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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 S基因可抑制喉癌细胞生长
Objective To explore the role of WAF1 S gene expression in laryngeal carcinoma cells and to provide a theoretical basis for the gene therapy of laryngeal carcinoma. Methods Subcloning technique was used to construct WAF1 S eukaryotic expression vector pcDNA3 WAF1 S. Western Blot and confocal laser scanning microscopy were used to detect the expression of WAF1 S gene mRNA in laryngeal carcinoma cell line Hep 2 by Lipofectamine. The positive clones were screened by dot blot hybridization. The protein expression of WAF1 S gene was analyzed. The growth of Hep 2 cells was detected by MTT and flow cytometry. At the same time, empty plasmid pcDNA3 was used as a control to analyze the effect of WAF1 gene on the growth of laryngeal carcinoma cells. Results Dot blotting confirmed that the expression of exogenous WAF1 S gene in Hep 2 cells transfected with WAF1 S gene and the expression of the foreign gene WAF1 S in Hep 2 cell line inhibited the growth of Hep 2 cell line. The protein expression of WAF1 S in laryngeal carcinoma cells after transfection was significantly higher than that in control group. Flow cytometry showed that WAF1 S could induce apoptosis of Hep 2 cell line and cause G1 arrest. Conclusion The introduction of wild-type WAF1 S gene can inhibit the growth of laryngeal carcinoma cells