简介:WehavedevelopedandtestedchimericT-cellreceptors(TCR)specificforp185HER2.Intheseexperiments,retroviralvectorsexpressingtheN297orN29ξreceptorswereconstructedinpRET6.AmphotropicviralproducercellswereestablishedintheGALV-basedPG13packagingcellline.Ficollpurifiedhumanperipheralbloodlymphocytes(PBL)werevitallytransducedusinganoptimizedprotocolincorporatingactivationwithimmobilizedanti-CD3/anti-CD28monoclonalantibodies,followedbyviralinfectioninthepresenceoffibronectinfragmentCH296.Transducedcellswereco-culturedwithhumantumorcelllinesthatoverexpress(SK-OV-3)orunderexpress(MCF7)p185HER2toassayforantigenspecificimmuneresponses.BothCD4^+andCD8^+T-cellstransducedwiththeN297orN29ξchTCRdemonstratedHER2-specificantigenresponses,asdeterminedbyreleaseofTh1likecytokines,andcellularcytotoxicityassays.OurresultssupportthefeasibilityofadoptiveimmunothempywithgeneticallymodifiedT-cellsexpressingachTCRspecificforp185HER2.
简介:Wehaveconfirmedefficientanti-tumoractivitiesoftheperipherallymphocytestransducedwithap185HER2-specificchimericT-cellreceptorgenebothinmurineandinhumaninourpreviousstudies.TofurthertestthefeasibilityofchimericT-cellreceptorinabonemarrowtransplantationmodel,wefirst,madetwomurinetumorcelllines:MT901andMCA-205,toexpresshumanp185HER2byretroviralgenetransduction.MurinebonemarrowcellswereretrovirallytransducedtoexpressthechimericT-cellreceptorandgene-modifiedbonemarrowcellsweretransplantedintolethallyirradiatedmouse.Sixmonthsposttransplantation,p185HER2-positivetumorcells:MT-901/HER2orMCA-205/HER2wassubcutaneouslyorintravenouslyinjectedtomakemousemodelssimulatingprimarybreastcancerorpulmonarymetastasis.Theinvivoanti-tumoreffectsweremonitoredbythesizeofthesubcutaneoustumororcountingthetumornodulesinthelungsafterIndiainkstaining.ThesizeofthesubcutaneoustumorwassignificantlyinhibitedandthenumberofpulmonarynodulesweresignificantlydecreasedinmouserecipientstransplantedwithchimericT-cellreceptormodifiedbonemarrowcellscomparedwiththecontrolgroup.Ourresultssuggesttheefficientinvivoanti-tumoractivitiesofchimericT-cellreceptorgenemodifiedbonemarrowcells.
简介:Thec-erbB-2proto-oncogeneencodesa185kDaproteinp185,whichbelongstoepidermalgrowthfactorreceptorfamily.Amplificationofthisgenehasbeenshowntocorrelatewithpoorclinicalprognosisforcertaincancerpatients.ThemonoclonalantibodyA21whichdirectedagainstp185specificallyinhibitsproliferationoftumorcellsoverexpressingp185,henceallowsittobeacandidatefortargetedtherapy.InordertoovercomeseveraldrawbacksofmurineMAb,wecloneditsVHandVLgenesandconstructedthesingle-chainFv(scFv)throughapeptidelinker.TherecombinantscFvA21wasexpressedinEscherichiacoliandpurifiedbytheaffinitycolumn.SubsequentlyitwascharacterizedbyELISA,Westernblot,cellimmunohistochemistryandFACS.Alltheseassaysshowedthebindingactivitytoextracellulardomain(ECD)ofp185.BasedonthosepropertiesofscFvA21,wefurtherconstructedthescFv-Fcfusionmoleculewithahomodimerformandtherecombinantproductwasexpressedinmammaliancells.Inaseriesofsubsequentanalysisthisfusionproteinshowedidenticalantigenbindingsiteandactivitywiththeparentantibody.Theseanti-p185engineeredantibodieshavepromisedtobefurthermodifiedasatumortargetingdrugs,withaviewofapplicationinthediagnosisandtreatmentofhumanbreastcancer.
