Study on vascular shutdown effects of carcinoma blood vessels and cytotoxic effects of carcinoma cells induced by photochemical reaction.

光化学反应诱导癌血管关闭效应及癌细胞细胞毒作用的研究。

• 批准号: 14380410
• 负责人: MINAMITANI Haruyuki
• 金额: $ 10.94万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380410/
• 关键词: Photodynamic therapy; Active oxygen; Apoptosis; Platelet thrombosis; Endothelial cell; Cancer cell; Porphyrin; Confocal laser microscopy

Photodynamic therapy (PDT) is a clinically effective cancer treatment which is based on cytotoxicity of reactive oxygen species (ROS) generated by an interaction between photosensitizer and light. PDT induces vascular shutdown (VSD) effects based on thrombus formation realized by administration of photosensitizing agent. This treatment results in a sequence of photochemical processes to produce cytotoxic ROSS which bring irreversible photo damage to tissue cells. ^1O_2 and O_2^-strongly activated adhesive function of platelet and leukocyte, and also induced endothelial cell retraction resulted in increase in sub endothelial area. Activation of both platelet and endothelial cells also released adhesion molecules that followed strong cell interaction indispensable for the platelet aggregation and thrombus formation. The platelet adhesion and thrombus formation in the microvessels accelerated under photodynamic reaction. For visualization of flow behavior of erythrocytes, leukocytes and p … More latelets, fluorescent dye probes were used and the flow images were observed under fluorescent microscope. Blood oxygen pressure was also measured by using phosphorescence quenching of Pd porphyrin with oxygen molecules. In order to measure erythrocyte velocity and to evaluate the degree of platelet adhesion and leukocyte sticking, high-speed CCD camera and image processing system were used and made possible to evaluate the contribution of ROSS to the VSD and pharmacological effect on the VSD. ROSs induced protein transformation of cytoplasm in the endothelial cells, especially actin filament polymerisation was observed that affected on increase in subendothelial area. These phenomena accelerated activation of platelets and leukocytes which progressively adhered on the endothelial cells and subendothelial area. On the other hand, cytotoxic effect of PDT on tumor cells, mainly apoptosis, was induced by ROSs attributed to photochemical reaction. Ca^<2+> is one of the most famous second messenger in cell function, especially, the cytoplasmic concentration of free calcium ions[Ca^<2+>]i has important role in apoptosis. In this study, we examined the role of Ca^<2+> in PDT-induced apoptosis of human lung small carcinoma (Ms-1) and human leukemia cell line (HL-60 cells). We performed the imaging of Ca^<2+> and monitoring Ca^<2+> concentration in apoptosis induced by PDT. With intracellular calcium indicator Fluo3-AM, increase of [Ca^<2+>]i was observed for 6 hours after PDT and was inhibited by extracellular Ca^<2+> chelator BAPTA. Agarose gel electrophresis revealed that PDT-induced apoptosis was inhibited by BAPTA. PDT induced loss of mitochondrial membrane potential and increase of Ca^<2+> in mitochondria. As a result, we have revealed that increase of [Ca^<2+>]i is induced by transcellular calcium influx from extracellular fluid to intracellular fluid, and induce apoptosis in Ms-1 and HL-60 cells. These results indicate that Ca^<2+> plays an important role in apoptosis induced by PDT. Less

