盐酸吉西他滨对非小细胞肺癌患者骨髓CD34+造血细胞的毒性研究

摘要

Objective To investigate human bone marrow hematopoietic CD34 + progenitor cells induced by gemcit-abine hydrochloride in patients with non-small cell lung cancer,the apoptotic response and gene expression change of DNA dam-age signaling pathway. Methods CD34+ hematopoietic cells were isolated and purified by MiniMACS immunomagnetic beads from mononuclear cells of bone marrow samples in 80 patients with non-small cell lung cancer. The purity of CD34 fractions was detected by flow cytometry. The CD34+ cells were treated by gemcitabine,after 0 h,4 h,8 h,12 h,the apoptosis rates were detec-ted by flow cytometry,total RNA were extracted from the cells,and gene expression of DNA repair signaling pathway were ana-lyzed by Oligo DNA damage signaling pathway microarray. Results The percentage of CD34+ cells from 80 samples were(6. 06 ± 1. 61)%;There was no significant difference in CD34 expression between patients with different TNM stages,P>0. 05. After immunomagnetic isolation,purity of CD34 +cells were (88. 63 ± 7. 29) %. when treated by gemcitabine for 0 hour,4hours, 8hours and 12 hours,the apoptosis rates of CD34+ cells were(5 ± 1. 37)%,(18 ± 4. 76)%,(39 ± 8. 15)%,(56 ± 9. 64)%, there were significant differences between them,P<0. 05. In the detection of genes related to DNA repair signaling pathway,over two-fold differences of 13 genes between bone marrow CD34+cells treated for 0 hour and 12 hours,including 9 kinds of genes ex-pression were higher in the latter than the former,4 kinds of genes expression were lower in the latter than the former. Conclusion Human bone marrow hematopoietic CD34+ progenitor cells,in patients with non-small cell lung cancer,can be induced to apop-tosis when treated by gemcitabine hydrochloride,the apoptosis rate can rise with time extended,accompanied by changes of gene expression levels of damage signaling pathway.%目的 探讨非小细胞肺癌患者骨髓CD34+前体造血细胞在细胞毒药物盐酸吉西他滨诱导下,发生细胞凋亡情况及DNA损伤修复信号通路上相关基因表达的变化.方法 采用免疫磁珠法分选出80例非小细胞肺癌患者骨髓单个核细胞中的CD34+细胞,并应用流式细胞术检测其纯度.用盐酸吉西他滨(终末浓度为10μg/ml)诱导CD34+细胞凋亡,检测诱导不同时间点的细胞凋亡率的差异.提取细胞RNA,利用基因芯片技术,将药物诱导前后的CD34+细胞在DNA损伤修复信号通路的相关基因表达情况进行检测,并分析比较.结果 80份骨髓样本CD34阳性率为(6.06±1.61)%;TNM分期不同的患者样本间CD34的表达无统计学差异,P>0.05.免疫磁珠法分选后,流式细胞术检测CD34+细胞的纯度为(88.63±7.29)%;CD34+细胞在经过盐酸吉西他滨诱导0 h、4 h、8 h、12 h后,凋亡率分别为(5±1.37)%、(18±4.76)%、(39±8.15)%、(56±9.64)%,各时间点细胞的凋亡率之间有统计学差异,P<0.05.基因芯片结果显示,骨髓CD34+细胞在盐酸吉西他滨诱导凋亡0 h与诱导12 h后相比,表达差异2倍以上的基因有13种,其中9种基因表达是升高的,4种基因表达降低.结论 盐酸吉西他滨可以诱导非小细胞肺癌患者骨髓前体CD34+细胞发生凋亡,随着药物作用时间延长,细胞凋亡率上升;DNA损伤修复系统中,部分基因表达水平出现变化.