【作者】 张军权；

【导师】 毛秉智；

【作者基本信息】 中国人民解放军军事医学科学院 ， 放射医学， 2003， 博士

【摘要】 目的：探索大剂量照射后造血功能的变化规律，寻找判断大剂量照射后造血损伤程度的分子生物学指标，探索急性放射病的诊断新指标和治疗新靶点。方法：观察不同剂量照射后C57BL／6J小鼠的生存率、外周血象、CFU-GM系集落培养、细胞周期和细胞凋亡的变化规律，小鼠骨髓组织部分造血生长因子与受体的表达规律，应用基因芯片技术筛选大剂量照射后小鼠骨髓组织的差异表达基因。结果：经生存率观察，选定了4个照射剂量组：6.5Gy、7.5Gy、8.5Gy和10Gy。发现以下指标存在一定的剂量相关性：外周血白细胞数最低值和血小板数降低发生时相；骨髓细胞照射后6小时的G2／M期阻滞细胞百分比和脾细胞照射后6小时S期阻滞细胞百分比；骨髓照射后12小时凋亡细胞百分比和脾脏与胸腺照射后6小时凋亡细胞百分比。应用RT-PCR技术从小鼠骨髓组织中扩增出干细胞因子(SCF)、粒细胞集落刺激因子受体(G-CSFR)、促红细胞生成素受体(EPOR)、促血小板生成素受体(c-mp1)和干细胞因子受体(c-kit)的部分序列，并经测序证实。用RT-PCR与Southern杂交联用法初步探索了大剂量照射后部分造血生长因子与受体的表达规律，发现大剂量照射后早期，各基因的表达水平未见明显变化，随着照射后时间的延长，由于造血处于枯竭状态，不能得到足量的总RNA用于分析。经过两次基因芯片杂交，发现功能未知的新基因NM-025383与辐射相关，并从小鼠骨髓组织扩增出该基因。结论：从细胞水平上观察到的部分剂量相关性指标，对大剂量照射后造血损伤程度的判断有参考意义。从小鼠骨髓组织克隆出部分造血生长因子与受体的cDNA序列，为从整体水平上研究这些低拷贝基因在大剂量照射后的表达规律奠定了基础。RT-PCR与Southern杂交联用法研究基因表达水平具有较好的可行性。发现功能未知的新基因NM-025383与辐射相关，为此基因的存在提供了实验证据，为此基因的功能研究提供了线索。更多还原

【Abstract】 Objective: To analyze the hematopoietic function after high dose irradiation. To seek molecular biology methods which can evaluate the severity of hematopoietic injury. To screen new biomarkers for the diagnosis and treatment of acute radiation sickness. Methods: To explore mortality rate, peripheral blood cell counts, CFU-GM counts, cell cycle retardation and apoptosis from mice strain C57BL/6J after y rays total body irradiation of different doses. To study the expression level of some hematopoietic growth factor and receptors of mouse bone marrow after high dose irradiation. To search differentially expressed genes from mouse bone marrow after high dose irradiation using DNA microarray. Results: 6.5Gy, 7.5Gy, 8.5Gy and lOGy were determined which represent the seventy of hematopoietic injury. The following criterions were found to be dose correlated: Nadir of white blood cell and reduction phase of platelet after high dose irradiation; Percentage of G2/M retardation cells in bone marrow and S retardation cells in spleen 6h post irradiation; Percentage of apoptosis cells 12h post irradiation in bone marrow and 6h post irradiation in spleen and thymus. RT-PCR was used to clone partial sequences of SCF, G-CSFR, EPOR, c-mpl and c-kit from mouse bone marrow. Combining RT-PCR and Southern hybridization, the post irradiation expression level of these hematopoietic growth factor and receptors was observed in mouse bone marrow. No significant changes were found within 24h after high dose irradiation. Since the severe depletion of hematopoietic cells, no sufficient amount of total RNA can be obtained for investigation 48h after high dose irradiation. New gene NM-025383 was found to be radiation associated through DNA microarray screening and was cloned from mouse bone marrow. Conclusion: The 7 dose correlated criterions may help evaluate the severity of hematopoietic injury. The successful cloning of 5 low copy genes make it possible to investigate their expression level in vivo after high dose irradiation. Combining RT-PCR and Southern hybridization is a practical method to analyze expression level of low copy genes in vivo. Functional research of radiation associated NM-025383 is feasible.更多还原