肝癌组织HNF4αmRNA定量检测方法的建立与初步应用

Establishment of RT-PCR for the detection of hepatocyte nuclear factor 4α in hepatocellular carcinoma

目的建立一种定量检测肝细胞癌(HCC)组织肝细胞核因子4α(HNF4α)m RNA的方法。方法取手术获得的HCC组织16例,提取总RNA,逆转录PCR扩增获得全长HNF4αm RNA对应c DNA产物,经TA克隆后,测序确认。根据获得序列,设计检测HNF4αm RNA引物及MGB荧光探针。以体外转录HNF4αm RNA为标准品,建立一步法逆转录荧光定量PCR检测方法,同时设定β-actin m RNA为内参对照,最终计算得到组织HNF4αm RNA水平。采用免疫组化染色检测HNF4α蛋白表达,比较HNF4αm RNA水平与HNF4α蛋白表达的对应结果。结果 HCC组织HNF4αm RNA定量标准曲线斜率为-3.237,相关系数r值达0.994,β-actin m RNA定量标准曲线斜率为-3.037,相关系数r值为0.996,表明建立的方法可用于检测HNF4αm RNA定量(V-4α值),16例HCC组织HNF4αm RNA与HNF4α蛋白表达结果呈一致性对应关系。结论我们初步成功建立了定量检测HCC组织HNF4αm RNA水平方法,其意义还需要进一步研究。

Objective To establish a quantified reverse transcription-polymerase chain reaction （RT-PCR） for the detection of hepatocyte nuclear factor 4α（HNF4α） mRNA in hepatocellular carcinoma tissues. Method Total RNA was extracted from 16 cancerous tissues of patients with hepatocellular carcinoma （HCC）. The full-length cDNA was obtained by RT-PCR and the products was sequenced after TA cloning. Then,the primer and MGB fluorescent probe were designed for HNF4α mRNA quantification. A one-step RT quantitative PCR assay was established and β-actin mRNA acted as internal control. The HNF4aα mRNA levels in the tissues was calculated. The expression of HNF4α protein was also detected in cancerous tissues by immunohistochemical staining. Results The quantitative detection of HNF4α mRN （V-4α） in HCC tissues was established successfully,and the slope of the standard curve was-3.237 with the correlation coefficient （r） being 0.994. The standard curve of β-actin mRNA was-3.037,and the correlation coefficient （r） was 0.996. The HNF4α mRNA levels were consistent with the HNF4α protein expression in the 16 HCC tissues. Conclusion We successfully establish a quantitative PCR for the detection of HNF4α mRNA in HCC patients,which warrants further study.