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背景与目的SYBRGreenⅠ是一种较为常用的非探针类定量PCR的方法,其主要优点在于操作过程简单,结果具有一定特异性。本研究的目的是建立检测色素上皮衍生因子(pigmentepitheliumderivedfactor,PEDF)mRNA的SYBRGreenⅠ实时定量PCR的方法,了解肺癌组织及其远癌肺组织中PEDF的表达水平,研究肺癌组织中PEDF的表达水平与各种临床病理特征之间的关系。方法利用RTPCR的方法扩增并纯化PEDF以及内对照GAPDHmRNA的目的片段,倍比稀释后作为定量模板。利用相对定量的方法,检测21例肺癌及相应远癌肺组织中PEDFmRNA相对于GAPDHmRNA的表达量,进行相对定量分析。结果21例肺癌组织及相应的远癌肺组织中均可检出PEDF的表达,肺癌组织中PEDF的相对表达量为0.5505±0.3590(0.11～1.11),远癌肺组织中为0.7219±0.2582(0.29～1.31)(P=0.024)。早期(Ⅰ～Ⅱ期)肺癌患者和肿瘤较小时(T1)PEDF的表达量显著高于晚期(Ⅲ～Ⅳ期,P=0.010)和肿瘤较大者(T2～4,P=0.007)。结论所建立的SYBRGreenⅠ实时定量PCR方法可以成功地检测PEDF基因的表达量。初步检测结果显示PEDF可能是一种肺癌发生发展过程中的保护因素。
BACKGROUND & OBJECTIVE: The SYBRGreen I is a commonly used non-probe quantitative PCR method. The main advantage of SYBRGreen I is that the operation is simple and the result has certain specificity. The aim of this study was to establish a SYBRGreen Ⅰ real-time quantitative PCR for the detection of PEDF mRNA in lung cancer tissues and their distant-lung tissues and to study the expression of PEDF in lung cancer tissues, The relationship between the clinicopathological features. Methods RTPCR method was used to amplify and purify the target fragment of PEDF and internal control GAPDH mRNA. Relative quantitative method was used to detect the expression of PEDF mRNA relative to GAPDH mRNA in 21 lung cancer and corresponding distant lung cancer tissues, and relative quantitative analysis was performed. Results The expression of PEDF was detectable in 21 cases of lung cancer and corresponding distant lung cancer. The relative expression of PEDF in lung cancer tissues was 0.5505 ± 0.3590 (0.11-1.11) and that in distant lung tissues was 0.7219 ± 0.2582 (0.29 ~ 1.31) (P = 0.024). The expression of PEDF was significantly higher in patients with stage Ⅰ-Ⅱ lung cancer and in younger stage (T1) than those in stage Ⅲ-Ⅳ (P = 0.010) and those with larger tumors (T2-4, P = 0.007). Conclusion The established SYBRGreen Ⅰ real-time quantitative PCR method can successfully detect the expression of PEDF gene. Preliminary test results show that PEDF may be a protective factor in the development of lung cancer.