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

在许多培养细胞中，细胞生长的对数期和平台期，以及单层和椭圆形等空间生长条件下，细胞对电离辐射(IR)的敏感性不同。由于这些事实使我们意识到细胞间的连接因素，特别是缝隙连接，如连接蛋白，我们开始了这个项目。平台期MDCK细胞或人肾癌UMRC-6细胞在加入100 uM油酰胺(缝隙连接抑制剂)的条件下接受2-10Gy射线照射后，用Alamar蓝比色法观察到细胞对IR的敏感性增加。虽然乙醇和二甲基亚砜(DMSO)都是油酰胺的溶剂，但只有使用前者才能检测到红外灵敏度的增强。另一种缝隙连接抑制剂18-β-甘草次酸具有很强的细胞毒性，很难维持细胞到平台期，于是我们放弃了使用它进行进一步的实验。接下来，我们构建了一个显性负性连接蛋白43表达载体(Δaa.130-136)。但使用LacZ表达载体的对照DNA转染实验不起作用，我们不能用上述突变体连接蛋白43载体进行实验，也不能获得抑制特定的缝隙连接因子(连接蛋白43)对辐射敏感性的影响的结果。

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.