外源性WAF_1-S基因表达对喉癌细胞生长的作用

Exogenous WAF_1-S gene inhibits growth of laryngeal cancer Hep-2 cell line

目的 探索WAF1 S基因在喉癌细胞内表达的作用 ,为喉癌的基因治疗提供理论依据。方法 采用亚克隆技术 ,构建WAF1 S真核表达载体pcDNA3 WAF1 S。利用lipofectamine介导 ,将外源野生型WAF1 S基因导入喉癌细胞系Hep 2 ,筛选阳性克隆 ,采用打点杂交技术观察WAF1 S基因mRNA在喉癌细胞中的表达 ,用Westernblot及激光共聚焦方法定量分析WAF1 S基因的蛋白表达产物 ,MTT及流式细胞仪检测Hep 2细胞的生长状态。同时以空载体质粒pcDNA3为对照 ,分析WAF1 基因对喉癌细胞生长的影响。结果 打点杂交证实转染WAF1 S基因的Hep 2细胞有外源WAF1 S基因的表达 ,外源基因WAF1 S在Hep 2细胞系中的表达能抑制Hep 2细胞系的生长。转染后喉癌细胞中WAF1 S的蛋白表达明显高于对照组。流式细胞仪计数证实WAF1 S能诱导喉癌细胞系Hep 2发生凋亡并导致其发生G1 期阻滞。结论 导入外源野生型WAF1

Objective To investigate the inhibitory effects of exogenous WAF 1 S gene on human laryngeal cancer Hep 2 cell line, and to explore the potential use of WAF 1 S in gene therapy for laryngeal cancer. Methods A eukaryotic expression vector containing 2 1kb human full length WAF 1 S cDNA was transfected into human laryngeal cancer Hep 2 cell line by using lipofectamine. Expression of exogenous WAF 1 S gene was detected by dot blot hybridization. By using Western blot and confocal microscope, expression of p21 protein was quantitatively analyzed in situ . The growth state of transfected Hep 2 cell was determined by flow cytometry and MTT. Results It was found by dot blot hybridization that WAF 1 S gene could express in Hep 2 cell. The expression of the exogenous p21 gene in Hep 2 cells was markedly higher than that in the control group. It was confirmed with flow cytometry that WAF 1 S gene could induce apoptosis of laryngeal cancer Hep 2 cell line, and the progression of cell cycle was arrested at G 1 phase. Conclusion Laryngeal cancer cells could be arrested at G 1 /S phase and the growth of the cells could be significantly suppressed by exogenous WAF 1 S gene