重组人可溶性凋亡素-2在人喉癌株中表达及其体外诱导凋亡作用的实验研究

Expression of recombinant human soluble apoptosis 2 ligand and vitro experimental study on laryngeal carcinoma cell line Hepa2

目的:研究凋亡因子TRAIL结合端粒酶启动子对肿瘤的特异性杀伤作用。方法:刺激人外周血淋巴细胞增殖,提取总RNA。采用RTPCR扩增IL2信号肽基因和TRAIL基因的融合基因,并克隆入真核表达载体pGL3181hTERT肿瘤特异性端粒酶启动子的下游,构建TRAIL基因的重组真核表达载体pGL3181hTERT/TRAIL。用Westernblot鉴定喉癌细胞株Hepa2中表达产物。将该重组载体经阳离子脂质体转染入人喉癌细胞株Hepa2中,用电镜观察细胞的形态变化,用流式细胞仪技术(FCM)分析转染后细胞凋亡率及细胞周期的变化。结果:PCR扩增得到了613bp的cDNA片断。与GenBank中报道的IL2信号肽和TRAIL凋亡诱导功能区cDNA序列完全一致,成功构建了重组真核表达载体pGL3181hTERT/TRAIL。Westernblot结果显示,获得的瞬时转染TRAIL在喉癌细胞Hepa2中特异表达,且能诱导其凋亡。结论:重组真核表达载体pGL3181hTERT/TRAIL的成功构建,为肿瘤基因治疗的靶向性提供了可行的理想工具。

OBJECTIVE：To investigate the specificity role of the apoptosis factor TRAIL gene with telomerase promoter in tumor gene therapy. METHODS： PBLs（Peripheral Blood lymphocyte） were stimulated for proliferation and the total RNA was extracted, shTRAIL （recombinant humam soluble APO2）, TRAIL gene with interleukin 2 signal peptide, was amplified by RT-PCR and cloned into the vector pGL3-181h TERT promoter downstream to form an eukaryotic expressing vector. Expressing protein was identified by Western blot on laryngeal carcinoma cell line Hepa2. The morphological changes were observed by transmission electron microscope （TEM）. The apoptotic rates and the changes of cell cycle were measured by flow cytometry （FCM） in cell line Hepa2 and HL-7702. RESULTS： The sequence of 613 bp cDNA was obtained and identified by enzyme digestion and sequencing analysis, and the recombinant eukaryotic expression vector for TRAIL gene was successfully constructed. The results of the Western blot showed the transfected Hepa2 cells expressed TRAIL protein and apoptosis were induced. CONCLUSION： Successful construction of eukaryotic expression vector pGL3-181hTERT/TRAIL provides the possibility for gene therapy of tumor.