The Exploration Of The Treatment To ED By Activating The Expression Of INOS In Smooth Muscle Cells Of Rat Corpus Cavernosum Through SaRNA

PartⅠStudy on up-regulation of iNOS in BRL rat liver cellby small double stranded RNAObjective: To activate the expression of iNOS gene by small activating RNA in BRL rat liver cell.and sereen a valid aceivation sequence.prove that the phenomenon exists in mammalian cells.Methods: the comlementary promoter sequence for iNOS was obtained from Ensemble genome databases(www.ensembl.org)and analyzed against several genome and sequcnce databases incluing dbTSS (Database of Transeriptional Start Sites. http://dbtss.hge.jp).AceView(http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly)and UCSC Genome Browser(http://genome.ucsc.edu)to determine transcription start sites. iNOS promoter sequence was inputted into an Excel macer template that implemented saRNA design rules derived from previous studies.Three different dsRNAs targeting the iNOS gene promoter and a non-speeifie dsRNA sequences as the control(saRNA-1. saRNA-2.saRNA-3.and saRNA-co)were designed and synthesized.and transfected into BRL cells with cationic liposome by lipofectamineTM2000.Expression of mRNA and protein was evaluated by semiquatitative RT-PCR and western-blot after it was transfected 72hResults:The green fluorescent expression was observed in the infected BRI.cells after transfected saRNA marked Hvdroxy fluorescein(FAM),over70% of cells expressing green fluorescent At 72h after the BRL cell was transected with saRNA-2.the results of semi-quantitative RT-PCR and Western-blot assay indicated iNOS mRNA and protein expression was activated.Other ds RNAs cause no induetion in iNOS expression after transfected.Conclusions:ThesaRNA can successfully activate the expression of the iNOS in Rat BRL cells.whieh laid a foundation for application of RNA active technology. PartⅡConstruction and identification of recombinat adenovirusexpressing short hairpin RNA of rat inducible nitric oxide synthaseObjectibe:To construet an adenovirus vector expressing short hairpin RNA of rat inducible oxide synthase(iNOS)marked enhanced green fluorescent protein(EGFP)and amplify the adenovirus vector in HEK-293 cellsMethods:According to saRNA-2 which targeting rai iNOS promoter sequenece and having induction role at an earlier research date.were designed and synthesized shRNA oligonucleotides.annealed to form double-stranded and cloned into the vector pDC316-EGFP-U6.recombinant plamid.for PCR identification.restriction enzyme digestion and sequencing.Uising AdMax adenovirus packaging system.the shuttle plasmid pDC316-iNOS-shRNA-EGFP marked enhanced green fluorescent protein(EGFP)which had been constructed and the adenovirus skeleton plasmid pBHGlox E1.3Cre were both transfeeted into HEK-293 cells to get the replication defective recombinant adenovirus veetor Ad5-iNOS-shRNA-EGFP by homologou recombination.Further the 293 cells infected with this virus for viros amplification.purified by ion-exchange method.then virus titer and purified particle were determined.Results:The recombinant shuttle plasmid pDC316-iNOS-shRNA-EGFP and the recombinant adenovirus vector Ad5-iNOS-shRNA-EGFP were successfully constructed. which was confirmed by the polymerase chain reaction(PCR)testing.determination of restriction analysis and sequencing.After amplification and purification.the virus particle count was 3.6×1011VP/ml.A260/A280 was1.25 and titer of recombinant adenovirus was 7.94×109IU/ml.Conclusions:The recombinant adenovirus vector Ad5-iNOS-shRNA-EGFP is correctllv constructed:the recombinant adenovirus vector can meet the need of in vivo and in vitro gene transfeetion in laboratory studies.which laid a foundation for gene active in erectile dvsfiunction treatment. Objective: To explore recombinant adenovirus expressing short hairpin RNA of rat inducible nitric oxide synthase could activate iNOS gene in rat corpus cavernosum smooth muscle cells. providing experimental evidence for erectile dysfunction gene therapy.Methods:Construction of the adenovirus Ad5-iNOS-shRNA-EGFP (AdU6/shiNOS) and the control virus AdU6/shControl transfected into rat cavernous smooth muscle cells.Cells were examined for iNOS expression by Real-time quantitative PCR and western blotting and different MOI(25,50,75) after 72 hours.Cells were then treated with 10mmol/L L-arginine.We analyzed for eyclic guanosine monophoshate(cGMP) with enzyme immunoassay (EIA) system at 72h ours after transfecting.record the effect of AdU6/shiNOS on the cGMP of smooth muscle cell.Results:Compared with mock group and control group. AdU6/shiNOS transfected rat corpus cavernosum smooth muscle cells after 72 hours.iNOS gene expression at the mRNA and protein were significantly increased in a dose-dependent(P<0.05).MOI=75 works best for RNAa. Corpus cavernosum smooth muscle cells transfected with AdlJ6/shiNOS showed an increase of cGMP level compared with corpus cavernosum smooth muscle cells transfected with control vector and the mock group. (P<0.05)Conclusions:Using adenovirus-mediated RNAa technology to improve the iNOS gene expression in corpus cavernosum smooth muscle cells to be successful, can increase the cGMP level in corpus cavernosum smooth muscle cell.and activative the NO/cGMP pathway.which opened a new research direction for the gene treatment of erectile dysfunction.