E1A基因对人宫颈癌细胞放射增敏的实验研究

Study on the radiosensitivity effect of E1A gene on human cervical carcinoma cell in vitro

目的探讨E1A基因对人宫颈癌细胞的放射增敏作用及其初步的作用机制。方法将人宫颈癌细胞(Hela)、转染空载体纳米粒的人宫颈癌细胞(Hela-vector)及转染E1A基因的人宫颈癌细胞(Hela-E1A)分别给以0、2、4、6和8Gy的6MV-X线照射,用集落形成法计算细胞存活分数及放射增敏比(SER),检测E1A基因对人宫颈癌细胞对放射线敏感性的影响;采用RT-PCR检测E1A基因的表达以及E1A基因对Her-2基因表达的影响,采用流式细胞术检测E1A基因对细胞周期的影响。结果集落形成实验结果显示:经不同剂量照射后Hela-E1A组平均细胞存活分数明显低于Hela和Hela-vector组,而后两组相近。Hela-E1A组D0值为1.23,Hela和Hela-vector组D0值分别为2.51和2.59,放射增敏比(SER)=(2.04);RT-PCR结果显示:E1A基因在Hela细胞中能稳定表达,E1A基因的转染明显降低了Her-2基因在Hela细胞中的表达水平;流式细胞术结果显示:转染E1A基因后,细胞周期出现S期抑制,G2期阻滞。结论E1A基因能够明显提高人宫颈癌细胞对放射线的敏感性,其作用机制可能与E1A基因抑制了该细胞Her-2基因的表达,使细胞周期再分布有关。

[Objective] To investigate the radiosensitivity effect of E1A gene to human cervical carcinoma cell in vitro and its mechanism of action. [Method] Human cervical carcinoma cell line of Hela was transfected with E1A gene or blank-vector followed by an irradiation by 6MV X ray at the dose of 0, 2, 4, 6, 8 Gy respectively. Radiosensitivity was determined by colony formation assay and quantified by calculating the cell survival rate and the sensitization enhancement ratio （SER）.The expression of the E1A and Her-2 gene was detected by RT-PCR. Cell cycle distribution was monitored by flow cytometry. [Results] Colony formation assay demonstrated the survival rate of Hela cell colony in Hela-E1A group was significantly lower than that in the control groups. There was no significant difference between Hela and Hela-vector groups. Do calculated from the dose-respense curve in Hela-E1A, Hela and Hela-vector groups were 1.23, 2.51 and 2.59 respectively, and corresponding SER was 2.04; RT-PCR showed that E1A gene had a stable transfection in Hela ceils and obviously down-regulated Her-2 expression. Flow cytometry revealed that E1A transfection induced S stage suppression and G2 stage arrest in Hela ceils. [Conclusion] E1A gene can effectively enhance the radiosensitivity of human cervical carcinoma ceils and its mechanisms of action may relate to the suppression of Her-2 expression and a redistribution of ceil cycles regulated by E1A gene.