光动力疗法（PDT）是一种临床上有效的癌症治疗方法，其基于光敏剂与光之间相互作用产生的活性氧（ROS）的细胞毒性。PDT基于通过施用光敏剂实现的血栓形成诱导血管关闭（VSD）效应。这种处理导致一系列光化学过程，产生细胞毒性ROSS，对组织细胞造成不可逆的光损伤。^1O_2和O_2^-能强烈激活血小板和白细胞的粘附功能，并诱导内皮细胞回缩，使内皮下面积增加。血小板和内皮细胞的活化也释放粘附分子，这些粘附分子遵循血小板聚集和血栓形成所必需的强烈细胞相互作用。光动力学反应加速了血小板在微血管内的粘附和血栓形成。为了显示红细胞、白细胞和血小板的流动行为， ...更多信息 采用荧光染料探针，在荧光显微镜下观察血流图像。用氧分子猝灭钯卟啉的磷光测定血氧分压。为了测定红细胞运动速度，评价血小板粘附和白细胞粘附的程度，采用高速CCD摄像机和图像处理系统，评价了ROSS对VSD的贡献和对VSD的药理作用。ROS诱导内皮细胞胞质蛋白质转化，尤其是肌动蛋白丝聚合，影响内皮下面积的增加。这些现象加速了血小板和白细胞的活化，并逐渐粘附在内皮细胞和内皮下区域。另一方面，PDT对肿瘤细胞的细胞毒作用，主要是通过光化学反应引起的ROS诱导的凋亡。Ca^2+是细胞功能中最重要的第二信使之一，尤其是胞浆内游离Ca ^2+浓度在细胞凋亡中起重要作用。本研究探讨了Ca^<2+>在PDT诱导人肺小细胞癌（Ms-1）和人白血病细胞系（HL-60）凋亡中的作用。我们对PDT诱导的细胞凋亡进行了Ca^<2+>显像和Ca^<2+>浓度监测。以Fluo 3-AM为细胞内钙指示剂，观察到PDT后6 h内细胞内[Ca^2+]i的增加，细胞外Ca^2+螯合剂BAPTA可抑制[Ca ^2+]i的增加。琼脂糖凝胶电泳显示BAPTA可抑制PDT诱导的细胞凋亡。PDT可引起线粒体膜电位降低和线粒体内Ca^<2+>增加。结果表明，[Ca^<2+>]i的增加是由钙离子从细胞外液跨细胞内流到细胞内液引起的，并诱导MS-1和HL-60细胞的凋亡。这些结果表明，Ca^<2+>在PDT诱导的细胞凋亡中起重要作用。少

项目成果

塚田孝祐, 緒方嘉貴, 辻岡克彦, 南谷晴之: "人工酸素運搬体Neo Red Cell交換輸血時の脳微小循環動態および酸素分圧計測"脳循環代謝. 15,4. 196-197 (2003)

Kosuke Tsukada、Yoshitaka Ogata、Katsuhiko Tsujioka、Haruyuki Minamitani：“人工氧载体 Neo Red Cell 换血过程中的脑微循环动力学和氧分压测量”《脑循环与代谢》15,4。

塚田孝祐, 緒形嘉貴, 辻岡克彦, 南谷晴之: "人工酸素運搬体Neo Red Cell交換輸血時の脳微小循環動態および酸素分圧計測"脳循環代謝. 15, 4. 196-197 (2003)

Kosuke Tsukada、Yoshitaka Ogata、Katsuhiko Tsujioka、Haruyuki Minamitani：“人工氧载体 Neo Red Cell 换血过程中的脑微循环动力学和氧分压测量”《脑循环与代谢》15, 4. 196-197 (2003)。

Nagao, K.Matsuzaki, Y.Imazeki, M.Takahashi, H.Minamitani: "Quantitative analysis for intracellular distribution of a photosensitizer using confocal laser scanning microscope"IEICE Trans. Information and Systems. 85D・5. 152-159 (2002)

Nagao、K.Matsuzaki、Y.Imazeki、M.Takahashi、H.Minamitani：“使用共焦激光扫描显微镜定量分析光敏剂的细胞内分布”IEICE Trans. 152-159（2002）。

Nagao M, Takahshi M, Matsuzaki K, Minamitani H: "Application of bioimaging techniques to mechanistic studies on photodynamic therapy"Bioimages. 11,2. 53-60 (2003)

Nagao M、Takahshi M、Matsuzaki K、Minamitani H：“生物成像技术在光动力治疗机制研究中的应用”生物图像。

Tsukada K, Ogata Y, Tsujioka K, Minamitani H: "Measurement of blood flow dynamics and blood oxygen tension in cerebrall microcirculation during exchange blood transfusion with hemoglobin encapsulated liposomes NRC."Journal of Brain Circulation and Metabol

Tsukada K、Ogata Y、Tsujioka K、Minamitani H：“用血红蛋白封装脂质体 NRC 进行换血时脑微循环血流动力学和血氧张力的测量。”脑循环与代谢杂志