EVALUATION OF APOPTOSIS RELATED GENE AND MUTATION INDUCTION FATER IRRADIATION.

为了评价重离子束和x射线对培养细胞的生物学效应，我们首先研究了hprt位点的细胞杀伤作用和突变诱导作用。我们使用的细胞系有中国仓鼠来源（V79）、人卵巢癌来源（RMG1）、人乳腺癌来源（MDA-MB231）和人食管癌来源（TE24）。细胞单层培养，x射线（150kVp）、碳束（20keV/ μ m、80keV/ μ m）、霓虹灯（80keV/ μ m）照射，通过菌落测定计算细胞存活率，并根据添加6-巯基鸟嘌呤的培养基中形成的菌落数计算hprt位点的突变频率。我们至少在三个细胞系（V79， RMG1和MDA-MB231）中获得了以下结果。(1)重离子束的细胞杀伤效果比x射线强得多，高LET的碳束的细胞杀伤效果比低LET的细胞杀伤效果强得多；(2)在相同LET水平下，碳束的细胞杀伤效果比氖束略高；(3)重离子束辐照的细胞突变频率比x射线辐照的细胞突变频率高得多。(4)随着碳束LET的增加，突变频率增加；(5)在相同LET水平下，氖束的突变频率小于碳束。我们在TE24上得到了几乎相同的结果，但还需要对这种细胞系进行更多的实验。我们还试图评估这四种细胞系在x射线和重离子束照射后凋亡细胞的频率。流式细胞仪检测凋亡细胞Apo2.7单克隆抗体。Apo2.7是一种PE标记的7A6抗原（蛋白38kD）单克隆抗体，该抗原出现在凋亡细胞早期的线粒体膜中。该方法对凋亡细胞的检测能力非常敏感，很难得到每个细胞系的最佳实验条件。这一次，我们可以明确这种方法的必要条件，这些细胞系照射x射线。我们将分析在获得的条件下，重离子束辐照后凋亡细胞的频率，并探讨其与细胞杀伤效应和诱变效应的关系。少

In order to evaluate the biological effectsu of heavy-ion beams and X-rays on cultured cells, we investigated the cell-killing effects and mutation induction at the hprt locus, at first. The cell-lines we used were Chinese hamster origin (V79), human ovarian cancer origin (RMG1), human breast cancer origin (MDA-MB231) and human esophageal cancer origine (TE24). Cells were cultured in monolayr condition and irradiated by X-rays (150kVp) or carbon beams (20keV/mu m, 80keV/mu m) or neon beam (80keV/mu m), and cell survivals were calculated by colony assay and the mutation frequencies at hprt locus were calculated from the numbers of colonies formed in media supplemented with 6-thioguanin. We obtained the following results at least in three cell lines (V79, RMG1 and MDA-MB231). (1) the heavy ion beams showed much more powerful cell-killing effects than X-rays and carbon beams with high LET showed more enhanced effects than low LET beams, (2) when the same LET level was used, carbon beam sh … More owed slightly higher cell-killing effect compared with neon beam, (3) extremely higher mutation frequency was observed for cells irradiated by the heavy ion beams compared with that irradiated by X-rays, (4) the mutation frequency was increased when the LET of carbon beam was increased, (5) the mutation frequency was less for neon than carbon when the same LET level was employed. We are getting almost the same results with TE24, but still more experiments will be needed with this cell-line.We also tried to evaluate the frequencies of apoptotic cells for these four cell-lines after irradiation by X-rays and heavy ion beams. Apoptotic cells were detected by Apo2.7 monoclonal antibody using the flow-cytometer. Apo2.7 is a PE labeled monoclonal antibody for the 7A6 antigen (protain of 38kD) which is appeared for the mitochondria membrain of apoptotic cells from eary stage of apoptosis. The detection ability of this method for the apoptotic cells were so sensitive that it was very difficult to get the best condition of experiment for each cell-line. This time, we could clearfy the indispensable condition of this nethod for these cell-lines irradiated by X-rays. We will analyze the frequencies of apoptotic cells after heavy ion beam irradiation under the obtained condition, and investigate the relationship with the cell-killing effects and the mutation induction effects. Less