摘要
目的观察吉西他滨对HL-60细胞增殖、凋亡及C-myc蛋白表达的影响,探讨吉西他滨对白血病细胞的作用及其与c-myc间的关系,为其作为新的抗白血病药物提供实验依据。方法体外培养HL-60细胞,吉西他滨及阿糖胞苷处理细胞0、24、48、72 h后倒置显微镜下观察细胞生长状态及形态学改变;锥虫蓝拒染法计数活细胞,计算细胞存活率,观察吉西他滨对细胞生长的影响;应用流式细胞仪进行细胞周期分析、检测凋亡率;免疫组织化学染色法检测C-myc蛋白表达改变。结果24 h时不同质量浓度吉西他滨组均显著抑制细胞存活,随药物质量浓度增加存活率减小,各组间比较有显著性差异(P<0.01);各组随作用时间延长存活率降低,在48、72 h抑制作用显著强于24 h;G0/G1期细胞比例升高而S期细胞比例降低,细胞阻滞在G0/G1期;凋亡率为(17.4 f1±4.88)%,较空白组(4.29±3.07)%和阿糖胞苷对照组(8.16±3.43)%显著升高(Pa<0.01);免疫组织化学染色示吉西他滨组C-myc蛋白在细胞中表达阳性平均积分为42.00±8.54,较空白组(248.33±20.74)和阿糖胞苷组(102.67±12.06)显著降低(Pa<0.01)。结论吉西他滨对HL-60细胞生长及C-myc蛋白表达有明显抑制作用;能使细胞周期发生重新分布,将细胞阻滞在G0/G1期,从而抑制细胞增殖,并可诱导白血病细胞凋亡;在抑制HL-60细胞增殖、诱导其凋亡及抑制C-myc表达方面,吉西他滨的作用显著强于相同剂量的阿糖胞苷,有望成为新药物用于白血病的治疗。
Objective To observe the effect of gemcitabine on the proliferation and the induction of apoptosis in HL-60 cells,assaye the expression of C-myc protein in HL-60 cells in gene level and explore the effect of gemcitabine on leukemia and the relationship between gemcitabine and c-myc expression,in order to provide laboratory evidence for the use of this agent in the treatment of leukemia.Methods HL-60 cells were cultured in vitro.Growth appearance and the change of cell morphology were observed with invert microscope after HL-60 cells was treated with gemcitabine and cytarabine for 0,24,48 and 72 hours.By Trypan blue exclusion tests,the number of viable cells was counted and the survival rate of HL-60 cells was evaluated.The cell cycle and apoptosis rate were analyzed by flow cytometry.The expression of the C-myc protein was assessed by immunohistochemistry.Results Treatment of HL-60 cells with a variety of concentrations of gemcitabine for 24 hours resulted in dose-dependent inhibition of cell growth,and among the different groups there was significant difference（P〈0.01）.The survival rate of HL-60 cells at 48 h and 72 h was significantly lower than that at 24 h at the same dosage（P〈0.01）.The cell growth inhibition was accompanied by G0/G1 cell cycle arrest and by a loss of cells in S phase.Apoptosis detected as a sub-G0 cell population and apoptotic DNA fragmentation was observed.The apoptosis rate was（17.41±4.88）% in gemcitabine treated cells,which was significantly higher than that in the untreated cells（4.29±3.07）% and the cytarabine treated cells（8.16±3.43）%（P〈0.01）.The result of immunohistochemistry showed that the mean score of C-myc protein in positive cells was 42.00±8.54,which was significantly lower than its correspondingly untreated cells（248.33±20.74） and Ara-c treated cells（102.67±12.06）（Pa〈0.01）.Conclusions The results suggest that gemcitabine can inhibit the growth and the C-myc protein expression of HL-60 cells significantly.Cell cycle regulation by gemcitabine in HL-60 cells was accompanied by G0/G1 cell cycle arrest.Gemcitabine inhibits the HL-60 cells proliferation and induces the cells apoptosis.It exerts stronger effects as compared with Ara-c in proliferation inhibition and apoptosis induction of HL-60 cells when treated with the same dosage.Gemcitabine may be a new therapeutic option in the treatment of leukemia.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2008年第15期1170-1172,共3页
Journal of Applied Clinical Pediatrics
基金
河南省医学科技攻关项目资助(200703023)
