CAJ | 학술논문

目的建立检测血管内皮生长因子-C(VEGF-C)mRNA和血管内皮生长因子受体-3(VEGFR-3)mRNA的实时荧光定量逆转录聚合酶链反应(FQ-RT-PCR)方法,并在食管癌组织中作初步应用.方法采用TaqMan荧光探针技术,分别以pMD18-VEGF-C和pMD18-VEGFR-3质粒作为定量模板,应用循环阈值(Ct)定量起始模板,建立检测食管癌组织的VEGF-C mRNA和VEG-FR-3 mRNA的实时FQ-RT-PCR方法.结果所建方法的线性范围:VEGF-C mRNA和VEGFR-3mRNA均为103～108拷贝/μg总RNA;测定VEGF-C mRNA低值的批内、批间变异系数(CV)分别为7.07%和9.04%,测定VDGF-C mRNA高值的批内、批间CV分别为7.55%和10.28%;测定VEGFR-3 mRNA低值的批内、批间CV分别为7.69%和12.49%,测定VEGFR-3 mRNA高值的批内、批间CV分别为7.31%和9.17%.24例淋巴结转移食管癌患者癌组织VEGF-C mRNA和VEG-FR-3 mRNA的测定范围分别为3.69×106～9.44×106拷贝/μg总RNA和2.54×104～8.03×106拷贝/μg总RNA,均值分别为2.18×106拷贝/μg总RNA和2.27 × 106拷贝/μg总RNA.16例无淋巴结转移食管癌患者癌组织VEGF-C mRNA和VEGFR-3 mRNA的测定范围分别为2.32×103～5.85×105拷贝/μg总RNA和7.31×102～8.21×104拷贝/μg总RNA,均值分别为1.08×105拷贝/μg总RNA和1.68×104拷贝/μg总RNA.提示有淋巴结转移的食管癌组织VEGF-C和VEG-FR-3基因表达水平上调.结论本组建立的检测VEGF-C mRNA和VEGFR-3 mRNA的实时FQ-RT-PCR方法灵敏、准确、稳定、重复性好,可供VEGF-C、VEGFR-3基因表达的临床检测和研究应用.
목적 건립검측혈관내피생장인자-C(VEGF-C)mRNA화혈관내피생장인자수체-3(VEGFR-3)mRNA적실시형광정량역전록취합매련반응(FQ-RT-PCR)방법,병재식관암조직중작초보응용.방법 채용TaqMan형광탐침기술,분별이pMD18-VEGF-C화pMD18-VEGFR-3질립작위정량모판,응용순배역치(Ct)정량기시모판,건립검측식관암조직적VEGF-C mRNA화VEG-FR-3 mRNA적실시FQ-RT-PCR방법.결과 소건방법적선성범위:VEGF-C mRNA화VEGFR-3mRNA균위103～108고패/μg총RNA;측정VEGF-C mRNA저치적비내、비간변이계수(CV)분별위7.07%화9.04%,측정VDGF-C mRNA고치적비내、비간CV분별위7.55%화10.28%;측정VEGFR-3 mRNA저치적비내、비간CV분별위7.69%화12.49%,측정VEGFR-3 mRNA고치적비내、비간CV분별위7.31%화9.17%.24례림파결전이식관암환자암조직VEGF-C mRNA화VEG-FR-3 mRNA적측정범위분별위3.69×106～9.44×106고패/μg총RNA화2.54×104～8.03×106고패/μg총RNA,균치분별위2.18×106고패/μg총RNA화2.27 × 106고패/μg총RNA.16례무림파결전이식관암환자암조직VEGF-C mRNA화VEGFR-3 mRNA적측정범위분별위2.32×103～5.85×105고패/μg총RNA화7.31×102～8.21×104고패/μg총RNA,균치분별위1.08×105고패/μg총RNA화1.68×104고패/μg총RNA.제시유림파결전이적식관암조직VEGF-C화VEG-FR-3기인표체수평상조.결론 본조건립적검측VEGF-C mRNA화VEGFR-3 mRNA적실시FQ-RT-PCR방법령민、준학、은정、중복성호,가공VEGF-C、VEGFR-3기인표체적림상검측화연구응용.
Objective To establish a real-time fluorescent quantitative reverse transcription pol-ymerase chain reaction (FQ-RT-PCR) method for detecting the expression levels of VEGF-C mRNA and VEGFR-3 mRNA,and explore its clinical application in esophageal carcinoma. Methods The real-time FQ-RT-PCR for detecting VEGF-C mRNA and VEGFR-3 mRNA was espabished based on Taq-Man fluorescent probe technology. In this method pMD18-VEGF-C and pMD18-VEGFR-3 were used as standard plasmid and RNA quantification was based on the threshold cycle (Ct) values to examine the specific expression of VEGF-C mRNA and VEGFR-3 mRNA in esophageal carcinoma. Results The detection linear range of the assay: VEGF-CmRNA and VEGFR-3 mRNA was both 103～ 108 copies/μg total RNA. The interassay and intraassay coefficient o{ variation of VEGF-C mRNA lower value was 7.07% and 9.04% respectively,7.55% and 10.28% for higher value respectively; the in-terassay and intraassay coefficient of variation of VEGFR-3 mRNA lower value was 7.69% and 12. 49% respectively,7.31% and 0.17%for higher value respectively. TheVEGF-C mRNA and theVEGFR-3 mRNA range in carcinoma tissue from 24 patients with lymphatic metastasis of esophageal carcinoma was 3.69×104-9.44 × 106 copies/μg total RNA and 2.54 × 104-8.03 × 106 copies/μg total RNA respectively, the mean value was 2.18 × 106 copies/μg total RNA and 2.27 × 106 copies/μg total RNA respectively. The VEGF-C mRNA and VEGFR-3 mRNA range in carcinoma tissue from 16 pa-tients without lymphatic metastasis of esophageal carcinoma was 2.32 × 103-5.85 × 105 copies/μg total RNA and 7.31 × 102-8.21 × 104 copies/μg total RNA respectively,the mean value was 1.08×105 cop-ies/μg total RNA and 1.68 ×104 copies/μg total RNA respectively. The VEGF-C mRNA and VEGFR-3 mRNA copy number per microgram of total RNA in carcinoma tissue from patients with lymphatic metastasis of esophageal carcinoma was significantly higher than that in carcinoma tissue from patients without lymphatic metastasis of esophageal carcinoma. Conclusion A sensive,accurate, stable and re-producible FQ-RT-PCR for the quantitative detection of VEGF-C mRNA and VEGFR-3 mRNA has been established. The method could be applied in clinical detection and research on the expression of VEGF-C and VEGFR-3 gene.