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Effects Of ~(32)P Labeled PDGFR-β Antisense Oligonucleotide On Cultured Vascular Smooth Muscle Cell

Posted on:2003-01-10Degree:MasterType:Thesis
Country:ChinaCandidate:C H GuFull Text:PDF
GTID:2144360062490692Subject:Medical imaging and nuclear medicine
Abstract/Summary:PDF Full Text Request
Restenosis remains the major limitation to the long term effectiveness of PTCA, with a rate of between 30%and 50%. Artery's mechnical injury of PTCA and CABG result in aexcessive healing response that is characterized by activation of VSMC migration and the proliferation and formation of extracellular matrix.Platelet derived growth factor and its P receptor play important roles in the development of restenosis. However, the effective therapy is still in exploring. Radiolabelled antisense oligonucleotides is not only a platform for the delivery of radiation, but also can inhibit targeted gene expression. Cultured rat aortic VSMC model was established in vitro.PDGFR- P antisense oligonucleotides were labelled by 32P. The effects of 32P-AODN on proliferation, migration, poptosis, cell cycle and targeted gene expression were observed to determine the feasibility of radiolabelled AODN.Materials and MethodsVascular smooth muscle cells were isolated from the thoracic aortas of Sprague-Dawley rats by enzymatic digestion or by explants of minced medial tissue.Cultured VSMCs model was established in vitro.Three different antisense oligonucleotides were synthesized, (antisense 1, 5 TAT CAC TCC TGG AAG CCC3 ,nucleotides 4 through 21; antisense 2, 5' AAG CCC TCT GAG CAC TAA3 ,nucleotides 14through 31; antisense 3, 5' TCT GAG CAC TAA AGC TGG3',nucleotides22 through 39). A scrambled oligonucleotides(5 GTG ATA GTA TGC CGA GCA3 ) and a sense oligonucleotides (5 GGG CTT CCA GGA GTG ATA3 ' )were used as controls. MTT assay , flow cytometry ,the TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-ended labeled) method and a modified Boyden's chamber method were used to determine the effects of PDGFR- 3 AODN on proliferation ,cell cycle ,apoptosis and the migration of VSMC, respectively. Expression of PDGFR- 3 was detected immunohistochemically. The oligonucleotides whichhas the most significant effects on VSMCs was choosen as the candidate for 32P radiolabeling. Interactive Laser Cytometer(ILC) was used to investigate fluoresceinisothiocyanate labeled PDGFR- P AODN cellular uptake and localization; MTT assay, flow cytometry ,the TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-ended labeled) method and a modified Boyden's chamber method were also used to determine the effects of 32P-labeled PDGFR-P AODN on proliferation , cell cycle ,apoptosis and the migration of VSMC,respectively. Immunohistochemistry procedures were performed to detect effects of 32P-labeIed AODN on PDGFR- P expression.Results1.24 hours and 48 hours after incubation with FITC labeled AODN , fluorescence was scattered in the cytoplasm and nucleus respectively.2. The MTT assay shows that the proliferation of VSMCs could be inhibited by PDGFR- P AODN in a dose and time-dependent fashion,yet the AODN1 was most potent at inhibiting proliferation of VSMCs. The percentage and the numble of quiescent cells (Go/G]) of AODN group at 48 hour were significantly increased compared with control group, sense ODN group, scrambled ODN(/><0.<0.05);The inhibitory effect in migration of VSMC was in a dose and time-dependent fashion.AODN1 was most potent at suppressing migration of VSMCs.Sense oligonucleotide (67.8?.0)and scrambled olgonucleotide(68.3?.2) could not significantly inhibit PDGF-induced migration of VSMC(70.2?.2, PX).05).4. No difference of morphology and ultrastructure was observed between control group and other groups at 24 hour;Compared with control group,cell morphology and ultrastructureof AODN...
Keywords/Search Tags:Platelet derived growth factor, Receptor, Restenosis, Vascular smooth muscle cells, Apoptosis, Antisense oligonucleotides, Terminal deoxynucleotidyl transferase-mediated dUTP nick-ended labeled
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