摘要目的 建立实时荧光定量聚合酶链反应(RFQ-PCR)检测非小细胞肺癌(NSCLC)患者痰中增殖诱导配体(APRIL)mRNA含量的方法,探讨痰脱落细胞APRIL mRNA表达在NSCLC诊断中的意义.方法 对2007年8月至2008年5月南通大学附属医院71例NSCLC患者和62例肺部良性病变患者痰中APRIL mRNA进行检测,同时取65名健康成人痰做对照.用RFQ-PCR技术实时检测PCR产物的荧光强度,由软件自动计算出待测样本中靶基因mRNA的准确含量,以靶基因和内参β2-微球蛋白(β2-M)mRNA含量的比值作为评价其表达水平的指标,并与细胞学检查结果进行比较.计量资料采用t检验,所得结果用x±s表示;对计数资料采用X2检验.结果 RFQ-PCR法检测APRIL mRNA含最的线性范围为38～3.8×106copies/ul,批内和批间变异系数(CV)分别为8.5%和13.6%.NSCLC组痰中APRIL mRNA表达水平显著高于肺部良性病变组和健康对照组(t值为10.50和11.32,P<0.01);良性病变组与健康组比较差异无统计学意义(t=0.379,P>0.05).以健康组APRIL mRNA的x±2s为cut-off值,肺癌组APRIL mRNA表达的阳性率为81.7%(58/71),明显高于肺部良性病变组的3.2%(2/62)和健康对照组的1.5%(1/65).NSCLC组痰中APRIL mRNA的表达与性别、年龄、吸烟史、TNM分期及淋巴结转移尤关(t值分别为1.700、1.014、1.484、1.298及1.186,均P>0.05),与病理分型和肿瘤部位有关(t值为1.650和1.873,均P<0.05).RFQ-PCR阳性率为82%(58/71),高于细胞形态学阳性率的14%(10/71),差异有统计学意义(X2=67.68,P<0.01).结论 RFQ-PCR检测痰中APRIL mRNA含量具有较高的敏感度和特异度,有助于NSCLC的临床诊断.

abstractsObjective To establish a real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) method for quantifying a proliferation-inducing ligand (APRIL) Mrna in sputum samples from patients with non-small cell lung cancer (NSCLC), and to evaluate its role in the diagnosis of NSCLC. Methods Seventy-one cases of NSCLC and 62 cases of benign pulmonary disease were enrolled in this study from August 2007 to May 2008 in Affiliated Hospital of Nantong University, Jiangsu. Sixty-five healthy volunteers served as the control. The fluorescence of the PCR products was detected continuously during the amplification by RFQ-PCR. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples was determined using software. The results were presented as the ratios of target genes to β2-microglobulin (β2-M ) Mrna, and compared with those obtained by conventional cytological method. Results The detection range of the assay was from 38 copies/ul to 3.8× 106 copies/ul. The coefficients of variation values of both intra-experimental and inter-experimental reproducibility were 8.5% and 13.6% ,respectively. The expression of APRIL Mrna in tumor sputum was higher than that in benign pulmonary disease and healthy volunteers (t=10.50, 11.32, P<0.01). The positive rate for APRIL Mrna expression was 81.7% (58 of 71) in sputum samples of NSCLC, 3.2% (2/62) in benign pulmonary disease and 1.5% (1/65) in healthy volunteers when cut-off values for positivity were set at the x±2s of Mrna expression in health volunteers. The level of APRIL Mrna of NSCLC was not related to sex, age, smoking status, TNM stage and lymph node metastasis (P>0.05, respectively), but was related to pathology subtype and the location of tumors (P<0.05, respectively). The APRIL Mrna assay (82%) produced a higher detection rate than conventional cytological method (14%) (X2= 67.68, P<0.01). Conclusion Measurement of the expression of APRIL Mrna in sputum by RFQPCR showed high sensitivity and specificity, which maybe useful in diagnosing NSCLC.