以支架为载体的TRADD 基因慢病毒转染对食管良性狭窄的抑制作用.docx

以支架为载体的TRADD基因慢病毒转染对食管良性狭窄的抑制作用以支架为载体的TRADD基因慢病毒转染对食管良性狭窄的抑制作用以支架为载体的TRADD基因慢病毒转染对食管良性狭窄的抑制作用刘云杰，袁月，童珂雅，陈瑶，代剑华，周学谦，彭贵勇（400038重庆，第三军医高校西南医院全军消化病探讨所）摘要：目的本试验视察TRADD基因慢病毒涂层食管支架置入后，TRADD基因对犬食管良性狭窄粘膜的转染效果及对食管狭窄的的抑制作用。方法将15只试验犬随机平均分为试验组、比照组及空白组，采纳氢离广凝固器制作良性食管狭窄模型，分别置入GEp-TRADD基因慢病毒涂层带膜食管支架、GFP-慢病毒涂层食管支架、一般带膜食管支架，1周后视察支架脱落状况，并取出支架，2,3、4周胃镜下视察狭窄程度。4周后处死试验犬，对狭窄食管组织行病理、免疫荧光检查，分别采纳western-b1.ot方法检测TRADD蛋白在三组食管组织中的表达,同时检测co1.1.agenI、Co1.1.agenIIR-SMA三种蛋白在食管组织中表达状况。结果置入食管支架1周后，胃镜检杳见试验组试验犬带膜支架全部移位进入胃，比照组和空白组试验犬分别有2只、3只试验犬其带膜支架移位进入胃中，胃镜取出全部试验犬未移位食管支架。此后每周测量其食管狭窄处直径，nJ见比照组及空白组试验犬再次出现食管明显狭窄，而试验组再狭窄较轻。采纳重复测量设计的多因素方差分析，P0.01,具有统计学意义。食管狭窄组织HE染色切片表明空白组及比照组肉芽组织及纤维组织增生，而试验组肉芽组织及纤维组织增生较不明显。免疫荧光镜检查可见绿色荧光蛋白在黏膜下组织，表明TRADD基因慢病毒及慢病毒均在食管黏膜下生长。WeStern-b1.ot检测试脸组可检测GFP-Tradd-F1.ag蛋白，而空白组和比照组未检测出该蛋白，表明TRADD基因慢病毒胜利转染至食管黏膜下组织。western-b1.ot检测显示试验组co1.1.agenI-kco1.IagenIIk-SMA三种蛋白较其他两组明显降低，具有统计学差异。结论以支架作为载体可胜利将TRADD慢病毒转入食管粘膜组织中，可抑制狭窄部位组织增生，削减食管狭窄部位再狭窄发生。关键词TRADD；慢病毒；食管支架；食管良性狭窄；基金项目国家H然科学基金面上项目(81070384,81470907)通讯作者彭贵勇，电话：Te1.:(023)68765185,邮箱：pgy63163TheinhibitoryeffectoftransfectionofTRDDgene-expressingIentivirusesusingstentsascarriersinbenignesophagea1.stricture1.iuYunjie,YuanYue,TongKeya,ChenYao,DaiJianhua,ZhouXueqian,PengGuiyong(InstituteofGastroentero1.ogy,SouthwestHospita1.,ThirdMi1.itaryMedica1.University,Chongqing,400038).Abstract:ObjectiveThisstudyobservedtheeffectofTRADDgenetransfectioninthemucosaofbenignesophagea1.strictureindogsanditsinhibitoryeffectonesophagea1.strictureafteresophagea1.stentscoatedwithTRDDgene-expressingIentiviruseswereinsta1.1.ed.MethodsFifteenexperimenta1.dogswererandom1.yandeven1.ydividedintoexperimenta1.,contro1.,andb1.ankgroups.Bcnignesophagea1.stricturemode1.swereconstructedusingargonp1.asmacoagu1.ation(PC).Coveredesophagea1.stentscoatedwithGFP-TRADDgene-expressingIentiviruses,esophagea1.stentscoatedwithgreenf1.uorescenceprotein(GFP)-expressingIentiviruses,andregu1.arcoveredesophagea1.stentswerep1.acedintodogs,andtheconditionsofstent1.osswereobservedafteroneweek.Stentswereremoved,andthedegreesofstricturewereobservedusinggastroscopyafter2,3,and4weeks.Experimenta1.dogsweresacrificedafter4weeks,andpatho1.ogica1.andimmunof1.uorescenceexaminationswereperformedonesophagea1.stricturetissues.Westernb1.ottingwasperformedtodetectTRDD,co1.1.agenI,co1.1.agen111.,and-smoothmusc1.eactin(-SMA)proteinexpressioninesophagea1.tissuesinthethreegroups.Resu1.tsOneweekafteresophagea1.stentp1.acement,gastroscopyresu1.tsshowedthata1.1coveredstentsindogsintheexperimenta1.grouphadmigratedintothestomach,whi1.ecoveredstentsmigratedintothestomachinon1.y2and3dogsinthecontro1.andb1.ankgroups,respective1.y.Non-migratedesophagea1.stentsinexperimenta1.dogswereremovedbygastroscopy.Afterremova1.,thediametersofesophagea1.stricturesitesweremeasuredeveryweek.Significantesophagea1.strictureoccurredagaininexperimentdogsinthecontro1.andb1.ankgroups,whi1ethere-strictureintheexperimenta1.groupwasmi1.der.Repeatedmeasuresdesignedmu1.tivariateana1.ysisofvariancewasperformed,andPO.O1.indicatedstatistica1.significance.Hematoxy1.inandeosin(HE)stainingresu1.tsofesophagea1.stricturetissuesindicatedthathyperp1.asiaofgranu1.ationtissuesandfibroustissuesoccurredintheb1.ankandcontro1.groups,whi1.ehyperp1.asiaofgranu1.ationtissuesandfibroustissueswasnotobviousintheexperimenta1.group.ImmunofIuorescencemicroscopyrevea1.edGFPexpressioninsubmucosa1.tissues,indicatingthatTRADDgene-expressingIentivirusesandIentivirusesgrewinesophagea1.submucosa.Westernb1.otdetectionresu1.tsshowedthattheGFP-TRADD-F1.agproteincou1.dbedetectedintheexperimenta1.group,whi1.etheproteinwasnotdetectedintheb1.ankandcontro1.groups,indicatingthatTRADDgene-expressingIentivirusesweresuccessfu1.1.ytransfectedintoesophagea1.submucosa1.tissues.Westernb1.otresu1.tsshowedthattheco1.1.agenI,co1.IagenIII,and-SMproteinexpression1.eve1.sintheexperimenta1.groupweresignificant1.y1.owerthanthoseintheothertwogroups：thedifferenceswerestatistica1.1.ysignificant.Conc1.usionUsingstentsascarriersresu1.tedinsuccessfu1.transferofTRADD-CxpressingIentivirusesintoesophagea1.mucosa1.tissues,whichcou1.dinhibittissuehyperp1.asiaatstricturesitesandcou1ddecreasere-strictureofesophagea1.stricturesites.KeywordsTRADDi1.entivirusiesophagea1.Stentibenignesophagea1.strictureThisworkwassupportedbytheNationa1.Natura1.ScienceFoundationofChina(81070384,81470907).Correspondingauthor:PcngGuiyong,Te1.:862368765185,E-mai1.:pgy63163食管良性狭窄引起的吞咽困难临床较难解决，严峻影响患者生活质量，并且引起严峻并发症11.其治疗常采纳探条扩张或球囊扩张，但其复发率较高23目前对难治性良性食管狭窄治疗主要是安装食管支架。食管支架可显著改善患者吞咽困难等症状4。但是各食管支架仍有缺点：自扩张金属支架(se1.f-expandab1.emeta1.stents,SEMS)两端因金属成分对食管黏膜的刺激作用，常导致肉芽组织增生而引起再狭窄，肉芽组织常将支架包埋，当要移除金属支架时，可导致食管裂开50自扩张塑料支架(se1.f-expandab1.ep1.asticstents,SEPS)能削减反应性组织增生及增加支架移除平安性，但是其具有较高的支架移位率062%)及缓解乔咽困难的时间较短1»可汲取支架(biodcgrdab1.estentsBDS)允许支架被组织包埋及不须要取出，但由于寿命一般只有6周，不能有效支撑到8-12周以上,再狭窄发生率仍较高61.因此，目前对难治性良性食管狭窄还缺乏满足的治疗手段。食管良性狭窄主要是由于瘢痕增生所致。目前的探讨表明，细胞凋亡与瘢痕增生、搬痕疙瘩的形成具有亲密的关系。细胞凋亡表现为凋亡信号通路的表达，其最主要的传导途径为死亡受体(Deat