摘要目的：将转染了人突变dhfr基因的第二代小鼠骨髓，移植给经致死剂量照射的第三代小鼠，观察该基因对小鼠造血功能的长期保护作用。方法：分离存活的第二代小鼠骨髓有核细胞，直接移植给经致死剂量照射的同系小鼠，以MTX筛选,观察小鼠血象和生存率变化，PCR和Southern印迹杂交分析目的基因在小鼠染色体DNA中的整合与表达情况。结果：在大剂量MTX筛选下，实验组小鼠造血功能逐渐恢复，对照组3周内全部死亡。实验组生存率和生存期明显高于对照组，但较前两代生存率低。PCR和Southern印迹分析结果提示，实验组脾脏和肝脏组织中均检测到前病毒标志基因neoR和dhfr基因的特异条带。结论：转染了人突变dhfr基因的第二代小鼠骨髓，能有效地重建经致死剂量照射的第三代小鼠造血功能。保护骨髓免遭大剂量MTX所致的严重骨髓抑制，dhfr基因在小鼠基因组DNA中的稳定整合是这种长期保护作用的物质基础。

AbstractObjective: To investigate the long-term protection of the bone marrow cells containing mutant dhfr gene on the reconstruction of murine hemacytogenesic function. Methods: The surviving mice with the tranduced marrow containing mutant hdhfr gene were used as donors for the third generation transplantation. The third transplant was carried out 14 weeks after secondary BMT(bone marrow transplantation). After irradiation(0.85～0.9 Gy) and BMT, the tertiary recipients, C57BL/6J, were treated with methotrexate (4 mg?kg -1 twice a week for the first week; 10 mg?kg -1 twice a week for the other weeks). The survival rate and blood pictures of murine as well as the integration and expression of target gene were studied. Results: The lethal irradiated murine hemacytogenesic function could be reinstituted and protected from lethal toxicity of high doses methotrexate selection after reinfusing the hematopoietic progenitor cells containing the hdhfr gene. The survival rate and survival time of experimental murine was higher than the control group. Survival rate of tertiary generation murine(40%) was less than the primary and secondary generations(87.5%). It was confirmed that hdhfr gene was still integrated into murine genomic DNA and expressed successfully using PCR and Southern blot analysis. Conclusion: he stable integration of human mutant dhfr gene may play an important role in this long term protection of murine hematopoietic function.