| Objective: This aim of the study is to detect the function of IL-10-modified EPCs and observe the effection of IL-10-modified EPCs in suppressing progression of nonproliferative diabetic retinopathy.Methods: Mononuclear cells were collected by density gradient centrifugation from the bone marrow of rats. The isolated cells were cultivated in dishes coated with fibronectin. Immuno-fluorescence staining were used to indentify EPCs. After being identified, EPCs were infected with LV-IL10-GFP or control lentvirus. Enzyme-linked immunosorbent assay(ELISA) was used to detect the concentrations of cytokines in supernatant with tumor necrosis factor-alpha(TNF-α) or without TNF-α. All kinds of cells were assessed the functions of tube formation, adhesion, migration and cell cycle. And then cells were transplanted into the streptozotocin-induced diabetic rats. Immunohistochemistry were used to track the transplanted cells. The blood-retinal barrier breakdown was detected by Evans blue dye. All eyeballs were examined using hematoxylin and eosin(HE) staining and transmission electron microscope. The expression of vascular endothelial growth factor(VEGF)、angiopoietin-1(Ang-1)、matrix metallproteinases-9(MMP-9)、inducible nitric oxide synthase(i NOS) and endothelial nitric oxide synthase(e NOS) in retina were measured by RT-PCR. Signal transducer and activator of transcription 3(STAT3) signal pathway in EPCs and nuclear factor-kappa B(NF-κB) signal pathway in retinas were examined by western blot.Results:(1)There were no significantly statistical difference in EPCs migration, adhesion and tube formation among EPC, EPC-LV-IL-10-GFP and EPC-LV-NC-GFP groups without TNF-α.(F=1.414, 1.817, 0.025, P=0.25, 0.169, 0.976) The percent of G1, S and G2 phase were similar among EPC, EPC-LV-IL-10-GFP and EPC-LV-NC-GFP groups without TNF-α. There were significantly statistical difference in EPCs migration, adhesion and tube formation among EPC, EPC-LV-IL-10-GFP and EPC-LV-NC-GFP groups with TNF-α.(F=8.260, 135.903, 13.246, P=0.001, 0, 0.011) EPC-LV-IL-10-GFP with TNF-α significantly enhancedEPCs migration, adhesion, and promoted tube formation.(P=0.001, 0.001; 0, 0; 0.013, 0.009)(2) ELISA analysis showed that there were significantly statistical difference in the levels of TNF-α, IL-10, Interleukin-8(IL-8) and vascular endothelial growth factor(VEGF) in supernatant among EPC, EPC-LV-IL-10-GFP and EPC-LV-NC-GFP groups without TNF-α.(F=28.910,14.242,10.795,26.097,P=0, 0, 0.002, 0) The levels of TNF-α and Interleukin-8 in supernatant without TNF-α significantly decreased in EPC-LV-IL-10-GFP group compare to EPC and EPC-LV-NC-GFP groups.(P=0, 0; 0.002, 0.002) The levels of IL-10 and VEGF increased in EPC-LV-IL-10-GFP group compare to EPC and EPC-LV-NC-GFP groups.(P=0.012, 0.025; 0, 0) The concertrations of cytokines in supernatant with TNF-α were consistent with in supernatant without TNF-α.(3) Western blot analysis revealed that the expression of VEGF, MMP-9 and phosphorylated-signal transducer and activator of transcription 3(p-STAT3) significantly increased in EPC-LV-IL-10-GFP group. STAT3 expression decreased in EPC-LV-IL-10-GFP group.(4) GFP expression was presented in the retina of EPC-IL-10-GFP and EPC-NC-GFP groups. Inner nuclear layer(INL) was in disorder and edema, ganglion cells decreased and some vessels significantly dilate for HE staining in DM group. Whereas, interventional groups presented structure of INL smoother than retina of DM group, and edema of INL better than retina of DM group. Retinal ultrastructure further confirmed that endothelial cells swelled and basement membranes were uneven, being thickened, split or lost in DM group. However, the number of occlusive capillaries decreased and ontogenetic healthy endothelial cells could be seen in interventional groups, especially in EPC-IL-10-GFP group.(5) EB angiography show that retinal vascular structure is clear and no leakage in CON group; there was diffuse leakage and vascular tortuosity in DM group; retinal vascular diffuse leakage is significantly reduced in interventional groups, especially in EPC-IL-10-GFP group. Retinal Evans blue permeability showed that there were significantly statistical difference among groups at 1 week, 2 week and 4 week after treatment.(F=35.316, 95.734, 101.608, P = 0, 0, 0) There were significantly increased in DM group compared with CON group,(P = 0, 0, 0) and there were significantly increased in EPC-IL-10-GFP group compared with DM and EPC-NC-GFP group.(P = 0, 0, 0; 0, 0, 0) There weresignificantly no significant difference in interventional groups compared with DM group and EPC-IL-10-GFP group at 1 week after treatment,(P=0.833) and there were significantly decreased in interventional groups compared with DM group and EPC-IL-10-GFP group at 2 week and 4 week after treatment.(P = 0.033, 0.043)(6) Western blotting analysis revealed that the expressions of TNF-α, interleukin 6(IL-6), inhibitor of nuclear factor kappa-B kinase(IKK-β), P65, i NOS and VEGF significantly increased in DM group compared with other groups. There were descended expressional trend in interventional groups compared with DM group, especially in EPC-IL-10-GFP group. The results were opposite to the expression of IL-10, inhibitor of nuclear factor kappa-B(IκB-α) and e NOS. The expressions of MMP-9 and Ang-1 elevated in DM group and interventional groups compared with CON group, and there were no statistically significant differences between three groups. The level of VEGF、Ang-1、MMP-9、i NOS and e NOS m RNA were silimar with the expression of protein.Conclusions: Our study demonstrates that overexpression of IL-10 has no effection on the cell migration, adhesion, tube formation and had no influence on growth of EPCs in vitro. Furthermore, overexpression of IL-10 improved EPCs function of migration, adhesion and tubu formation through activating STAT3 signal pathway in vitro. IL-10-modified EPCs suppressed the pathogenesis of NPDR through inhibiting NF-κB signal pathway in vivo. |
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