ObjectiveTo observe the inhibitory effect of intraocular injection of recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor (rAAV-PSF) in oxygen induced retinopathy mice model. MethodsEighteen C57BL/6J mice were divided into 3 groups randomly, including normal group, rAAV-PSF injection group, rAAV injection group. Western blot analysis was applied to detect the transfection expression level of PSF. The other 48 C57BL/6J mice were randomly divided into 4 groups: normal group, ischemia-induced retinopathy (OIR) group, rAAV-PSF treated OIR group, rAAV treated OIR group, 12 mice in each group. Placed all mice (excepted the mice in control group) in cages of (75±2)% oxygen concentration environment for 5 days then moved to a normal environment for 5 days to induced the OIR model. At the 12th day the OIR rAAV-PSF treated OIR group was intravitreal injected with 2 μl 5×1013 pfu/ml rAAV-PSF and the rAAV treated OIR group was treated with intravitreal injection of 2 μl 5×1013 pfu/ml rAAV. The mice in OIR group were left intact after moved out of oxygen cages. Five days after injection, the eyeballs were harvested and retinal sections were stained to count the nuclear of retinal endothelium cells. Western blot analysis was applied to detect the protein level of vascular endothelial growth factor (VEGF) in retina. ResultsThere was a significant difference of the expression PSF between normal group and rAAV-PSF treated group (F=16.05,P=0.001). There was no significant difference of the PSF expression between normal group and rAAV treated group(F=16.05,P=0.890). There was a significant difference of the number of retinal endothelium cells nuclear between normal group and OIR group (F=101.00,P=0.007). There is a significant difference of the number of retinal endothelium cells nuclear between rAAV-PSF treated group and OIR group (F=101.00,P=0v002). There was no significant difference of the number of retinal endothelium cells nuclear between OIR rAAV treated group and OIR group (F=101.00,P=0.550). There was a significant difference of VEGF protein expression between normal group and OIR group (F=13.20,P=0.005), OIR group and rAAV treated OIR group (F=13.20,P=0.001). There was no difference of VEGF protein expression between OIR rAAV treated group and OIR group (F=13.20,P=0.071). ConclusionThe rAAV-PSF vitreous injection can inhibit the expression of VEGF in OIR mice, hence to restrain the proliferation of neovascularization.
Objective To observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro. Methods RPE cells were cultured and divided into a normal group, normal+H2O2 group, Vec+H2O2group, PSF+H2O2 group according to the experimental design. Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells, then RPE cells were exposed to H2O2. The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay. The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit. Meanwhile, intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method. Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining, and effectively reduce dead cells number shown by Live/Dead staining. After H2O2 stimulation, the survival rate, apoptosis rate and ROS production level in PSF overexpression group were 0.68±0.12, 0.44±0.08 and 18 616±3 382.54 respectively, showing significant difference in comparison with the vector plasmid group and normal group (P＜0.05). Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.
ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.