丝裂霉素诱发Jurkat细胞DNA损伤修复机制中blm基因的作用研究

本研究通过丝裂霉素(mitomycin C,MMC)诱发Jurkat细胞凋亡,了解Jurkat细胞DNA损伤后blm mRNA水平变化与细胞周期和凋亡之间关系及意义。用流式细胞术检测凋亡率和细胞周期变化;用半定量RT-PCR检测blm mRNA的表达水平。结果表明:0.4 g/L MMC分别诱导Jurkat细胞和正常成纤维细胞后,Jurkat细胞于24小时凋亡率为(11.42±0.013)%,此时(66.08±1.60)%的Jurkat细胞受阻于G2/M期;48小时时Jurkat细胞凋亡率反而下降(8.08±0.27)%,停滞于G2/M期Jurkat细胞下降为(33.96±1.05)%;正常成纤维细胞诱导后24小时与48小时凋亡率变化不明显,G2/M期细胞、G0-G1、S期细胞在24小时和48小时时变化不显著。2组细胞凋亡率和各期细胞所占比例的变化幅度比较显示,其差异显著有统计学意义(p0.01)。Jurkat细胞blm mRNA表达水平不仅明显高于正常成纤维细胞(p0.01),诱导48小时后blm mRNA表达水平明显较诱导24小时后增高,2组细胞间blm mRNA表达水平变化幅度比较差异显著(p0.01)。结论:blm基因在MMC诱发Jurkat细胞DNA损伤修复过程中发挥重要作用,其mRNA表达异常可能是导致白血病耐药的原因之一。

Mechanism Involving blm Gene Underlies Repair of DNA Damage of Jurkat Cells Induced by Mitomycin C

The defect or block of apoptosis is an important factor involved in the drug resistance of tumor cells.Blm gene plays a great role in DNA damage and repair. This study was aimed to explore the relationship of blm gene expression with cell cycle and apoptosis after Jurkat DNA damage.The apoptosis rate and change of cell cycle were detected by flow cytometry,the expression level of blm mRNA in Jurkat cells was determined by semi-quantitative RT-PCR.The results indicated that after induction with 0.4 g/L of mitomycin C（MMC） for 24 hours the apoptosis rate of Jurkat cells were（11.42±0.013）%,and（66.08±1.60）% Jurkat cells were arrested in G2/M phase.After induction for 48 hours,the apoptosis rate of Jurkat cells declined from（11.42±0.013）% to（8.08±0.27）%,and cell count of Jurkat cells arrested in G2/M phase decreased from（66.08±1.60）% to（33.96±1.05）%.When induced with 0.4 g/L of MMC for 24 hours,the apoptosis rate of fibroblasts and the percentage of fibroblasts in G2/M,G0-G1 and S phase all showed no significant change until 48 hours.The range of apoptosis rate and the change of cell percentage in three phases were significantly different between Jurkat cells and fibroblasts（p0.01）.Expression level of blm mRNA in Jurkat cells was remarkably higher than that in normal fibroblasts（p0.01）,at 48 hours expression level of blm mRNA was remarkably higher than that at 24 hours.The 2 groups showed clear difference of blm mRNA expression after treated by MMC（p0.01）.It is concluded that the blm gene may play a significant role in repair of DNA damage of Jurkat cells after MMC induction.Abnormal expression of blm is correlated to the drug resistance of leukemia cells.