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，我们首先研究了重离子束和X射线对HPRT基因座细胞的杀伤作用和诱变作用。我们使用的细胞系是中国仓鼠来源(V79)、人卵巢癌来源(RMG1)、人乳腺癌来源(MDA-MB231)和人食道癌来源(TE24)。在单层培养条件下，用X射线(150kVp)、碳束(20keV/mU、80keV/mUm)或霓虹灯(80keV/mUm)照射细胞，用集落试验计算细胞存活率，根据添加6-硫鸟苷的培养液中形成的集落数计算HPRT位点突变频率。我们至少在三种细胞系(V79、RMG1和MDA-MB231)上获得了以下结果。(1)重离子束对细胞的杀伤作用比X射线强得多，高LET的碳束比低LET的碳束表现出更强的增强效应。(2)当使用相同的LET能级时，碳束的…(3)重离子束照射的细胞诱变率明显高于X射线照射的细胞；(4)随着碳束LET的增加，细胞的突变率增加；(5)在相同的LET水平下，霓虹灯的诱变率低于碳素。我们用TE24得到了几乎相同的结果，但对这种细胞系还需要更多的实验。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的细胞凋亡率。Apo2.7单抗流式细胞仪检测细胞凋亡率。Apo2.7是一种PE标记的针对7A6抗原(38kD蛋白)的单抗，它出现在细胞凋亡早期的线粒体记忆中。该方法对凋亡细胞的检测能力非常灵敏，很难得到每种细胞系的最佳实验条件。这一次，我们可以明确这种方法对于这些X射线照射的细胞系来说是必不可少的条件。我们将在所获得的条件下分析重离子束照射后的细胞凋亡率，并探讨其与细胞杀伤效应和诱变效应的关系。较少

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