实时荧光定量PCR检测原发性肝癌中PRL-2基因的表达_医学论文.doc免费

实时荧光定量PCR检测原发性肝癌中PRL-2基因的表达_医学论文 实时荧光定量PCR检测原发性肝癌中PRL-2基因的表达_医学论文 作者：程超， 郭爱林， 巫国勇， 谢春玲， 吴伟康【摘要】 【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-2 mRNA表达水平显著高于肝癌及癌旁组织（ 0.01），肝癌组织表达显著高于癌旁组织（ 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。 【关键词】 肝细胞癌； 聚合酶链反应 基因表达； PRL-2基因 Abstract：【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 (PVTT) 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 1×102 copies to 1×106 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 ( 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 PVTT suggesting that it may play an important role in the development and metastasis of the HCC. Key words: hepatocellular carcinoma polymerase chain reaction gene expression PRL-2 Gene [J SUN Yat-sen Univ(Med Sci), 2007, 28（６）:702-705] 肝细胞再生磷酸酯酶 (phosphatase of regenerating liver, PRL)是一类新发现的小分子酪氨酸蛋白磷酸酯酶，由PRL-1、PRL-2和PRL-3三个成员组成[1]。它们具有酪氨酸蛋白磷酸酯酶共同活化序列HCXXGXXR，分子量约为20 ku，氨基酸同源性超过76％[2,3]。最近的研究表明，PRL-1、PRL-3与肿瘤的转移密切相关，特