Rb94对人肺腺癌细胞系A549放射增敏作用的实验研究

目的 构建人视网膜母细胞瘤基因(Rb94)真核表达载体,观察其对人肺腺癌细胞系A549的放射增敏作用并探讨其机制.方法 构建重组表达质粒pIPES-Rb94,经脂质体转染A549细胞、G418筛选获得稳定转染的细胞系.细胞计数法绘制生长曲线、计算细胞倍增时间;实时RT-PCR检测hTERT、Bcl-2mRNA的表达;成克隆实验检测pIRES-Rb94对A549细胞放射敏感性影响,计算放射增敏比(SER);流式细胞仪分析细胞周期.结果 成功构建pIRES-Rb94质粒,转染A549细胞后获得稳定转染细胞系pIRES-Rb94-A549.与A549细胞相比,pIRES-Rb94-A549细胞倍增时间延长(31.5h:39.5 h;t=5.15,P<0.01);hTERT、Bcl-2 mRNA表达下降(0.02:1.00;t=18.99,P<0.01;0.01:1.00;t=13.73,P<0.01);G2+M期细胞增加(35.91%:4.53%;t=36.78,P=0.00),G0+G1期和S期细胞减少(47.02%:74.07%;t=11.71,P=0.00和17.07%:22.32%;t=2.30,P<0.05);由D0值计算的SER为1.30.结论 Rb94对A549细胞具有明显放射增敏作用,其机制可能是G2+M期阻滞作用以及下调hTERT、Bcl-2 mRNA表达,并可能通过调节细胞周期来影响细胞增殖.

Radiosensitising effect of retinoblastoma gene (Rb94) on human lung adenocarcinoma cell line A549

Objective To construct an eukaryotic expression vector of retinoblastoma gene (pIRES-Rb94), and investigate the radiosensitising effect of Rb94 on lung adenocarcinoma cell line A549 and the mechanism. Methods Recombinant expression plasmid pIRES-Rb94 was constructed and then transfected into A549 cells using lipofectamine 2000. Steadily transfected cells were obtained using G418 se-lecting system. Cell counting method was used to produce the growth curve and the population doubling time was then calculated. The radiosensitivity of A549 cells was assessed by clonogenic assay. The expression of bTERT and Bcl-2 mRNA was detected by real-time RT-PCR. Cell cycle distribution was measured by flow cytometry. Results Steadily transfected cell line pIRES-Rb94-A549 was aquired. Compared with A549 cells, the population doubling time of pIRES-Rb94-A549 cells was increased from 31.5 h to 39.5 h (t=5.15, P<0.01). The expression of hTERT and Bcl-2 mRNA was both down-regulated (0.02:1.00, t= 18.99,P<0.01,0.01:1.00,t=13.73,P<0.01). The number of cells was increased in G2/M phases (35.91%:4.53%, t =36.78,P=0.00), whereas decreased in G0/G1 and S phases (47.02%:74.07%, t =11.71,P=0.00;17.07%:22.32%, t =2.30,P<0.05). The sensitizing enhancement ratio was 1.30. Conclusions Rb94 has marked radiosensitizing effect on A549 cells by G2/M phase blockage and down-regulation of hTERT and Bcl-2 mRNA expression. Rb94 may also inhibit the ability of cell proliferation by regulating cell cycle distribution.