ObjectiveTo observe the effects on the function and structure of retina in diabetic rats by intravitreal transplantation of retinal nerve stem cells (NSC) differentiated from human umbilical cord mesenchymal stem cells (hUCMSCs). MethodsFifty clean male Sprague-Dawley rats were randomly divided into normal control with 9 rats (group A) and diabetes mellitus (DM) group with 31 rats. The DM models were induced by intraperitoneal injection of streptozocin. The rats of DM group were randomly divided into four groups after 10 weeks: rats with DM only (group B), diabetic rats with saline intravitreal injection (group C), diabetic rats with NSC intravitreal injection (group D), and 9 rats for each. The rats in the group A and B received no treatment. The retinal function was examined by the flash-electroretinogram on 2, 4, 6 weeks after intervention, the latency and amplitude of a-wave, b-wave of Rod, a-wave, b-wave of Max reactions (Max-R) and the total amplitudes of OPs were recorded. The morphological changes of retina were observed by hematoxylin-eosin staining. ResultsOn 2 and 4 weeks after the intervention, the differences of latency and amplitude of b-wave of Rod, a-wave, b-wave of Max-R and the total amplitudes of OPs among group A-D were significant (P<0.05). Compared group D with group B, C, the amplitude of b-wave of Rod, Max-R and the total amplitudes of OPs were increased (P<0.05); latency of b-wave of Max-R was decreased (P<0.05). On 6 weeks after the intervention, the amplitude of b-wave of Rod and the amplitude of a-wave, b-wave of Max-R and the total amplitudes of OPs in group D were increased compared with group B and C (P<0.05), the latency of b-wave of Rod and Max-R in group D were decreased compared with group C (P<0.05). On 10 weeks after molding, each retinal layers were disordered in diabetes mellitus group. On 2 weeks after the intervention, the number of cells in the retinal layers in group B and C were reduced compared with group A, and the structure was more disorder. On 4 weeks after the intervention, the structure of each retina layer in group D arranged less disordered, and the number of retinal ganglion cells was more than group B and C. It was also found that the retinal vascular endothelial expanded and retinal blood vessels cells proliferated. ConclusionThe function of retina in diabetes mellitus rats is improved by intravitreal injection of retinal NSCs differentiated from hUCMSCs.
ObjectiveTo observe the immunological regulation effects of human umbilical cord mesenchymal stem cells (hUCMSC) on glucose-damaged rhesus retinal vascular endothelial cells (RF/6A). MethodshUCMSC and RF/6A were co-culture according to 1:1 ratio in the co-culture system (Transwell plates), hUCMSC cells were added to upper chamber, while the lower chamber containing 25mmol/L glucose and RF/6A. There were three groups including RF/6A blank control group, high glucose treated RF/6A group, and high glucose treated RF/6A with hUCMSC co-culture group. MTT was used to measure the RF/6A cell viability. Western blot was used to to detect protein level of Foxp3. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of interleukin (IL)-17. ResultsMTT assay revealed that at the first day, the survival rate of the three groups had no significant difference (F=0.030, P > 0.05). On day 3 and day 7, the cell viability of the high glucose group was significantly lower than that of the control group (t=36.072, 27.890; P < 0.05), the cell viability of the high glucose treated RF/6A with hUCMSC co-culture group was higher than that of high glucose group (t=36.072, 19.650; P < 0.05).Western blot analysis showed that Foxp3 in high glucose RF/6A group was significantly lower than that in the control group at day 7 after culture (t=7.826, P < 0.05) and high glucose RF/6A with hUCMSC group (t=19.936, P < 0.05). ELISA showed that IL-17 in the high glucose group, high glucose with hUCMSC co-culture group was significantly higher than that of the control group (F=1 267.503, P < 0.05), while IL-17 in the hUCMSC co-culture group was significantly lower than that in high glucose group (t=17.386, P < 0.05). ConclusionhUCMSC can regulate the expression of Foxp3 and IL-17 to increase the proliferative ability of RF/6A, which was suppressed by high glucose.
Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, −7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.