E1A基因对喉癌细胞系放射敏感性的影响及机制的初步实验研究

Effect of E1A gene on radiosensitivity of human laryngeal carcinoma cells and its correlated mechanisms

目的探讨E1A基因对喉癌细胞系（Hep-2细胞）放射敏感性的影响并初步探讨其机制。方法以腺病毒为载体将E1A基因及其空载体转染至人Hep-2细胞，经RT—PCR法鉴定获得含E1A的阳性克隆。设未转染组（PBS）、转染空载体组（Ad—β—gal）和转染组（Ad—E1A），分别给予6MVx线单次照射0、1、2、4、6、8、10Gy，成克隆实验绘制细胞存活曲线并计算D0、Dq、α、β值以观察E1A基因对Hep-2细胞放射敏感性的影响。设PBS组、Ad—B—gal组、Ad—E1A组、PBS＋照射组、Ad—β—gal＋照射组、Ad-E1A＋照射组，单次照射6Gy，流式细胞仪检测细胞凋亡及RT-PCR检测血管内皮生长因子（VEGF）水平并半定量VEGF以初步探讨机制。结果转染后的阳性克隆细胞RT—PCR结果显示E1A已整合到细胞基因组中并且稳定表达。Ad—E1A组、Ad—β—gal组、PBS组细胞的D0值分别为0．86、1．96、1．98Gy，Dq值分别为1．02、1．97、1．99Gy，d值分别为0．536、0．112、0．104（Gy^-1）和13值分别为0．521、0．137、0．125（Gy^-2）。、PBS组、Ad-β-gal组、Ad-E1A组、PBS＋照射组、Ad-β-gal＋照射组、AdE1A＋照射组的细胞凋亡率分别为1．26％、1．18％、2．16％、2．55％、2．96％、4．96％。Ad-E1A组、PBS＋照射组、Ad-β-gal＋照射组及Ad-E1A＋照射组VEGF的表达水平较PBS组及Ad—β—gal组低，且Ad-E1A＋照射组表达最低。结论成功构建了稳定表达E1A基因的喉癌细胞系。E1A基因对人喉癌细胞有放射增敏作用，其机制可能与降低VEGF表达和促进细胞凋亡有关。

Objective To investigate the effect of El A gene on the radiosensitivity of human laryngeal carcinoma cells and its correlated mechanisms. Methods The Ad-E1A and Ad-β-gal were amplificated in Hek293 cells, extracted by freezing（ -80℃ ） and thawing（ 37℃） repeatedly （3 times） , purificated by the method of density gradient of CsCI and titrated by plaque assay method. Then they were transfected into human laryngeal carcinoma cells （ Hep-2 ） and authenticated by RT-PCR. The radiosensitivity of Hep-2 cells transfected with or without E1A were studied by cell surviral curve. Finally we investigated the correlated mechanisms including cell apoptosis studied by flow cytometry and VEGF content studied by RT-PCR. Resuits The radiosensitivity of Hep-2 cells transfected with E1A was intensified, Do and Dq were lowered and α was increased. Flow cytometry showed that the apoptosis rate of cells with E1A or with E1A and radiotherapy was increased. The VEGF content of the cells transfected with E1 A or treated by radiotherapy was decreased, which reached the lowest level when the cells were treated with the both mathods. Conclusions E1A gene can intensify the radiosensitivity and contribute to the apoptosis of human laryngeal carcinoma ceils. E1A gene and radiotherapy can markedly decrease the VEGF content.