ABSTRACT
Background It has been determined that dipeptidyl peptidase Ⅲ (DPPⅢ) plays an important role in the metabolism and modification of proteins and DPPⅢ of human has highly homologous to rat.Researches have shown that DPPⅢ is associated with the formation of cataract.However,few relevant studies have been reported.ObjectiveThe present study is to find out the relationship between the expression of DPPⅢ in rat lenses and age-related cataract.Methods Lenses were obtained from general Wistar rats of ages 3,6,9,or 12 weeks old (10 lenses each ) and homogenized with different concentrations of standard bovine serum.The proteins were resolved using SDS-PAGE gel electrophoresis.Peak concentration and total concentration of DPPⅢ protein in lenses from rats of different ages were detected by Western blot.Enzyme activity of DPPⅢ was determined by monitoring the amount of dipeptides removed from a special substrate (Arg-Arg-4mb NA) by measuring the absorbance with UV-2500PC at 525 nm.The relationship between the enzyme activity of DPPⅢ in lenses and age of rats was evaluated using regression analysis.Results DPPⅢ was detected at a molecular weight of 82000 Da.The peak concentration and total concentration of DPPⅢ in normal rat lenses increased with the growth of age.The total protease activity of DPPⅢ in the lenses of rats was correlated with the ages of the rats (r=0.99,P＜0.05).Conclusion DPPⅢ may be involved in the alteration of crystallin during the development of lenses,and it may play an important role in the formation and aggravation of age-related cataract.
ABSTRACT
Objective To study the vascular endothelial growth factor(VEGF) expression and secretion of mesenchymal stem cells(MSCs) of rabbit transfected with pcDNA3-VEGF165 expression plasmid in order to construct a kind of tissue engineering artificial skull with more blood supply.Methods By gene reconstruction method,the VEGF165 gene was cloned into eukaryotic expression plasmid pcDNA3 and recombined eukaryotic expression plasmid pcDNA3-VEGF165 was constructed;By lipofectamine transfection method,pcDNA3-VEGF165 expression plasmid was transfected into MSCs of rabbit;By RT-PCR and Western blotting methods,the VEGF mRNA expression and VEGF protein secretion in the MSCs were detected.Results Recombined eukaryotic expression plasmid pcDNA3-VEGF165 was confirmed to be true by double enzyme digestion,two strips came to appear in 5400 bp and 600 bp of gelose electrophoresis and their sizes accorded with pcDNA3 plasmid and VEGF165 accordingly;Primarily cultured and subcultured MSCs of rabbit were successfully performed and the MSCs storehouse of rabbit was established,primary MSCs presented lymphoid form at first and then the morphology of them became circulara,polygonal or irregular forms,they were more and more like fibroblastic cells after subcultured cultivation.The expressions of VEGF mRNA and VEGF protein in the MSCs were found after transiently transfection by RT-PCR and Western blotting methods.Conclusion Recombined eukaryotic expression plasmid pcDNA3VEGF165 can be transfected into MSCs of rabbit effectively by lipofectamine and the VEGF expression can be detected in the MSCs after transfection.