Gene therapy using lympho-epithelial Kazal-type-inhibitor (LEKTI) for head and neck cancer

1.腺病毒载体转导人鳞状细胞系的功效和体外细胞毒性将全长LEKTI cDNA亚克隆到腺病毒载体（Ad-lacZ-LEKTI）中。使用人口腔鳞状细胞系SAS、HSC-2、3、4。将两万个细胞接种到8孔载玻片的每个孔中。24小时后，将Ad-lacZ-LEKTI载体以不同的感染复数（MOI）添加到每个孔中。将X-gal溶液加入每个孔中，并测定转导效率。在24孔板中进行腺病毒载体的毒性实验。24小时后，将Ad-lacZ-LEKTI载体以不同的MOI加入每个孔中。在Ad-lacZ-LEKTI载体感染后96小时，使用台盼蓝染料排除法计数活细胞数。GCV的毒性实验类似于用腺病毒载体的实验进行。通过X-gal染色将转导的细胞染成蓝色。到转导后24小时，几乎100%的 ...更多信息 所有细胞以10的MOI转导。在MOI 1下的转导效率几乎为20%。Ad-lacZ-LEKTI在MOI大于100时对所有细胞系均显示出毒性，但在MOI为10或更低时没有明显的毒性。在所有细胞系中评价了对GCV的细胞毒性，发现其为25 μg/ml或更高。24小时后，将Ad-lacZ-LEKTI载体以不同的MOI加入每个孔中。向每个孔中加入10 μg GCV。在GCV给药后72小时，使用台盼蓝染料排除法计数活细胞数。当MOI为1时，细胞死亡率超过60%，当MOI为10.3时，几乎100%的癌细胞死亡。Ad-lacZ-LEKTI和GCV处理口腔鳞状细胞系的形态学变化Ad-lacZ-LEKTI以MOI为10通过GCV转导细胞。GCV作用24 h后，细胞出现染色质浓缩、细胞皱缩、细胞膜起泡、气球样变等凋亡现象。少

1.efficacy of adenovirus vector transduction into human squamous cell lines and cytotoxicity in vitroFull length LEKTI cDNA was subcloned into adenovirus vector (Ad-lacZ-LEKTI). Human oral squamous cell lines, SAS, HSC-2, 3, 4, were used. Twenty thousand cells were inoculated into each well of an 8-well glass slide. 24 hours later, Ad-lacZ-LEKTI vectors were added to each well at varying multiplicities of infection (MOI). A solution of X-gal was added to each well, and transduction efficacy was determined. Experiments for toxicity of the adenovirus vector were performed in 24-well plates. 24 hours later, Ad-lacZ-LEKTI vectors were added to each well at varying MOI. Numbers of living cells were counted 96 hours after Ad-lacZ-LEKTI vector infection, using trypan blue dye exclusion method. Experiments for toxicity of GCV were performed similar to the experiments with adenovirus vector. The transducted cells were stained blue by X-gal staining. By 24hours after transduction, nearly 100% of … More all cells were transducted at a MOI of 10. Transduction efficacies at an MOI 1 were almost 20%. Ad-lacZ-LEKTI showed toxicity in all cell lines at an MOI of more than 100, but there was no evident toxicity at an MOI of 10 or less. The cell toxicity to GCV was evaluated in all cell lines, and found to be 25 μg/ml or more.2.effects on cancer cell death in vitroFifty thousand cells were inoculated into each well of a 24-well plate. 24 hours later, Ad-lacZ-LEKTI vectors were added to each well at varying MOI. 10 μg of GCV was added to each well. Numbers of living cells were counted 72 hours after GCV administration, using trypan blue dye exclusion method. More than 60% cell death was induced at an MOI of 1, and almost 100% cancer cell death was obtained at an MOI of 10.3.morphological changes in oral squamous cell lines treated with Ad-lacZ-LEKTI and GCVThe cells were transduced at an MOI of 10 with Ad-lacZ-LEKTI by GCV. At 24 hours after administration of GCV, the cultured cells showed signed apoptosis, such as chromatin condensation, cell shrinkage, blebbing of cell membrane, and ballooning formation. Less