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.
ABSTRACT
Objective To investigate the protective effects of intravitreal injection of pigment epithelial-derived factor (PEDF) gene-modified human umbilical cord mesenchymal stem cells (PEDF-MSCs) on the pathological changes in retinal tissue of diabetic rats.Methods hUCMSCs were isolated from human umbilical cord tissue using tissue culture methods,and transfected with lentiviral vectors at a infection multiplicity of 50.Then diabetic model in rats was successfully induced by intraperitoneal injection of streptozotocin.And the rats were divided into normal control (N),PBS treatment (D1),hUCMSCs treatment (D2) and PEDF-MSC treatment (D3) group according to different treatment methods.Three months after modeling,treatment began in D1,D2 and D3 group,but N group left untreated.Two weeks after treatment,the expression of PEDF-MSCs in the eye of rats was detected by fluorescence microscopy,and HE staining was performed to observe the changes in retinal structure and the full-thickness of the retina in each group.Results The expression of CD105,CD73,CD90 was observed,while the expression of CD34,CD45,CD11b,CD19 and HLA-DR did not present.After 2 weeks of treatment,it was in the vitreous cavity not the retina that clusters of red fluorescence appeared in D2 group with fluorescence microscope.There were clusters of green fluorescence in the vitreous cavity not in the retina of D3 group.HE staining showed that the retina had intact structure and clear layers as well as neatly arranged and stained evenly cells in N group.In D1 group,the nerve fibers layer (NFL) showed obvious edema,the blood vessels were dilated,the inner plexiform layer (IPL) were loose and the inner nuclear layer (INL) cells were disordered.In D2 group,the edema of NFL relieved.In D3 group,NFL edema was significantly alleviated,and the cells of INL and outer nuclear layer (ONL) arranged in regular.Full-thickness of retina was (103.82 ±4.15) μm in N group,(138.86 ±4.71) μm in D1 group,(131.17 ±3.89) μm in D2 group,and (112.24 ±4.22) μm in D3 group,respectively,and the differences were statistically significant (all P ＜ 0.05).Conclusion PEDF-MSCs can survive and continue to express in the vitreous cavity of diabetic rats for a long time.Meanwhile,intravitreal injection of PEDF-MSCs can ameliorate retinal edema and the retinal injury in diabetic rats.