摘要
目的探讨过表达的RbAp46基因对白血病细胞系U937生物学特性的影响。方法运用电穿孔介导的转基因技术,在U937中建立稳定表达外源RbAp46基因的细胞株。用锥虫蓝拒染法判定细胞活力,细胞计数判定细胞生长速率。将处在对数生长期U937/RbAp46和U937/CMV各以2×105/ml浓度接种,加入TPA50ng/ml,同时以培养液作为空白对照,培养72h后收集细胞,流式细胞术测定细胞表面分化抗原CD11b的表达,RT-PCR分析p21WAF1/CIP1mRNA的表达,流式细胞术检测细胞周期分布情况。结果转染RbAp46的U937细胞的生长速率要比对照组低1倍左右。无论TPA诱导与否,RbAp46都能增加U937细胞表面CD11b的表达。相对于对照组,正常条件下,RbAp46转染的细胞p21WAF1/CIP1mRNA水平没有明显增加,细胞在G1期分布增加,但是经TPA诱导后,p21WAF1/CIP1mRNA水平的明显增加,细胞阻滞在G1期。结论过表达的外源RbAp46基因能抑制U937的增殖,并为细胞分化准备了条件,使细胞更易于启动分化,p21WAF1/CIP1可能参与该过程。
Objective To investigate the impact of overexpression of RbAp46 on Proliferation and Differentiation of monoblastic cell line U937.Methods Electroporation was performed to establish U937 cells stably overexperssing RbAp46 gene.Viability of transfected cells was assayed by trypan blue exclusion.Cell number was counted daily to measure the growth rate.Cells at an initial concentration of 2×105 cells/ml were incubated with or without 50 ng/ml TPA.After 72h,cell surface antigen CD11b,mRNA p21WAF1/CIP1 and cell cycle distribution were determined by flow cytometry,RT-PCR,and flow cytometry,respectively.Results The U937 cells expressing exogenous RbAp46 showed about 1-fold lower growth rate than control cells without exogenous.The expression of CD11b in RbAp46-transfected U937 cell line was upregulated regardless the presence of TPA.Furthermore,the expression level of p21WAF1/CIP1 in RbAp46 transfected cells was comparable to that in control cells at mRNA level,while U937/RbAp46 cells treated with TPA had a marked increase in p21WAF1/CIP1 mRNA expression and a G1 phase arrest.Conclusion Overexpression of RbAp46 inhibited the proliferation of U937 and triggered several differentiation programs in which p21WAF1/CIP1 may be involved.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2010年第10期1113-1116,共4页
Cancer Research on Prevention and Treatment
基金
国家自然科学基金资助项目(39770306)
