Objective To investigate if insulin can affect the expression of vascular endothelial growth factor (VEGF) in the retina of streptozotocin-induced diabetic rats. Methods A total of 60 male SpragueDawley rats were randomly divided into sodium citrate buffer control group (CIT-CON, n=30) and STZ-induced diabetic group (STZ-DM, n=30). At the 16th week, 24 rats from CIT-CON group at random were randomly divided to group A (sodium citrate buffer control group, n=12) and group B (sodium citrate buffer plus insulin group, n=12). The remaining 6 rats from as CIT-CON group served as negative control. At the same time, 24 rats from STZDM group at random were randomly divided to group C (STZinduced diabetic group, n=12) and group D (STZ-induced diabetic plus insulin group, n=12). The remaining 6 rats from STZ-DM group also served as negative control. 4 IU of insulin was injected subcutaneously to rats of group B and D. Immunohistochemistry, Western blot and Real-time polymerase chain reaction (RT-PCR) were used to measure the expression level of VEGF protein and mRNA respectively. RESULTS Insulin significantly increased the VEGF mRNA (7.71plusmn;0.25 vs 5.36plusmn;0.37, t test Plt;0.05) and protein expression (0.4925plusmn;0.0122 vs 0.4272plusmn;0.0110, t test Plt;0.05) in the retina of CITCON rats. However, in retina of STZDM rats, insulin had no effect on VEGF mRNA (8.92plusmn;0.27 vs 9.05plusmn;0.28, t test, Pgt;0.05) and protein expression (0.5152plusmn;0.0109 vs 0.5099plusmn;0.0100, t test Pgt;0.05). Conclusions Insulin had no effect on VEGF expression in the retina of STZ-DM rats.
Objective To study the effects of advanced glycation end (AGEs) products induced by bovine serum albumin (BSA) on the survival and the morphology of bovine retinal endothelial cells (BREC) and pericytes (BRP). Methods BSA with the final concentration of 50 mg/ml was incubated in PBS, containing 500 mmol/L D-glucose, for 12 weeks under 37℃. AGEs-BSA was purified by Sephacryl S-300 column chromatography and was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of AGEs-BSA was determined by the method of commassie protein assay. In order to detect the toxic effects of AGEs-BSA on cultured BREC and BRP, groups of AGEs-BSA and BSA with different concentration and untreated control were set up. Phase contrast microscope was used to observe the effect of AGEs-BSA and BSA (with the concentration of 500mu;g/ml and actuation duration of 48 hours) on morphology of BREC and BRP. Results As the dosage of AGEs-BSA increased, the number of inhibited cells increased. When the concentration of AGEs-BSA was 500mu;g/ml, the inhibited BREC in AGEs-BSA group was (72.8plusmn;15.9)% of which in untreated control group, and the inhibited BRP was (64.8plusmn;9) % of which in untreated control group. AGEs-BSA with low concentration promoted the proliferation of endothelial cells, but there was no significant difference between AGEs-BSA and the control group (P=0.231). Inhibited proliferation and abnormal morphology were seen under the phase contrast microscope while the normal morphology of cells was found in BSA and control group. Conclusion AGEs-BSA with the high concentration may inhibit the growth of both BREC and BRP, which leads the loss of BRP and damage of vascular function. These results suggest that nonenzymatic glycosylation plays a major role in diabetic complications. (Chin J Ocul Fundus Dis, 2006, 22: 11-15)
Objective To probe a selective cultural method for bovine retinal endothelial cells (BREC) and pericytes (BRP) in vitro.Methods With the isolation of active retinal blood vessels, BREC were cultured in a fibronectin coated substrate and Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% human serum and 100μg/ml heparin, while homogeneous cultures of retinal pericytes were obtained when isolated microvessels were seeded to uncoated dishes and grown in DMEM supplemented with 20% fetal bovine serum. BREC were identified by acetylated-low density lipoprotein (Dil-Ac-LDL) incorporation and immunohistochemical method of Von Willebrand factor, while BRP were identified by the immunohist ochemical method of α-isoform of smooth-muscle actin. Results The purity of selectively cultured BREC and BRP was more than 98%, being reproducible. BREC got together around the microvessel fragments with the small-cyprinoid-like configuration at first，and could phagocytize Dil-Ac-LDL with the expression of fluorescence in cytoplasm. The expressions of Von Wllebrand factor and α-isoform of smooth-muscle actin were positive and negative in BREC respectively, while were negative and positive in BRP respectively.Conclusion BREC and BRP with high purity can be obtained by using selective culture and coated-dishes respectively which are simple and repeatable methods. (Chin J Ocul Fundus Dis，2004，20：23-26)