TOHNAI Iwai Nagoya University, School of Medicine, Professor, 医学部, 教授 (50172127)
MIZUNO Masaaki Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (70283439)
UEDA Minoru Nagoya University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (00151803)
|Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
1.efficacy of adenovirus vector transduction into human squamous cell lines and cytotoxicity in vitro
Full 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
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 vitro
Fifty 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 GCV
The 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