| Objective1. To characterize the gene expression time course of CTGF and VEGF in STZ-induced diabetic retina and detect the changes of microcirculation ultrastructure by transmission electron microscope (TEM).2. To study the expression changes of these two genes in response and the microcirculation ultrastructure changes to intravitreal injection of VEGF inhibitor Lucentis and short hairpin RNA (shRNA) against CTGF in diabetic rats retina.Methods1.Forty-four adult male rats were randomly divided to diabetes mellitus group (DM group) and normal group. Retinas were obtained at8,10,12weeks after DM induction from both groups. CTGF and VEGF mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). Rat eye cups were collected at each time points, fixed with Glutaraldehyde, then observed by TEM.2. Based on the results of part I experiments, DM rats were randomly divided into Lucentis single-target intervention group, CTGF shRNA single-target intervention group, Lucentis and CTGF shRNA dual-target intervention group and the group without intervention. Also, normal rats were included as control gro-up. Rat retinas were examined by RT-PCR and TEM.Results1.1Transcript levels of CTGF were significantly higher in diabetic reti-nas than in normal retina at the8th week following STZ induction (P=0.037), and this up-regulation was maintained through the12th week of diabetes (P=0.032, P=0.039).VEGF mRNA levels in the diabetic retina started to be signify-cantly elevated over those in the normal ones at the10th week of DM (P=0.043),and remained to be higher at the12th week of DM(P=0.036).1.2TEM showed no change in CON group. However, it showed swelling of e-ndothelium, finger like process into the capillary cavity, uneven distribution of heterochromatin in pericytes in8weeks. Obvious fragmental thickening and s-plitting of basement membrane, swelling and deformation, finger like process to the capillary cavity, and concentration and margination of heterochromatin in endothelial cells, and swelling and deformation of rough surfaced endoplasmic reticulum and mitochondrion in pericytes were observed in10weeks and12weeks groups, especially in the latter.2.1The VEGF gene expression of lucentis single-target group was significantly lower than that in nonintervention group (P=0.014), however, the CTGF mRNA level was significantly increased as compared to the non-intervention group (P=0.048). In the CTGF shRNA intervention group, CTGF mRNA in the diabetic retina was reduced to the level similar to that displayed in the normal control group (P=0.825), the level of VEGF transcript was also significantly decreased (P=0.026) in comparison to that obs-erved in non-intervention DM group. In the dual-target intervention group, the level of VEGF and CTGF were both significantly lower than that in noninter-vention DM group (P<0.001,P=0.001), which was similar to normal control group (P=0.292,P=0.287)2.2The retinal Capillary wall in CON group was smooth and intact, flat endothelium were attachment to integrate basement membrane. The capillary endothelium in DM without intervention group showed swelling, heterochromatin uneven distribution, vascular lumen was constricted. In dual-target group, the vascular wall was rather intact, endothelium was slightly edema, pericytes was clear and integrity, the ultrastructure was close to CON group. Both single-target intervention groups showed the ultrastructure integrity worse than dual-target group.Conclusion Both CTGF and VEGF gene expression were up-regulated in early diabetic rat retina, and the transcript level of CTGF increased earlier than VEGF. Lucentis single-target intervention caused abnormal up-regulation of CTGF expression, although it did significantly down-regulate VEGF transcript level in diabetic rat retina. In contrast, dual-target intervention could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina, and the retina microvessel ultrastructure also showed the advantage of double-target block. |
