CAJ | 학술논문

目的构建含有表皮生长因子受体Ⅲ型突变体(epidermal growth factor receptor variantⅢ,EGFRv Ⅲ)的重组质粒,建立实时荧光定量聚合酶链反应(PCR)技术测定EGFRv Ⅲ的标准曲线,以定量检测喉鳞癌组织中EGFRv Ⅲ的表达水平.方法以重叠延伸PCR法构建含EGFR第1外显子及第8至部分第9外显子的融合序列.再以融合序列为模板,以高特异性的引物扩增EGFRv Ⅲ的目的片段,构建含有EGFRv Ⅲ的重组质粒,进行测序鉴定.将此质粒作为标准品进行梯度稀释,以TaqMan探针实时荧光定量PCR进行检测,建立标准曲线.并以此技术对32例喉鳞癌组织中EGFRvⅢ mRNA表达水平进行测定.结果经测序鉴定,含EGFRv Ⅲ的重组质粒标准品构建成功,以不同稀释水平的标准品质粒进行荧光定量PCR扩增所得的标准曲线,可用于EGFRv Ⅲ定量分析.32例喉鳞癌组织中共有5例检测到EGFRv Ⅲ的表达,阳性率为15.6%.除1例癌旁组织中存在极少量的EGFRv Ⅲ表达外,EGFRv Ⅲ均在肿瘤组织中表达.结论用TaqMan探针实时荧光定量PCR技术定量研究EGFRv Ⅲ的方法已构建成功,在喉癌组织中的成功运用已证明该方法切实可行,并且较为准确可靠.EGFRv Ⅲ在喉癌组织中有表达,但是阳性率及含量不高,是否能成为喉癌生物治疗的特异性攻击靶点尚待进一步探索.
목적 구건함유표피생장인자수체Ⅲ형돌변체(epidermal growth factor receptor variantⅢ,EGFRv Ⅲ)적중조질립,건립실시형광정량취합매련반응(PCR)기술측정EGFRv Ⅲ적표준곡선,이정량검측후린암조직중EGFRv Ⅲ적표체수평.방법이중첩연신PCR법구건함EGFR제1외현자급제8지부분제9외현자적융합서렬.재이융합서렬위모판,이고특이성적인물확증EGFRv Ⅲ적목적편단,구건함유EGFRv Ⅲ적중조질립,진행측서감정.장차질립작위표준품진행제도희석,이TaqMan탐침실시형광정량PCR진행검측,건립표준곡선.병이차기술대32례후린암조직중EGFRvⅢ mRNA표체수평진행측정.결과 경측서감정,함EGFRv Ⅲ적중조질립표준품구건성공,이불동희석수평적표준품질립진행형광정량PCR확증소득적표준곡선,가용우EGFRv Ⅲ정량분석.32례후린암조직중공유5례검측도EGFRv Ⅲ적표체,양성솔위15.6%.제1례암방조직중존재겁소량적EGFRv Ⅲ표체외,EGFRv Ⅲ균재종류조직중표체.결론 용TaqMan탐침실시형광정량PCR기술정량연구EGFRv Ⅲ적방법이구건성공,재후암조직중적성공운용이증명해방법절실가행,병차교위준학가고.EGFRv Ⅲ재후암조직중유표체,단시양성솔급함량불고,시부능성위후암생물치료적특이성공격파점상대진일보탐색.
Objecfive To construct the recombinant plasmid and standard curve for detection of epidermal growth factor receptor variant Ⅲ (EGFRv Ⅲ) by real-time quantitive polymerase chain reaction (PCR) and establish the RT-PCR assay for accurate detection of EGFRv Ⅲ.Methods To amplify the DNA sequences of exon 1 and exon 8 to exon 9 of EGFR seperately and fuse the above sequences by overhang extension PCR.Then constructing the recombinant plasmid of EGFRv Ⅲ by highly specific primers with the fused sequence as its template.The recombinant plasmid of EGFRv Ⅲ was then sent for sequence analysis.The gradient diluted recombinant plasmid were used as the template to amplify the EGFRv Ⅲ by RT-PCR with TaqMan probe and then applied it in 32 laryngeal carcinoma tissues.Results The recombinant plasmid of EGFRv Ⅲ was successfully constructed.The gradient diluted recombinant plasmid could be used in RT-PCR for quantitative analysis of EGFRv Ⅲ.In all,5 tumor samples expressing EGFRv Ⅲ were detected,with its positive rate of about 15.6%.The expression of EGFRv Ⅲ was tumor specific with only one exception for extraordinarily low expression of EGFRv Ⅲ in macroscopically normal laryngeal mucosas adiacent to the tumor.Conclusions RT-PCR with TaqMan probe was good at sensitivity,specificity,quantification and linear function.It can be a standard method for quantitative detection for EGFRv Ⅲ.Expression of EGFRv Ⅲ was detected in laryngeal carconoma tissues with relatively low level.Whether EGFRv Ⅲ was a suitable target for biological therapy in laryngeal carcinomas remained to be discussed.