X射线和喜树碱对外周血淋巴细胞不同细胞亚群生物学效应的分析-生物化学与分子生物学专业论文.docx

华中科技大学博士学位论文 华 中 科 技 大 学 博 士 学 位 论 文 4 4 human peripheral blood lymphocytes as a model to construct different cell cycle state and respectively to study the DNA damage repair response and the law of cell death with chemoradiation in different subset of tumor cells. Over the past three decades, few studies have compared the difference in the response to the external stimuli of resting and proliferating peripheral blood lymphocytes, and some studies just used the traditional alkaline comet assay to simply analyse DNA damage in epidemiological studies. In this study, γH2AX biomarker was used to examine DNA damage response in different cell cycle states of peripheral blood lymphocytes (PBLs) and its sensitivity to chemoradiation. The formation of γH2AX surely reflects DNA damage, when chemical drug camptothecin and X-ray function in the G0 phase and the proliferation of PBLs induced by PHA, the results of immunoblotting and flow cytometry exhibited that proliferating lymphocytes treated with chemical or ionizing radiation had higher γH2AX formation compared to resting lymphocytes. The results indicated that stimulated PBLs are more vulnerable to DNA fragmentation and more sensitive to chemoradiation than their quiescent counterparts. Comparison of the apoptotic rates of quiescent and proliferating PBLs revealed increased apoptosis ratio of proliferating cells. The expressions of apoptosis- related proteins Bcl-2, caspase-3 and caspase-9 were also consistent with the findings. To further elucidate the roles of H2AX in the biological processes, RNA interference technique was used to reduce its expression in 293T cells. It was found that H2AX siRNA had no significant effect on the CPT- and X-ray-induced apoptosis. Therefore, H2AX is only the marked protein involved in DNA damage but not in the regulation of apoptosis pathway. This study aims to use PBLs as a model to simulate the different cell cycle status of tumor cells (G0 phase and proliferative phase) and further to s