奥沙利铂诱导人骨髓造血细胞凋亡及DNA损伤修复

Oxaliplatin Induces Apoptotic Response and DNA Damage Repair of Human Hematopoietic Cells

目的探讨在细胞毒药物奥沙利铂诱导下,人骨髓CD34^+前体造血细胞与CD34^-成熟造血细胞发生细胞凋亡及DNA损伤修复信号通路上相关基因表达的情况。方法采用免疫磁珠法分选出16例人骨髓单个核细胞中的CD34^+及CD34^-细胞,并应用流式细胞术检测其纯度。用奥沙利铂(终末浓度为10μg/mL)诱导2种细胞凋亡,检测诱导不同时间的2种细胞凋亡率的差异。提取细胞RNA,并利用基因芯片技术,分析比较药物诱导前后的CD34^+及CD34^-细胞中DNA损伤修复信号通路相关基因的表达情况。结果16份骨髓样本CD34^+阳性率为(4.75±1.82)%;免疫磁珠法分选后,流式细胞术检测CD34^+细胞的纯度为(81.24±5.68)%,所得CD34^-细胞的纯度为(97.67±1.21)%。CD34^+细胞在经过奥沙利铂诱导后4、8、12h,凋亡率分别为(27.44±4.12)%、(53.58±7.89)%,(84.65±6.20)%,CD34^-细胞在诱导后4、8、12h,凋亡率分别为(18.40±3.07)%、(39.11±5.28)%、(71.65±7.73)%,各时间点2种细胞的凋亡率差异均有统计学意义(均P<0.05)。基因芯片结果显示,在加入奥沙利铂诱导前,CD34^+细胞与CD34^-细胞表达差异2倍以上的基因有9种,其中前者有7种基因表达高于后者,2种基因表达低于后者,在奥沙利铂诱导8h后,CD34^+细胞与CD34^-细胞表达差异2倍以上的基因有14种,均为前者高于后者。结论在奥沙利铂诱导下,骨髓前体CD34^+细胞和成熟的CD34^-细胞相比,其DNA损伤修复系统相关基因表达水平较高,但更易于发生细胞凋亡。

Objective To investigate the apoptotic response and the expression of DNA damage repair signaling pathwayrelated genes of human bone marrow hematopoietic CD34~＋progenitor cells and mature CD34-cells and compare the differences between the two kinds of cells.Methods CD34＋and CD34-cells were isolated by MiniMACS immunomagnetic beads from mononuclear cells of 16 human bone marrow samples and their purity was detected by flow cytometry.The two populations of cells were treated by oxaliplatin（10μg/mL）,and the difference in the apoptosis rate between the two cells at different inducing time was detected.Total RNA was extracted from the cells for comparing the expression of DNA damage repair signaling pathway-related genes by Oligo DNA Damage Signaling Pathway Microarray.Results The percentage of CD34~＋cells from the 16 samples was（4.75±1.82）%.After immunomagnetic isolation,the purity of CD34~＋cells was found to be（81.24±5.68）% and that of CD34-cells was（97.67±1.21）%.The apoptosis rates of CD34＋cells treated with oxaliplatin for 4,8and 12hwere（27.44±4.12）%,（53.58±7.89）%,and（84.65±6.20）%,and those of CD34^-cells were（18.40±3.07）%,（39.11±5.28）%,and（71.65±7.73）%,respectively.There were significant differences between the two populations（P0.05）.Gene microarray showed that there were 9genes that had over two-fold differences between CD34~＋cells and CD34^-cells before oxaliplatin treatment,including seven genes upregulated and two genes downregulated in CD34~＋cells.After oxaliplatin treatment,the expression levels of 14 genes that had over two-fold differences were significantly higher in CD34~＋cells than in CD34^-cells.Conclusion Human bone marrow hematopoietic CD34~＋progenitor cells have higher expression levels of DNA damage repair signaling pathway-related genes and are more likely to develop apoptosis after oxaliplatin treatment,when compared with mature CD34^-cells.