Metalloproteinase Inhibitory Factor-1 (timp-1), Ag3340 Islet Function And Raav-of Timp-1, Lv-timp-1 Construction And Identification

Objective(1)To evaluate the effect of tissue inhibitor of metalloproteinase-1 (TIMP-1) and AG3340 on the insμlin secretion in interleukin-1β treatedpancreatic islets.(2)To construct and identify recombinant adeno-associated virusrAAV-TIMP-1 and lentivirus vector LV-TIMP-1 which encoding fμlllength cDNA of TIMP-1.Methods(1)Rat islets were pretreated with TIMP-1 or AG3340 1h beforeadditional IL-1β were treated and maintained 24 hours at 37℃, theglusose stimulated insulin secretion(GSIS)test were performed and thenthe medium was collected for insulin measurement.(2)Using the technique of gene recombination, the main plasmid ofpAAV-TIMP-1 was constructed. Recombinant AAV-TIMP-1 wasobtained from HEK293 cells, which were cotransfected with AAV vectorplasmids by calcium phosphate precipitation method. The titer ofrecombinant AAV was determined by counting positive HT1080 cellswhich were transduced with rAAV-GFP, finally, Western blots wereperformed to confirm the expression of rAAV-TIMP-1 in HT1080 cells.(3)Using the technique of gene recombination, the main plasmid ofpLenti6/V5-TIMP-1 was constructed. Viral supernatant was obtainedfrom 293FT cells, which were cotransfected with plasmids of lentivirusexpression system by Lipofectamine precipitation method. The titers ofthe lentiviral vector were determined by scoring GFP positive RIN5Fcells following serial dilutions of the viral supernatant transduction,finally, Western blots were performed to confirm the expression ofLV-TIMP-1 in 293FT cells.Results(1)Insμlin secreation was decreased by interleukin-1β . Islets treated withTIMP-1 or AG3340 did not show any alteration in GSIS compared withcontrol group. TIMP-1,but not AG3340 was able to restore GSIS ininterleukin-1β treated islets.(2)The titer of rAAV viral stock could be reached up to 10~9 TU/mL.Western blot results showed that rAAV-TIMP-1 expressed TIMP-1proteins in HT1080 cells.(3)The titer of lentivirus supernatant could be reached up to 10~4TU/mL.Western blot showed recombinant Lentivirus expressed TIMP-1 proteinsin 293FT cells.Conclusion(l)Tissue inhibitor of metalloproteinase-1, not AG3340, could preventinterleukin-1β mediated dysfunction in pancreatic Islets.(2)The development of recombinant rAAV-TIMP-1 and LV-TIMP-1provided a preliminary tool for TIMP-1 gene therapy in islets.