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      • Photodynamically induced endothelial cell injury and neutrophil-like HL-60 adhesion

        Takahashi, Miho,Nagao, Tomokazu,Matsuzaki, Kazuki,Nishimura, Toshihiko,Minamitani, Haruyuki Korean Society of Photoscience 2002 Journal of Photosciences Vol.9 No.2

        Photodynamic therapy (PDT) is a treatment modality based on photochemical reaction and the resultant cytotoxic reactive oxygen species. The platelet thrombus formation leading to stasis observed in vivo during PDT is called vascular shut down (VSD) effect. To investigate the mechanism of the VSD effect, we observed Human Umblical Vein Endothelial Cell (HUVEC) injury induced by photochemical reaction. We observed cell retraction and blebbing after PDT. It seems that the injury was not fetal and only morphological change. Then, the cytoplasm was stained by Calcein-AM and subendothelial area was evaluated from fluorescence microscopy. The rate of subendothelial area after PDT increased significantly. Second, we investigated interaction between neutrophils and HUVEC. Human promyelocytic leukemia cells (HL-60) were differentiated into neutrophil by incubation with all-trans retinoic acid. Calcein-AM labeled neutrophil adhesion to HUVEC was evaluated from fluorescence microscopy. PDT-induced neutrophil adhesion to HUVEC depended more on the exposure of subendothlial area than on neutrophil activation. This result suggests that there is a certain interaction between neutrophil and HUVEC during PDT.

      • Apoptotic Signaling Cascade of 5-aminolaevulinic Acid-based Photodynamic Therapy in Human Promyelocytic Leukemia HL-60 Cells

        Nagao, Tomokazu,Matsuzaki, Kazuki,Takahashi, Miho,Minamitani, Haruyuki Korean Society of Photoscience 2002 Journal of Photosciences Vol.9 No.2

        In this study, we investigated apoptotic cell death induced by photodynamic therapy using 5-aminolaevulinic acid (ALA-PDT) in human promyelocytic leukemia cells (HL-60). ALA-PDT induced apoptosis in HL-60 cells as confirmed by DNA agarose gel electrophoresis and nuclear staining with Hoechst 33342. The apoptotic cell death was inhibited by addition of broad-spectrum caspase inhibitor Z-Asp-CH$_2$-DCB, indicating that the apoptotic cell death was induced in a caspase-dependent manner. Actually, western blotting analysis revealed that caspase-3 was processed as early as 1.5 h after ALA-PDT. Cytoplasmic cytochrome c released from mitochondria was detected by western blotting. However, inhibitor of caspase-9, a cysteine protease located in the downstream of cytochrome c release, was not able to reduce the apoptotic cell death. Therefore, the mitochondrial apoptotic pathway was not involved in the ALA-PDT-induced apoptosis. On the other hand, it was found that ALA-PDT-induced apoptosis was clearly inhibited by pretreatment of caspase-8 inhibitor. These data suggest that caspase-8-mediated apoptotic pathway is important in ALA-PDT-induced cell death.

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        Twice Stretched Fabrication of Polylactic Acid Microneedle Arrays Using Drawing Lithography

        Shingo Terashima,Chikako Tatsukawa,Masato Suzuki,Tomokazu Takahashi,Seiji Aoyagi 한국정밀공학회 2020 International Journal of Precision Engineering and Vol.21 No.10

        A biodegradable poly-lactic acid (PLA) microneedle array composed of needles with a maximum length of 3 mm and an aspect ratio of up to approximately 18 was fabricated by drawing lithography. In the proposed fabrication method, the melted polymer is stretched twice. The approximate length is set in the first stretch, and the tip is sharpened in the second stretch. In the first stretch, by changing the thickness of the PLA sheet, PLA pillars with various lengths are fabricated. By defining the initial length of the needle, it is possible to set a wide range of aspect ratios and lengths. In the second stretch, it is possible to control the final aspect ratio and length of the needle, as well as the shape of its tip by changing the temperature and stretch speed of the PLA pillars. Finally, it was confirmed that the needle can pierce the surface of artificial skin and porcine skin.

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