VEGF-C mRNA和VEGFR-3 mRNA实时荧光定量检测方法的建立及初步应用

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（FO-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-CmRNA和VEG—FR-3tuRNA的实时FQ-RT-PCR方法。结果所建方法的线性范围：VEGF-CmRNA和VEGFR-3mRNA均为10^3～10^8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-CmRNA高值的批内、批间CV分别为7．55％和10．28％;测定VEGFR-3mRNA低值的批内、批间CV分别为7．69％和12．49％,测定VEGFR-3mRNA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-CmRNA和VEG—FR-3 mRNA的测定范围分别为3．69×10^4～9．44×10^6拷贝／μg总RNA和2．54×10^4～8．03×10^6拷贝／μg总RNA,均值分别为2．18×10^6拷贝／μg总RNA和2．27×10^6拷贝／μg总RNA。16例无淋巴结转移食管癌患者癌组织VEGF-CmRNA和VEGFR-3mRNA的测定范围分别为2．32×10^3～5．85×10^5拷贝／μg总RNA和7．31×10^2～8．21×10^4拷贝／μg总RNA,均值分别为1．08×10^5拷贝／μg总RNA和1．68×10^4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-CmRNA和VEGFR-3mRNA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。

Establishmant of a real-time fluorescent quantitative method far detection of VEGF-C mRNA/VEGFR-3mRNA expression and its application

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.