ABSTRACT
Background Diabetic retinopathy (DR) leads to blindness because of the retinal angiogenesis caused by the ischemia of retina.Vascular endothelial growth inhibitor (VEGI) is a recently identified anti-angiogenic cytokine,which can suppress endothelial cell proliferation and angiogenesis.Objective The aim of this study was to detect the change of serum and vitreous VEGI/TL1A and its relative cytokines in patients with DR.Methods A non-randomized controlled clinical trial was performed.Fifty-five DR patients were enrolled in Tianjin Medical University General Hospital from November 2012 to March 2013 with the informed consent.The patients were divided into non-proliferative DR (NPDR) group (20 cases) and PDR group (35 cases).Eleven cataract patients served as normal control group,and 15 patients with diabetic mellitus (DM) were included as DM group.The demography was matched among the groups,but the course of DM and the blood glucose level were elevated in the PDR group and the DM group compared with DR group (all at P＜0.05).We collected the serum of all the patients above.Another 23 PDR patients (25 eyes) were enrolled in Tianjin Medical University General Hospital from November 2012 to March 2013 with the informed consent and served as PDR group,healthy corpse's eyes (n=7) as control group,the patients were assigned to the retinal photocoagulation group,surgery group and photocoagulation +surgery group according to different treatment procedures.Vitreous samples were collected during the progress of vitrectomy.TL1A/VEGI 251,VEGF,TNF-α,IL-1β and NF-κB p65 concentrations in the serum and vitreous specimens were detected using ELISA.The differences of serum and vitreous TL1A/VEGI 251,VEGF,TNF-α,IL-1β and NF-κB p65 in various groups were statistically analyzed by ANOVA and independent sample t test,respectively.The correlation between TL1A/VEGI 251 and VEGF,TNF-α,IL-1β,NF-κB p65 were calculated by Pearson correlation analysis.Results TL1A/VEGI 251 concentration was elevated in the DM group,NPDR group and PDR group compared with the normal control group,with significant difference among the 4 group (F =27.431,P =0.009),and TL1A/VEGI 251 concentration was higher in the PDR group than that in the DM group or the NPDR group (P＜0.05).VEGF,TNF-α,IL-1 β and NF-κB p65 concentrations in serum were increased in the PDR group in comparison with the DM group,NPDR group and the normal control group (P＜0.05).However,no significant difference among the DM group,NPDR group and the normal control group (P＞0.05).Serum TL1A/VEGI 251 concentration was significant correlated with VEGF,TNF-α,IL-1β and NF-κB p65 concentration (r=0.951,0.951,0.851,0.944,all at P＜0.01).Vitreous TL1A/VEGI 251,VEGF,TNF-α,IL-1 β concentrations were ascended in the PDR group compared with the normal control group (P =0.024,0.001,0.000,0.037),but there was no significantly difference in vitreous NF-κB p65 concentration between the two groups (P =0.073).Vitreous TL1A/VEGI 251 concentrations declined in the retinal photocoagulation group and the surgery group compared with the normal group (all at P＜ 0.05),and significant positive correlations were found between vitreous TL1A/VEGI 251 concentration and VEGF or TNF-α concentration (r =0.675,0.950,P ＜ 0.01) ;while Pearson correlation coefficient was not statistically significant between vitreous TL1A/VEGI 251 concentration and IL-1β or NF-κB p65 concentration (r=0.233,0.318,P＞0.05).Conclusions VEGI is involved in the pathogenesis of DR,and it interacts with VEGF,TNF-α,IL-1β and NF-κB to affect the development of DR.These results provide a new clue for the further study of DR.
ABSTRACT
Objective: To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI+, so as to lay a basis for studying its biological activity. Methods: Chimeric molecule VEGI+ was constructed by grafting oligopeptide CTTH-WGFTLC to extracellular region of VEGI (VEGI23-174). Before ligation into pET30a(+) expression vector, PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing, then pET30a-VEGI was used to transfect BL21 (modified E. coli strain). The chimeric protein was purified by metal affinity chromatography. Western blotting and coomassie blue staining were used for protein identification. Results: The chimeric molecule VEGI+ was confirmed by restriction enzyme digestion and DNA sequencing. The constructed pET30a-VEGI was confirmed by enzymatic digestion. The expression was mainly in the form of inclusion body. SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23 000, with a purity of about 90%. Conclusion: We have successfully constructed the recombinant plasmid pET30a-VEGI+ and expressed it in E. coli. And we have obtained high purity of soluble chimeric protein VEGI+ through affinity chromatography.
ABSTRACT
Objective:To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI~+,so as to lay a basis for studying its biological activity.Methods:Chimeric molecule VEGI~+ was constructed by grafting oligopeptide CTTH- WGFTLC to extraeellular region of VEGI(VEGI_(23-174)).Before ligation into pET30a(+)expression vector,PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing,then pET30a-VEGI was used to transfect BL21(modified E.coli strain).The chimeric protein was purified by metal affinity chro- matography.Western blotting and coomassie blue staining were used for protein identification.Results:The chimeric molecule VEGI~+ was confirmed by restriction enzyme digestion and DNA sequencing.The constructed pET30a-VEGI was confirmed by enzymatic digestion.The expression was mainly in the form of inclusion body.SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23000,with a purity of about 90%.Conclusion:We have successfully constructed the recom- binant plasmid pET30a-VEGI~+ and expressed it in E.coll.And we have obtained high purity of soluble chimeric protein VEGI~+ through affinity chromatography.