实时荧光定量PCR检测原发性肝癌中PRL-2基因的表达

Real-time Fluorescence Quantitative PCR in Detecting PRL-2 Gene Expression in Hepatocellular Carcinoma

【目的】建立TaqMan实时荧光定量逆转录聚合酶链反应(RT-PCR)法检测肝细胞再生磷酸酯酶2(PRL-2)mRNA表达水平,并应用该法检测原发性肝细胞癌中PRL-2基因的表达。【方法】构建含PRL-2基因开放阅读框架的T载体,制作标准曲线。提取手术切除12例人肝癌、门静脉癌栓(PVTT)和癌旁组织总RNA并逆转录为cDNA。应用实时荧光定量PCR法观察人肝细胞癌组织和门静脉癌栓及癌旁组织中PRL-2的表达水平。【结果】线性检测范围达5个数量级,最低检测下限为1×102拷贝,最高检测上限为1×106拷贝。PRL-2在所有的门脉癌栓及10例肝癌组织表达,仅在4例癌旁组织中表达。门脉癌栓PRL-2mRNA表达水平显著高于肝癌及癌旁组织(P<0.01),肝癌组织表达显著高于癌旁组织(P<0.01)。【结论】实时荧光定量PCR可以准确定量测定PRL-2基因的表达;PRL-2基因在门脉癌栓的更高表达提示其在肝细胞癌的发生、转移中可能起重要作用。

[Objective] To quantitatively detect the mRNA expression level of PRL-2 in primary hepatocellular carcinoma with RT-PCR method. [Methods] T vector including open reading frame of PRL-2 gene was constructed to make standard curve. The total RNA isolated from human HCC, portal vein tumor thrombosis （PVTF） and adjacent liver tissue was reversely transcribed into cDNA. The RT-PCR method was used to analyze the expression level of PRL-2 gene. [Results] The RT-PCR method was performed successfully to precisely detect RNA level ranging from 1×10^2 copies to 1×10^6 copies. PRL-2 was expressed by all the portal vein tumor thrombosis （PVTT） and 10 cases HCC, but only by 4 cases paratumor tissue. The expression level of PRL- 2 gene was higher in PVTT than that in paratumor liver tissues and in HCC （P〈 0.01）, and it was higher in HCC than that in paratumor liver tissues. [Conclusion] The RT-PCR is the precise method to quantitatively detect PRL- 2 gene RNA level. The higher expression of PRL- 2 gene in PVTY suggesting that it may play an important role in the development and metastasis of the HCC.