The Role And Mechanism Of Bone Marrow Mesenchymal Stem Cells In Oxygen-induced Retinopathy

Retinal neovascular disease is a kind of disease characterized by abnormalpathological vascular hyperplasia of the retina.The current treatment methods mainly include anti-vascular endothelial growth factor?VEGF?drugs,but anti-VEGF drugs have their limitations.Some patients have no response or even retinal atrophy.Studies have shown that bone marrow-derived mesenchymal stem cells?BMSCs?can differentiate into retinal cells and participate in microenvironment regulation related to angiogenesis in the eye.At the same time,they can secrete neurotrophic factors and inhibit retinal cell apoptosis,which plays a role in retinal protection.Oxygen-induced retinopathy?OIR?is a good model for studying retinal neovascular diseases.In this study,the modified OIR modeling method was used to evaluate the effect of intravitreal injection of BMSCs on OIR,and the mechanism of BMSCs in retinal neovascularization was investigated by immunofluorescence tracing.The first part of the protective effect of bone marrow mesenchymal stem cells on oxygen-induced retinopathyOBJECTIVE:To establish a stable OIR model,to identify the retinal-related pathological damage indicators in the disease model,and to explore the intervention effect of BMSCs on OIR through intravitreal injection.METHODS:Newborn mice were divided into five groups:Healthy group;Oxygen-induced retinopathy?OIR?group?OIR-blank group?;Oxygen-induced retinopathy?OIR?combined with injection of DMEM group(OIR-DMEM group;Oxygen-induced retinopathy?OIR?combined with injection of BMSCs group?OIR-BMSCs group?;Oxygen-induced retinopathy?OIR?combined with injection of Conbercept group,?OIR-CON group?.In addition to the Healthy group,the mice on the seventh day of birth were placed in a 75%oxygen chamber for 5 days,returned to the normal air oxygen environment,and the group was treated with intravitreal injection in the day of regression.5 days later,retinal vascular was observed by fluorescence retinal angiography,paraffin sections combined with hematoxylin-eosin staining?HE staining?were used to observe the neovascularization.TUNEL was used to evaluate retinal apoptosis,and retinal periodic acid schiff staining was performed?PAS staining?to evaluate the density and occlusion of vascular network,and the expression of retinal apoptosis protein was detected by Western blot.RESULTS:After intervention of OIR in BMSCs,the ratio of ischemic perfusion area and neovascular area was significantly reduced?OIR group:10.39±0.99%and 9.63±0.47%,after intervention:0.85±0.25%and 0.78±0.55%,P<0.05?The number of vascular endothelial cells in the inner limiting membrane was reduced ?148.00±46.27/eye and 11.00±8.70/eye,P<0.05?.At the same time,Acellular capillaries/mm² of the retinal after BMSCs intervention was significantly reduced ?35.33±3.68/mm² and 15.33±1.25/mm²,P<0.05?,the number of endothelial cells and pericyte cell ratio?E/P?/mm² were down-regulated?1.56±0.08/mm²,2.36±0.22/mm²,P<0.05?.Finally,TUNEL showed BMSCs reduced the apoptosis of retinal cells ?0.61±0.20%and 0.17±0.02%,P<0.05?,and the level of Caspase-3 decreased and the difference was statistically significant(F_OIR=0.97±0.03,F_BMSCs=0.74±0.03,P<0.05).CONCLUSION:BMSCs can inhibit the development of neovascularization in OIR,increase the density of retinal vascular network,reduce the ischemic perfusion zone,reduce apoptosis of retinal cells,and play a role in protection on retina.The second part of the mechanism of bone marrow mesenchymal stem cells intervention in oxygen-induced retinopathyOBJECTIVE:To investigate the migration and differentiation of BMSCs and the mechanisms of effect in OIR.METHODS:BMSCs?RFP-BMSCs?expressing red fluorescent protein?RFP?were intravitreally injected in OIR mice.The migration of BMSCs was observed by fluorescence retinal angiography.The frozen sections combined with immuno-fluorescence experiments were used to observe the homing and differentiation of BMSCs in the retina.Western blotting was used to detect the expression of angiogenesis-related regulatory factors and neurotrophic factors.RESULTS:The expression of pigment epithelium-derived factor?PEDF?protein was increased after BMSCs intervention(F_OIR=0.225,F_BMSCs=0.3275,P<0.05).After injection,BMSC were able to migrate and integrate into the host retinal blood vessels,differentiate into vascular endothelial-like cells,and express the vascular endothelial cell marker CD31.CONCLUSION:BMSCs have an inhibitory effect on OIR pathological neovascularization,which can be related to the increase of PEDF expression and homing as well as differentiation of BMSCs.