摘要
目的建立测定血管内皮生长因子-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）,探讨其在食管癌组织及其癌旁组织中的表达水平。方法分别以自行构建和克隆的pMD18-VEGF-C和pMD18-VEGFR-3质粒作为定量模板,用循环阈值（Ct）定量起始的模板,在荧光TaqMan方法的基础上建立了测定VEGF-C mRNA和VEGFR-3 mRNA的实时FQ-RT-PCR,并分别测定了40例食管癌组织及其癌旁组织中VEGF-C mRNA和VEGFR-3 mRNA的表达水平。结果VEGF-CmRNA和VEGFR-3 mRNA测定的线性范围均为10^3-10^8拷贝/μg总RNA;VEGF-C mRNA和VEGFR-3 mRNA高、低值的批内、批间变异系数（CV）在7.07%-12.49%之间。VEGF-C mRNA主要表达在癌组织中,而VEGFR-3mRNA在癌组织与癌旁组织中表达无差异;VEGF-C mRNA与VEGFR-3 mRNA在癌组织与癌旁组织中表达存在相关性;24例淋巴结转移食管癌患者癌组织及其癌旁组织VEGF-C mRNA和VEGFR-3 mRNA水平与16例无淋巴结转移食管癌患者癌组织及其癌旁组织存在显著差异,提示有淋巴结转移的食管癌组织及其癌旁组织VEGF-C和VEGFR-3基因表达水平上调。VEGF-C mRNA和VEGFR-3 mRNA的表达水平与食管癌临床病理分期有密切关系。结论我们建立的测定VEGF-C mRNA和VEGFR-3 mRNA的实时FQ-RT-PCR灵敏、稳定、重现性好,可供VEGF-C、VEGFR-3基因表达的临床检测和研究。VEGF-C mRNA和VEGFR-3 mRNA基因表达水平可作为食管癌淋巴结转移的预测指标,亦可作为分子水平上判定食管癌临床分期的辅助指标之一。
Objective To establish a real-time fluorescent quantitative reverse transcription polymerase chain reaction （FQ-RT-PCR） method for the detection of vascular endothelial growth factor-C（VEGF-C） mRNA and vascular endothelial growth factor receptor-3 （ VEGFR-3 ） mRNA, with which VEGF-C mRNA and VEGFR-3 mRNA level in esophageal carcinoma and pericarcinoma tissue can be analyzed. Methods The self-constructed and cloned plasmids pMD18-VEGF-C and pMD18-VEGFR-3 were used as quantitative templates respectively and the quantification of initial cDNA was based on the cycle threshold （Ct） values, the real-time for quantitative detecting VEGF-C mRNA and VEGFR-3 mRNA was set up based on fluorescent TaqMan methodology to examine the specific expression level of VEGF-C mRNA and VEGFR-3 mRNA in esophageal carcinoma and pericarcinoma tissue. Results The detection linear range of the VEGF-C mRNA and VEGFR-3 mRNA assay was 10^3-10^8 copies/μg total RNA respectively. The inter-assay and intra-assay coefficient of variation （CV） of VEGF-C mRNA and VEGFR-3 mRNA for lower value and higher value was from 7.07% to 12.49% . The VEGF-C mRNA level in esophageal carcinoma tissue was markedly higher than that in pericarcinoma tissue, there was no significant difference of the VEGFR-3 mRNA level between carcinoma and pericarcinoma tissue. There was correlation between the level of VEGF-C mRNA and VEGFR-3 mRNA in carcinoma or pericarcinoma tissue. The levels of VEGF-C mRNA and VEGFR-3 mRNA in esophageal carcinoma and pericarcinoma tissue from 24 patients with lymphatic metastasis of esophageal carcinoma were significantly higher than that from 16 patients without lymphatic metastasis of esophageal carcinoma. The levels of VEGF-C mRNA and VEGFR-3 mRNA were associated with the clinicopathological stage of esophageal carcinoma. Conclusions A sensive, accurate,stable and reproducible 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 an
出处
《检验医学》
CAS
北大核心
2009年第5期355-360,共6页
Laboratory Medicine
基金
上海市科技攻关医学临床研究(064119638)
关键词
血管内皮生长因子-C
血管内皮生长因子受体-3
食管癌
淋巴结转移
实时荧光定量逆转录聚合酶链反应
Vascular endothelial growth factor-C
Vascular endothelial growth factor receptor-3
Esophageal carcinoma
Lymphatic metastasis
Real-time fluorescent quantitative reverse transcription polymerase chain reaction
