三种癌细胞对Ad-E1A的敏感程度研究

Three Human Carcinoma Cell Lines with Different Sensitivity to Adenovirus Expressed E1A Gene

目的研究腺病毒介导的E1A基因(Ad-E1A)体外对Hep-2人喉癌细胞、A375人黑色素瘤细胞、SMMC-7721人肝癌细胞的生长抑制作用及敏感程度。方法 RT-PCR鉴定E1A基因的转录;MTT法检测细胞的生长抑制作用;Hoechst染色法检测细胞核形态改变;流式细胞术(FCM)检测细胞周期和细胞凋亡。结果 Ad-E1A在三种癌细胞内均有效表达,在其感染72h和96h后对Hep-2、A375、SMMC-7721细胞的生长抑制率达分别为18.65%和29.95%;33.02%和45.36%;36.32%和54.11%;其中对SMMC-7721细胞的作用最强,FCM检测发现其凋亡率为36.92%。Hoechst染色表明Ad-E1A诱导肿瘤细胞凋亡,使其呈现典型的核固缩、断裂并出现凋亡小体等核形态改变。结论 Ad-E1A对Hep-2、A375、SMMC-7721三种癌细胞均有生长抑制作用,尤以对SMMC-7721细胞作用最强、A375细胞次之,Hep-2细胞的敏感性相对较低;此外,Ad-E1A还能诱导肿瘤细胞凋亡。

Objective To investigate the different effects of adenovirus-expressed E1A gene （Ad-E1A） on human laryngocarcinoma cell Hep-2,human melanoma cell A375 and human hepatocarcinoma cell SMMC-7721 in vitro,and to select the differential sensitivity of these cell lines to Ad-E1A.Methods RT-PCR was applied to examine the transcription of E1A gene in the infected carcinoma cells.Cell growth inhibition was detected by MTT assay.Hochest staining was applied to the observation of morphological changes of nucleus.Apoptosis and cell cycle were tested by flow cytometry.Results E1A gene is expressed in all these carcinoma cells,which significantly inhibited cell growth.In 72 hs the growth inhibiting rate of Ad-E1A on Hep-2,A375,SMMC-7721 cells are 18.65 %,33.02% and 36.32% respectively,and the counterparts are 29.95 %,45.36% and 54.11% in 96 hs.Among the three cell lines SMMC-7721 cell demonstrated the highest sensitivity to E1A which also induced apoptosis of SMMC-7721 cells at a rate of 36.92%.Hoechst staining finds that Ad-E1A can induce apoptosis of human carcinoma cell as shown by classical karyopycnosis,ruptured apoptotic body and morphological changes of the nuclei.Conclusion Ad-E1A inhibits the growth of these three carcinoma cells,among which SMMC-7721 cell is the most sensitive and A375 cells the second and Hep-2 cells the least. Furthermore Ad-E1A can also induce apoptosis of these carcinoma cells significantly.