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目的将转染了人突变ＤＨＦＲ基因的小鼠骨髓移植给经致死剂量照射的小鼠，观察其对受者造血功能的重建和保护作用。方法分离转染人突变ＤＨＦＲ基因小鼠骨髓有核细胞，移植给经致死剂量照射的同系小鼠，以甲氨蝶呤（ＭＴＸ）筛选。观察受体小鼠血象、生存率和ＣＦＵＧＭ的改变，并用ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹杂交分析外源基因在小鼠染色体ＤＮＡ中的整合与表达情况。对照组以正常同系小鼠为供体。结果在大剂量ＭＴＸ筛选下，实验组小鼠造血功能逐渐恢复，对照组在３０天内全部死亡。实验组骨髓、脾脏和部分肝脏及肾脏组织中均检测到了前病毒标志基因Ｎｅｏｒ和ＤＨＦＲ基因的特异条带。ＣＦＵＧＭ分析提示在不同Ｇ４１８和ＭＴＸ浓度中均有集落形成。结论转染了人突变ＤＨＦＲ基因的小鼠骨髓能有效地重建和保护受致死量照射受体小鼠的造血功能。
OBJECTIVE: To transplant mouse bone marrow cells that have been transfected with human mutant DHFR gene into lethally irradiated mice and observe their effects on reconstitution and protection of hematopoietic function. METHODS: Mice bone marrow nucleated cells were transfected with human mutant DHFR gene and transplanted into lethally irradiated syngeneic mice for screening with methotrexate (MTX). The hemogram, survival rate and CFUGM of recipient mice were observed, and the integration and expression of foreign genes in mouse chromosomal DNA were analyzed by PCR and Southern blot analysis. In the control group, normal syngeneic mice were used as donors. Results Under high-dose MTX screening, the hematopoietic function of the experimental group gradually recovered, and all the control group died within 30 days. The specific bands of the provirus marker gene Neor and DHFR genes were detected in bone marrow, spleen and part of liver and kidney tissues in the experimental group. CFU GM analysis suggested colony formation in different concentrations of G418 and MTX. Conclusion The bone marrow of mice transfected with human mutant DHFR gene can effectively reconstruct and protect the hematopoietic function of lethally irradiated recipient mice.