| Objective To construct and identify the recombinant Ad-RP105vector, which madethe high expression of RP105gene in ischemia-reperfusion myocardium.To observe theeffects of RP105gene on myocardial after transfection of adenovirus-mediated RP105vector in the ischemia-reperfusion injury rats models. We investigate the protection andmechanism of RP105gene on myocardial ischemia-reperfusion from levels of geneexpression and protein synthesis through TLR-4signaling pathway, which supply a newapproach to ischemia-reperfusion injury and experience of gene therapy.Methods40male SD rats were divided into4groups (n=10) in random: saline+sham group (Sham group), saline+ischemia-reperfusion group (I/R group), Ad-RP105+ischemia-reperfusion group (Ad-R group), Ad-GFP+ischemia-reperfusion group (Ad-Ggroup). The recombinant adenovirus vector was injected in myocardial coronary artery,established myocardial ischemia-reperfusion model after3d. Killed the rats at2hpostoperative. Light microscope were used to observe structural changes of myocardium;Evans blue and TTC staining method were used to estimate myocardial infarct size; ELISAanalyze myocardial TNF-α and IL-6levels; myocardial RP105, TLR-4, MyD88, NF-κBwere detected by Real-time PCR or Western blot.Results (1) The green fluorescent protein was seen in myocardium and transfectionefficiency was91.5%,the rats survival condition keep stable.(2) Pathologicalmorphological examination showed normal morphology of the myocardial ultrastructureand organization of the Sham group; IR and Ad-G group myocardial structural wereseriously damaged, while R group was significantly improved.(3)Infarct size of Ad-Rgroup was significantly reduced compared to IR group (41.3±1.5%vs26.7±1.3%, P<0.05);the changes of Ad-G group(39.9±1.2%) was not statistically significant.(4) Comparedwith Sham group, RP105mRNA and protein expression of Ad-R group of weresignificantly increased (4.080vs0.374vs0.149, P <0.05), while Ad-G Group and I/Rgroup were down-regulated (P <0.05).(5) The levels of TLR-4, MyD88, NF-κB mRNAand protein were higher in I/R group than Sham group, however, compared with I/R group,Ad-R group was significantly down-regulated(P<0.05).(6) TNF-α and IL-6levels weresignificantly secretion in myocardium after ischemia and reperfusion group compared withthe Sham group; Ad-R group wse inhibited partly compared with I/R group (P <0.05).Conclusion (1) Recombinant Ad-RP105was safety and effective;(2) The RP10gene carried by adenovirus can be effectively transfected into the myocardial tissue and expressstably in vivo and significantly enhance the level of RP105mRNA and protein inmyocardial tissue;(3) the high expression of RP105gene can significantly reducemyocardial ischemia-reperfusion injury;(4) TLR-4signaling pathway was activated in theprocess of myocardial ischemia-reperfusion injury, made TLR-4,RP105, MyD88, NF-κBgene and protein expression levels obviously increased, and promoted the secretion ofinflammatory cytokines TNF-α and IL-6that mediated inflammation cascade reaction, toparticipate ischemia-reperfusion injury pathophysiology process;(5) RP105playingprotective effect on myocardial ischemia-reperfusion injury was likely to inhibitTLR-4-mediated signal transduction pathway, and then reduce production of downstreaminflammatory cytokine. |
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