外源性p16基因稳定转染对喉癌细胞周期和增殖的影响

目的：探索p16基因在喉癌细胞内的表达作用及基因治疗方面的应用前景。方法：采用亚克隆技术，构建p16真核表达载体pCDNA3-p16.利用lipofectamine介导，将外源野生型p16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、免疫荧光、免疫组化、图像分析法观察和分析p16基因的表达。同时以空载体质粒pCDNA3为对照，用流式细胞仪分析瘤细胞生长周期、荧光染色检测细胞凋亡。结果：打点杂交、免疫荧光、免疫组化及图像分析法分析证实转染p16基因的Ｈep-2细胞有外源p16基因的表达，外源基因p16在Hep-2细胞系中的表达能抑制Hep-2细胞系的生长。流式细胞仪计数和免疫荧光检测证实p16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p16基因可抑制喉癌细胞恶性增殖。

Effects of exogenous p16 gene on the cell cycle and proliferation of Hep-2 laryngeal cancer cell line

AIM: To investigate the suppressive effects of exogenous p16 gene on human laryngeal cancer Hep 2 cell line and to explore the potential use of p16 in gene therapy for laryngeal cancer. METHODS: A eukaryotic expression vector containing 570 bp human full length p16 cDNA was transfected into human laryngeal cancer Hep 2 cell line using lipofectamine. Expression of exogenous p16 gene was detected by dot blot hybridization and immunohistochemistry. Flow cytometry, immunohistochemical technique and image analysis system were adopted to measure the effects of exogenous p16 gene on cell cycle progression, morphology and cell growth features of the transfected Hep 2. RESULTS: After the transfected cell was selected 2 wk by G418, we found by dot blot hybridization that p16 gene could express in Hep 2 cell. Using immunohistochemical technique and image analysis system, we observed that the level of p16 expressed in endocellular was increased. The progression of cell cycle was arrested from phase G1 to S. CONCLUSION: Laryngeal cancer cells can be arrested at G1/S phase and the growth of cells can be significantly suppressed by exogenous p16 gene.