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Puma为一种凋亡调控因子。本研究探讨慢性淋巴细胞白血病(CLL)患者p53上调puma mRNA的表达情况,并探索其在评估CLL预后中的意义。采用基于SYBR GreenI荧光染料法的实时定量逆转录PCR(qRT-PCR)方法检测100例CLL患者及11名健康对照者外周血标本中puma基因的表达,以β-actin为内参照,用2(-△Ct)方法计算puma的相对定量值,组间分析采用秩和检验。结果显示,qRT-PCR标准曲线的R2均≥0.990,批内差及批间差变异系数(CV)均小于5%,灵敏度可达102copies/μgRNA。100例CLL患者puma基因表达水平中位数为1.038×10-3(4.106×10-4-2.806×10-3);11名健康对照者puma表达水平中位数为1.220×10-3(7.233×10-4-1.405×10-3),二者无显著性差异(U=544.5,p=0.957)。临床分期较早(Binet分期A期)的患者puma表达水平明显高于临床分期较晚(Binet分期B期、C期)的患者(p<0.001);具有CD38表达阳性、ZAP-70蛋白表达阳性、血清乳酸脱氢酶水平增高、血清β2-微球蛋白水平增高的患者,其puma基因表达水平均明显低于无以上不良预后因素的患者,差异显著(p值分别为0.002,0.012,0.009和0.046);FISH检测显示存在12号染色体三体和14q32易位的患者puma表达水平明显低于无以上分子遗传学异常者,其差异显著(p值分别为0.003和0.045)。结论:qRT-PCR方法检测puma表达灵敏可靠,puma表达水平与CLL多种预后因素存在相关性,在评估CLL预后中具有重要价值。
Puma is a regulator of apoptosis. This study was aimed to investigate the upregulation of puma mRNA expression in patients with chronic lymphocytic leukemia (CLL) and its significance in assessing the prognosis of CLL. The expression of puma gene in peripheral blood samples from 100 CLL patients and 11 healthy controls was detected by real-time quantitative reverse transcription PCR (qRT-PCR) based on SYBR GreenI fluorescent dye method. Β-actin was used as internal reference and 2 - △ Ct) method to calculate the relative quantitative value puma, group analysis using rank sum test. The results showed that the R2 of the qRT-PCR standard curve was ≥0.990, the intra-assay difference and the inter-assay coefficient of variation (CV) were less than 5%, and the sensitivity was up to 102 copies / μgRNA. The median puma gene expression level in 100 CLL patients was 1.038 × 10-3 (4.106 × 10-4-2.806 × 10-3). The median puma expression level in 11 healthy controls was 1.220 × 10-3 (7.233 × 10-4-1.405 × 10-3), no significant difference between the two (U = 544.5, p = 0.957). Patients with earlier clinical stage (Binet stage A) had significantly higher puma expression than patients with late stage (Binet stage B and C) (p <0.001); those with positive CD38 expression and positive ZAP-70 protein , The levels of serum lactate dehydrogenase and the level of serum β2-microglobulin in patients with puma expression were significantly lower than those without adverse prognostic factors, the difference was significant (p = 0.002,0.012,0.009 and 0.046). FISH showed that puma expression in patients with chromosome 12 trisomy 12 and 14q32 translocation was significantly lower than those without molecular cytogenetic abnormalities (p = 0.003 and 0.045, respectively). Conclusion: qRT-PCR method is sensitive and reliable for detecting puma expression. The correlation between puma expression and prognosis of CLL is of great value in evaluating the prognosis of CLL.