简介:目的:探讨nm23,mdm2,p185,p21及p53在骨巨细胞瘤(GCT)的表达及与GCT病理分级和复发的关系。方法:应用SP免疫组织化学方法检测nm23,mdm2,p185,p21p53在52例GCT中的表达(GCT按Jaffe分级,I级15例,II例25例,Ⅲ级12例),结果:52GCT中nm23,mdm2,p185,p21andp53的阳性表达率分别为40.4%(21/52),34.6%(18/52),21.2%(11/52),13.5%(7/52),26.9%(14/52)。其阳性表达与病理分级差异均无显著性(P值分别为0.797,0.871,0.441;0.658;0.699)。nm23,mdm2,p185,p21,p53在复发和无复发的病例中,阳性表达率分别为69.2%(9/13),30.8%(12/39),61.5%(8/13),25.6%(10/39),38.5%(5/13),15.4%(6/39);23.1%(3/13),10.3%(4/29),46.2%(6/13),20.5%(8/39)。nm23,mdm2表达与GCT复发率差异有显著性(P值分别为0.003,0.018),而p185,p21,p53的阳性表达与GCT复发差异无显著性(P>0.05),结论:nm23,mdm2,p185,p21及p53在GCT中的表达与病理分级差异无关,而nm23,mdm2的表达与其复发有关。
简介:无
简介:摘要HER2的基因扩增和(或)蛋白过表达与多种肿瘤的发生、发展关系密切。它通过基因突变或基因扩增及激活下游的信号转导途径等参与肿瘤细胞的增殖、凋亡、浸润与侵袭等基因的调控。HER2有望成为肿瘤治疗的一个新靶点。
简介:随着分子生物学技术的发展肿瘤治疗进入了分子靶向治疗时代,高效、选择性地杀伤肿瘤细胞,能减少对正常组织损伤,不良反应小。我们回顾性分析148例HER2蛋白(2+)乳腺癌的HER2基因扩增情况及临床病理特征,并探讨与HR、P53、KI67之间的相互关系,进而为更好的实施乳腺癌的个体化治疗提供依据。1材料与方法1.1临床资料:筛选山西省肿瘤医院2013年4月至2015年4月手术切除标本中经病理确诊及术前未行放化疗、免疫组织化学(IHC)法检测HER2蛋白(2+)的乳腺浸润性导管癌标本148例,患者的平均年龄为47.5岁(范围28~83岁),同时收集雌激素受体(ER)、孕激素受体(PR)、P53、KI67的免疫组织化学标记结果及组织学分级、淋巴结转移情况。
简介:目的构建携带融合基因SLC-Her-2/neu-P53-Fc(SLC-HP-Fc)的重组腺病毒AdEasy^TM-SLC-HP-Fc表达载体。方法提取SLC-HP基因及含Fc段基因的质粒构建携带目的基因的腺病毒穿梭载体pShuttle-CMVSLC-HP-Fc,经酶切线性化后转入含有pAdEasy-1质粒的E.coliBJ5183中进行同源重组,得到重组腺病毒真核表达载体AdEasy^TM-SLC-HP-Fc。线性化后的病毒表达载体通过脂质体Lipofectamine2000转染293细胞,通过包装扩增并经PCR鉴定后得到大量复制病毒并纯化保存。结果得到重组腺病毒AdEasy^TM-SLC-HP-Fc,成功转染293细胞出现细胞病变反应,经酶切及PCR法确定目的基因的表达。结论成功构建了SLC-Her-2/neu-P53-Fc融合基因重组腺病毒表达载体,并建立了重组腺病毒AdEasy^TM-SLC-HP-Fc种子病毒库和工作病毒库,为进一步研究该融合基因与肿瘤相关作用奠定了实验基础。
简介:摘要目的探究Her-2、p53、Ki-67在结直肠腺癌组织中的表达及其临床病理特征,为结直肠腺癌患者的预后评估研究提供数据支持。方法本次研究对象选取我院2014年1月至2018年1月经手术切除的结直肠腺癌标本84例,采用免疫组化法测定患者组织内Her-2、p53和Ki-67的表达,并将其与临床病理学特征进行相关性分析。结果(1)Her-2在不同浸润深度、淋巴结转移及改良Dukes分期组中表达中差异显著差异(P<0.01),p53在肿瘤大小、不同分化程度及淋巴结转移组中的表达差异显著差异(P<0.01),Ki-67在肿瘤大小、不同浸润深度及改良Dukes分期组中呈差异显著(P<0.01)。(2)在结直肠癌患者组织中,Ki-67与p53的表达呈正相关(r=0.202,P<0.01),Her-2与p53的表达无相关性(r=0.121,P>0.05),Her-2与Ki-67的表达无相关性(r=0.126,P>0.05)。结论免疫组化方法检测结直肠腺癌中Her-2、p53、Ki-67的表达,明确其与临床病理特征的相关性,为后期结直肠癌的诊疗与预后评估提供依据,同时为后续其靶向治疗的药物研究奠定基础。