人肝癌组织中APRIL及其受体mRNA水平的定量测定

Measurement of the expression level of APRIL and its receptors in liver cancer tissue by real-time fluorescence quantitative method

目的采用实时荧光定量聚合酶链反应(RFQPCR)技术分析肝癌组织中增殖诱导配体(APRIL)及其受体mRNA的表达水平。方法在APRIL及其受体基因的高保守区设计相应引物和荧光探针,实时检测PCR产物的荧光强度,根据标准品建立的标准曲线,由软件自动计算出待测样本中靶基因mRNA准确含量,并以靶基因和内参β2MmRNA含量的比值作为评价靶基因表达水平的指标。结果RFQPCR检测靶基因mRNA含量的线性范围为10～109pg/ml,批内和批间重复性测定的变异系数v分别为3.87%～10.12%和6.86%～12.29%。20例肝癌组织样本的检测结果显示肝癌及癌旁组织中APRIL和BCMA的水平均显著高于远端正常组织(P<0.01),而TACI的表达水平在三组间差异无统计学意义(P>0.05)。结论成功建立RFQPCR检测APRIL及其受体mRNA含量的方法,具有较好的检测灵敏度和重复性;而且发现APRIL在肝癌发生、发展过程中可能起了重要作用,有可能还参与了肿瘤细胞的转移,并主要是通过受体BCMA而不是TACI发挥作用。

Objective To establish a real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) for measurement of the expression level of APRIL and its receptors in liver cancer tissue. Methods Specific primers and TaqMan probes of APRIL and its receptors have been designed. The fluorescence of the PCR products were detected continuously during the amplification. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples have been determined using software. The results were presented as the ratios of target genes’ mRNA to β2-microgluobulin(β2M)’s mRNA. Results The detection range of RFQ-PCR was from 109 pg/ml to 101 pg/ml. The coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 3.87% to 10.12% and 6.86% to 12.29%, respectively. The results from 20 samples showed that the expression level of APRIL and BCMA in cancer and cancroid tissue were significantly higher than that in normal tissue (P<0.01). The expression level of TACI had no significant difference within three groups (P>0.05). Conclusions The results showed that RFQ-PCR had high sensitivity and reproducibility. It was suitable for clinical practice. There was a possibility that APRIL maybe play a great role in development, progress and metastasis of liver cancer through its receptor B cell maturation antigen (BCMA) rather than transmembrane activator and CAML inter actor (TACI).