INVESTIGATION OF EFFECT OF THE CELL-CELL CONTACT INHIBITION ON RADIATION SENSITIVITY AND DETERMINATION OF THE RELATING FACTORS

细胞-细胞接触抑制对辐射敏感性影响的研究及相关因素的测定

• 批准号: 15591291
• 负责人: INOUE Tomio
• 金额: $ 1.79万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591291/
• 关键词: CULTURED CELLS; IONIZING RADIATION; GAP JUNCTION; OLEAMIDE; oleamide; MDCK; UMRC-6; connexin43; 感受性; olemide; 細胞間接着因子; 薬剤

In many cultured cells, sensitivity to ionizing radiation (IR) is different between in log-phase and plateau phase of cell growth or spatial growing condition such as in monolayer and spheroid. Since these facts occur to us the cell-to-cell connecting factors, particularly gap junctions such as connexins, we started this, project. When plateau phase MDCK cells or UMRC-6 (human renal cell carcinoma) cells were given 2-10Gy of gamma ray with 100uM oleamide (a gap junction inhibitor) in the culture medium, augmentation of the IR sensitivity was observed using alamar blue assay. Although both ethanol and DMSO (dimethylsulfoxide) are known as solvent for oleamide, augmentation of the IR sensitivity could be detected only when using the former. Another gap junction inhibitor, 18-beta-glycyrrhetinic acid had strong cytotoxicity and maintaining cells to plateau phase was difficult, then we abandoned to using it for further experiments. Next we made a dominant negative connexin 43 expression vector (Δaa.130-136). But a control DNA transfection experiment using lacZ-expression vector did not work, we could not perform the experiment using above-mentioned mutant connexin 43 vector and obtain any results for the effect of inhibiting a specific gap junction factor (=connexin 43) upon the radiation sensitivity.

在许多培养细胞中，在细胞生长的对数期和平台期或在单层和球形等空间生长条件下，对电离辐射（IR）的敏感性是不同的。因为我们知道细胞间的连接因子，特别是像连接蛋白这样的间隙连接，我们开始了这个项目。当平台期MDCK细胞或UMRC-6（人肾细胞癌）细胞在培养基中给予2-10Gy的伽马射线和100uM的油酰胺（一种间隙连接抑制剂）时，用阿拉马尔蓝法观察到IR敏感性的增强。虽然乙醇和二甲基亚砜（DMSO）都是已知的油酰胺溶剂，但只有当使用前者时才能检测到红外灵敏度的增强。另一种间隙连接抑制剂18- β -glycyrrhetinic acid具有很强的细胞毒性，很难将细胞维持到平台期，因此我们放弃使用它进行进一步的实验。接下来，我们制作了显性阴性连接蛋白43表达载体（Δaa.130-136）。但使用lacz表达载体的对照DNA转染实验没有成功，我们无法使用上述突变体connexin 43载体进行实验，也无法获得抑制特定间隙连接因子（=connexin 43）对辐射敏感性影响的结果。