteristics related to the high-affinity xAG- system (EAAT1), and the Na+-independent uptake system presented characteristics related to the xCG- system, which highly contributes to glutamate transport in the retina. Glutamate shares the xCG- system with another amino acid, L-cysteine, suggesting the possible involvement of glutathione. Both transporter proteins are present mainly in Müller glial cells. GABA transporters (GATs) mediate high-affinity GABA uptake from the extracellular space and terminate the synaptic action of GABA in the central nervous system. GABA transporters can be modulated by molecules that act on specific sites to promote transporter phosphorylation and dephosphorylation. In addition to a role in the clearance of GABA, GATs may also release GABA through a reverse transport mechanism. In the chicken retina, a GAT-1 blocker, but not GAT2/3 blocker, was shown to inhibit GABA uptake, suggesting that GABA release from retina cells is mainly mediated by a GAT-1-like transporter.
Assuntos
Ácido gama-Aminobutírico , Ácido Glutâmico , RetinaRESUMO
Objective To investigate the possibility that transplanting the full-thickness neonatal piggy retinas that are completely differentiated but immature improves the retinal function after light-induced retinal degeneration in pigs.Methods Retinas from normal Guangxi Bama pigs aged 1-6 days were used as donor tissues.Neuroretina-RPE cografts were obtained from newborn pigs by using excimer laser for microablation of choroidal tissue and transplanted into the subretinal cavity of adult Bama pigs after light-induced retinal degeneration through vitrectomy and retinotomy.On days 5-7 and in 1st to 5th month after retinal transplantation,the survival of the cografts in the recipients and whether the host retinas have rejection were observed by ophthalmoscope,colour fundus photography and fundus fluorescein angiography,and the amplitude and lantency of N1,P1 waves between different periods were measured by mfERG.Results The retinal transplantation was performed in 15 eyes of 8 Bama pigs after light-induced retinal degeneration.The subretinal transplantation of the cografts was performed successfully in 11 eyes,with the operation success rate of 84.6%(11/15).In host retina,the gray-black graft inside transplantation bed could be seen clearly in 1st to 2nd month after transplantation and the leaked fluorescence in transplants was checked with FFA.The vertical comparison between different periods showed the amplitude of N1,P1 waves of retinal transplants rose with the extension of the survival time,and the areas where active response was observed were ring 2 and ring 3;but the latency of N1,P1 waves was shortened significantly in each ring as compared with that before operation,especially in late survival period.Conclusion The function measurement and the observation of living body together confirm the transplantation of completely differentiated retina from newborn pigs improves the retinal function of pigs after light-induced retinal degeneration.