Therapeutic Effect Of Neural Epithelial Stem Cells On Sodium Iodate Induced Retinal Pigment Epithelial Cells And Retinal Injury

ObjectiveObjective to investigate the therapeutic effect of neuroepithelial stem cells(NESC)on retinal pigment epithelial cells(RPE)and retinal injury rats treated with sodium iodate(SI).MethodsImmunofluorescence identification of specific proteins expressed in NESC and observation of NESC differentiation characteristics.In vitro experiment:Human retinal pigment epithelial cells(ARPE-19)were divided into normal group,sodium iodate model group(SI group)and NESC conditioned medium treatment group(SI+NESC-CM).Cell viability was measured by CCK-8,intracellular oxidative stress level was detected by DCFH-DA staining,apoptosis was detected by Annexin FITC staining and flow cytometry,and mitochondrial membrane potential was detected by JC-1 staining.In vivo experiment:1.Model establishment:SD rats were randomly divided into a normal group and a model group,and SI(50mg/kg)was injected into the tail vein.Each group underwent optical tomography(OCT)to record retinal morphology at various time periods(injection 3D,7D,14D,21D,28D).2.NESC intravitreal injection:On day 0 of SI(50mg/kg)modeling,SD rats were randomly divided into a normal group,a sham operated group(Sham group),and a NESC group.Neurobasal-A was injected into the vitreous cavity of the right eye in the Sham group,NESC was injected into the vitreous cavity of the right eye in the NESC group,and no injection was given to the left eye control group.Each group underwent OCT recording of retinal morphology at various time periods(3D,7D,14D,21D,28D after injection);On the 28th day,the eyeballs were taken for HE staining to observe the retinal morphology.Immunofluorescence was used to observe the distribution of NESC retina..ResultIn vitro experiments:The identification results showed that NESC can express Nestin and Ki-67 proteins and differentiate into structures similar to neurons.CCK-8 shows that the absorbance of the SI+NESC-CM group is higher than that of the SI group;The results of DCFH-DA staining showed that the level of oxidative stress in the SI+NESC-CM group was lower than that in the SI group;The results of Annexin FITC staining and flow cytometry showed that the number and rate of apoptotic cells in the SI+NESC-CM group were lower than those in the SI group;JC-1 staining showed that the mitochondrial membrane potential in SI+NESC-CM group was higher than that in SI group.The Western blotting test results showed that the SI group had p-ERK and ERK ratios,p-p38 to p38 ratios,INOS and TNF-α The relative expression levels were higher than those of the normal group,and the NESC-CM group had pERK and ERK ratios,p-p38 to p38 ratios,INOS and TNF-α The relative expression level was lower than that of the SI group.In vivo experiments:1.Model establishment:SI injection for 0 days showed the appearance of typical SD rat OCT images of retinal structure;On the 7th day,verrucous processes in RPE layer and outer nuclear layer(ONL)were observed for the first time,and then the degree of retinal injury was timedependent.2.NESC intravitreal injection:OCT results showed that at 28 days of injection,compared with the Sham group and the left eye control group,the NESC group had a lighter degree of retinal injury and decrease in retinal thickness.HE staining results showed that compared with Sham group and left eye control group,the ONL and inner nuclear layer of NESC group had less fold disorder,the RPE layer had less verrucous process area,and the outer nuclear layer had more cells.The ERG results showed that there was no significant difference between the NESC group and the Sham group and the left eye control group.The distribution of NESC vitreous cavity:OCT results showed that on the 7th day of NESC injection,the vitreous was found to be dotted hyperreflective opacification of NESC,and on the 28th day,the dotted hyperreflective opacification in the vitreous cavity disappeared,while on the surface of the inner limiting membrane of the retina,it was found to be dotted hyperreflective signal of NESC.Retinal section immunofluorescence showed that labeled NESC cells were found in the inner nuclear layer of the retina on the 28th day.ConclusionsIn vitro,NESC-CM can reduce RPE inflammatory protein expression by reducing ERK1/2 and p38 hyperphosphorylation,thereby protecting RPE under oxidative stress.In vivo,NESCs survive for at least 28 days in the vitreous cavity,and some NESCs can migrate into the rat retina,delaying structural damage to the retina caused by SI,but without significant improvement in retinal function.