稳定表达外源RbAp46基因的U937白血病细胞株的建立和初步特性分析

为建立RbAp46基因稳定转染的白血病细胞株，对悬浮培养的白血病细胞的电穿孔条件进行优化之后，运用电穿孔介导的转基因技术，在低电压、高电容的条件下将含RbAp46基因cDNA的表达质粒转染白血病细胞系U937，经G418筛选获得稳定转染的克隆，用PCR检测RbAp46基因的整合，用Western blot分析蛋白质的表达水平，然后筛选出表达外源RbAp46蛋白水平最高的亚克隆。用台盼蓝拒染法判定细胞活力，用细胞计数判定细胞生长速率。结果发现，转染RbAp46基因的U937细胞的生长速率比对照组低50％左右。结论：在白血病细胞系U937中建立了稳定表达外源RbAp46基因的细胞株，并发现过表达的外源RbAp46基因能抑制U937细胞的增殖。

Establishment and Characterization of Leukemic Cell Line U937 Stably Expressing Exogenous RbAp46

To establish leukemic cell lines stably transfected by RbAp46 gene,electroporation was performed after optimizing the transfection condition for suspended cells. Under conditions of low voltage and high capacitance,RbAp46 was transfected into U937 by electroporation. Individual clones selected with G418 for 3 weeks were isolated. The integration and the protein levels of the exogenous RbAp46 in transfectants were determined by PCR and Western blot analysis,respectively. The subclone expressing high level of RbAp46 was then established. Viability of transfected cells was assayed by trypan blue exclusion. Cell number was counted daily to determine the growth rate. The results showed that growth rate of U937 cell lines expressing exogenous RbAp46 was about 50% lower than that in control. It is concluded that leukemic cell lines stably expressing exogenous RbAp46 were established and overexpression of RbAp46 inhibits the growth of U937 leukemic cells.