Diabetic retinopathy is a serious complication of diabetes and is the leading cause of blindness in people with diabetes. At present, there are many views on the pathogenesis of diabetic retinopathy, including the changes of retinal microenvironment caused by high glucose, the formation of advanced glycation end products, oxidative stress injury, inflammatory reaction and angiogenesis factor. These mechanisms produce a common pathway that leads to retinal degeneration and microvascular injury in the retina. In recent years, cell regeneration therapy plays an increasingly important role in the process of repairing diseases. Different types of stem cells have neurological and vascular protection for the retina, but the focus of the target is different. It has been reported that stem cells can regulate the retinal microenvironment and protect the retinal nerve cells by paracrine production, and can also reduce immune damage through potential immunoregulation, and can also differentiate into damaged cells by regenerative function. Combined with the above characteristics, stem cells show the potential for the repair of diabetic retinopathy, this stem cell-based regenerative therapy for clinical application provides a pre-based evident. However, in the process of stem cell transplantation, homogeneity of stem cells, cell delivery, effective homing and transplantation to damaged tissue is still a problem of cell therapy.
Objective To observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplanted into the tail vein of diabetic rats on apoptosis of retinal neurons and the retinal expression level of glial fibrillary acidic protein (GFAP). Methods Seventy clean male Sprague-Dawley rats were randomly divided into the normal control group (group A), diabetes mellitus (DM) only group (group B), DM + balanced salt solution (BSS) group (group C), DM + hUCMSC group (group D), with 10 rats in each group. DM rats were induced by intraperitoneal injection of streptozotocin. Apoptosis of retinal cells was assayed by dUTP nick end labeling. Immunohistochemistry and Western blot was performed to detect the retinal expressions of GFAP in rats. Results Compared with group A, large numbers of apoptotic cells could be found in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) of group B and group C, however the apoptotic cells in group D were significantly reduced than group B and C. The expression of GFAP was mainly located in the retinal GCL and retinal nerve fibre layer (RNFL) in group A, throughout the inner plexiform layer (IPL) in group B and C, only distributed in RNFL and GCL in group D. It was obvious that the expression of GFAP in group B and C was higher than group A. Compared with group B and C, the expression of GFAP in group D was significantly reduced. The difference of GFAP expression among the 4 groups was significant (F=79.635, P<0.05). Conclusion hUCMSC could inhibit the apoptosis of retinal cells and activation of glial cells in early DM rats.
ObjectiveTo observe the effect of Delta-like ligand 4 (Dll-4) on the pathological structure of retina in early diabetic rats (DM) and its relationship with vascular endothelial growth receptor-2 (VEGFR-2).MethodsA total of 70 male Sprague-Dawley rats were randomly divided into normal group and DM group, with 10 and 60 rats in each group, respectively. The rats of DM group was induced by intraperitoneal injection of streptozotocin to established DM model. The rats with blood glucose recovery and death were excluded, and the final 60 rats were included in the statistics. Rats in the normal group were injected with an equal volume of citric acid-sodium citrate buffer. Rats in the DM group were divided into DM 1 month (DM 1m) group, DM 2 months (DM 2m) group, DM 3 months (DM 3m) group and DM 3m + Anti group, DM 3m + phosphate buffer solution (PBS) group by random number table method, and 10 rats in each group. In the DM 3m+Anti group, 4 μl of anti-Dll-4 polyclonal antibody was injected into the vitreous cavity, and the antibody concentration was 0.25 mg/ml. The DM 3m+PBS group was intravitreally injected with an equal volume of PBS. Five days after the injection, the rats were sacrificed. Rats in the DM 3m group and the normal group were not treated, and were sacrificed 3 months after the model was established. The structure and microvascular changes of the retina were observed by hematoxylin-eosin staining, and the total thickness of the retina was measured. The expression of Dll-4 and VEGFR-2 in the retina was detected by immunohistochemistry and fluorescence quantitative polymerase chain reaction (PCR). One-way analysis of variance was used to compare the expression of Dll-4 and VEGFR-2 in the retina of each group. The least significant difference t test was used to compare the two groups.ResultsLight microscopy showed that the retinal ganglion cells layer in the DM 3m group were obviously edematous, the inner and outer nuclear layers were thinner, the number of cells was reduced, the arrangement was disordered, the edema of outer plexiform layer was obvious, and the microvessels were abnormally dilated. In the DM 3m+Anti group, the edema of outer plexiform layer was lessened than that of the DM 3m group, and the other layers were not significantly different from the DM 3m group. Compared with the normal group, the total retinal thickness of the DM 3m group, the DM 3m+Anti group and the DM 3m+PBS group increased (t=5.596, 3.290, 4.286; P=0.000, 0.008, 0.002). Immunohistochemical staining showed that a small amount of Dll4 was positively expressed in the retinal ganglion cell layer of the normal group; a small amount of VEGFR-2 was positively expressed in the ganglion cell layer and the inner and outer nuclear layers. The positive expression of Dll-4 and VEGFR-2 in retinal vascular endothelial cells of DM 3m group increased significantly. The expression of Dll-4 was significantly decreased in the retinal layers and vascular endothelial cells of DM 3m+Anti group, while the expression of VEGFR-2 was significantly increased. There was no significant difference between the positive expression of Dll4 and VEGFR-2 in the DM 3m+PBS group and the DM 3m group. The results of real-time PCR showed that the relative expression of Dll-4 and VEGFR-2 mRNA in the DM 3m group was significantly higher than that in the normal group (t=6.705, 20.871; P＜0.05). Compared with DM 3m group, the relative expression of Dll-4 mRNA in DM 3m+Anti group decreased, and the relative expression of VEGFR-2 mRNA increased (t=2.681, 3.639;P＜0.05). The relative expressions of Dll-4 and VEGFR-2 mRNA in the DM 3m+PBS group and DM 3m group were not statistically significant (t=0.513, 0.657; P＜0.05).ConclusionsThe expression of Dll-4 in retinal vascular endothelial cells is gradually increased during the early retinopathy of DM rats. The expression of Dll-4 is inhibited, the expression of VEGFR-2 is up-regulated, and the plexus edema is alleviated.