逆转录病毒载体介导Neo^R基因转入正常人造血祖细胞  

RETROVIRUS-MEDIATED TRANSFER OF NEO^R GENE INTO NORMAL HUMAN HEMATOPOIETIC PROGENITOR CELLS

作　　者：王建安[1] 赖春宁[1] 马宏进 汲言山 沈倍奋[1] 

机构地区：[1]北京基础医学研究所,北京100850

出　　处：《中国实验血液学杂志》1993年第2期159-165,共7页Journal of Experimental Hematology

摘　　要：本文应用逆转录病毒载体介导基因转移法将含有新霉素抗性(neo^R)基因的双拷贝逆转录病毒载体pN2A转入正常人造血祖细胞。应用Ficoll分层液(比重1.064)富集人造血干、祖细胞,预培养后与已经照射的生产重组病毒包装细胞株共同培养24小时,经含G418(1000μg/ml)体外半固体培养,可见具有抗性的CFU-GM集落生长。应用PCR方法检测转染细胞中neo^R基因的转移和表达,在生产逆转录病毒的辅助细胞株PA317/N2及体外液体培养的骨髓细胞中均可扩增出neo^R基因的DNA片段,进一步证实了neo^R基因在正常人造血干、祖细胞的有效转移和表达。Retroviral vectors containing bacterial neomycin-resistant (neoR) gene were transfered into primary human hematopoietic progenitor cells (HHPC). To obtain HHPC rich populations, mononuclear bone marrow cells were collected using Ficoll density gradient centrifugation. These cells were preincubated and cocultivated with irradiated (1500 cGy) viral producer cells for 24 hours, then plated in semisolid medium with increasing G418 concentrations (0 to 1000μg/ml). The results showed 9.7%- 17.8% of them were G418 resistant CFU-GM colonies. Furthermore,the neoR gene from viral producer cells and long-term marrow cultures was directly detected by polymerase chain reaction (PCR),and the results showed the presence of neoR gene in the transfected cells. These studies demonstrate that the neoR gene can be successfully transfected into normal hematopoietic stem / progenitor cells at high efficiency using the retrovirus vectors. Therefore, it would be an important step towards human gene therapy and the study of human hematopoiesis.

关 键 词：逆转录病毒载体 Neo^R基因 造血祖细胞 基因转移 基因表达 

分 类 号：R4[医药卫生—临床医学]