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

在许多培养的细胞中，细胞生长的对数期和平台期或空间生长条件（如单层和球体）对电离辐射（IR）的敏感性是不同的。由于这些事实使我们想到细胞与细胞之间的连接因子，特别是间隙连接，如连接蛋白，我们开始了这个项目。当给予平台期MDCK细胞或UMRC-6（人肾细胞癌）细胞2- 10 Gy的γ射线和100 μ M的油酰胺（间隙连接抑制剂）时，用阿利兰法观察到IR敏感性增加。虽然乙醇和DMSO（二甲基亚砜）都是油酰胺的溶剂，但只有使用前者时才能检测到IR灵敏度的增强。另一种缝隙连接抑制剂18-β-大黄酸细胞毒性强，维持细胞进入平台期困难，因此我们放弃使用它进行进一步的实验。接下来，我们制备了显性阴性连接蛋白43表达载体（Δ aa. 130 -136）。但是使用lacZ-表达载体的对照DNA转染实验不起作用，我们不能使用上述突变连接蛋白43载体进行实验并获得抑制特异性间隙连接因子（=连接蛋白43）对辐射敏感性的影响的任何结果。

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.