实时定量聚合酶链反应法检测人周围血淋巴细胞受照后GADD45基因表达的改变

Real-time PCR detection of the change of GADD45 gene expression in irradiated lymphocytes

目的建立辐射诱导人淋巴细胞GADD45基因表达定量检测技术,探讨GADD45基因表达变化作为辐射生物剂量计的可行性。方法健康人周围血经X射线照射后分离淋巴细胞,提取总RNA,反转录成cDNA,利用实时荧光定量聚合酶链反应(PCR)法检测GADD45基因经不同剂量照射(0～8 Gy)、2 Gy照后不同时间(0～72 h)的mRNA表达水平,以管家基因GAPDH为内参进行定量分析。结果照射后GADD45基因在转录水平表达呈剂量依赖性增加,4 Gy照射时达到峰值,8 Gy仍然保持在较高水平,GADD45基因表达改变存在线性剂量-效应关系,其线性回归方程为y^=3.408+1.528x,R2=0.787。2 Gy照射后GADD45基因mRNA表达在照射后1 h呈现上升趋势,在4 h时达峰值,照射后72 h基因表达仍未恢复到初始水平。结论实时荧光定量PCR检测GADD45基因表达方法具有良好的敏感性、重复性和剂量-效应关系,有可能成为新的辐射生物剂量计。

Objective To establish the quantifying technology for gene expression change （GEC） of GADD45 after X-ray irradiation, and to discuss the feasibility of the GEC of gene GADD45 as a radiation biodosimeter. Methods Healthy whole-blood model was used to separate lymphocytes after irradiation, total mRNA was extracted and reverse transeripted to get cDNA, SYBR Green real-time PCR technology was used to assay gene GADD45 expression at mRNA level exposed to different doses. Then housekeeping gene GAPDH was chosen and applied to rectify the quantity of every dose. Results The up-regulation of gene GADD45 was dose-dependent in transcription level after irradiation. It reached the peak at 4 Gy irradiaton and still stayed in relatively high level at 8 Gy. A linear regression equation was established as y=3.408＋1.528x, R^2=0.787. After its being exposed to 2Gy X-ray, mRNA expression showed up-regulated at 1 hour, reached the peak at the 4th hour, but still not returned to the beginning level. Conclusion GEC of GADD45 analysis using SYBR Green real-time PCR is rapid, sensitive and reproducible.