逆转录病毒介导高效的白血病细胞基因转移

目的建立安全高效的逆转录病毒介导的基因转移系统,为白血病基因治疗提供实验基础。方法分别用脂质体转染和小鼠逆转录病毒转导方法,将含标志基因ＮｅｏＲ的逆转录病毒载体ｐＬＸＳＮ导入双嗜型包装细胞ＧＰ＋ｅｎｖＡｍ１２,获得重组逆转录病毒;用含病毒上清感染白血病细胞系（ＮＢ４、Ｕ９３７和ＴＨＰ－１）,并分析对Ｋ５６２细胞的转导效率。结果脂质体ＤＯＳＰＥＲ转染法获得病毒滴度为８．０×１０５ＣＦＵ／ｍｌ,而病毒感染法高达１．６×１０７ＣＦＵ／ｍｌ。经Ｇ４１８选择,ＰＣＲ证实ＮｅｏＲ基因被整合到白血病细胞基因组中,巢式ＰＣＲ与补救分析均未检测到辅助病毒存在。单个集落Ｋ５６２细胞的ＰＣＲ分析证实,基因转移效率可达９３．３％～１００％。结论逆转录病毒介导的基因转移系统是高效、安全的,有助于人类白血病的基因治疗研究。

Efficient gene transfer mediated by retrovirus vector in leukemic cells

OBJECTIVE: To establish an efficient and safe gene transfer system mediated by retrovirus for providing an experimental basis of gene therapy in human leukemia. METHODS: The retrovirus vector pLXSN, carrying the NeoR, was transferred into amphotropic packaging cell line GP + envAm12 by liposome method or by transduction. Retrovirus with higher titer was used to infect human leukemic cell lines NB4, U937 and THP-1. The efficiency of gene transduction was tesed on colonies formed by K562 cells after gene transduction. RESULTS: The titer of DOSPER liposome transfected GP + envAm12 pool on NIH3T3 cells was 8.0 x 10(5) CFU/ml while that of producer pool infected with mouse retrovirus was 1.6 x 10(7) CFU/ml. Integration of NeoR into human leukemic cells was confirmed by PCR. By both nested PCR and NeoR rescue assay, no helper virus was found. NeoR gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by semi-nested PCR on the colonies after seven days' culture. CONCLUSION: The efficiency and safety of the gene transfer system may provide an optimal experimental system in gene therapy for human leukemia.