靶向表皮生长因子受体的RNA干扰技术对非小细胞肺癌细胞株A549细胞抑制及顺铂敏感性的研究

Study of RNAi targeting epidermal growth factor receptor on lung adenocarcinoma A549 cell line growth and sensitivity to eisplatin

目的利用RNA干扰（RNAi）技术，将携带表皮生长因子受体（epidermal growth factor receptor，EGFR）同源基因的重组质粒稳定转染非小细胞肺癌细胞株A549细胞，观察其对A549细胞生长抑制作用及对顺铂敏感性的变化。方法根据RNAi原理，体外设计并合成靶向EGFR特异性干扰片段，即h-EGFR，以pGensil-1为载体，构建重组质粒pGensil-1-EGFR，以Lipofectamine 2000介导转染肺腺癌细胞株A549细胞，经G418筛选后挑选出稳定转染的单克隆细胞。将所挑选出的单克隆细胞大量培养后加入不同浓度顺铂（40mg／L、10mg／L、2．5mg／L），采用四甲基偶氮唑蓝比色法检测细胞活力，绘制生长曲线，流式细胞仪分析细胞周期及细胞凋亡的变化，观察RNAi对A549细胞抑制作用及是否与顺铂有协同作用。结果与对照组、阴性对照组相比，从第2天起实验组生长缓慢，吸光度有统计学差异（P〈0．05）；顺铂或联合dsRNA-EGFR对A549细胞有抑制作用，且随着浓度的增加和时间的延长抑制作用增强，dsRNA—EGFR联合顺铂与等浓度顺铂吸光度有统计学差异（P〈0．05），顺铂作用24h后40mg／L、10mg／L、2．5mg／L顺铂抑制率分别提高了7．8％、30．70％、34．42％。实验组G0／G1细胞百分比较对照组增加了13．84％，较阴性对照组增加了14．65％，进入S期的细胞百分比较对照组减少了19．10％，较阴性对照组减少了18．68％；40mg／L、10mg／L、2．5mg／L顺铂处理细胞24h后，与对照组相比，凋亡率明显增高（P〈0．05），且浓度越高，凋亡率越高；同一浓度顺铂作用下转染pGensil-1-EGFR的A549细胞凋亡率较A549细胞增高（P〈0．05）。结论dsRNA-EGFR可有效抑制A549细胞生长，使细胞阻滞在G0-G1期，顺铂可有效诱导细胞凋亡，RNAi与顺铂具有协同效应，提高细胞对顺铂的敏感性，RNAi技术为非小细胞肺癌基因治疗提供了新策略。

Objective Using RNA interference（RNAi） technique,to observe the effect of recombinant plasmid carrying epidermal growth factor receptor（EGFR） homologous gene on the biological characteristics of A549 cell and sensitivity to cisplatin after being transfected to non-small cell lung cancer A549 cell. Methods According to the principle of RNAi,a strip of interfering fragment targeting EGFR gene,h-EGFR was designed and synthesized,then pGensil-1 was used as vector, the recombinant plasmid pGensil-1-EGFR was constructed and transfected into human lung cancer A549 cell line mediated by Lipofectamine 2000. Then the transfected cells were selected with G418 and the stable transfected monoclone cells were picked out. After being collected and cultured, cells were treated with different concentrations of cisplatin（2.5mg/L, 10 mg/ L,40mg/L） for different time （24 h,48 h, 72 h）. The antiproliferative effects of dsRNA and cisplatin were assessed by MTT. Cell cycle analysis and the apoptosis rate were carried out via flow cytometry. Results Compared with control group, experiment group grew more slowly, the optical density value was downregulated significantly （ P〈0.05）. Treated with cisplatin of 40 mg/L, 10 mg/L, 2.5 mg/L, the inhibition ratio of pGensil-1-EGFR-3 increased by 7.8% ,30.70% ,34. 42% compared with un-transfected control. Cell cycle analysis showed that dsRNA-EOFR induced accumulation of cells in G0-G1 phase by 13.84% and decreased in the percentage of cells in S-phase by 19.10% compared with control group. After 24 hours treatment with cisplatin of 40 mg/L, 10 mg/L, 2.5 mg/L, the apoptosis rates increased compared with that of control group. Conclusions Via stable transfection,recombinant plasmid targeting EGFR gene could effectively inhibit cell growth,make more cells block in the G0-G1 phase and could increase the sensitivity of A549 cell to cisplatin. This provides an experimental basis for RNAi technique in gene therapy on non-small cell lung cancer.