Protective effects of PEDF gene modified human umbilical cord mesenchymal stem cells in rat with retinal ischemia-reperfusion injury / 眼科新进展

ABSTRACT

Objective To observe the effects of pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells (PEDF-MSCs) on the expression of pigment epithelial-derived factor (PEDF) and vascular endothehal growth factor (VEGF) in a rat model of retinal ischemia-reperfusion injury (RIRI) and its protection on retinal ganglion cells.Methods Lentivirus (LV) labeled with green fluorescent protein (GFP) was used as a vector to transfect the PEDF gene into human umbilical cord mesenchymal stem cells (hUCMSCs) at a multiplicity of infection (MOI) of 1,10,20,and 50 in vitro,and then the expression of PEDF gene and protein in cells transfected with the best MOI value was detected by RT-PCR and ELISA.The healthy male SD rats were randomly divided into normal group (N group) and experimental group.The RIRI model was made by high intraocular pressure in the experimental group,and the RIRI rats with PBS treatment were allocated as the PBS group,with hUC-MSCs treatment as M group and LV-PEDF-MSCs treatment as P group,and the N group was left untreated.All rats were sacrificed on day 5,and the number of retinal ganglion cells were counted by Nissl staining,the thickness of the retina was calculated,as well as the expression of PEDF and VEGF mRNA in rat retina was detected by RT-PCR.Results The transfection efficiency was as high as 75.8％ under fluorescence microscope.The results of RT-PCR showed that the relative expression of PEDF mRNA in PEDF-MSCs supernatant (4.34 ± 0.29) was significantly higher than that in hUCMSCs (1.08 ± 0.15),and the difference was statistically significant (P ＜ 0.05);moreover,the results of ELISA showed that PEDF protein expression in PEDF-MSCs supernatant [(83.09 ± 7.58)μg · L-1] was significantly higher than that in hUCMSCs [(12.30 ±1.24) μg · L-1],and the difference was statistically significant (P ＜ 0.05).Nissl staining results showed that the number of ganglion cells in group PBS decreased after model establishment.After 5 days of treatment,the number of ganglion cells in P group and M group was higher than that in PBS group,and the difference was statistically significant (both P ＜ 0.05);and P group was higher than M group,with the significant difference (P ＜ 0.05).And this was true of the thickness of the retina.RT-PCR showed that the expression of PEDF mRNA in P group was significantly up-regulated,but VEGF mRNA expression was significantly down-regulated,and the differences were statistically significant when compared with PBS and M group (both P ＜ 0.05).Conclusion Intravitreal injection of PEDF-MSCs can up-regulate the expression of PEDF but down-regulate the expression of VEGF in the retina of RIRI rats,which can protect the retinal ganglion cells against RIRI.