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

1.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2万个细胞接种到8孔玻片的每孔中。24小时后，将Ad-lacZ-LEKTI载体加入不同感染倍数（MOI）的每孔中。每孔加入X-gal溶液，测定转导效果。在24孔板上进行腺病毒载体毒性实验。24小时后，以不同的MOI将Ad-lacZ-LEKTI载体添加到每口井中。感染Ad-lacZ-LEKTI载体96 h后，用台盼蓝染色法计数活细胞数。GCV毒性实验与腺病毒实验相似。转导后的细胞用X-gal染色成蓝色。在MOI为10的情况下，所有细胞均被转导。MOI 1的转导效率几乎为20%。当MOI大于100时，Ad-lacZ-LEKTI对所有细胞系均有毒性，但当MOI小于等于10时，无明显毒性。所有细胞系对GCV的细胞毒性均为25 μg/ml以上。在24孔板的每孔中接种5万个细胞。24小时后，以不同的MOI将Ad-lacZ-LEKTI载体添加到每口井中。每孔加GCV 10 μg。用台盼蓝染色法测定给药72h后的活细胞数。当MOI为1时，可诱导60%以上的细胞死亡，当MOI为10.3时，几乎100%的癌细胞死亡。Ad-lacZ-LEKTI和GCV对口腔鳞状细胞形态学的影响。GCV给药24小时后，培养的细胞出现明显的凋亡，如染色质凝集、细胞收缩、细胞膜起泡和气球形成。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