外源性WAF1-S基因表达对喉癌细胞生长的作用

目的 探索WA1F-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Western blot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-S基因可抑制喉癌细胞生长。

Exogenous WAF1-S gene inhibits growth of laryngeal cancer Hep-2 cell line

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