Bipolar Cell Proliferation And Its Mechanism After Inhibiting Sox2 Protein Expression In The Retina

Part 1: After inhibiting Sox2 protein in the retina of newborn mice,the number of retinal bipolar cells increasedPurpose: To detect the effect of inhibiting Sox2 protein expression on the development and differentiation of retinal bipolar cells and amacrine cells in the retina of newborn mice.Methods: CD1 newborn mice were randomly selected and divided into control group and experimental group.1 μl of sh RNA to knockdown Sox2(p Sico-sh SOX2+IRES+GFP)(3 μg/μl)or vehicle only was injected into the subretinal space using a microliter syringe.Immediately following injection,electric pulses(100 V,five 50 ms pulses with 950 ms pause intervals)were applied through tweezer-type electrodes.After electroporation for 12.5 days and 30 days,immunohistochemistry cell staining with type-specific markers were performed to identify retinal neurons number change.Results: After 12.5 days of inhibiting Sox2 protein expression in the retina of newborn mice,the number of bipolar cells increased significantly,and the increased bipolar cells were cone bipolar cells,while the number of amacrine cells decreased significantly.However,after 30 days of inhibition of Sox2 protein by electroporation,the number of bipolar cells increased significantly,while the number of amacrine cells did not change significantly.Conclusion: Inhibition of Sox2 protein expression in the retina of newborn mice increased the number of cone bipolar cells and decreased the number of amacrine cells.Part2: Changes in retinal function after inhibiting Sox2 protein expression in the retina of newborn micePurpose: To observe the effect of inhibiting Sox2 protein expression on retinal function.Methods: CD1 newborn mice were randomly selected and divided into control group and experimental group.1 μl of sh RNA to knockdown Sox2(p Sico-sh SOX2+ IRES+GFP)(3 μg/μl)or vehicle only was injected into the subretinal space using a microliter syringe.Immediately following injection,electric pulses(100 V,five 50 ms pulses with 950 ms pause intervals)were applied through tweezer-type electrodes.Scotopic and photopic electroretinogram experiments were carried out in mice to observe the overall function of the mice retina.The visual acuity of the mice was observed through the optomotor response.By monitoring the intraocular pressure of mice,the intraocular pressure of mice was determined.Results: The results of scotopic ERG showed that the peak time of a wave and b wave and the amplitude of wave b in the experimental group were significantly delayed and decreased.There was no significant difference between the two groups of mice in the results of photopic ERG.The visual acuity of mice in the experimental group decreased compared with that in the control group.There was no significant difference in IOP between the two groups.Conclusion: Inhibition of Sox2 protein expression in the retina of newborn mice could inhibit the overall function of the retina.Part 3: To investigate the mechanism of retinal cells and function changes induced by inhibition of Sox2 expression in the retina of newborn micePurpose: To investigate the mechanism of retinal cell and function changes induced by inhibition of Sox2 expression in the retina of newborn mice.Methods: CD1 newborn mice were randomly selected and divided into control group and experimental group.1 μl of sh RNA to knockdown Sox2(p Sico-sh SOX2+ IRES+GFP)(3 μg/μl)or vehicle only was injected into the subretinal space using a microliter syringe.Immediately following injection,electric pulses(100 V,five 50 ms pulses with 950 ms pause intervals)were applied through tweezer-type electrodes.After electroporation operation in the retina of newborn mice,immunofluorescent cell staining was performed on the retina sections after 6 days,and then the changes in cell number were statistically analyzed.The m RNA levels of transcription factors associated with the development and differentiation of bipolar cells and amacrine cells were detected by polymerase chain reaction(PCR)in single cells selected under patch clamp at the time points of 6 and 12.5 days.Results: After Sox2 protein was inhibited in the retina of newborn mice for 6 days,there existed a specific antibody co-labeled cells of bipolar cells and amacrine cells.The single cell rt-PCR result revealed that the transcription factors associated with retinal bipolar cells development Ascl1,Ebf and Crx m RNA level increased compared with control group on 12.5 days after Sox2 protein inhibited.On 6 days the m RNA level of Bhlhe22 have significantly increased and decreased on 12.5 days.The m RNA level of Ascl1 has a significant decline on 6 days compared with the control group.The m RNA levels of the transcription factors Foxn4 and Ptf1 a,which are related to the development of retinal amacrine cells,decreased significantly on both 6 and 12.5 day compared with the control group.Conclusion: After the inhibition of retinal Sox2 protein,the retinal progenitor cells that originally developed to amacrine cells developed into cone bipolar cells by changing the expression of various related transcription factors.